Eckert Animal Physiology: Mechanisms and Adaptations (Fourth Edition)

F O U R T H E D I T I O N ECKERT ANIMAL PHYSIOLOGY MECHANISMS AND ADAPTATIONS DAVID R A N D A L L U N I V E A S ~...

Author: David J. Randall | Warren W. Burggren | Kathleen French | Roger Eckert

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F O U R T H

E D I T I O N

ECKERT

ANIMAL PHYSIOLOGY MECHANISMS AND ADAPTATIONS

DAVID

R A N D A L L

U N I V E A S ~O T FW B R I T I S HC O L U M B I A

1

WARREN

B U R G G R E N

KATHLEEN

FRENCH

U N I V E R S I TOYF C A L I F O R N I A S, A N D L E G O

W I T H C O N T R I B U T I O NBSY

RUSSELL FERNALD S T A N F O R DU N I V E R S I T Y

W. H. Freeman and Company New Ynrk

ACQUISITIONS EDITOR: Deborah Allen DEVELOPMENT EDITOR: Kay Ueno PROJECT EDITOR: Kate Ahr PHOTO RESEARCH: Larry Marcus COVER DESIGNER: Michael Mendelsohn, Design 2000, Inc FRONTCOVER PHOTOGRAPH: Arctic

fox, Canada O Daniel J. Cox/Tony Stone Images

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'

TEXT DESIGNERS: Michael Mendelsohn, Design 2000, Inc.; Victoria Tomaselli

ILLUSTRATION COORDINATOR: Bill Page ILLUSTRATION: Fine Line Illustrations; Medical and Scientific Illustration, William C. Ober, M D and Claire Garrison, RN, BA PRODUCTION COORDINATOR: Maura Studley COMPOSITION: Progressive Information Technologies MANUFACTURING: RR Donnelley & Sons Company

L~braryof Congress Cataloging-in-PublicationData Randall, Dav~dJ., 1938Eckert an~malphys~ology:mechanisms and adaptations/Dav~d Randall, Warren Burggren, Kathleen French.-4th ed. P. cm. references and ~ndex. Includes blbl~ograph~cal ISBN 0-7167-2414-6 (hardcover) 1.Physiology. I. Burggren, Warren. 11. French, Kathleen. 111. Tltle. QP31.2.R36 1997 591.1-dc20 96-31713 CIP Copyright O 1978, 1983, 1988, 1997 by W. H. Freeman and Company. All rights reserved. No part of this book may be reproduced by any mechanical, photographic, or electronic process, or in the form of a phonographic recording, nor may it be stored in a retrieval system, transmitted, or otherwise copied for public or private use, without written permission from the publisher. Printed in the United States of America Second printing, 1997, RRD

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ABOUT THE AUTHORS

DAVID RANDALL

contributions to the field of fish physiology. A frequent symposium lecturer on fish physiology and other subjects, most recently in Brazil, France, Germany, Italy, the People's Republic of China, Russia, and the United States. He has worked with both the World Health Organization and the United States Environmental Protection Agency in developing ammonia criteria. Widely published as author and co-author in leading journals, Randall is co-editor of the noted series Fish Physiolog?, (AcademicPress), of which 15 volumes are in print. Volume 16, subtitled "Deep-sea Fish," will appear in 1997. Along with his other duties, Randall co-teaches third year courses in vertebrate physiology and environmental physiology. His research interests concern the interactions between gas and ion exchange across fish gills. ......................................... ....................................... WARREN BURGGREN late them change over the course of development. Warren Burggren has taught in physiology for 23 years, Burggren has been actively involved in symposia, seminars, and formal extramural researchltraining activities and has been a professor of biological sciences at the University of Nevada at Las Vegas since 1992. Courses he in many countries. A co-author of The Evolution of Air has taught at UNLV and at the University of MassachuBreathing in Vertebrates (Cambridge University Press, 1981),Burggren has been a frequent contributor since setts, where he was Professor of Zoology from 1987 1980 to edited collections of physiology, including through 1991, include Human Anatomy and Physiology, Bioenergetics, Introductory Zoology, and Comparative Presser's Comparative Animal Physiology, Fourth EdiPhysiology. Burggren's research interest include develoption (Wiley-Liss, 1991). Burggren co-edited Environmental physiology, comparative animal physiology, and mental Physiology of the Amphibia (University of environmental and ecological physiology. In particular, Chicago Press, 1992), and more recently co-edited Dehis research focuses on the ontogeny of respiratory and velopment of Cardiovascular Systems; Molecules to Orcardiovascular systems, and how the systems that reguganisms (CambridgeUniversity Press, 1997). A prominent fish physiologist and a leading expert in respiratory and circulatory physiology, David Randall collaborated with the late Roger Eckert on the earlier editions of Animal Physiology and continues his contribution in the fourth. A faculty member at the University of British Columbia in Vancouver, Canada, since 1963, and full professor since 1973, Randall was appointed Associate Dean of Graduate Studies in 1990. Elected a fellow of the Royal Society of Canada in 1981, Randall has been both a Guggenheim and a Killam fellow, and was awarded the prestigious Fry Medal for research contributions to zoology by the Canadian Society of Zoology in 1993. In 1995, he received the Award of Excellence from the American Fisheries Society for

........................................ .......................................

KATHLEEN FRENCH

.

A neurobiologist at the University of California at San Diego since 1985, Kathleen French has for 10 years taught upper division courses in embryology, mammalian physiology for premedical students, and cellular neurobiology. In addition, at UCSD, French participates in a training program to instruct science teaching assistants in the techniques and philosophy of teaching. She also serves on the faculty of the Neuroscience and Behavior Course at the Marine Biological Laboratories in Woods Hole, Massachusetts, an intensive course designed primarily for graduate students and post-doctoral fellows. French brings her expertise in-and love of-teaching to her

role as co-author of the current edition of Animal Physiology, along with a lifelong interest in the nervous systems of organisms from a broad range of phyla. As an Associate Project Scienrist at UCSD, French's research focuses on the control of neuronal development, a topic that she has studied in various invertebrate species. Her current research concerns the cellular events that control differentiation of identified neurons in the medicinal leech, with an emphasis on the cellular physiology of embryonic neurons and the effects of cell-cell contacts. She has been the author and co-author of numerous published research and review articles in journals including the Journal of Neuroscience and Journal of Neurophysiology.

PART I PRINCIPLES OF PHYSIOLOGY 1 STUDYING ANIMAL PHYSIOLOGY

2 EXPERIMENTAL METHODS FOR EXPLORING PHYSIOLOGY 3 MOLECULES, ENERGY, AND BIOSYNTHESIS

4 MEMBRANES, CHANNELS, AND TRANSPORT

PART II PHYSIOLOGICAL PROCESSES 5 THE PHYSICAL BASIS OF NEURONAL FUNCTION 6 COMMUNICATION ALONG AND BETWEEN NEURONS

7 SENSING THE ENVIRONMENT 8 GLANDS: MECHANISMS AND COSTS OF SECRETION 9 HORMONES: REGULATION AND ACTION 10 MUSCLES AND ANIMAL MOVEMENT

351

11 BEHAVIOR: INITIATION, PATTERNS, AND CONTROL

405

PART Ill INTEGRATION OF PHYSIOLOGICAL SYSTEMS 465 12 CIRCULATION

467

13 GAS EXCHANGE AND ACID-BASE BALANCE

517

14 IONIC AND OSMOTIC BALANCE

571

15 ACQUIRING ENERGY: FEEDING, DIGESTION. AND METABOLISM

627

16 USING ENERGY: MEETING ENVIRONMENTAL CHALLENGES

665

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CONTENTS

i I

I

I

Preface Acknowledgments

ix xvii

Structural Analys~sof Cells Cell Culture

BIOCHEMICAL ANALYSIS Measurlng Composit~on:What Is Present Measurlng Concentration: How Much IS Present

PART I PRINCIPLES OF PHYSIOLOGY CHAPTER 1 STUDYING ANIMAL PHYSIOLOGY

3

THE SUBDISCIPLINES OF ANIMAL PHYSIOLOGY WHY STUDY ANIMAL PHYSIOLOGY?

4 4

Sc~ent~fic Curlos~ty Commerc~aYAgr~cultural Appl~cat~ons Inslghts into Human Physiology

CENTRAL THEMES IN ANIMAL PHYSIOLOGY Structure-Funct~onRelat~onsh~ps Adaptation, Accl~mat~zat~on, and Accl~mat~on Homeostas~s Feedback-Control Systems C o n f o r m ~and t ~ Regulation

LITERATURE OF PHYSIOLOGICAL SCIENCES SPOTLIGHT 1-1 THE CONCEPT OF FEEDBACK ANIMAL EXPERIMENTATION IN PHYSIOLOGY Summary Rev~ewQuest~ons Suggested Readlngs

4 4 4

4 5 5 7 8 9 10

12 13 13 14 14

EXPERIMENTS WITH ISOLATED ORGANS AND ORGAN SYSTEMS OBSERVING AND MEASURING ANIMAL BEHAVIOR The Power of Behav~oralExperiments Methods In Behav~oralResearch

IMPORTANCE OF PHYSIOLOGICAL STATE IN RESEARCH Summary Revlew Quest~ons Suggested Readlngs

CHAPTER 3 MOLECULES, ENERGY, AND BIOSYNTHESIS ORIGIN OF KEY BIOCHEMICAL MOLECULES ATOMS, BONDS, AND MOLECULES THE SPECIAL ROLES OF H, 0, N, AND C IN LIFE PROCESSES WATER: THE UNIQUE SOLVENT The Water Molecule Propert~esof Water Water as a Solvent

PROPERTIES OF SOLUTIONS

CHAPTER 2 EXPERIMENTAL METHODS FOR EXPLORING PHYSIOLOGY FORMULATING AND TESTING HYPOTHESES The August Krogh Prlnc~ple Experimental Deslgn and Phys~olog~cal Level

MOLECULAR TECHNIQUES Traclng Molecules w ~ t hRadlolsotopes Traclng Molecules wlth Monoclonal Antlbod~es Genetic Englneerlng

CELLULAR TECHNIQUES Uses of M~croelectrodesand Mlcroplpettes

Concentration, Coll~gatlveProperties, and Activ~ty Ionizat~onof Water Ac~dsand Bases The B~olog~cal Importance of p H Henderson-Hasselbalch Equat~on Buffer Systems Electrlc Current ~nAqueous Solutions

SPOTLIGHT 3-1 ELECTRICAL TERMINOLOGY AND CONVENTIONS Blndlng of Ions to Macromolecules

X

CONTENTS

..............................

.................................

BIOLOGICAL MOLECULES Llplds Carbohydrates Protelns Nucle~cAc~ds ENERGETICS O F LIVING CELLS Energy: Concepts and Defin~t~ons Transfer of Chem~calEnergy by Coupled React~ons ATP: Energy Carrier of the Cell Temperature and React~onRates ENZYMES: GENERAL PROPERTIES Enzyme Speclficlty and Actlve S~tes Mechanism of Catalys~sby Enzymes Effect of Temperature and pH on Enzymat~cReact~ons Cofactors Enzyme Klnetlcs Enzyme Inhlb~t~on REGULATION O F METABOLIC REACTIONS Control of Enzyme Synthes~s Control of Enzyme Actlvlty METABOLIC PRODUCTION O F ATP Oxldat~on,Phosphorylatlon, and Energy Transfer Glycolys~s Cltr~cAc~dCycle Effic~encyof Energy Metabol~sm Oxygen Debt Summary Rev~ewQuest~ons Suggested Readlngs

Ion Gradients as a Source of Cell Energy Coupled Transport MEMBRANE SELECTIVITY Selectlvlty for Electrolytes Selectlv~tyfor Nonelectrolytes ENDOCYTOSIS AND EXOCYTOSIS Mechanlsms of Endocytosis Mechanlsms of Exocytosls

CHAPTER 4 MEMBRANES, CHANNELS, AND TRANSPORT MEMBRANE STRUCTURE AND ORGANIZATION Membrane Composlt~on Flu~dMosa~cMembranes SPOTLIGHT 4-1 THE CASE FOR A LIPID BILAYER MEMBRANE Var~at~on In Membrane Form CROSSING THE MEMBRANE: AN OVERVIEW D~ffus~on Membrane Flux Osmos~s Osmolarlty and Tonlclty Electr~calInfluences on Ion D ~ s t r ~ b u t ~ o n Donnan Equll~brlurn OSMOTIC PROPERTIES OF CELLS Ion~cSteady State Cell Volume PASSIVE TRANSMEMBRANE MOVEMENTS Slmple D~ffus~on through the Llp~dBllayer through Membrane Channels D~ffus~on SPOTLIGHT 4-2 ARTIFICIAL BILAYERS Facllltated Transport across Membranes ACTIVE TRANSPORT The Na+lK+Pump as a Model of A a v e Transport

JUNCTIONS BETWEEN CELLS

Gap Junct~ons Tlght Junct~ons EPITHELIAL TRANSPORT Actlve Salt Transport across an Ep~thellum Transport of Water Summary Rev~ewQuest~ons Suggested Readlngs

PART II PHYSIOLOGICAL PROCESSES CHAPTER 5 THE PHYSICAL BASIS OF NEURONAL FUNCTION OVERVIEW OF NEURONAL STRUCTURE, FUNCTION, AND ORGANIZATION Transmlss~onof S~gnalsIn a Slngle Neuron Transm~ss~on of Slgnals Between Neurons Organ~zat~on of the Nervous System MEMBRANE EXCITATION Measuring Membrane Potentials Dlstlngu~shlngPasslve and Actlve Membrane Electr~calPropert~es SPOTLIGHT 5-1 THE DISCOVERY OF "ANIMAL ELECTRICITY" Role of Ion Channels PASSIVE ELECTRICAL PROPERTIES O F MEMBRANES Membrane Res~stanceand Conductance Membrane Capac~tance ELECTROCHEMICAL POTENTIALS The Nernst Equat~on:Calculatlng the Equ~l~brlum Potent~alfor Slngle Ions SPOTLIGHT 5-2 A QUANTITATIVE CONSIDERATION OF CHARGE SEPARATION ACROSS MEMBRANES The Goldman Equation: Calculatlng the Equll~brlumPotent~alfor Mult~pleIons THE RESTING POTENTIAL Role of Ion Grad~entsand Channels Role of Actlve Transport ACTION POTENTIALS General Propert~esof Act~onPotent~als Ion~cBaas of the Act~onPotent~al SPOTLIGHT 5-3 THE VOLTAGE-CLAMP METHOD Changes In Ion Concentrat~ondurlng Exc~tat~on OTHER ELECTRICALLY EXCITED CHANNELS Summary

CONTENTS

xi

...................................... Review Questions Suggested Readings

CHAPTER 6 COMMUNICATION ALONG AND BETWEEN NEURONS TRANSMISSION OF SIGNALS IN THE NERVOUS SYSTEM: AN OVERVIEW TRANSMISSION OF INFORMATION WITHIN A SINGLE NEURON Pass~veSpread of Electr~calS~gnals Propagatlon of Actlon Potentials Speed of Propagat~on Rapid, Saltatory Conduction In Myelmated Axons SPOTLIGHT 6-1 EXTRACELLULAR SIGNS OF IMPULSE CONDUCTION SPOTLIGHT 6-2 AXON DIAMETER AND CONDUCTION VELOCITY TRANSMISSION OF INFORMATION BETWEEN NEURONS: SYNAPSES / Synapt~cStructure and Function: Electr~calSynapses Synaptlc Structure and Function: Chermcal Synapses Fast Chemical Synapses SPOTLIGHT 6-3 PHARMACOLOGICAL AGENTS USEFUL IN SYNAPTIC STUDIES SPOTLIGHT 6-4 CALCULATION OF REVERSAL POTENTIAL PRESYNAFTIC RELEASE OF NEUROTRANSMITTERS Quanta1 Release of Neurotransm~tters Depolarizat~on-ReleaseCoupllng Nonsplking Release THE CHEMICAL NATURE OF NEUROTRANSMITTERS Fast, D~rectNeurotransm~ss~on Slow, Indirect Neurotransmission POSTSYNAPTIC MECHANISMS Receptors and Channels in Fast, Direct Neurotransmission Receptors m Slow, Ind~rectNeurotransm~ss~on Neuromodulat~on INTEGRATION AT SYNAPSES SYNAPTIC PLASTICITY Homosynaptlc Modulation: Facil~tatlon Homosynapt~cModulat~on:Posttetan~cPotentlation Heterosynapt~cModulation " Long Term Potent~at~on Summary Rev~ewQuestions Suggested Read~ngs

Encod~ngStlmulus Intensities Input-Output Relations Range Fractionation Control of Sensory Senslt~v~ty THE CHEMICAL SENSES: TASTE AND SMELL Mechan~smsof Taste Recept~on Mechanisms of Olfactory Reception MECHANORECEPTION H a ~ Cells r Organs of Equ~librlum The Vertebrate Ear An Insect Ear ELECTRORECEPTION THERMORECEPTION VISION Optlc Mechan~sms:Evolut~onand Funct~on Compound Eyes SPOTLIGHT 7-1 SUBJECTIVE CORRELATES OF PRIMARY PHOTORESPONSES The Vertebrate Eye Photoreception: Convert~ngPhotons into Neuronal Signals SPOTLIGHT 7-2 THE ELECTRORETINOGRAM SPOTLIGHT 7-3 LIGHT, PAINT, AND COLOR VISION LIMITATIONS O N SENSORY RECEPTION Summary Revlew Quest~ons Suggested Read~ngs

224 225 226 226 231 232 235 238 238 241 242 248 248 250 251 252 253

256 257 261 263 268 269 270 271 271

CHAPTER 8 GLANDS: MECHANISMS AND COSTS OF SECRETION CELLULAR SECRETION Types and Funct~onsof Secretions Surface Secretions: The Cell Coat and Mucus Packag~ngand Transport of Secreted Material SPOTLIGHT 8-1 SUBSTANCES WITH SIMILAR STRUCTURES AND FUNCTIONS SECRETED BY DIFFERENT ORGANISMS Storage of Secreted Substances Secretory Mechanlsms GLANDULAR SECRETIONS Types and General Properties of Glands Endocrine Glands Exocr~neGlands ENERGY COST OF GLANDULAR ACTIVITY Summary Rev~ewQuestions Suggested Read~ngs

CHAPTER 7 SENSING THE ENVIRONMENT

CHAPTER 9 HORMONES: REGULATION 301 AND ACTION

GENERAL PROPERTIES OF SENSORY RECEPTION Properties of Receptor Cells Common Mechanisms and Molecules of Sensory Transduction From Transduction to Neuronal Output

ENDOCRINE SYSTEMS: OVERVIEW Chem~calTypes and General Functions of Hormones Regulat~onof Hormone Secretion NEUROENDOCRINE SYSTEMS Hypothalmlc Control of the Anterlor Pitu~tary Gland

302 3 02 303 303 304

xii

CONTENTS

........................................

Glandular Hormones Released from the Anterlor P~tuitaryGland 305 Neurohormones Released from the Posterlor P~tu~tary Gland 308 SPOTLIGHT 9-1 PEPTIDE HORMONES 310 CELLULAR MECHANISMS O F HORMONE ACTION 311 Lipld-Soluble Hormones and Cytoplasmic Receptors 311 Lipld-Insoluble Hormones and Intracellular Slgnal~ng 312 SPOTLIGHT 9-2 AMPLIFICATION BY ENZYME CASCADES 322 PHYSIOLOGICAL EFFECTS O F HORMONES 328 Metabolic and Developmental Hormones 328 Hormones That Regulate Water and Electrolyte Balance 336 Reproduct~veHormones 338 Prostagland~ns 342 HORMONAL ACTION IN INVERTEBRATES 343 Summary 346 Revlew Quest~ons 348 Suggested Readings 349

CHAPTER 10 MUSCLES AND ANIMAL MOVEMENT STRUCTURAL BASIS O F MUSCLE CONTRACTION Myofilament Substructure Contraction of Sarcomeres: The Slldlng Fllament Theory Cross-Bridges and the Productlon of Force SPOTLIGHT 10-1 PARALLEL AND SERIES ARRANGEMENTS: THE GEOMETRY OF MUSCLE MECHANICS OF MUSCLE CONTRACTION Relatlon Between Force and Shortening Veloclty SPOTLIGHT 10-2 SKINNED MUSCLE FIBERS Effect of Cross-Brldges on Force-Veloclty Relatlon REGULATION OE CONTRACTION Role of Calclum In Cross-Brldge Attachment Excltatlon-Contraction Coupllng Contraction-Relaxat~on Cycle THE TRANSICNT PRODUCTION O F FORCE Serles Elast~cComponent The Act~veState Twitches and Tetanus ENERGETICS O F MUSCLE CONTRACTION ATP Usage by Myosln ATPase and Calclum Pumps Regeneration of ATP durlng Muscle Actlvlty FIBER TYPES I N VERTEBRATE SKELETAL MUSCLE Class~ficat~on of Flber Types Functional Rat~onalefor Different Flber Types ADAPTATION OE MUSCLES FOR VARIOUS ACTIVITIES Adaptatlon for Power: Jumplng Frogs Diversity of Function: Swlmmmg Fish Adaptatlon for Speed: Sound Productlon Hlgh-Power, Hlgh Frequency Muscles:Asynchronous Fllght Muscles NEURONAL CONTROL OF MUSCLE CONTRACTION

Motor Control in Vertebrates Motor Control ln Arthropods CARDIAC MUSCLE SMOOTH MUSCLE Summary Revlew Questions Suggested Readings

CHAPTER 11 BEHAVIOR: INITIATION, PATERNS, AND CONTROL

405

SPOTLIGHT 11-1 BEHAVIOR IN ANIMALS THAT LACK A NERVOUS SYSTEM 403 EVOLUTION O F NERVOUS SYSTEMS 408 ORGANIZATION O F THE VERTEBRATE NERVOUS SYSTEM 412 Major Dlv~slonsof the Central Nervous System 413 420 The Automatic Nervous System ANIMAL BEHAVIOR 423 Bas~cBehavioral Concepts 423 426 Examples of Behavlor PROPERTIES OF NEURONAL CIRCUITS 432 Pieces of the Neuronal Puzzle 433 Sensory Networks 434 $SPOTLIGHT 11-2 TUNING CURVES: THE RESPONSE OF A NEURON PLOllED AGAINST THE PARAMETERS OF A STIMULUS 436 SPOTLIGHT 11-3 SPECIFICITY OF NEURONAL CONNECTIONS AND INTERACTIONS 447 Motor Networks 453 Summary 461 Revlew Questions 462 462 Suggested Reahngs

PART Ill INTEGRATION OF PHYSIOLOGICAL SYSTEMS CHAPTER 12 CIRCULATION GENERAL PLAN O F T H E CIRCULATORY SYSTEM Open C~rculat~ons Closed C~rculatlons T H E HEART Electrical Act~v~ty of the Heart Coronary Clrculat~on Mechanical Properties of the Heart SPOTLIGHT 12-1 THE FRANK-STARLING MECHANISM The Pericardlum Vertebrate Hearts: Comparative Functional Morphology HEMODYNAMICS Laminar and Turbulent Flow Relat~onshipbetween Pressure and Flow T H E PERIPHERAL CIRCULATION Arterlal System Venous System Capillaries and the M~croc~rculat~on T H E LYMPHATIC SYSTEM

... CONTENTS xi11 ...................................... CIRCULATION AND THE IMMUNE RESPONSE REGULATION OF CIRCULATION Control of the Central Cardiovascular System Control of the M~croc~rculat~on CARDIOVASCULAR RESPONSE T O EXTREME CONDITIONS Exerc~se D~vmg Hemorrhage Summary Rev~ewQuestions Suggested Readings

CHAPTER 13 GAS EXCHANGE AND ACID-BASE BALANCE GENERAL CONSIDERATIONS SPOTLIGHT 13-1 EARLY EXPERIMENTS ON GAS EXCHANGE IN ANIMALS OXYGEN AND CARBON DIOXIDE IN BLOOD Resp~ratoryP~gments SPOTLIGHT 13-2 THE GAS LAWS Oxygen Transport In Blood de m Blood Carbon D ~ o x ~ Transport Transfer of Gases to and from the Blood REGULATION O F BODY pH Hydrogen Ion Production and Excret~on Hydrogen Ion D~str~but~on between Compartments Factors Influenc~ngIntracellular pH Factors Influenc~ngBody pH GAS TRANSFER I N AIR: LUNGS AND OTHER SYSTEMS Funct~onalAnatomy of the Lung Pulmonary C~rculat~on SPOTLIGHT 13-3 LUNG VOLUMES Vent~lationof the Lung Pulmonary Surfactants Heat and Water Loss across the Lung Gas Transfer In B~rdEggs Insect Tracheal Systems GAS TRANSFER IN WATER: GILLS Flow and Gas Exchange across G~lls Funct~onalAnatomy of the G~ll REGULATION OF GAS TRANSFER AND RESPIRATION Ventdat~on-to-Perfus~on Rat~os Neural Regulat~onof Breath~ng RESPIRATORY RESPONSES T O EXTREME CONDITIONS Reduced Oxygen Levels (Hypox~a) Increased Carbon D ~ o x ~ Levels de (Hypercapma) D ~ v ~ by n gAir-Breathmg An~mals Exerc~se SWIMBLADDERS: OXYGEN ACCUMULATION AGAINST LARGE GRADIENTS The Rete M~rab~le Oxygen Secret~on

Summary Review Questions Suggested Readings

CHAPTER 14 IONIC AND OSMOTIC BALANCE PROBLEMS OF OSMOREGULATION OBLIGATORY EXCHANGE OF IONS AND WATER Grad~entsBetween the An~maland the Env~ronment Surface-to-VolumeRatlo Permeab~l~ty of the Integument Feedmg, Metabol~cFactors, and Excret~on Temperature, Exerc~se,and Resp~rat~on OSMOREGULATORS AND OSMOCONFORMERS OSMOREGULATION IN AQUEOUS AN TERRESTRIAL ENVIRONMENTS Water-Breathmg An~mals hr-breathmg Animals OSMOREGULATORY ORGANS MAMMALIAN KIDNEY Anatomy of the Mammakn l d n e y Ur~neProduct~on SPOTLIGHT 14-1 RENAL CLEARANCE Regulat~onof pH by the K~dney Ur~ne-ConcentratmgMechan~sm SPOTLIGHT 14-2 COUNTERCURRENTSYSTEMS Control of Water Reabsorpt~on NONMAMMALIAN VERTEBRATE KIDNEYS EXTRARENAL OSMOREGULATORY ORGANS IN VERTEBRATES Salt Glands F~shG~lls INVERTEBRATE OSMOREGULATORY ORGANS F~ltrat~on-Reabsorpt~on Systems Secretory-Reabsorpt~onSystems EXCRETION OF NITROGENOUS WASTES Ammon~a-Excretmg(Ammonotel~c)An~mals Urea-Excret~ng(Ureotehc)An~mals Ur~cAc~d-Excretmng(Ur~cotel~c) An~mals Summary Rev~ewQuest~ons Suggested Read~ngs

CHAPTER 15 ACQUIRING ENERGY: FEEDING, DIGESTION, AND METABOLISM FEEDING METHODS Food Absorpt~onthrough Extenor Body Surfaces Endocytos~s Filter Feeding Fluid Feed~ng of Prey Se~z~ng Herb~voryand Graz~ngTo Collect Food OVERVIEW OF ALIMENTARY SYSTEMS Headgut: Food Recept~on Foregut: Food Conduct~on,Storage, and Digestion M~dgut:Chemical D~gest~on and Absorpt~on

571 571 574 574 574 575 577 5 78 580 581 581 584 587 587 588 590 595 601 603

604 606 608 608 608 613 616 616 617 620 621 623 624 624 625 625

xiv

CONTENTS

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Hindgut: Water and Ion Absorption and Defecation Dynamics of Gut Structure-Influence of Diet MOTILITY OF THE ALIMENTARY CANAL Muscular and Ciliary Mot~lity Peristalsis Control of Motility GASTROINTESTINAL SECRETIONS Exocr~neSecret~onsof the Ahrnentary Canal Control of Dlgestlve Secret~ons SPOTLIGHT 15-1 BEHAVIORAL CONDITIONING IN FEEDING AND DIGESTION ABSORPTION Nutnent Uptake in the Intestme Blood Transport of Nutr~ents Water and Electrolyte Balance tn the Gut NUTRITIONAL REQUIREMENTS Energy Balance Nutr~entMolecules Summary Review Questions Suggested Readlngs

643 644 644 645 645 646 649 650 653

654 657 657 658 659 66 1 66 1 66 1 663 664 664

CHAPTER 16 USING ENERGY MEETING ENVIRONMENTAL CHALLENGES 665 THE CONCEPT O F ENERGY METABOLISM MEASURING METABOLIC RATE Basal and Standard Metabol~cRate Metabol~cScope D~rectCalor~rnetry SPOTLIGHT 16-1 ENERGY UNITS (OR WHEN IS A CALORIE NOTA CALORIE?) Indlrect Calorimetry-Measurement from Food Intake and Waste Excretion Ind~rectMeasures of Metabol~cRate Resp~ratoryQuohent Energy Storage Spec~ficDynam~cAct~on BODY SIZE AND METABOLIC RATE SPOTLIGHT 16-2 THE REYNOLDS NUMBER: IMPLICATIONS FOR BIG AND SMALL ANIMALS TEMPERATURE AND ANIMAL ENERGETICS Temperature Dependence of Metabohc Rate Determ~nantsof Body Heat and Temperature Temperature Classlficat~onsof Anlmals

665 666 666 667 668

668 668 669 670 671 672 672

676 677 677 680 682

TEMPERATURE RELATIONS OF ECTOTHERMS Ectotherms In Freezlng and Cold Env~ronments, Ectotherms ln Water and Hot Envlronments Costs and Benefits of Ectothermy: A Compar~son w ~ t hEndothermy TEMPERATURE RELATIONS O F HETEROTHERMS TEMPERATURE RELATIONS O F ENDOTHERMS Mechan~smsfor Body Temperature Regulation Thermostatic Regulat~onof Body Temperature Fever DORMANCY: SPECIALIZED METABOLIC STATES Sleep Torpor H~bernat~on and Wlnter Sleep Estlvat~on ENERGETICS O F LOCOMOTION An~malSue, Velocity, and Cost of Locomohon Physlcal Factors Affecting Locomotlon Aquat~c,Aer~al,and Terrestrial Locomotlon BODY RHYTHMS AND ENERGETICS C~rcadlanRhythms Nonclrcad~anEndogenous Rhythms Temperature Regulat~on,Metabohsm, and B~olog~cal Rhythms ENERGETICS O F REPRODUCTION Patterns of Energet~cInvestment In Reproduct~on The "Cost" of Gamete Product~on Parental Care as an Energy Cost of Reproduct~on ENERGY, ENVIRONMENT, AND EVOLUTION Summary Rev~ewQuest~ons Suggested Readings

Appendzx 1: SI Units Appendlx 2: Logs and Exponentials Appendrx 3: Conversions, Formulas, Physzcal and Chemrcal Constants, Definitions References Czted Glossary Index

I

t is nearly ten years since the third edition of Animal

Physiology first appeared, written by Roger Eckert with the help of David Randall. Roger died in 1986 while revising the third edition, which was completed by George Augustine and David Randall. That book formed the basis for the fourth edition, which is fittingly referred to as Eckert Animal Physiology. Although this new edition has been extensively revised and redesigned, the approach that so successfully characterized earlier editions has been maintained: the use of comparative examples to illustrate general principles, often supported by experimental data. In addition, we have emphasized the principle of homeostasis, and we have updated the molecular and cellular coverage. Retained in this edition is the comprehensivecoverage of tissues, organs, and organ systems. Cellular and molecular topics are integrated early in the book so that common threads are developed to explain and compare the interactions between regulated physiological systems that produce coordinated responses to environmental change in a wide variety of animal groups. The basic principles and mechanisms of animal physiology and the adaptations of animals that enable them to exist in so many different environments form the central theme of this book. The diversity and adaptations of the several million species that make up the animal kingdom provide endless fascination and delight to those who love nature. Not the least of this pleasure derives from a consideration of how the bodies of animals function. At first it might appear that with so many kinds of animals adapted to such a variety of lifestyles and environments, the task of understanding and appreciating the physiology of animals would be overwhelming. Fortunately (for scientist and student alike), the concepts and principles that provide a basis for understanding animal function are relatively few, for evolution has been conservative as well as inventive. A beginning course in physiology is a challenge for both teacher and student because of the interdisciplinary nature of the subject, which integrates chemistry, physics, and

biology. Most students are eager to come to grips with the subject and get on with the more exciting levels of modern scientific insight. For this reason, Eckert Animal Physiology has been organized to present the essential background material in a way that allows students to review it on their own and go on quickly to consider animal function and to understand its experimental elucidation. Eckert Animal Physiology develops the major concepts in a simple and direct manner, stressing principles and mechanisms over the compilation of information and illustrating the functional strategies of animals that have evolved within the bounds of chemical and physical possibility. Common principles and patterns, rather than exceptions, are emphasized. Examples are selected from the broad spectrum of animal life, consciously illustrating similarities between organisms; for example, similar compounds are associated with reproduction in both humans and yeast. Thus, the more esoteric and peripheral details receives only passing attention, or none at all, so as not to distract from central ideas. We use the device of a narrative, describing experiments, to provide a feeling for methods of investigation while presenting information.

ORGANIZATION OF THE BOOK For the first time, the chapters are organized into three parts, which we feel will promote an understanding of animals as integrated systems at every level of organization. Each part is introduced by an opening statement that gives students an overview of the material to follow. Part I contains four chapters and is concerned with the central principles of physiology. Part I1 (Chapters 5 - 11)deals with physiological processes, while Part I11 (Chapters 12-16) discusses how these basic processes are integrated in animals living in a variety of environments. All 16 chapters have been extensively reworked and reorganized to stay abreast of new scienthc developments.

xvi

PREFACE

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NEW TO THIS EDITION A new chapter on methodology (Chapter 2) in Part I, in which some of the latest molecular techniques are discussed and illustrated, along with traditional methods. This emphasis on molecular coverage continues throughout the book; Chapters 5,6, and 7, for example, are updated with recent insights into the cellular and molecular underpinnings of membrane excitation, synaptic transmission, and sensory transduction. Part I1 features a new chapter (Chapter 8, Glands: Mechanisms and Costs of Secretion), which brings together information on an important, but frequently neglected, effector system. In Part 11, Chapter 11 (Behavior: Initiation, Patterns, and Control) preserves and expands the descriptions of vertebrate and invertebrate nervous systems found in previous editions, presenting an up-to-date view of systems neurobiology, one of the fastest-growing areas of neurobiology. Several concepts from neuroethology, which bridges the gap between the pure study of behavior and the study of cellular function in the nervous system, are introduced, along with examples of important recent neuroethological studies. The role of the nervous system in maintaining homeostasis through the modulation of all systems has been incorporated into Part 111, which further advances the integrated approach of the book. There is an increased emphasis throughout the book on environmental adaptations, and specific examples of environmental adaptation (such as water balance in elephant seals in Chapter 14)illustrate the general principles of comparative physiology. Some of the new topics introduced in the fourth edition include a section on the immune response in Chapter 12 (Circulation), and a section on biorhythms in Chapter 13 (Using Energy: Meeting Environmental Challenges).

PEDAGOGY The ideas developed in the text are illuminated and .augmented by liberal use of illustrations and figure

legends. For the first time, full color drawings have been added, creating a high quality visual program to further motivate students. Spotlights provide in-depth information about the experiments and individuals associated with important advances in the subject matter, the derivation of some equations, or simply historical background on a topic under discussion. Thought questions within chapter text (look for the a)encourage problem-based learning and stimulate discussion on various aspects of the material presented. The text narrative includes effective, integrated examples to support principles; while presenting information, it provides consistent thematic coverage and a feeling for methods of investigation. References to the literature within the body of the text and in figure legends are made unobtrusively, but with sufficient frequency that students can become aware of the role of scientists and their literature as a subject is developed. Further pedagogical aids include key terms that are explained and appear in boldface type at their first mention in the text, and that are formally defined in a useful, comprehensiveglossary. End of chapter materials include a summary, which provides the student with a quick review of important points covered in the chapter, review questions, and an annotated list of suggested further readings. Students will find the following resources at the back of the book: appendixes that provide information on units, equations, and formulas; the glossary; and a bibliography that includes the full citations of all references cited in the chapters. Our goal has been to produce a balanced, up-to-date treatment of animal function that is characterized by its clarity of exposition. We hope that readers will find Eckert Animal Physiology valuable, and we welcome your constructive criticism and suggestions.

September 1996

DAVIDRANDALL BURGGREN WARREN KATHLEEN FRENCH

E

Bill Kristan, University of California at Sun Diego Paul Lennard, Emory University Jon E. Levine, Northwestern University Harvey B. Lillywhite, University of Florida, Gainesville Duane R. McPherson, SUNY at Geneseo Duncan S. MacKenzie, Texas A&M University Eric Mundall, late, University of California at Los Angeles Kenneth Nagy, University of California at Los Angeles Richard A. Nyhoff, Calvin College Richard W. Olsen, UCLA School of Medicine C. Leo Ortiz, University of California at Santa Cruz Harry Peery, University of Toronto J. Larry Renfro, University of Connecticut Marc M. Roy, Beloit College Roland Roy, Brain Research Institute, UCLA School of Medicine Jonathon Scholey, University of California at Davis C. Eugene Settle, University of Arizona Michael P. Sheetz, Washington University School o f Medicine Gregory Snyder, University of Colorado at Boulder Joe Henry Steinbach, Washington University School o f Medicine Curt Swanson, Wayne State University Malcolm H . Taylor, University of Delaware-School o f Life and Health Sciences Ulrich A. Walker, Columbia University-College o f Physicians Eric P. Widmaier, Boston University Andrea H. Worthington, Siena College Ernest M . Wright, UCLA School of Medicine-Center for the Health Sciences

ckert Animal Physiology has benefitted greatly from the contributions of several people whose efforts we gratefully acknowledge. Russell Fernald, Stanford University, participated in the planning and reorganization of the book and the initial revision of many of the chapters. Lawrence C. Rome, University of Pennsylvania, wrote the initial draft of Chapter 10 (Muscles and Animal Movement), which contained most of the updated material except for the sections on cardiac and smooth muscle and on neuronal control. Harold Atwood, University of Toronto, was involved in some early discusions of the revision. Further, we are grateful for the informal comments of associates around the world, and for the formal reviews of manuscript, which were provided by the follow~ngcolleagues from across the country:

'

Joseph Bastian, University of Oklahoma Robert B. Barlow, Institute for Sensory Research, Syracuse University Francisco Bezanilla, UCLA School of MedicineCenter for the Health Sciences Phillip Brownell, Oregon State University Richard Bruch, Louisiana State University Wayne W. Carley, National Association of Biology, Teachers Ingrith Deyrup-Olsen, University of Washington Dale Erskine, Lebanon Valley College A. Verdi Farmanfarmaian, Rutgers University Robert Full, University of California at Berkeley Carl Gans, University of Michigan Edwin R. Griff, University of Cincinnati Kimberly Hammond, University of California at Riverside David F. Hanes, Sonoma State University Michael Hedrick, California State University- Hayward James W. Hicks, University of California at Irvine Sara M . Hiebert, Swarthmore College William H. Karasov, University of Wisconsin at Madison Mark Konishi, California Institute of Technology

Finally, the good sense and kind words of Kay Ueno and Kate Ahr, our editors, have much improved the book and ensured its publication. DAVIDRANDALL WARREN BURGGREN FRENCH KATHLEEN xvii

0

ur knowledge of animal physiology is based on information (data) derived from experimentation. Since the ultimate goal of animal physiology is to understand how a process operates within an organism, experiments must be designed to allow the measurement of key variables (e.g., metabolic rate, blood flow, urine production, muscle contraction) in the animal (or its cells or tissues) while it is in a known state such as resting, exercising, digesting, or sleeping. This kind of experimentation is particularly challenging and requires the use of a variety of techniques and methods. Many of the experimental techniques and measuring devices common in animal physiology are "time-honored." These include pressure transducers to measure pressure, catheter implantation to draw blood or inject samples, respirometers for determining metabolic rates, and numerous others. A description of each of these is beyond the scope of this chapter, especially since such fundamental techniques are well described in texts such as J. N. Cameron's Principles of Physiological Measurement. In this chapter, we will focus on a few of the many molecular and cellular techniques that have recently been added to the physiologist's tool box, briefly describing them and illustrating their use in physiological research. First, however, we consider the nature of hypotheses and the general principles that apply in testing them. By knowing why and how experiments in animal physiology are performed-whether they employ traditional or emerging methods-you will be much better able to evaluate the strengths and limitations of the information you will learn in this book.

FORMULATING AND TESTING HYPOTHESES Scientists use experimental data to create general laws of physiology-some literally centuries old, and some still emerging. These general laws, in turn, serve as the basis for formulating new hypotheses, which are specific predictions that can be tested by performing further experiments. An example of a general "law" supported by much existing

data is that water-breathing animals regulate acid-base balance by modifying the excretion of HCO,- in exhaled water, while air-breathing animals regulate acid-base balance by modifying the elimination of CO, gas in exhaled air. The following testable hypothesis could be derived from this general law: A transition from H C O , elimination to CO, elimination occurs when water-breathing tadpoles metamorphose into air-breathing frogs. Although hypotheses are framed as statements rather than as questions, the goal of experimentation is to test the validity of hypotheses, and thus answer the implied questions. Physiological experiments should begin with a wellformed, specific hypothesis that focuses on a particular level of analysis and is amenable to a verifiable experimental approach. Although a hypothesis such as killer whales have a very high cardiac output while in pursuit of seals may be interesting and in fact true, it is merely an intellectual exercise to suggest this hypothesis unless a feasible experimental approach exists for gathering data necessary to accept (prove) or reject (disprove) it. However, the search for means to test novel hypotheses has been an important stimulus for development of new experimental techniques and measuring instruments. For example, telemetry devices currently available for gathering data on blood flow in small to medium-sized animals like ducks, fishes, and seals are being modified for use on even larger animals.

The August Krogh Principle August Krogh was a Danish animal physiologist with extremely broad interests in comparative physiology. Dozens of key research articles bearing his name have served as the basis for whole areas of further experimentation in the area of respiration and gas exchange. Indeed, Krogh's work in the late 1800s and early 1900s eventually led to his winning the Nobel Prize for physiology. One of the reasons for Krogh's extraordinary success as a physiologist was his uncanny ability to choose just the right experimental animal with which to test his hypotheses. His view was that for every defined physiological problem, there was an optimally suited animal that would most efficiently yield an answer.

\

16

P R I N C I P L E S OF P H Y S I O L O G Y

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The design of experiments based on the unusual characteristics of an animal has come to be known as the August Krogh principle (Krebs, 1975). Illustrations of this principle abound in this book and throughout modern animal physiology. For example, in the 1970s a group of animal physiologists, interested in the evolution of air breathing in crustaceans, were studying relatively tiny intertidal crabs, but they were frustrated because the small size of these animals kept them from "giving up" their physiological secrets. Evoking the August Krogh principle, which suggested that there was an ideal animal with which to carry out their studies, these physiologists organized an expedition to the Palau Islands in the South Pacific. These islands are home to the "coconut," or "robber," crab, a terrestrial hermit crab weighing up to 3 kg. The monstrous size of these animals (for a terrestrial crab) allowed numerous experiments yielding important new data during the one-month expedition. As another example, animal physiologists interested in cardiac performance in fishes often have a difficult time measuring pressure and flow and sampling blood from the heart because of its typical location in bony fish (i.e., teleosts). Yet, the sea robm, a deep-water (benthic)marine teleost that is quite unremarkable in most respects (although it is down-right ugly!), has an unusually large heart, which is much easier to access than in other fishes. By following the August Krogh principle and using the sea robin as the basis for their experiments, comparative cardiovascular physiologists now know more about heart function in fishes than they would if they had continued to struggle with the relatively unforgiving anatomy of the trout, salmon, or catfish. Experimental Design and Physiological Level

In designing an experiment, the first and most important decision a physiologist must make is about the level at which the physiological problem will be analyzed. This choice of level determines the methodology (and choice of animal) appropriate for measuring the experimental variables of interest. Historically, techniques for exploring physiological problems at the level of the whole animal were developed first; subsequently, and with increasing rapidity in recent decades, have come new techniques for experimenting at the cellular and now at the molecular level. Conceptually, however, we generally operate in the reverse order: starting at the molecular level, then moving successively to the cellular, tissue, organ, and finally whole-animal levels, much as outlined in Figure 1-1. Consequently, in the following sections, we describe some representative experimental methods for studying physiological processes, beginning at the molecular level. Much of the information presented in other chapters of this book is based on experimental results obtained with these various techniques. Only by learning how and why these methods work, as well as some of their limitations, can you adequately assess the information presented.

Note that no level of analysis is intrinsically more valuable or important than any other. Indeed, the best understanding of animal physiology comes from integrating knowledge about the contributing components from the molecular through organ-system level. Having said this, we recognize the strong trend in animal physiology (as in all of biology) during the last decade towards "reductionism," the study of cellular and molecular mechanisms in an attempt to explain more complex processes at higher organizational levels. Ultimately, some of the most valuable experiments are those at a level of analysis that allows insights about processes at adjacent organizational levels. Although researchers and students often are fascinated by new and frequently expensive methodologies, incisive results can be obtained with well-designed experiments using relatively simple instruments and techniques. In other words, a well-conceived experimental design often can compensate for the lack of the latest, cutting-edge equipment and techniques.

MOLECULAR TECHNIQUES The past few decades have seen a veritable explosion in the number and sophistication of available techniques for probing molecular events, with new methods and refinements constantly emerging. The variety of molecular techniques available have had major implications for biological research in general, and animal physiology has certainly benefited from molecular approaches. In this section we describe just a few of the powerful molecular techniques that have been used to answer questions in animal physiology. More detailed discussion of these and related techniques are presented in textbooks such as Molecular Cell Biology by H. D. Lodish et al. Tracing Molecules with Radioisotopes

Greater understanding of physiological processes can often be achieved by knowing the movements of molecules within and between cells. For example, we can more easily understand the role of a particular neurohormone in regulating physiological processes if its movements can be traced from its site of synthesis to its site of release and on to its site of action. Many types of experiments that follow the movement of physiologically important molecules employ radioisotopes, the relatively unstable, disintegrating radioactive isotopes of the chemical elements. The natural disintegration of radioisotopes is accompanied by release of high-energy particles, which can be detected by appropriate instruments. With the exception of 12jI,which emits y particles, the isotopes commonly used in biological research emit p particles. Although radioisotopes occur naturally, those normally used in experimental studies are produced in nuclear reactors. The most commonly used isotopes in biological research are 32P,12jI,35S,14C,4SCa,and 3H.A radioisotope of an element normally present in the molecule of interesttan be incorporated in vitro or in vivo either directly into the

molecule or into a precursor molecule that will eventually be converted into the molecule of interest. The resulting radiolabeled molecule has the same chemical and biochemical properties as the unlabeled molecule. An amazing array of so-called radiolabeled biologically active molecules (e.g., amino acids, sugars, hormones, proteins) are now readily available (at a substantial price) from companies that specialize in their production. Once a molecule has been radiolabeled, the particles emitted from the radioisotope can be used to detect the presence of the molecule, even at very low concentrations. In one type of tracing experiment, the radiolabeled molecule of interest or its precursor is administered to an animal, isolated organ, or cells growing in uitro culture, and then samples are removed periodically for measurement of particle emission. Two types of instruments are used to detect emitted particles. A Geiger counter detects ionization produced in a gas by emitted energy. A scintillation counter detects and counts tiny flashes of light that these particles create as they pass through a specialized "scintillation fluid." The amount of radiation detected by either instrument is related directly to the amount of the radiolabeled molecule present in the sample. In another type of experiment, the location of radiolabeled molecules within a tissue section is pinpointed by autoradiography. In this technique, which literally "takes a picture" of the radioisotopes in tissues, a thin tissue slice containing a radioisotope is laid on a photographic emulsion. Over the course of days or weeks, particles emitted from the radioisotope expose the photographic emulsion, producing black grains that correspond to the location of the labeled molecules in the tissue (Figure2-1).This qualitative record can be quantitated by measuring the amount of exposure of the emulsion in a densitometer and comparing it with exposures caused by standards of known concentration; in this way, the actual concentration of a radiolabeled molecule in the tissue or portions of it can be determined. Autoradiography has been particularly useful in neurobiology, endocrinology, immunology, and other areas of physiology involving cell-to-cell communication. Tracing Molecules with MonoclonalAntibodies Examination of a biological structure in a fixed tissue slice on a microscope slide can be daunting. Even when the tissue has been stained so that the cell nuclei are dark purple and the cell membranes a somewhat lighter shade, for example, it remains difficult to discern much about the details of the tissue. Much better visualization of the structural details of cells is possible with antibody staining. This remarkable technique permits localization of molecules present in such extremely low concentrations that they are difficult to study by other techniques. Antibody staining generally involves covalently linking a flourescent dye to an antibody that recognizes a specific determinant on an antigen molecule. (Althoughwe often think of antigens as disease-causing microbes or invading foreign materials like pollen, normal, biologically active molecules,

Caudate-putamen

Figure 2-1 Autoradiograms can reveal biochemical and structural details that cannot be seen with traditional techniques for tissue fixation and staining. This autoradiograph shows a frontal section through the rat brain after cannabinoid receptors have been bound by a radiolabelled synthetic cannabinoid (closely resembling the active ingredient of marijuana). The most radioactive areas (that is, the areas with the most cannabinoid receptors)have most heavily exposed the photograph film on which the brain slice was laid, and show up primarily as the dark areas in the striaturn (caudate-putamen),which mediate motor functions. [Courtesy of Miles Herkenham, NIMH.]

such as neurotransmitters and cell growth regulators, can act as antigens and induce production of specific antibodies when injected into an appropriate animal.) Identical antibodies produced in response to an antigen are called monoclonal antibodies; however, most natural antigens have multiple, rather than single determinants, thus the production of several different antibodies is likely. A mixture of antibodies that recognize different determinants on the same antigen is called polyclonal. Once antibodies that recognize discrete sites on a molecule of interest have been produced and linked to a flourescent dye, thay can be injected into the cells or tissues under study. Over the past decade, researchers increasingly have used a combination of monoclonal and polyclonal antibodies for antibody staining, particularly in irnmunofluorescent microscopy (Figure2-2). Alternatively, radiolabeled monoclonal antibodies can be used and the location of any antigen-antibody complexes that form in a sample detected by autoradiography. This approach has been used to localize the hormones epinephrine and norepinephrine within certain cells of the adrenal medulla, as described in Chapter 8 on glands. Monoclonal antibodies can be used not only to track down specific molecules within cells but also to purify them, as described in a later section. Such purified molecules are suitable for detailed studies on their structure and function. The crucial advance that made antibody staining feasible was development of a method for producing large amounts of monoclonal antibody. Isolation and purification of a single type of monoclonal antibody from antiserum taken from animals exposed to the corresponding antigen is not practical, because each type of antibody is present only in very small amounts. Moreover, the B

Figure 2-2 Both monoclonal and polyclonal antibodies are frequently used in antibody staining. In this irnrnunofluorescent micrograph of rat spinal cord cultured 10 days, a mouse monoclonal antibody (green)and a rabbit polyclonal antibody (red) that is specific for a single protein, along with a blue fluorescent dye that binds DNA directly, are used. Here we see neurons (red), astrocytes (green), and DNA (blue). [Courtesy of Nancy L. Kedersha/lrnrnuno Gen.]

lymphocytes (or B cells) that produce antibodies normally die within a few days, and thus cannot be grown for extended periods in culture. In the mid-1970s, G. Kohler and C. Milstein discovered that normal B cells could be fused with cancerous lymphocytes, called myeloma cells, which grow indefinitely in culture (i.e., they form an "immortal" cell line). The resulting hybrid cells, termed hybridomas, are spread out on a solid growth medium in a culture dish. Each cell grows into a clone of identical cells, with each clone secreting a single monoclonal antibody. Clones are then screened to identify those that secrete the desired antibody; these self-perpetuating cell lines can be maintained in culture and used to obtain large quantities of homogeneous monoclonal antibody (Figure 2-3). Although individual investigators can make and maintain their own hybridoma cell lines, many now choose to have specific monoclonal antibodies prepared by companies specializing in their production. (The next time you are in your university or college library, find the journal Science and take a look at the classified ads in the back.) The development of monoclonal antibody technology by Kohler and Milstein so revolutionized molecular studies that they received the Nobel Prize for their research. Genetic Engineering

Genetic engineering encompasses various techniques for manipulating the genetic material of an organism. This approach is increasingly used in both agriculture and medicine, and it offers considerable promise for investigators in animal physiology. These techniques make it possible to produce large quantities of biologically important molecules (e.g., hormones) normally present at very low con-

centrations, animals with mutations that affect specific physiological processes, and animals that synthesize aboveor below-normal amounts of specific gene products. Genetic engineering begins with identification of the structural gene that codes for a specific protein within the DNA isolated from an organism of interest. For example, the gene that encodes human insulin can be identified in DNA isolated from human cells. The section of DNA containing the insulin gene of interest can be "clipped out" of the original very long human DNA strands and then inserted into a cloning vector, which is a DNA element that can replicate within appropriate host cells independently of the host cells' DNA. Insertion of a fragment of foreign DNA (e.g., the human insulin gene) into a cloning vector yields a recombinant DNA, which is any DNA molecule containing DNA from two or more different sources. Bacterial plasmids are a common type of cloning vector. These are extrachromosomal circular DNA molecules that replicate themselves within bacterial cells. Under certain conditions a recombinant plasmid containing a gene of interest is taken up by the common bacterium Escherichia coli, a process called transformation (Figure 2-4). Normally, only a single plasmid molecule is taken up by any one bacterial cell. Within a transformed cell, the incorporated plasmid can replicate, and as the cell divides a group of identical cells, or clone, develops. Each cell in a clone contains at least one plasmid with the gene of interest. This general genetic engineering procedure, called DNA or gene cloning, can be used to obtain a DNA "library" consisting of multiple bacterial clones, each of which contains a specific gene from humans or other species. Several variations of DNA cloning are used depending on the size and number of the genes in the organism being studied. Clonal populations for medicine and research Under appropriate environmental conditions, the recombinant DNA in an "engineered" E. coli clone is transcribed into messenger RNA, which is used to direct synthesis of the encoded protein. Commercial companies, for example, grow E. coli cells carrying recombinant DNA containing the gene for human insulin or other hormones in huge vats; after the bacterial cells are harvested, large quantities of the human hormone can be isolated relatively easily. In the past, hormones needed for treating humans with endocrine disorders were extracted from the tissues of other mammals such as cows and pigs. Because hormones are present in quite low concentrations, this is a time-consuming and expensive process. Producing these hormones with genetically engineered bacteria has proven to be far less expensive and yields a purer product. Moreover, hormones isolated from other mammalian species often induce an immune response in humans, a complication not encountered with human hormones obtained from engineered bacteria. Recombinant DNA technology is also a powerful tool in basic research on human genetic disorders. By isolating and studying genes associated with hereditary diseases, scientists can determine the molecular basis of these diseases. This will

Figure 2-3 Hybrldoma cell llnes secrete

W Cell-culture

Fuse In polyethylene glycol n \ n

00

000

-

@

Spleen cells

Q

"pure" (homogeneous) monoclonal antibodles To prepare monoclonal antlbodles, antlbody-producing spleen cells flrst are fused w ~ t hmyeloma cells orlglnally derlved from B lymphocytes The hybr~dcells, or hybrldomas, that secrete antlbody speclflc for the proteln of Interest are separated out They can be malntalned In cell culture, where they secrete large quantites of the speclflc antibody, or in jected into a host mouse, where they induce the production of the antlbody.

@

@@ Myeloma cells

I 1

Transfer cells to selective medium

Select hybrid cells

1

Select cells that produce a desired antibody

Grow in mass culture

Antibody

certainly lead to better methods of controlling or even curing them. Over the last several years numerous laboratories worldwide have been engaged in a massive project to "map" the locations of all human genes on the long strands of DNA in human chromosomes and determine their nucleotide sequences. This Human Genome Project is providing invaluable data for researchers studying genetic diseases. DNA cloning and recombinant DNA technology also form the basis for gene therapy. In this approach to treating those with genetic disorders, the normal form of the gene that is missing or defective is introduced into patients. For example, persons with cystic fibrosis have a defective CFTR gene and thus cannot produce the normal protein encoded by this gene. One result of this defect is production of a very thick mucus in the lungs' airways, which leads to potentially lethal breathing problems. Molecular biologists have engineered common cold viruses with the normal CFTR gene. When some cystic fibrosis patients were infected with an engineered cold virus, the viral particles carried the normal human gene into the patients' lung cells, where it became established. Subsequent synthesis of the

Antibody

normal gene product helped alleviate most of the symptoms of cystic fibrosis in the treated patients.

20

PRINCIPLES OF PHYSIOLOGY

............................................................................... Plasmid vector

DNA fragment

Bacterial chromosome

Figure 2-4 DNA cloning is a way to isolate and maintain individual genes. In the cloning procedure illustrated, a specific DNA fragment to be cloned is inserted Into a plasmid vector, whichalso contains a gene conferring resistance to the antibiotic ampicill~n.When the resulting recombinant plasm~dsare mixed with E. colicells, a few cells take up a plasmid, which can replicate within the cells. If the cells are placed in media containing ampicillin, only those that have taken up the vectorwill grow. As each selected cell multiplies, it eventually forms a colony of cells (clone) all containing the same recombinant plasmid.

"Made-to-order" mutants As mentioned in Chapter 1, mutations are permanent changes in the nucleotide sequence of DNA. Mutations, which can occur spontaneously or be induced experimentally, are duplicated and passed on to daughter cells at the time of cell division. Mutant genes can tell us a great deal about how physiological processes work. The specific disruption in a physiological process resulting from a single mutant gene can pinpolnt the functions controlled by particular genes, information that may not be revealed by conventional physiological techniques. For example, cardlovascular physiologists are producing and analyzing the effects of mutations in zebrafish to understand heart development. In research described by J-N. Chen and M. Fishman (1997),dozens of specific cardiovascular mutations have been produced in zebrafish. The process starts when adult zebrafish are exposed to powerful mutagens-compounds that produce permanent mutations in the germ cell line. Subsequent matings of the F, and F2 generations lead to embryos with large numbers of mutations. Very rarely, an embryo will appear with just one specific mutation in a structure or process of interest. The Fishman group, for instance, has identified

mutations that cause a heart with abnormallv thin ventricular walls and another with a constriction of the arterial outflow tract of the heart. Both of these conditions mimic human disease states. Mutations often produce abnormal effects only in the homozygous state (i.e., when an individual receives a mutated form of a gene from each parent). Even when a mutation causes a lethal condition incompatible with longterm survival, it can be "preserved" in the parents, who are heterozygous for the mutation, carrying one normal and one mutated form of the gene. Each time these parents breed, some of the offspring will be homozygous and show the abnormal effects. Thus, the heterozygous parents are a "living gene library" of these mutations. Transgenic animals Transgenic animals are another type of genetically engineered organism with the potential for making great contributions to physiology. A transgenic animal is one whose genetic constitution has been experimentally altered by the addition or substitution of genes from other animals of the same or other species. Transgenic animals (especially, mice) are at the forefront of the menagerie of animal models that are helping researchers understand basic physiological processes and the disease states that results from their dysfunction. Numerous techniques have been employed to produce transgenic animals. In one method, "foreign" DNA containing a gene of interest, called a transgene, is injected into a pronucleus of fertilized eggs (commonly from mice), which then are implanted into pseudopregnant females. At a relatively low frequency, the transgene is incorporated into the chromosomal DNA of the developing embryos, leading to offspring that carry the transgene in all their germ-line cells and somatic cells (Figure 2-5). Mice expressing the transgene then are mated to produce a transgenic line. This approach is used to add functional genes, either extra copies of a gene already present in the animal or a gene not normally present, leading to overexpression of the gene product. Subsequent analysis of the morphology and physiology of the transgenic animals can provide considerable insight into physiological processes that cannot easily be investigated in other ways. Transgenic animals characterized by underexpression or complete lack of expression of a particular gene can be equally informative. M. R. Capecchi (1994)has reviewed a procedure for replacing a functional gene with a defective one, thereby producing so-called knockout mice. These mice cannot express the protein originally coded for by the replaced gene and thus lack the functions mediated by the missing protein. The molecular and genetic basis of physiological processes can be determined by examining the effect of such functional ablation of genes. Knockout mice are used extensively to unravel human physiological processes, because human and mouse genes are greater than 98% identical. To cite a couple of examples, researchers are investigating the normal genes that regulate

EXPERIMENTAL METHODS FOR EXPLORING PHYSIOLOGY

21

...............................................................................

I

Fertilized eggs collected from female

I

Injected eggs implanted into oviduct of new female

10-30% of offspring contain transgene

1

individual cells, are used to measure various properties of cells or inject materials into them. Although cellular physiologists employ these devices in a variety of ways, the technology used to make them is decades old. Essentially, a region in the middle of a glass capillary tube is heated to the point of melting. The ends of the tube are then pulled apart, which draws the soft spot in the glass down to an invisibly small diameter before it breaks and separates. Two micropipettes, each with a drawn-down tip as small as just a micron in diameter, are produced as a result. When a micropipette is filled with an appropriate solution, it can function as a microelectrode. Typically, a micropipette (or microelectrode) is mounted in a micromanipulator, a mechanical device that holds the pipette steady and allows its tip to be moved incrementally in three different planes. Measuring electrzcalproperties Since neurons communicate via electrical signals, microelectrodes can be used to "eavesdrop" on their communication by measuring the electrical signals across the cell membrane and changes In these signals under different conditions. The microelectrodes used to measure the electric potential (voltage)across the cell membrane cause virtually no flow of current from the cell into the electrode. Thus little or no disruption of the nerve cell occurs even as its com-

axQ Breed transgenics to ma~ntainDNA in germ line

Figure 2-5 A transgenic animal is produced by adding or substituting genes from another animal of the same or different species.To introduce a transgene into mice, cloned "foreign" DNA is injected into fertilized eggs, which then are implanted into a female. A proportionof the viable offspring will retain the transgene, which can be maintained in the germ line by selective breeding.

early heart development in the embryo and the oncogenes s cancer in studies with knockresponsible for some t y p ~ of out mice.

CELLULAR TECHNIQUES Understanding cells and cellular behavior is a goal of many experiments in physiology. With a knowledge of cellular behavior and communication, we can begin to understand how communities of cells function as tissues, and tissues as organs. Physiological analysis at the cellular level has been pursued most vigorously using several now-standard techniques. In this section we discuss three very common and productive cellular techniques: recording with microelectrodes, microscopy, and cell culture. Uses of Microelectrodes and Micropipettes

Many experiments in cellular physiology make use of micropipettes or various types of microelectrodes. These tiny glass "needles," which can be inserted into tissues or even

munication A microelectrode with neighboring for recording cells iselectrical being detected. signals from neurons or muscle cells is made by filling a micropipette with an ionlc conducting solution (typicallyKCl) and connecting it to an appropriate amplifier. A second electrode connected to the amplifier is placed in the fluid or organism in the vicinity of the first electrode. When the tip of the first electrode is pushed through the cell membrane into the cytoplasm, it completes an electric circuit whose properties (voltage, current flow) can be measured. Since microelectrode recording techniques were introduced in the 1950s, our understanding about the electrical activities within a cell have increased dramatically. One of the most revolutionary advances in microelectrode recording methodology is patch clamping. With this technique, the behavior of a single protein molecule constituting an ion channel can be recorded in situ (Latin for "in its normal place"), as illustrated in Figure 2-6. This method lies at the heart of the recent explosion of knowledge about membranes, including their channels and how they regulate the movement of materials across the membrane (see Chapters 4-6). Measuring ion and gas concentrations Specially constructed microelectrodes can be used to probe the intracellular concentration of common inorganic ions including H+,Na+, K+, C1- ,Ca2+,and Mg2+.Because cells use movements of ions across cell membranes to communicate and to do work, the magnitude, direction, and time course of ion movements provide important information about certain processes. ~croelectrodesthat measure the

22

PRINCIPLES O F PHYSIOLOGY

...............................................................................

A

Flu~dto conduct

sides of the ion-exchange barrier in the tip. Proton-selective microelectrodes are particularly useful for measuring the pH of blood and other body fluids.

Measuring intracellular and blood pressure Microelectrodes are now being used to measure hydrostatic pressures within individual cells and microscopic blood vessels-indeed, in any fluid-filled space into which the tip of a microelectrode can be inserted. To understand the principle of such micropressure systems, let's consider a small blood vessel. A microelectrode, filled with at least a 0.5 M NaCl solution and mounted in a micromanipulator, is inserted into the vessel of interest. The higher pressure inside the vessel causes the interface between the plasma and the solution filling the electrode to move into the electrode. This results in increased resistance across the electrode tip, because the resistance of plasma is higher than that of the NaCl solution. The change in resistance is measured and is proportional to the change in blood pressure. A motor-driven pump associated with the micropressure system produces a pressure in the microelectrode that just offsets the pressure in the vessel. This opposing pressure keeps the interface at a stationary position; therefore, it is called a servo-null system. The required offsetting pressure generated in the micropressure system is then monitored with a conventional pressure transducer such as would be used for measuring blood pressure in much larger vessels. Micropressure systems have greatly extended our knowledge of the development of cardiovascular function in developing embryos and larvae. These techniques have also allowed direct cardiovascular measurements in adults of very small animals like insects.

Figure 2-6 Patch-clamp recording permits determination of ion movement across a small patch of membrane containing transmembrane ion channels. (A) Diagram of patch clamp in place. When a fire-polished microelectrode is placed against the cell surface, a very high resistance seal t forms between the electrode tip and the membrane This t ~ g h seal allows direct measurement of the membrane features beneath the tip Typically, only a few transmembrane ion channels lie beneath the t ~ pal, lowing the current flowthrough them to be measured directly (0)Photomicrograph showing tip of a patch mlcropipette abutting the cell body of a nerve cell The tip has a diameter of about 0 5 p m [Part B from Sakmann, 1992.1

partial pressure of gases (e.g., 0, and CO,) dissolved in a fluid also are now available. The tip of a microelectrode for measuring the concentration of a particular ion (e.g., Na+) is plugged with an ion-exchange resin that is permeable only to that ion. The remainder of the electrode (the "barrel") is filled with a known concentration of the same ion. The electrical potential measured by the microelectrode when no current flows reflects the ratio of the ion concentrations on the two

Microinjecting materials into cells In addition to their use as microelectrodes, micropipettes also can be used to inject substances into individual cells. These substances may be active molecules that produce a measurable change in cell or tissue function. For instance, drugs that influence blood pressure and heart rate can be injected into very small blood vessels (e.g., those lining the shell of a bird egg) or into the microscopic heart of a frog embryo. Alternatively, the injected substance may be a dye used to mark injected cells, helping to reveal cell processes or to trace cells as they divide. A classic variation of this technique involves horseradish peroxidase, an enzyme derived from the horseradish plant that forms a colored product from specific colorless substrates. When this enzyme is injected via a micropipette into the extensions (especiallyaxo n ~of) neurons, it is taken up and transported back to the neuron's cell body; subsequent injection of the substrate generates a colored "trail" between the injection site and cell body. By this technique, peripheral nerves can be traced back to their origin in the central nervous system, a task that would defy even the most skilled neuroanatomist using more traditional techniques.

Structural Analysis of Cells

Cellular function is dependent on cellular structure, reaffirming the central theme discussed in Chapter 1that strong structure-function relationships govern animal physiology. Physiologists commonly use structural analyses at the cellular level to complement physiological measurements in order to discover how animals function. Such analyses depend on various types of microscopy, because animal cells are typically about 10-30 p m in diameter, which is well below the smallest particle visible to the human eye.

Light microscopy Light microscopy, as its name implies, uses the photons of visible or near visible light to illuminate specially prepared cells. Under optimal conditions, the resolution, or resolving power, of light microscopes is a few microns; two objects that are located closer to each other than a microscope's resolution will appear as one. As the resolution of microscopes has been improved, our understanding of the structure of cells and their components has increased. Since cells removed from a living animal rapidly die, tissue must be prepared quickly to prevent degradation of cellular constituents. Fixation is the addition of a specialized chemical (e.g., formalin) that kills the cells and immobilizes

their constituents, typically by cross-linking amino groups of proteins with covalent bonds. The fixed cells then are treated with dyes or other reagents that stain particular cellular features, allowing visualization of the cells, which otherwise are colorless and translucent. Fixation and staining of large blocks of tissue is impractical and does not allow visualization of individual cells. Typically, small blocks of tissue are cut into ultrathin sections or slices just 1- 10 p m thick using a special knife called a microtome. Because most tissue is fragile even when fixed, it is embedded in some medium (e.g., wax, plastic, gelatin) to support it while it is sectioned. Such media surround and infiltrate the tissue and then harden to make sectioning possible. The tissue sections are then placed on glass slides for staining and subsequent viewing in a microscope (Figure2-7A). In some instances, tissue embedding compromises the structure of the cell or its contents such that they can no longer be stained or labeled with special compounds prior to viewing. An alternative method is to freeze the tissue rather than embed it, allowing the ice to provide support for the tissue as it is subsequently sectioned. Once prepared, tissue is viewed with a compound optical microscope, the simplest type of light microscope (Figure 2-7B).

Specimen embedded in paraffin wax or plastic resin and mounted on arm of microtome

Metal or glass blade

'2W"

Ribbon of thin sections

Ribbon of sections on glass slide, stained and mounted under a cover slip

I-

Reflecting prism

Objective lenses Specmen >Condenser lenses Path of light

3ase with ight source

Figure 2-7 Specimens are preparedfor light microscopy by cutting them into thin sections and staining. (A) Cells and tissue removed from living organisms first are fixed to preserve their structure and then cut ~ n t o thin sections using a metal or glass knife. These sections are mounted on a glass slide, where they can then be stained for subsequent viewing

through a.compound light microscope. (B) The compound optical microscopetransrnitslight vertically up through a condenser lens, the specimen on the slide, an objective lens, and finally the ocular lens in the eyepiece, from which the specimen is viewed. [Adapted from Lodish et al., 1995.1

As improvements have been made in the available optics, staining techniques also have improved. Many organic dyes, originally developed for use in the textile industry, were discovered through trial and error to selectively stain particular cellular constituents. Some of these dyes stain according to the charge, such as hematoxylin, which marks negatively charged molecules like DNA, RNA, and acidic proteins. Howevel; the basis of the specificity of many dyes is not known. Staining with fluorescent-labeled reagents, rather than traditional dyes, increases the sensitivity of visualization. Fluorescent molecular labels absorb light of one wavelength and emit it at another, longer wavelength. When a specimen treated with a fluorescent reagent is viewed through a fluorescence microscope, only those cells or cellular constituents to which the label has bound are visualized (Figure 2-8). Probably the most common and useful type of fluorescence microscopy is immunofluorescencemicroscopy in which specimens are treated with fluorescentlabeled monoclonal and polyclonal antibodies. A good example of the images obtained with this technique is shown in Figure 2-2. Because immunofluorescence microscopy gives poor results with fixed thin sections, this technique usually is applied to whole cells. However, the images obtained by standard fluorescence microscopy of whole cells represent a supposition of emitted light coming from labeled molecules located at many depths in the cell. For this reason, the images often are blurred. The confocal scanning microscope eliminates this problem, providing sharp images of fluores-

cent-labeled specimens without the need for thin sectioning. In this microscope, the specimen is illuminated with exciting light from a focused laser beam, which rapidly scans different areas of the specimen in a single plane. The light emitted from that plane is assembled by a computer into a composite image. Repeated scanning of a specimen in different planes provides data with which the computer can then create serial sections of the fluorescent images. Figure 2-9 compares the images obtained with conventional and confocal fluorescence microscopes. Visualization by other types of microscopy depends on the specimen changing one or more properties of the light passing through the tissue on the slide, rather than on fixation and staining. Since these methods do not re-

>

Eyepiece

Incident light source

d

Barrier filter

Dlchromatlc filter

Specimen

I

Figure 2-8 Aspecimen stained with a fluorescent label is viewed through a fluorescence microscope, which produces an image only of structures that bind the label-The incident light source is passed through an exc~ter filterthat passes blue light (450-490 nm) to provide optimal illumination for the specimen.The incident light is directed towards the specimen by a beam-splitting mirror that reflects light below 510 nm downwards but transmits light above 510 nm upwards. The fluorescent signals emitted from the labeled specimen pass upward through a barrier filter that removes unwanted fluorescent signals not corresponding to the wavelengths emitted by the label used to stain the specimen.

Figure 2-9 Conventional and confocal microscopy provide different images of biological material. These photomicrographs are of a lysed mitotic fertilized egg from a sea urchin.A fluorescein-tagged antibody was used to bind an antibody for tubulin, a major structural component of the mitotic spindle. (A) Conventional fluorescence microscopy shows a blurred image as a result of fluorescein molecule above and below the plane of focus. (B) Confocal microscopy,which detects fluorescence only from within the plane of focus, produces a much sharper image of the same sea urchin egg. [From White et al., 1987.1

E X P E R I M E N T A L M E T H O D S FOR E X P L O R I N G P H Y S I O L O G Y

25

............................................................................... quire staining, they can be used on living tissue, provided it is thin enough to allow sufficient light to pass through. Bright field microscopy (Figure 2-10A) reveals few details compared to phase-contrast microscopy, in which the image has varying degrees of brightness and directness due to differential light refraction by different components of the specimen (Figure 2-10B). In Nomarski microscopy, also called differential-interference contrast microscopr, an illuminating beam of plane polarized light is split into closely parallel beams before it passes through the tissue specimen and the exiting beams are reassembled into a single image. Slight differences in the refractive index or thickness of adjacent parts of the specimen are converted into a bright image, if the beams are in phase when they recombine, or a dark image, if they are out of phase. The final image gives an illusion of depth to the specimen (Figure 2-1OC). In dark-field microscopy, light is directed towards the specimen from the side so the observer sees only light scattered from cellular constituents. The image therefore appears as if the specimen has numerous sources of light within it. In addition to direct viewing through the microscope, images can be stored electronically after collection by digital or video cameras. With a digital camera, a color image is collected in its entirety on a two-dimensional array of photosensitive elements. Although digital cameras provide a very high resolution, the required light intensity can be high. Alternatively, a video camera, which has lower light requirements, can be used to sample the image according to a preset scanning pattern. Because of the high light sensitivity of the video camera, it permits viewing of cells for long periods without associated light damage. Such image intensification is particularly important for viewing live cells that contain fluorescent labels, which can be toxic to cells at high light intensities. A

Bright field

B Phase

contrast

Figure 2-10 Different light microscopictechniques give strikingly different images. (A) Bright-field image of a cell, typical of that obtained with an unstained specimen viewed through a compound light microscope, exhibits l~ttlecontrast and few details. (B) Phase-contrast image height-

Electron microscopy For all imaging devices, the limit of resolution is directly related to the wavelength of the illuminating light. That is, the shorter the wavelength of the illumination, the shorter the minimal distance between two distinguishable objects (i.e., the greater the resolution). In electron microscopy, a highvelocity electron beam, rather than visible light, is used for illumination. Because the wavelength of electron beams is much shorter than that of visible light, electron microscopes have much better resolution. Indeed, modern transmission electron microscopes typically have a resolution of 0.5 nm (5 angstroms, A), whereas light microscopes have a resolution of no less than about 1000 nrn (1pm). Because the effective wavelength of an electron beam decreases as its velocity increases, the limit of resolution of an electron microscope depends on the voltage available to accelerate the illuminating electrons. The transmission electron microscope forms images by sending electrons through a specimen and focusing the resulting image on an electron-sensitive fluorescent screen or photographic film (Figure 2-11). The electron beam is modified by magnets, which bring the electrons into alignment and focuses them on the specimen, much like the condenser lens in a compound light microscope. Image formation depends on the differential scattering of electrons by different regions of the specimen; scattered electrons cannot be focused by the objective lenses and thus do not impinge on the viewing screen. Because the electron beam passes through an unstained sample nearly uniformly, little differentiation of its components is possible without staining. The most common stains for electron microscopy are salts of heavy metals (e.g., osmium, lead, or uranium), which increase electron scattering. In photographs of an electron microscope image, components stained with such electrondense materials appear dark. c Nomarski

ens the visual contrast between different regions of the specimen. (C) Nomarski (differential-interferencecontrast) microscopy provides the sensation of depth to the image. [Courtesy of Matthew J. Footer.]

P R I N C I P L E S OF P H Y S I O L O G Y

................................... Tungsten filament

Condenser lens

Viewing screen

or

Figure 2-11 Electron microscopes share features such as lenses with compound opt~calmicroscopes, but use an electron beam rather than a light beam to illuminate the specimen. In a transmission electron microscope, shown here, an image is formed by passing electrons through an object and projecting them onto a fluorescent screen. In a scanning electron microscope, electrons reflected from the surface of a specimen coated with a reflect~vemetal film are collected by lenses and viewed on a cathode ray tube.

Since air would deflect the focused electron beam aimed at the sample, the specimen, must be held in a vacuum during imaging. Specimens must be very well fixed to preserve their biological structure during viewing in the electron microscope. Glutaraldehyde is used to covalently cross-link proteins and osmium tetroxide to stabilize lipid bilayers. After fixing, the specimens are infiltrated with a plastic resin. Thin sections cut from the resin block then are stained and finally placed on a metal grid in the transmission electron microscope. Specimens must be sectioned into extremely thin slices (50-100 nm thick) to allow penetration by the electron beam. Only diamond or glass knives are sharp enough to cut tissue sections into such thin slices. Glass knives are formed by breaking on the diagonal a 2.5-cm glass square that is about 5 mm thick. Because glass is actually a slowmoving liquid, the edge formed is only sharp enough to cut tissue for a few hours before molecular flow of glass dulls the edge. Although extremely expensive, diamond knives do not suffer from this problem and thus are the preferred tool for cutting thin sections.

The exquisite detail available from the transmission electron microscope can provide important insights into the structure of biological tissue (Figure 2-12A). Unfortunately, the size of the specimen that can be examined is very small, because it must be thin sectioned. Consequently, it is difficult to develop an understanding of the three-dimensional structure without the truly tedious procedure of reconstructing an image from a series of individual sections. Development of various techniques for preparing specimens for transmission electron microscopy have extended the range of objects that can be visualized and the information available from images. The scanning electron microscope, like the transmission electron microscope, uses electrons rather than photons to form images of the specimen. However, the scanning electron microscope collects electrons scattered from the surface of a specially prepared specimen. This instrument provides excellent three-dimensional images of the surface of cells and tissues, but it cannot reveal features beneath the surface (Figure 2-12B). Before examination in a scanning electron microscope, the specimen is coated in an extraordinarily thin film of a heavy metal like platinum. The tissue is then dissolved away with acid, leaving a metal replica of the tissue's surface, which is viewed in the microscope. Scanning electron microscopes have a resolution of about 10 nm, considerably less than the resolution of transmission electron microscopes.

Cell Culture The rearing of cells in uitro (Latin for "in glass") in glass or plastic containers is known as cell culture. This technique has revolutionized our ability to study cells and the physiological processes they support at the tissue and organ level. Historically, explants (small pieces of tissue removed from a donor animal) were kept alive and grown in a flask filled with an appropriate mixture of nutrients and other chemicals. Today, the most common procedure is to break up (dissociate) small pieces of tissues and then suspend the dissociated cells in a nourishing chemical broth in which they grow and divide as separate entities. Successfully growing cells in vitro requires the right culture medium, the liquid in which the cells are suspended. Up until the early 1970s, cells from all animals were routinely grown in liquid medium consisting largely of either serum (a clear component of blood plasma) from horses or fetal calves or of an unrefined chemical extract made from ground-up chick embryos. However, these media were poorly defined chemically, containing numerous unidentified compounds. Moreover, it was difficult to predict whether cells from a particular source would grow in one of these media, or what components might be added if the first attempt was unsuccessful. Growing cells in vitro was largely a matter of trial and error (and luck). Today, defined culture media manufactured according to precise chemical formulas are available for research. However, the successful culture of many cell types requires addition of a small trace (less than 5%) of horse serum to such defined media. This observation

tion, many types of animal cells have not yet been successfully cultured. However, the list of cells that can be cultured is constantly growing, the result of refinements in media and culture techniques. For example, cell strains derived from the following tissues and organs can be grown in culture: Bone and connective tissue Skeletal, cardiac, and smooth muscle Epithelial tissue from liver, lung, breast, skin, bladder, and kidney Some neural tissue Some endocrine glands (e.g., adrenal, pituitary, islets of Langerhans in pancreas ) M

0.2 pm

In contrast to normal animal cells, cancer cells commonly exhibit rapid, uncontrolled growth in the body and are c a ~ a b l eof indefinite growth in culture. Treatment of some normal cultured cells with certain agents may cause transformation, a process that makes them behave like cancer cells that have been isolated from tumors. Such transformed cells also can be cultured indefinitely. Homogeneous populations of such "immortal" cells are termed cell lines. Although normal cells differ from cancer cells and transformed cells in many ways, cell culture of the latter has permitted certain types of studies that are not feasible with primary cell cultures of normal cells. Cell culture has numerous potential uses in animal physiology. New devices such as silicon wafer sensors for measuring acidity and other variables have been combined with cell culture techniques to provide important insights into cellular and organismal physiology. For example, the hormonal regulation of H+secretion from a variety of cells grown in vitro can be studied by stimulating the cultured cells with agonists and antagonists and measuring the changes in the rate of medium acidification. This approach has also been used to study tissues and organs with unusual rates or ~ r o ~ e r t iof e sH + secretion, such as the swim bladder tissues of fishes. 1

'

L

Figure 2-12 Transmission electron mic~oscopyprovides a view of the interior of biological tissue, while scanning electron microscopy emphasizes surface features (A) Transmission electron micrograph of cilia in the mouse oviduct. (B) Scanning electron micrograph of cilia in the mouse oviduct. [Courtesy of E. R. Dirksen.]

Complete medium

n

suggests that some growth factor in blood is necessary for the growth and division of animal cells in vitro (Figure 2-13). Even with the availability of defined culture media, growing animal cells in vitro is a demanding technique. Normal animal cells generally can grow only for a few days in vitro, then stop multiplying and eventually die out. A relatively homogeneous population of such cells is referred to as a cell strain. Cultured cell strains are useful for many kinds of experiments but their limited life span makes them unsuitable for other studies. In addi-

C

EGF absent from medium

P a,

EGF added I

0

2

4 Days in culture

I

6

Figure 2-13 Cells grown in culture often require specific factors to stimulate maximal rates of division and growth. In the culture indicated, maximal cell numbers are reached only in the presence of ep~dermalgrowth factor (EGF). Addition of EGF (arrow)to a culture lacking this substance results in immediate further growth of the cell colony. [Adapted from Lod~shet al., 1995.1

28

PRINCIPLES O F P H Y S I O L O G Y

..............................................................................

BIOCHEMICAL ANALYSIS Most biochemical processes occur in aqueous solution and require the exchange of gases. For this reason, physiologists often need to measure the chemical composition of the fluid in various body compartments and/or the concentration of its constituents. For example, to assess whether a crab can regulate its internal salt concentration when swimming in dilute estuarine waters, a physiologist would need to know the salt concentration in the water surrounding the crab, in the crab's hemolymph (blood), and in the urine produced by the crab. With these data, the ability of the crab to maintain homeostasis can be evaluated. Biochemical analyses of biologically relevant fluids, gases, and structures typically are based on some physical or chemical attribute of the materials of interest (e.g., Na+ in the crab's urine). The substantial increase in the sensitivity and accuracy of such measurements in the recent past has allowed physiologists to ask questions about subtle physiological functions that previously could not even be measured. Both qualitative and quantitative analyses can be important for physiological studies. The objective of the first is to determine the composition of some fluid or structurethat is, the elements, ions, and compounds that compose it. The objective of the second analytical approach is to measure the concentration of particular substances in the fluid or structure of interest. Many analytical instruments and techniques provide both composition and concentration data. Measuring Composition: What Is Present Numerous time-honored and emerging methods are available for measuring chemical composition. Sometimes, animal physiologists are interested only in knowing whether a particular substance (e.g., ammonia or hemoglobin) is present in a sample. At other times, they may want to identify all the different proteins or carbohydrates or other molecular species in a sample. In other words, the nature of the problem being studied determines which compositional data are relevant. Rarely is a biological sample subjected to a full compositional analysis similar to that assigned in a beginning chemistry laboratory course. A wide variety of colorimetric assays have been developed for determining the presence or absence of specific substances in a solution. These assays depend on the substance of interest undergoing a chemical reaction that changes its ability to absorb visible light or ultraviolet (W)

radiation at different wavelengths. As a result, the transmission of light or W radiation by the solution changes, which can be detected by a spectrophotometer. Many biochemical assays employ an enzyme that catalyzes a reaction involving the substance of interest. For example, a common assay for lactate (a product of the anaerobic metabolism of glucose) makes use of an enzyme that converts lactate into products with different W absorption properties. To perform this assay, a solution suspected of containing- lactate is placed in a small reaction vial along with the enzyme and other reaction components. After a short time, the vial is placed in a spectrophotometer, and the UV transmission of the solution is measured. The transmission of a control reaction vial lacking the enzyme also is measured. A difference in UV transmission through the control and assay vial indicates that lactate is present in the sample. Chromatography is a widely used technique for separating proteins, nucleic acids, sugars, and other molecules present in a mixture. In its simplest form, paper chromatography, the components of the sample move at different rates in chromatography paper, depending on their relative solubility in the solvent, as illustrated in Figure 2-14A. In order to visualize the separated components, the chromatogram commonly is sprayed with a colorimetric reagent that produces a visible color with the components of interest. More complex mixtures can be separated by column chromatography, in which the sample solution is passed through a column packed with a porous matrix of beads (Figure2-14B). The different components of the sample pass through the column at varying rates, and the resulting fractions are collected in series of tubes. Depending on the nature of the sample, different assays are used to determine the presence of the separate components in the fractions collected. Many different kinds of matrix are employed in column chromatography depending on the composition of the solution being separated. For example, matrices are available that sort components according to their charge, size, insolubility in water (hydrophobicity),or binding affinity for the matrix. The last type of matrix is used in afinity chromatography, in which the matrix beads are coated with molecules (e.g., antibodies or receptors) that bind to the component of interest. When a sample mixture is applied to the column, all the components except the one recognized by the affinity matrix pass through. This is a very powerful technique for purifying proteins and other biological molecules present at very low concentrations. Electrophoresis is a general technique for separating molecules in a mixture based on their rate of movement in an applied electric field. The net charge of a molecule, as well as its size and shape, determines its rate of migration during electrophoresis. Small molecules such as amino acids and nucleotides are well separated by this technique, but by far the most common use of electrophoresis is to separate mixtures of proteins or nucleic acids. In this case, the sample is placed at one end of an agarose or polyacrylamide gel, an inert matrix with fixed diameter pores that

impedes or allows migration of molecules when an electric field is applied. Protein mixtures usually are exposed to SDS, a negatively charged detergent, before and during electrophoresis. The rate of migration of the resulting SDScoated proteins in the gel is proportional to their molecular weight; the lower the molecular weight of a protein, the faster it moves through the gel ( ~ i ~ u2-15). r e when a protein-binding stain is applied to the gel, the separated proteins are visualized as distinct bands. A

Three slightly different, but basically similar, procedures employing gel electrophoresis are used to separate and detect specific DNA fragments, messenger RNAs (mRNAs), or proteins. Each of these procedures involves three steps (Figure 2-16): I. Separation of the sample mixture by gel electrophoresis 2. Transfer of the separated bands to a nitrocellulose or other type of polymer sheet, a process called blotting .3. Treatment of the sheet (or blot) kith a "probe" that reacts specifically with the component of interest The first of these procedures to be developed, named Southern blotting after its inventor Edward Southern, is used to identify DNA fragments containing specific nucleotide sequences. Northern blotting is used to detect a particular mRNA within a mixture of mRNAs. Specific proteins

Chromatography paper Sample at origin

A

condiner with solvent B

sepa;ated components w ~ t hSDS

Add solvent through column

Partially separated proteins

I

Stain to visualize separated bands

Collect fractions; larger components pass more quickly Figure 2-14 Chromatography is a powerful technique for separating the components of a mixture in solution. (A) In paper chromatography, the sample is applied to one end of a piece of chromatographic paper and dried. The paper is then placed into a solution containing two or more solvents, which flow upward through the paper via capillary action. Different components of the sample move at different rates in the paper because they have different relative solubilities in the solvent mixture. After several hours, the paper is dried and stained to determine the location and relative amounts of the separated components. (B) In column chromatography,the sample is applied to the top of a column that contains a permeable matrix of beads through which a solvent flows. Then solvent is pumped slowly through the column and is collected in separate tubes (calledfractions) as ~temerges from the bottom. Components of the sample travel at d~fferentrates through the column and are thus sorted Into d~fferentfract~ons.

II @ @$ @ @

Ij

Proteins separated by size

Figure 2-1 5 Gel electrophoresisseparates the components of a mixture based on their charge and their mass. Proteins commonly are separated by SDS-polyacrylam~de gel electrophoresis, as illustrated here (A) SDS, a negatively charged detergent, is added to the sample to coat the proteins. (B) The sample then is placed in a well in the polyacrylamide gel and an electricf~eldis applied. Small proteins move more easily and faster along the length of the gel than larger ones. (C)After a period of time, the rotei ins in the mixture are separated into bands composed of proteins of different sizes. These can be visualized by various proteinstaining reagents. [Adapted from Lodish et al., 1995.1

30


-

Band corresponding to component A

Electrophoretic transfer

Electric current

reagent spec~flc

corresponding t o component B

Gel after electrophoresis

Polymer sheet

Audioradiograms

Figure 2-16 Southern, Northern, and Western blotting are similar proceduresfor separating and identifying specific DNA fragments, mRNAs, and proteins, respectively,within a mixture. In each method, the components of a sample first are separated by gel electrophoresis; the separate bands are transferred to a polymer sheet, which then is flooded w~tha radiolabeled reagent specific for the component of interest. The presence and in some cases the quantity of the labeled component is determined by autoradiography.See Table 2-1 for details of each procedure.

be the deflection of the ions from a standard path as they head towards the analyzer. The degree of deflection is detected by an array of detectors, which then allows determination of the presence and quantity of gases in the gas sample introduced into the mass spectrometer. The various techniques for measuring chemical composition described in this section are widely used by physiologists, but many others also are employed in physiological research. To learn about additional methods and further details about those discussed here, you can refer to chemistry and biochemistry textbooks.

within a complex mixture can be detected by Western blotting, also known as irnrnunoblotting. (As yet, there are no Eastern, South-Western, etc., blots, but it is probably just a matter of time.) Table 2-1 summarizes the unique features of the three blotting procedures. Many of the common methods of determining composition are applicable to solutions but not gases. The mass spectrometer, however, can distinguish the different gases composing a gaseous mixture based on their mass and charge. Animal physiologists most often use this instrument to determine the composition of respiratory gases while an animal is resting or exercising in an experimental setting. Figure 2-17 illustrates the basic design of a mass spectrometer. The gas sample first is ionized by intense heating and passage through an electron beam. The charged ions then are focused and accelerated by an electric field into an analyzer where the beam of ions is deflected by either an applied magnetic field or by passage through tuned rods emitting specific radio frequencies that will deflect ions. The lighter the ion mass and smaller its charge, the smaller will

Measuring Concentration: How Much Is Present

Most instruments or analytical techniques used to determine the composition of a fluid or gas mixture also provide data about the concentration of the components present. For example, the degree of color change produced in a colorimetric assay depends on how much of the substance being measured is present in the sample. Likewise, the output signal from a mass spectrometer depends not only on the

TABLE 2-1 Electrophoreticblotting procedures - -

-

-

Molecules detected

Separation and detection procedure*

DNA fragments produced by cleavage of DNA wrth restrlctlon enzymes

Electrophorese mlxture of dsDNA fragments on agarose or polyacrylamlde gel, denature separated fragments Into ssDNA and transfer bands t o polymer sheet, use radlolabeled ssDNA or RNA t o label fragment of Interest, detect labeled band wlth autorad~ography

Northern blotting

Messenger RNAs

Denature sample mixture; electrophorese on polyacrylamide gel and transfer separated bands t o polymer sheet; use radiolabeled ssDNA t o label mRNA of Interest; detect labeled band with autoradiography

Western blotting

Proteins

Electrophorese sample mixture on SDS-polyacrylamide gel and transfer separated bands t o polymer sheet; use radiolabeled monoclonal antibody t o label protein of interest; detect labeled band with autoradiographyt

Southern blottlng

'

%

*dsDNA

=

double-stranded DNA, ssDNA = single-stranded DNA.

+Ifradiolabeled monoclonal antibody is not available, then the band contaming the antibody-protein complexes can be detected by addlng a secondary antibody that binds to any monoclonal antibody Th~ssecondary antibody is covalently linked to an enzyme, such as alkaline phosphatase, that catalyzes a colorimetric reaction. When substrate is added, a colored product forms over the band with the protein of interest, generating a visible colored stain in this region of the blot.

Applied magnetic field

I

,,,

To vacuum

Accelerator

1 beam

To amplifier and recorder

ion~zation Sample

B

py$:.~Fs

fi

particles Ion source

Ion collector

Ion source

Ion collector

Ion source

Ion collector

Decreasing ionization voltage

Figure 2-17 The identity of gases in a mixture and their concentrations can be determined by mass spectrometry. (A) The fixed-collector mass spectrometer, which detects how much an ionized sample is deflected by an imposed magnetic field, has four essential parts. First is a carefully constructed inlet device (I) through which the sample is delivered to the system with a constant viscous flow. Second is an ionization chamber (Z), kept under vacuum and at high temperature (about 19O0C),where the sample passes through an electron beam and is accelerated vla application of an electric field. The gas molecules leave this chamber as negatively charged ions. Third is an analyzer tube in which the accelerated beam of ions are subjected to a magnetic field that causes the ions to

flow in a curved path (3). Finally, the ion beam is detected using an ion collector situated at the end of the analyzer tube (4).The extent to which an ion is bent in the applied magnetic field depends on the strength of the field and the ion's mass, charge, and velocity. Only those Ion species that are bent so their ~ a t h s parallel the sides of analyzer tube will reach the ion collectors and be detected. (B) At a constant magnetic field, the specific type of particles detected in the mass spectrometer is determined by the strength of the ion~zation voltage, which can be varied. The lower the ionizing voltage, the heavier is the particle detected. [Adapted from Fessenden and Fessenden, 1982.1

types of gases present in a mixture, but how much of each is present. Thus the output signal produced by an analytical instrument- be it a transmission spectrophotometer, densitometer, or mass spectrometer-is directly related to the concentration of the substance responsible for the signal. Typically, the analytical technique being used to determine the concentration of a particular substance is carried out on several samples of the substance at different known concentrations'; the output signals then are plotted against the concentrations, yielding a standard curve. The actual concentration of an experimental sample corresponding to the output signal it produces is determined by comparison with this standard curve.

difficult, if not impossible, to do by studying the intact organs in situ; instead, experiments are conducted on isolated organs removed from the animal by surgery and maintained in an in uitro artificial environment. Two examples will illustrate the power of this experimental approach. When the heart of almost all vertebrates, including mammals, is isolated and placed in a bath of saline, it continues to beat and perform work by pumping saline or other fluid supplied to it. The isolated vertebrate heart will continue to beat in the absence o f neural input if it is kept at an appropriate temperature and is perfused with an oxygenated solution that has the correct ionic composition and contains an energy source such as glucose. With the heart isolated, physiologists can measure the effect of chemical stimulation by drugs and hormones or electrical stimulation of nerves within the heart on the heart rate, amplitude, flow rate, and mechanical movements. Experiments performed on isolated hearts have been fundamental in advancing our knowledge of the cardiovascular system. A second example is the vertebrate pineal gland, a small organ found at the top of the vertebrate brain. The pineal, which plays a key role in regulating daily (circadian) rhythms in physiological processes, is sensitive to light-

EXPERIMENTS WITH ISOLATED ORGANS AND ORGAN SYSTEMS All animals have several differentmajor organ systems that must be coordinated and controlled to help maintain homeostasis. As we'll examine in later chapters, the functions of these organ systems are regulated primarily by neural and/or hormonal inputs. To understand physiological control mechanisms, the key controlling inputs and their sources must be characterized. In many cases, this is

32


.........................................

related stimuli and releases various amounts of regulatory chemicals into the bloodstream as a function of the time of day. When the pineal gland is isolated and placed into an appropriate culture system, it continues to exhibit a circadian rhythm. Direct experimentation with this in vitro preparation has provided answers to specific questions concerning pineal gland regulation of physiological systems.

OBSERVING AND MEASURING ANIMAL BEHAVIOR Scientists studying animal ~ h ~ s i o l frequently og~ supplement their experiments with observations of animal behavior. Useful behavioral experiments are difficult to perform, however, because the animals must be in an appropriate physiological state (e.g., breeding, rearing young, digesting a meal, to name a few). Further, the experiment must exploit natural behavioral tendencies in the animal. Despite these difficulties, experimental methods to control and stimulate specific behavioral states can provide important insights into physiological processes that are not always amenable to direct physiological investigation. The prerequisite for such experimentation is a thorough knowledge of the natural behavior of the animal in its habitat. The Power of Behavioral Experiments

Research in the 1950s and 1960s on the retrieval behavior of ground-nesting birds illustrates how behavioral studies can contribute to physiological knowledge. K. Z. Lorenz and N. Tinbergen discovered that geese not only recognized their eggs and recovered them if they lay outside the nest, but also would retrieve a wide variety of objects (e.g., grapefruits, light bulbs, baseballs) laying near their nest. Tinbergen and his students subsequently conducted ingenious experiments with gulls in which they offered pairs of objects to the birds and recorded which one was recovered first. By exploiting the process of painvise comparison, they could define the properties that gulls use to choose what to retrieve. Although the birds would retrieve many different objects, these experiments showed that real eggs are always preferred over unnatural objects. The relative size, color, and speckling of an egg were found to contribute independently to the likelihood that an egg would be retrieved. Taken together, these experiments revealed that for gulls, eggs provide a powerful natural stimulus that induces specialized retrieval behavior. Armed with knowledge of the exact properties of the stimuli causing this behavior, physiologists have been better able to conduct physiological experiments about the nature of vision in birds. Behavioral experiments often analyze the total time the animal under study spends performing each behavior and the temporal sequence of behaviors. These data, in conjunction with information about the behavior of other animals and key environmental variables, frequently reveal how closely behavior is related to the internal state of the animal. The great majority of information about animal behavior collected in this way pertains to reproduction and

feeding, two of the most important behaviors engaged in by any animal; both reproductive and feeding behaviors are greatly affected by an animal's physiological state. Careful observations usually can reveal which behavioral patterns of one individual influence another and may suggest why this might be so. For example, in stickleback fish, the display of a red belly by a male signals to other male sticklebacks that he is defending a nest and to females that he is interested in spawning. Thus, the meaning of this signal depends on the sex of the receiver. The red belly arises from physiological processes triggered by the onset of the breeding season. The coordination between behavior and physiology in this species was investigated using behavioral analysis to guide physiological investigation. Methods in Behavioral Research

A variety of instruments are used to record and analyze the physiological basis of specific behavioral acts. In some experiments, high-speed video cameras are used in conjunction with electrophysiological detectors of neural or muscular activity to capture simultaneously both the behavior and its physiological underpinnings. Since behavioral acts of interest are often rapid and fleeting, these events typically are recorded at high speed and the video tape played back at slow speed to aid analysis. The use of x-ray cameras allows examination of the interaction of skeletal components during specific behaviors (e.g., feeding, running on a treadmill). As in so many other aspects of physiology, the availability of inexpensive, fast computers with ever-increasing data storage capabilities has revolutionized the acquisition and analysis of data. Figure 2-18 illustrates how many of the commonly used techniques to study animal behavior and its underlying physiological processes can be brought to bear on a single behavior-the feeding strike of a venomous snake. To discover how such a strike proceeds, the motion of the body and jaws must be related to the forces exerted by contraction of the jaw muscles. The rapid strike is recorded in two views, dorsal and lateral, using a video camera viewing the animal directly and via a mirror set at 45' above the snake. Quantification of the position of the animal is possible because of a grid image in the background that is included in the video images. The snake is placed on a platform that records the force exerted along three orthogonal axes. By measuring this set of external parameters, the investigator can record the forces associated with the snake's movement across the surface. The force exerted by the jaws of the snake are recorded by a strain gauge mounted on the head, and muscle activity is measured by electrodes into the four lateral jaw muscles. All of the data are recorded on both tape and in a computer using data-acquisition hardware and software. The values of the measured variables are typically displayed as a function of time and related to the behavioral analysis recorded on videotape. Data from such an experiment reveals how contraction of the muscles results in positioning of the fang and closing of the jaws around the prey.

EXPERIMENTAL METHODS FOR EXPLORING PHYSIOLOGY

33

...............................................................................

Bipolar electrode

Strain gauge

Force platform Oscilloscope

Highspeed camera

recorder Tape recorder

Figure 2-18 The feeding strike of a venomous snake can be analyzed to determine the muscles used and the pattern in which they contract. (A) To record electrical potentials from the jaw musculature, fine bipolar wire electrodes are placed surgically into the four lateral jaw muscles in a procedure that is performed under anesthesia. Astrain gauge is also attached to the top of the snake's head t o measurethe motion of underiy-

ing skull bones. (B) The snake is placed on a force-recording table and videotaped as it strikes its prey. The leads from the electrodes and strain gauge are connected t o electronic amplifiers, which boost their lowvoltage signals. The amplified signals are displayed on an oscilloscope and chart recorder and stored on a tape recorder and computer.

These experimental measurements can be used to test hypotheses about which structures and muscles are involved in a strike and how their temporal relationships change during the behavior. The experiment also suggests how many physiological systems contribute to production of a complex behavioral act. A more complete functional analysis of this prey-capture behavior and a greater understanding of the performance of the animal is possible if other variables are measured in repeat experiments performed under identical conditions. This experimental setup could be used to measure differences in the strike behavior as a function of the size and type of prey species. Such measurements also can form the basis for formulation of hypotheses about the neural control of muscle activity, visual feedback guiding the behavior, and a host of other interesting topics.

IMPORTANCE OF PHYSIOLOGICAL STATE IN RESEARCH

an b e instructed t o behave ways during physiolo breathe deeply, t o run, o r t o flex muscles). Some animals can b e trained t o perform as needed for a particular experiment (e.g., t o run o n a tread-

perform, is trained t o perform, o r is simply allowed t o behave spontaneously?

a

Research studies at all physiological levels-molecular to behavioral-must take into account the animal's physiological state at the time of experimentation (or tissue sampling). Some physiological states are quite obvious to the investigator, as when an animal is diving (breath holding), actively moving, or hibernating. Other physiological states may be far more subtle, but have just as great an influence on physiological processes. Of course, the obvious or subtle nature of a physiological state depends on the animal. For instance, a mouse that is curled up with eyes closed and is showing relatively regular breathing with no locomotor activity can probably be assumed to be asleep. But what about a relatively sluggish species of fish that is motionless? Is it asleep, or merely not showing locomotor activity? Physiological state may be greatly influenced by environmental variables such as the season and time of day. To illustrate, stimulation of the vagus nerve causes a much greater slowing of the heart in temperate frogs examined at night in the spring than in frogs examined in the afternoon in the autumn. Thus the outcome of an experiment can be greatly influenced by the time of day and year when it is performed experiments are carried out. To characterize the physiological states of an animal, one or more variables can be measured while the animal is in different behavioral states and their values compared. For example, blood pressure, pulse rate, and skeletal musclc aiti\,~tymight all be measured simultaneously while the animal is observed in several different states such as sleeping, moving, digesting a meal, or hibernating. Such

34

PRINCIPLES OF P H Y S I O L O G Y

.........................................

measurements usually do not allow identification of cause-and-effect relationships among the measured variables. However, inferences can be drawn and testable hypotheses about the relationships among the measured variables can be formulated based on such data. Because multiple physiological states can exist simultaneously (e.g., sleeping in winter, breath holding during hibernation), experiments to determine physiological states are often complex and time consuming. However, such experiments, if carefully planned to reveal the influences of phpsiological state on the basic physiological processes of an animal, can greatly elevate our knowledge of physiological systems. A typical experiment, for example, might measure key physiological variables during intermittent bouts of hibernation of the ground squirrel. Comparison of body temperature and metabolic rate recorded over time with observed behavioral activity reveals that the increased activity in the awake state is correlated with increased body temperature and metabolic rate. These correlations suggest that as the animal becomes active, key physiological systems also become active at about the same time. Although it makes intuitive sense that the animal will need more blood circulating when it is physically active, it is not clear from these data how that increase in blood flow is achieved or how it is regulated. Does the physiological change precede the behavioral activity or result from it? Distinguishing among these and other possible explanations requires experiments that focus on the causal relations,hips between specific behaviors and the physiological systems that underlie changes in physiological state. Observations of correlations between physiology and behavior such as those discussed here usually form the basis for such subsequent experiments. Perhaps as important, they can provide a way to characterize specific physiological states. In a study designed to probe the causal relationship between blood flow and heart rate during hibernation, for example, variables such as body temperature or metabolic rate could be used to assure that the animal was indeed hibernating during the experimental tests.

SUMMARY Physiological research should begin with a well-formed, specific hypothesis related to a particular level of analysis and capable of being tested experimentally. Testing of hypotheses is greatly facilitated by employing the August Krogh principle, that is, choosing the optimally suited animal for carrying out those experiments needed to answer particular questions. A key issue in designing physiological experiments is the level at which each physiological problem studied will be analyzed. The choice of level determines the methodology and experimental animal appropriate for measuring the physiological variables of interest. Techniques that detect or analyze events at the molecular level have greatly benefited animal physiology. Radioisotopes can be incorporated into physiologically im-

portant molecules or their precursors. After a radiolabeled molecule is injected into the animal, its movements can be determined by subsequently sampling tissue and measuring the particles emitted by the radioisotope using either a Geiger counter or scintillation counter. The presence and location of radiolabeled molecules in thin tissue slices can be detected by autoradiography. Monoclonal antibodies covalently labeled with a fluorescent 'dye or radioisotope are another powerful tool for tracing the movement of specific proteins within physiological systems. Because of their great specificity, monoclonal antibodies permit detection of a single protein (e.g., nerve growth factor or a neurotransmitter) even when it is present at a very low concentration in the cells or tissues under study. Genetic engineering, which involves recombinant DNA technology and gene cloning, is also revolutionizing animal physiology. Genes cloned in easily grown bacterial cells can be used to produce large quantities of the gene products (e.g., human insulin and other hormones). Genetic engineering techniques also allow production of transgenic animals (commonly mice) that contain additional copies of a gene of interest. In knockout mice, a normal gene is replaced with a mutant form of the same gene, so the animals cannot produce a functional protein. Analysis of the effects of either the addition or deletion of specific genes can provide insights into the mechanism and regulation of a physiological process. Microelectrodes and micropipettes have many uses in cellular physiology. The most common use of microelectrodes is to record electrical signals from neurons or muscle cells. The concentration of ions and some gases and the fluid pressure within cells or blood vessels can be determined with specially constructed microelectrodes. Micropipettes are used to inject materials (e.g., dyes, radiolabeled compounds) into individual cells or fluid-filled tissue spaces. Structural analysis of cells, and the physiological processes that derive from these cells, depends heavily on microscopy. Light microscopy uses photons of visible or near visible light to illuminate specially prepared tissue samples. Specimens are first fixed (preserved),embedded in plastic or wax, and then cut into extremely thin slices (sections) with a microtome. Finally, the sections are treated with organic dyes or fluorescent-labeled antibodies that differentially bind to and stain various cell components. Once prepared, tissue is typically viewed with one of a variety of light microscopes. The advent of electron microscopes, which use electrons to form images, greatly increased the resolution of microscopic analysis, permitting visualization of intracellular structural details not apparent with light microscopes. In transmission electron microscopes, a beam of electrons is directed straight through ultrathin tissue slices stained with electron-dense heavy metals. In scanning electron microscopes, electrons are reflected from the surface of the specimen, producing a three-dimensional image of the surface features of cells and other structures.

E X P E R I M E N T A L M E T H O D S FOR E X P L O R I N G P H Y S I O L O G Y 35 ...............................................................................

Cell culture, the rearing of cells in vitro, allows the propagation of relatively short-lived cell strains and "immortal" cell lines, which can grow indefinitely. Cultured cells, which usually are quite homogeneous, are very useful in experiments designed to examine the functions, secretions, responses, and other properties of particular cell types. Such experiments depend on biochemical analysis to determine the composition of sample mixtures derived from cells as well as the concentration of the constituents present. Among the most commonly used techniques in biochemical analyses are colorimetric assays, transmission spectrophotometry, paper and column chromatography, electrophoresis, and mass spectrometry. At an increasing level of organizational structure, maintenance of isolated organs or entire organ systems in uitro allows the function of intact tissues to be examined in an artificial, controlled environment. Important variables such as temperature, oxygen availability, and nutrient levels can be controlled, mimicking homeostasis, or can be varied to test particular hypotheses. .Animal physiologists frequently supplement their experiments with observations of animal behavior. Experimental methods to control and stimulate specific behaviors can provide important insights into physiological processes that are not always amenable to direct physiological investigation. Also, analysis of the total time spent performing each behavior and the temporal sequence of behaviors in conjunction with information about the behavior of other animals and key environmental variables may reveal how closely behavior is related to the internal physiological state of the animal. Finally, in all experimental approaches, from those conducted at the simplest (molecular) level to those suitable at most complex (behavioral)level, the animal's physiological state at the time of experimentation (or tissue sampling) is an important consideration. Physiological state can depend upon internally regulated factors (sleep, hibernation, activity, etc.) or environmental influences. To characterize the physiological states of an animal, one or more variables can be measured and the values of these key variables correlated with different behavioral states.

REVIEW QUESTIONS 1. What is the difference between a scientific question, a hypothesis, a theory, and a law? 2. An investigator carries out experiments on crickets, bullfrogs, and rattlesnakes, but is testing a single hypothesis related to a single physiological process. Explain how this investigator could be embracing the August Krogh principle.

3. 4. 5.

6.

7.

8.

9.

What are radioisotopes and monoclonal antibodies?What common feature makes them of use to physiologists? What is a clone, and how is it produced? If an interesting and useful mutation to a physiological system is ultimately lethal before an animal reaches the reproductive stage of its life cycle, how can it be perpetuated in the laboratory to allow repeated experiments for its long-term study? Why would an air bubble within a microelectrode used in recording nerve action potentials disrupt the recording? What are the major differences between light and electron microscopy?What are the advantages and disadvantages of each? Describe the difference between an experiment done in vivo, in uitro, and in situ. What are the advantages and disadvantages of each experimental approach? How would you determine if the resting heart rate of an animal was influenced by daily rhythms?

SUGGESTED READINGS Burggren, W. W. 1987. Invasive and noninvasive methodologies in physiological ecology: a plea for integration. In M. E. Feder, A. F. Bennett, W. W. Burggren, and R. Huey, eds., New Directions in Physiological Ecology. New York: Cambridge University Press, pp. 251 -272. (Descriptionof two major approaches to animal experimentation.) Burggren, W. W., and R. Fritsche. 1995. Cardiovascular measurements in animals in the milligram body mass range. Brazil.]. Med. Biol. Res. 28:1291-1305. (Description of methods for extending cardiovascular techniques to microscopic animals.) Cameron, J. N. 1986. Principles of Physiological Measurement. New York: Academic Press. (A short but detailed introduction to several important methods of physiological measurement.) Hall, Z. 1992. An Introduction to Molecular Neurobiology. Sunderland, Mass.: Sinauer Associates. (A comprehensive discussion of how a molecular approach can provide rich insight into a vitally important organ system.) Lodish, H., et al. 1995. Molecular Cell Biology. 3d ed. New York: Scientific American Books. (A well-written, very comprehensive text that describes many techniques used for molecular analyses of the cell.) Lorenz, K. Z. 1970. Studies in Animal and Human Behauior. Vol. 1.Cambridge, Mass.: Harvard University Press. (Collectionof research papers, translated from the original German, describing the early research of Lorenz, who won the Noble Prize in physiology in 1973.)

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3 MOLECULES, ENERGY, AND BIOSYNTHESIS

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he living organisms found on our planet form a vast and varied array, ranging from viruses, bacteria, and protozoa to flowering plants, invertebrates, and the "higher" animals. In spite of this immense diversity, all forms of life as we know it consist of the same chemical elements and share similar types of organic molecules. Moreover, all life processes take place in a milieu of water and depend on the physicochemical properties of this extremely abundant and very special solvent. That all living organisms share a common biochemistry is one of the powerful evidences in support of their evolutionary kinship, the common thread that runs through all areas of biological study.

ORIGIN OF KEY BIOCHEMICAL MOLECULES

'

Biologists generally agree that life arose through processes of chance and natural selection under appropriate environmental conditions on the primitive Earth. Experiments first performed by Stanley Miller in 1953 show that certain molecules essential for primitive life (e.g., amino acids, peptides, nucleic acids) can be formed by the action of lightning-like electric discharges on an experimental atmosphere of methane, ammonia, and water. This simple atmosphere is believed to be similar in composition to that of the primitive atmosphere of Earth about 4 billion years ago. Earth's early atmosphere was modified during subsequent eons by photosynthetic plants, which added the currently immense quantities of oxygen and which took up nitrogen compounds for incorporation into nitrogenous biological compounds. The experimental formation of simple organic molecules under conditions similar to those that may have prevailed in the primeval atmosphere suggests that such molecules may have accumulated in ancient shallow seas, forming an "organic soup" in which life may then have undergone its first evolutionary stages of organization. The combination and recombination of these molecules eventually led to the most simple life forms capable of producing and arranging more complex molecules into informa-

tional assemblages like nucleic acids and enzymes. Critical in the process of producing primitive cell-like organisms was the formation of small liquid droplets with membranes around them. Lipid (fat) molecules will spontaneously form a double layered "molecular skin" around microscopic fluid droplets. When these skins began to incorporate other materials (simple nucleotides, etc.), then the first steps were under way for the formation of a true cell membrane-thin structures that enclose the contents of a cell, control movement of molecules between the cell interior and the surrounding environment, and provide a potential structure for organizing its contents. Many, many such additional steps defined the path towards the current vast array of species in the more than 35 phyla now found on Earth. This hypothetical scenario of the first stages towards the evolution of life raises many questions. To what degree did the origin of life depend on the "right" conditions? Would life of another sort have appeared on Earth if the chemical and physical environment had been quite different? What if there had been no carbon atom? As we will see shortly, the occurrence of life as we know it (and can imagine it) depends heavily on the chemical nature of the Earth's environment. Life would be either nonexistent or at least vastly different if some of the fundamental properties of matter of the early atmosphere had been different. A controversyonce raged between the vitalists, who beprinciples not found lieved life was based on special LLvital" in the inanimate world, and the mechanists, who maintained that life can ultimately be explained in physical and chemical terms. Until the early part of the nineteenth century, students of the natural world supposed that the chemical composition of living matter differed fundamentally from that of inanimate minerals. The vitalist view held that "organic" substances can be produced only by living organisms, setting them apart in a mysterious way from the inorganic world. This concept met its end in 1828, when Friedrich Wohler reacted lead cyanate and ammonia, both obtained from nonliving mineral sources, to synthesize the simple organic molecule urea:

ciples of chemical reactions apply to the assembly of macromolecules and more complex cellular organelles that constitute the cell. His successful organic synthesis set the stage for modern chemical and physical studies aimed at elucidating the mechanisms of life processes. Modern biochemists can now duplicate in vitro in isolated cell-free systems nearly every synthetic and metabolic reaction normally performed by living cells. The biochemical and physiological processes of the living organism ultimately depend on the physical and chemical properties of the elements and compounds it contains. At first glance, the properties of living systems seem far too marvelous and complex to be explained by a mere mixture of elements and compounds. Yet, living systems are not simple chemical "soups"; rather, they are highly organized structures composed of often very large and complex molecules called macromolecules. Macromolecules of many kinds participate in the regulation and direction of chemical activities within living cells. Organelles such as the plasma membrane, lysosomes, and mitochondria lend structural organization to the cell, the basic unit of living systems, by differentiating it from the surrounding environment and internally separating it into compartments and subcompartments. Organelles also hold molecules in functionally important spatial relations to one another. Cells are organized into tissues, tissues into organs, and those into interacting systems. Thus, the organism consists of an organizational hierarchy with each higher level imparting further functional complexity to the whole (see Figure 1-1).In this chapter, we begin with the most basic level-the chemical level-and learn how simple prin-

ATOMS, BONDS, AND MOLECULES All matter is composed of chemical elements, which can be arranged into the familiar periodic table of the naturally occurring elements and dozens of artificially synthesized elements created fleetingly in the laboratory (Figure 3-1). Of all the chemical elements, only a very small subset naturally occurs in animal tissue. Table 3-1 compares the major components of the Earth's mineral crust and seawater with those in the human body. About 99% of the human body is made up of just four elements: hydrogen, oxygen, nitrogen, and carbon. This holds true for all organisms. Is the preponderance of these elements in living systems simply a matter of chance, or is there a mechanistic explanation for their uniform prevalence in the great diversity of organisms that have evolved over the past 3 billion years? George Wald, a biologist who contributed much to our understanding of the chemical basis of vision, suggested that the biological predominance of hydrogen, oxygen, nitrogen, and carbon is not at all a matter of chance, but is the inevitable result of certain fundamental atomic properties of these elements-properties that render them especially suited for the chemistry of life. We will review briefly the factors that influence the chemical behavior of atoms, and then return to consider Wald's ideas. Atomic structure is far more complex and subtle than can be fully described here; for our purposes, we need consider only some basic features that affect the formation of chemical bonds between atoms and molecules. The basic

First shell

1 H

Second shell

3 LI

4 Be

5 B

6 C

Thlrd shell

11 Na

13 Al

14 Si

15 P

16

S

17 CI

18 Ar

Fovrlh shell

19 K

12 Mg 20 Ca

21 Sc

22 Ti

23 V

24 Cr

25 Mn

26 Fe

27 Co

28 NI

29 Cu

30 Zn

31 Ga

32 Ge

33 As

34 Se

35 Br

36 Kr

Flfth shell

37 Rb

38 Sr

39 Y

40 Zr

41 Nb

42 Mo

43 Tc

44 Ru

45 Rh

46 Pd

47 Ag

48 Cd

49 In

50 Sn

51 Sb

52 Te

53 I

54 Xe

Slxth shell

55 Cs

56 Ba

57 La

72 Hf

73 Ta

74 W

75 Re

76 0s

77 Ir

78 Pt

79 Au

80 Hg

81 TI

82 Pb

83 BI

84 Po

85 At

86 Rn

Seventh shell

87 Fr

88 Ra

89 Ac

104

105

106

58 Ce

59 Pr

60 Nd

61 Pm

62 Sm

63 Eu

64 Gd

65 Tb

66 Dy

67 Ho

68 Er

69 Tm

70 Yb

71 Lu

90 Th

91 Pa

92

93 Np

94 Pu

95 Am

96 Cm

97 Bk

98 Cf

99 E3

100 Fm

101 Md

102 No

103 Lw

2 He

U

7 N

8 O

9 F

Figure 3-1 In the periodic table of the elements, each row corresponds to a different electron orbital shell. The elements in colored lettering are physiologically important in their ~onicforrns.

1 N

0 e

\ MOLECULES, ENERGY, A N D BIOSYNTHESIS

39

............................................................................ TABLE 3-1 Comparison of the chemical composition of the human body with that of seawater and the Earth's crust* .

-

Human body

Seawater

Earth's crust

portant to animal physiology because they dominate interactions among elements central to organic life. Indeed, the interactions amongst these three particles dictate the attraction among elements necessary for life itself. Each atom consists of a dense nucleus of protons and neutrons surrounded by a "cloud" of electrons equal in number to the protons in the nucleus. The atomic particles have the following charge and mass (in daltons, Da): Proton: Neutron:

+ 1; 1.672 Da 0; 1.674 Da

Electron: -1; 0.001 Da

All others

AH be endergonic or exergonic?

Under what conditions will an endergonic reaction proceed? What is AG for a system at equilibrium? How does ATP "donate" stored chemical energy to an endergonic reaction? What is meant by the term coupled reaction? How does increased temperature increase the rate of a chemical reaction? What factors can influence the temperature optimal for an enzymatic reaction? How does a catalyst increase the rate of a reaction? Why is catalysis necessary in living organisms? How do enzymes exhibit substrate or bond specificity? How does pH affect the activity of an enzyme? How was the "steric-fit" theory of active-site specificity shown to be correct? What factors can influence the rate of enzymecatalyzed reactions? The Michaelis-Menten constant, KM, is equal to the substrate concentration at which a particular reaction proceeds at half its maximum velocity, V,,,,,. Does a high K , indicate a greater or a lesser enzymesubstrate affinity? Why does a high substrate concentration reverse the effects of a competitive inhibitor and yet have no effect on a noncompetitive inhibitor? How does each type of inhibition affect the MichaelisMenten constant, K , ? Explain why. Why does aerobic metabolism yield much more energy per glucose molecule than anaerobic metabolism? What is the advantage of incremental drops in electron pressure compared with a single large drop in electron pressure in the electron-transport chain? How is energy liberated in discrete amounts in the electron-transport chain? How does the mechanism of energy release by the citric acid cycle differ from that during glycolysis?

SUGGESTED READINGS Atkins, P. W. 1994. Physical Chemistry. New York :W. H. Freeman and Company. ( A complete treatment at the undergraduate level of many of the basic concepts introduced in this chapter.) Lehninger, A. L., et al. 1993. Principles of Biochemistry. 2d ed. New York: Worth. (A short, straightforward book about biochemical principles.) Lodish, H. D., et al. 1995. Molecular Cell Biology. 3d ed. New York: Scientific American Books. (Comprehensive textbook describing many of the basic biochemical processes that occur in the cell.) Stryer, L. 1995. Biochemistry. 4th ed. New York: W. H. Freeman and Co. (Highly readable reference for information about biochemical structures and mechanism.)

i

T

he complex chemical reactions that ultimately are responsible for animal life will proceed only under stable, restricted conditions. Such constancy is maintained within cells largely through the action of biological membranes, which form a protective barrier that allows only certain materials to pass into or out of the cell. Animal tissue contains an astounding amount of biological membrane. The chimpanzee brain, for example, is estimated to have about 100,000 m2 of cell membrane, an area equal to three full-sized soccer fields. Though cell membranes are a major constituent of all living matter and essential to all life

process, their existence was questioned up until the 1930s. There was little or no direct anatomical evidence for biological membranes at the time, so their existence could only be inferred from physiological studies. The first important observations on the diffusion-limiting properties of the cell surface were made in the mid-nineteenth century by Karl Wilhelm von Nageli, who noticed that the cell surface acted as a barrier to free diffusion of dyes into the cell from the extracellular fluid. From these experiments he deduced the presence of a "plasma membrane." He also discovered the osmotic behavior of cells, noting that they swell when placed in dilute solutions and shrink in concentrated solutions. Structural evidence for the existence of a distinct cell membrane was first available after the development of electron microscopy (see Chapter 2). At the surface of every cell type is a continuous double-layered membrane ranging in thickness from 6 to 23 nm (Figure 4-1). Understanding membrane structure and function is critical to the study of animal physiology. In this chapter we discuss membrane structural features and their critical role in maintaining cell integrity and controlling cell activities. In the next chapter, we discuss the electrical behavior of cell membranes that is responsible for cell-to-cell signaling, which, in turn, coordinates action in animals.

MEMBRANE STRUCTURE AND ORGANIZATION

10 nm

Figure 4-1 The plasma membrane creates a barrier between the interior and exterior of the cell, as revealed in this electron micrograph. The cell interior (lower right) is separated from the cell exterior by the surface membrane bilayer, which is seen in cross-sectionas a dark-light profile about 10 nm thick. The dark-light-dark sandwich-likeappearance is due to the differential staining of the "unit membrane" by an electronopaque substance during preparation of the tissue. [Courtesy of J. D. Robertson.]

At their surfaces, cells are surrounded by a plasma membrane, an extraordinarily thin, complex, lipid-based structure that encloses the cytoplasm (including the cytosol and all cell organelles) and the cell nucleus. internal organelles, such as the An-producing mitochondria, which We ~ ~ S C U in S SChapter ~ ~ 3, have their own surface membranes.) This enclosing feature of the plasma membrane is its most obvious function, and also its most critical. With the help of various metabolic mechanisms, described latel; the membrane regulates molecular traffic between the orderly interior ofthe cell and themore disorderly, potentially disruptive external environment.

he

94

PRINCIPLES O F P H Y S I O L O G Y

........................................

Membrane Composition

The cell membrane is spanned by integral proteins. These proteins act as both selective filters and active transport devices responsible for getting nutrients into and cellular products and waste out of the cell. Other proteins contained within the membrane sense external signals that direct the cell responses to environmental changes. Cell membranes sustain different concentrations of certain ions on their two sides, leading to concentration gradients of several ionic species across membranes. The channel proteins contained in cell membranes actively participate in the translocation of substances between compartments and ultimately regulate the cytoplasmic concentration of dissolved ions and other molecules rather precisely. This allows maintenance of an intracellular milieu required for the finely balanced metabolic and synthetic chemical reactions of the cell. All biological membranes, including the internal membranes of organelles of eukaryotic cells, have essentially the same structure: lipid and protein molecules kept together by noncovalent interactions. The lipid molecules are arranged in a continuous double layer, called the lipid bilayer, which is relatively impermeable to passage of most water-soluble molecules. In 1925, using a simple but elegant experiment, Gorter and Grendel provided the first evidence that cell membranes are lipid bilayers. First, they dissolved the lipids from red blood cell ghosts, the empty membrane sacs left when red blood cells have been induced to burst open. The extracted membrane lipids were then allowed to spread out on the surface of water in a trough. Because of their asymmetry, the lipid molecules became oriented so that their po-

lar head groups formed hydrogen bonds with the water and their hydrophobic hydrocarbon chains stuck up into the air. When the dispersed film of lipid molecules was gently compressed into a continuous monomolecular film, it occupied an area about twice the surface area of the original red blood cells. Since the only membrane in mammalian red blood cells is the plasma membrane, it was concluded that the lipid molecules in the membrane must be a continuous bilayer. As we saw in Figure 4-1, the bilayer has since been visualized in cross-section by the electron microscope, as well as with freeze fracture methods, in which the membrane is split through the center of the bilayer (see Chapter 2). The chemical properties of lipid molecules, which cause them to assemble spontaneously into bilayers even under artificial conditions (see Chapter 3), are responsible for the structure of the membrane. Membranes are remarkably fluid structures, in which most of the lipid and protein molecules "float" around in the plane of the bilayer (Figure 4-2). The relative proportion of lipids and proteins present in a membrane depends on the kind of cell or organelle the membrane encloses. Lipids, which are far smaller and simpler molecules than proteins, provide the primary structure of the membrane. Integral proteins embedded in the membrane play more specialized roles such as transporting molecules through the membrane, catalyzing reactions, and transducing chemical signals. Other proteins connect the membrane to the cytoskeleton or to adjacent cells. Some proteins are intimately associated with lipid molecules because of lipophilic groups exposed on the surface of the protein molecule. The protein-lipid complex is called a lipoprotein.

Figure 4-2 The Singer-Nicolson fluid mosaic model of the membrane is widely accepted. The globular integral proteins embedded in the lipid bilayer provide a mechanism fortransmembrane transport The inner mitochondrial membrane would have an even higher protein content and thus less lipid bilayer than this figure represents. The glycoproteins bear

oligosaccharide side chains and are vital for cell recognition and communication. Cholesterol molecules lie close to the heads of the phospholip~dmolecules, where they reduce membrane flexibility. The inner ends of the phospholipid tails are highly mobile, giving the membrane fluidity.

MEMBRANES, CHANNELS, A N D TRANSPORT

It

Lipid molecules are insoluble in water but can be dissolved in organic solvents. They comprise about half the mass of plasma membranes in animal cells, the rest being essentially protein. Each square micron of membrane has about 106lipid molecules, meaning typical small cells have about lo9lipid molecules. The three primary types of lipids in cell membranes are Phosphoglycerides,characterized by a glycerol backbone Sphingolipids, which have backbones made of sphingosine bases

* Sterols, such as cholesterol, which are nonpolar and only slightly soluble in water

If

It

The first two lipid types are amphipathic, meaning that they have a hydrophilic (water-soluble)and a hydrophobic (water-insoluble)end (Figure4-3). The dual nature of these amphipathic membrane lipids, with their hydrophilic heads

95

and hydrophobic tails, is crucial to the organization of biological membranes. Their polar heads seek water and their nonpolar tails seek one another (see Figure 3-14), being mutually attracted by van der Waals forces. Thus, these molecules are ideally suited to form an interface between a nonaqueous lipid environment (phase)within the membrane itself and the aqueous intra- and extracellular phases in contact with the inner and outer membrane surfaces. These same forces cause lipid bilayers to reseal themselves when they are torn, which gives cells a self-repair capability. Differences in the length of the two fatty acid chains and in their composition (see Figure 3-20) influence lipid packing and hence fluidity, causing subtle differences in lipid bilayer characteristics. The hydrophobic properties of the phospholipid hydrocarbon tails are responsible for the low permeability of membranes to polar substances (e.g., inorganic ions and polar nonelectrolytes such as sucrose and inulin) and for their correspondingly greater permeability to nonpolar substances (e.g., steroid hormones).

Figure 4-3 Phosphatidyl choline, a phosphoglyc-

Polar head

Nonpolar tails

eride, has charges that give the head group its polar character. Note that theleft hydrocarbon chain in this figure is unsaturated. In order t o distinguish the unsaturated fatty a c ~ dchain from the saturated one, in this figure (and in those that follow) the unsaturated fatty acid chain is drawn with a distinct bend in it rather than with a small kink. Actually, only the double b o n d is rigid in the unsaturated fatty acid. Because the single carbon-carbon bonds in the rest of the chain are free to rotate, both the saturated and unsaturated fatty acid chains tend t o pack in parallel arrays in each phospholipid monolayer. [Stryer, 1988.1

96


.......................................

I

The third class of membrane lipids, the sterols, are largely nonpolar and only slightly soluble in water (Figure 4-4). In aqueous solution they form complexes with proteins that are far more water-soluble than the sterols are alone. Once in the membrane, the sterol molecule fits snugly between the hydrocarbon tails of the phospholipids and glycolipids (Figure 4-5) and increases the viscosity of the hydrocarbon core of the membrane. Fluid Mosaic Membranes The concept of a lipid bilayer membrane enclosing most cells gained wide acceptance by the early 1950s because of compelling evidence from a variety of measurement techniques (Spotlight 4-1). Chemical iractionation of membranes and immunochemical studies confirmed that proteins are also an important component of membranes. Moreover, the enzymatic properties of membranes, such as active transport and other metabolic functions, require the participation of proteins. An example is the protein complexes responsible for electron transport and oxidative phosphorylation described in Chapter 3. Despite this early progress in characterizing the membrane, it wasn't until 20 years later that researchers recognized how fluid and heterogeneous membranes really are. It was discovered that some of the protein molecules are free to diffuse laterally along the membrane, presumably because of the fluidity of the lipid matrix. In addition, labeling studies demonstrated that protein molecules or parts of molecules facing one side of the membrane differ from those facing the other side, and that they normally do not "flip-flop" across the membrane as previously suspected.

Nonpolar hvdrocarbon talk of p h ~ s p h o ~ ~ p ~ d s and sphingoliplds

Sterol Figure 4-5 Nonpolar sterols insert themselves between the hydrocarbon tails and the polar head groups of the phospholipids in the membrane.

Additionally, in many membranes, the distribution of lipid species differs in the two lipid layers.

The fluid mosaic model On the basis of evidence that had emerged during the 1950s and especially the 1960s, Singer and Nicolson (1972) proposed the fluid mosaic model of the membrane, in which globular proteins are integrated with the lipid bilayer, with some protein molecules penetrating the bilayer completely and others penetrating only partially (see Figure 4-2). These integral proteins are thought to be amphipathic, their nonpolar portions buried in the hydrocarbon core of the bilayer and their polar portions protruding from the core to form a hydrophilic surface with charged amino acid side groups in the aqueous phase. Uncharged hydrophobic side groups, on the other hand, are associated with the hydrocarbon bilayer (Figure 4-6). The hydrophobic nature of these side groups is important in keeping the integral proteins from leaving the lipid bilayer. Evidence continues to emerge in support of this model, which is now widely accepted a quarter of a century after first being described. Membrane fluidity A variety of techniques have been used to demonstrate that lipid molecules in the membrane very rarely move from one Integral proteins

CH3

- +

+

I HC-CH, I CH2 I CH2 Chotesterol

I

HC-CH,

Figure 4-4 Cholesterol, a sterol, IS an Important component of the llpld membrane [From Lehnlnger, 19751

Figure 4-6 A cross-sectional view shows the complexity of the mosaic bilayer model. The charged hydrophilic amino acid side groups of the proteins project into the aqueous phase, and the uncharged hydrophobic groups are buried in the lipid phase of the bilayer.

MEMBRANES,

C H A N N E L S , AND TRANSPORT

................................ side of the membrane to the other {ahout once a month), but exchange places with adjacent molecules in a monolayer about 10- times per second. This brisk lipid exchange within a membrane results in rapid migration along the plane of the membrane hut not across it. Memhcane fluidly depends on its composition, and cholesterol plavs an important role in governing this rnembrane charactezist~c.Plasma membranes in eukarvotic organisms contain lots of cholesterol, up to one molecule for every phospholipid. Cholesterol, when present, binds weakly to adjacent phospholipids, making lipid hilapers significantly less fluid, but stronger (Figure 4-7). The incorporation of too much cholesterol into cell membranes, however, causes the membranes to lose flexibility. This ir the mechanism underlying "hardening of the arteries," a major cause of cardiovascular disease, in which the ceEl membranes of the endochelia! cells lining the arteries become abnormally rigid (with additional cholesterol plaques stored in the intirnal lavers, as well). Lipid composition of biological membranes varies among tissue tvpes. While most membranes contain a significant fraction of cholesterol ( > I S % ) , other types of lipids may he present in much higher or lower fractions. Lipids also differ in their head groups (see Figure 3-1 3), which in turn influences their interactions with proteins. Indeed, some integral proteins function only in the presence of a specific ratio of lipid types. Hence, cells must regulate the distribution of lipid species in their membranes during cell development and rearrange lipid concentrations according to specific functional needs.

97

......

Hetuogenei9 of ffieintegral membrane proteins The integral proteins found in the plasma membrane (see Figure 4-2)take manv functional forms, including the ion channels, various carriers and membrane pumps, receptor molecules, and recognition molecules. The number of integral proteins varies, but in some membranes the protein content is so high that only about three lipid molecules separate the proteins a t the point of closest approach. .Morphological evidence for the hererogeneous mosaic arrangement of globular proteins in a lipid bilaper is seen in freeze-etch electron micrographs of the surface of a membrane (Figure 4-8). When subjected to digestion by

Phospholipid

Cholesterol

Figure 4-7 Cholesterol interacts weakly with adjacent phospholip~dsIn the membrane, partially irnmob~l~z~ng their fatty acyl chams. As a result. stronger The amount of chcthe membrane IS less fluid but mechan~cally lesterol present In the lipid bilayer vanes widely with cell type. In some cells, the membranes have nearly a5 many cholesterol moleculesasphospholip~ds,while the membranes of other cells are almost devoid of chlemerol The structural formula of cholesterol is shown In Flgure4-4

Figure 4-8 Freeze-etchmethods yield morphological evrdence for the mosarcmembranemodel. In these freeze-etchelectron m~crographs,the plasma membrane has been spl~talongthe middleof the bilayer, exposing membrane-embedded pan~cleswith diameters of 5 t o 8 nm Digestion with a proteolyt~cenzyme produced progressiveloss of these particles, rndicating that they are globular protelns inserted Into the l ~ p i d phase of the membrane (A) Control (B) 45% of the partrcle digested. (C)70% digested [Courtesy of L. H Engstrom and D Branton.]

4. When fixed with permanganate, membranes appear as

SPOTLIGHT 4-1

THE CASE FOR A LIPID

BILAYER M E M B R A N E

triple-layered profiles: a lightly staining central zone sandwiched between two electron-dense outer layers (see Figure 4-I), with a total thickness of about 7.5 nm. In 1955,

J. David Robertson (1960) named this three-layered structure

There is a large body of accumulated evidence that points to the existence of the lipid bilayer membrane:

the unit membrane. The unit-membrane concept is consistent with a bimolecular layer of lipid between two layers of protein.

1. The lipid content of membranes 1s conslstent wlth a b~layer

5. The thickness of a lipid bilayer, calculated as twice the length of a single membrane lipid molecule, agrees roughly

of orlented llpld molecules, as flrst shown by Gorter and

with the dimensions of the unit membrane seen in electron

Grendel In 1925 2. The ease of passage of nonelectrolytesthrough the mem-

6. Freeze-etch electron microscopy shows that membranes

brane IS conslstent wlth the presence of a llpld membrane barrler, glven the tendency of such molecules to leave an

have a preferential plane of splitting down the middle, which is consistent with separation of a bilayer into two

aqueous phase for a llpld phase, as when

011and

water

separate The greater thls tendency, the more permeant

micrographs.

monolayers.

7. Artificial lipid bilayers (see Spotlight 4-2), reconstituted

the molecule Moreover, certaln Ilpld-~nsolublesubstances

lipid bilayers of similar thickness and presumed structure to

must flrst be converted to a lip~d-solubleform (by attach-

the bimolecular lipid core of the fluid mosaic membrane

ment of a Ilpld-soluble molecule) before they can cross the

model, have permeabilities and electrical properties funda-

membrane.

mentally similar to those of cell membranes. Those differ-

3. The capacitance of b~ologlcal membranes, typically lo-' Facm-', 1s the same as that of a layer of llpld the thick-

ences that exist can be attributed to special channels and carriers present in natural membranes.

ness of two phosphol~p~d molecules placed end to end (r.e., 6.0-7.5 nm).

proteolytic (protein-digesting) enzymes, the globular units seen in the membrane are progressively removed, demonstrating that they are indeed proteins. Variation in Membrane Form

Membrane composition varies greatly between cell types. At one extreme is the metabolically inert myelin sheath surrounding the axons of some nerve cells, in which the lipid bilayer is largely uninterrupted. At the other extreme are cells with membranes that have structures of repeating nonlipid macromolecular units that nearly obliterate the lipid bilayers. Such membranes evolved for highly specialized purposes such as signaling or enzymatic activity. In visual receptor cells, for example, the repeating macromolecular unlts are molecules of the visual pigment opsin. Mitochondrial membranes, specialized for enzymatic activity, are composed almost entirely of repeating subunits of ordered enzymatic aggregates. Between these extremes are the plasma membrane and most intracellular membranes, in which the bilayer is interrupted frequently by integral protein molecules. Thus the basic structure of the lipid bilayer with integral proteins is highly modified as required for functional specialization.

CROSSING THE MEMBRANE: AN OVERVIEW The structure of membranes makes them quite selective about which molecules can pass through them. The hy-

drophobic interior of the lipid bilayer makes membranes highly impermeable to most polar molecules. This prevents water-soluble components of the cell from easily entering or escaping. However, such movement may at times be necessary or desirable, so mechanisms for transferring these molecules across membranes have evolved in all cells. Macromolecules like proteins and large particles must also be transported across plasma membranes using specialized mechanisms. To understand these special means of membrane transport in living cells, we will first review the physical principles of solute and solvent displacement in solution and across semipermeable membranes. Such membranes closely resemble those found in living cells, and the principles explained here apply in many physiological situations. Diffusion

Random thermal motion of suspended or dissolved molecules causes their dispersion from regions of higher concentration to regions of lower concentration, a process called diffusion. Diffusion is extremely slow when viewed on a tissue, rather than a cellular, scale. For example, a crystal of copper sulfate dissolves in unstirred water so slowly that it may take a whole day to color a liter of water completely. When viewed in the microscopic dimensions of the cell, however, diffusion times can be as short as a fraction of a millisecond. The rate of diffusion of a solute s can be defined by the Fick diffusion equation:

MEMBRANES,

C H A N N E L S , A N D TRANSPORT

99

............................................................................. Semipermeable

in which dQSldtis the rate of diffusion (i.e., quantity of s diffusing per unit time), D, is the diffusion coefficient of s, A is the cross-sectional area through which s is diffusing, and dC,ldx is the concentration gradient of s (i.e., the change in concentration with distance).The gradient factor dCs/dx is clearly very important, because it determines the rate at which s will diffuse down the gradient. D,varies with the nature and molecular weight of s and of the solvent, which is water in most physiological situations.

Unidirectional flux

Membrane Flux

If a solute occurs on both sides of a membrane through which it can diffuse, it will exhibit a unidirectional flux in each direction (Figure 4-9A). The flux, or rate of diffusion, Jis the amount of the solute that passes through a unit area of membrane every second in one direction, so that

Net flux

where J would typically have units of moles per square centimeter per second (M cm-2. s-l). The flux in one direction (say,from cell exterior to cell interior) is considered independent of the flux in the opposite direction. Thus, if the influx and efflux are equal, the net flux is zero. If the unidirectional flux is greater in one direction, there is a net flux, which is the difference between the two unidirectional fluxes (Figure 4-9B). The permeability of the membrane to a substance is the rate at which that substance passively penetrates the membrane under a specified set of conditions. A greater permeability will be accompanied by a greater flux if other factors remain equal. If we assume that the membrane is a homogeneous barrier and that a continuous concentration gradient exists for a nonelectrolyte substance between the side of high concentration (I)and the side of low concentration (II),then - PIC, - C,)

dQs -

dt

in which dQ,ldt is, again, the amount of substance s crossing a unit area of membrane per unit time (e.g., moles per square centimeter per second), C, and C,, are the respective ) substance on the two concentrations (e.g., M ~ m - of~ the sides of the membrane, and P is the permeability constant of the substance, with the dimension of velocity (cm-s-I). Note that equation 4-3 applies only to molecules that are not being actively transported or influenced by any forces other than simple diffusion. This excludes electrolytes, since they are electrically charged when dissociated, and consequently their flux depends not only on the

Figure 4-9 Solutes can move through a membrane in either dired~on, depending on prevailing physical and chemical conditions. (A) The arrows represent the actual fluxes of a substance between compartments I and II. (B) The single arrow indicates the resulting net flux, from compartment Ito II.

concentration gradient, but also on the electrical gradient (i.e., the electric potential difference across the membrane). As is evident from equation 4-3, the flux of a nonelectrolyte should be a linear function of the concentration gradient (C, - C,). This linear relationship is characteristic of simple diffusion, and can be used in experiments to distinguish between passive diffusion of a substance and any other mechanism. The permeability constant incorporates all the factors inherent in the membrane and the substance in question. These factors will determine the probability that a molecule of a particular substance will cross the membrane. This relationship can be expressed formally as

.

where Dmis the diffusion coefficient of the substance within the membrane, K is the partition coefficient of the substance, and x is the thickness of the membrane. The more viscous the membrane or the larger the molecule, the lower the value of Dm. Permeability constants for different substances vary greatly. For example, the permeability of red blood

100


..........................................

cells to different solutes ranges from 10-12 cm-s-I to cm s-l. Importantly, the permeability of some membranes to certain substances can be altered greatly by hormones and other molecules that react with receptor sites on the membrane and thereby influence channel size or carrier mechanisms. Antidiuretic hormone, for example, can increase the water permeability of the renal collecting duct in mammals by as much as 10 times. Similarly, neurotransmitters, acting on specialized integral membrane proteins in nerve and muscle cells, induce large increases in permeability to ions such as Nac, KC, Ca2+,or C1-.

-

Osmosis

In 1748 AbbC Jean Antoine Nollet noted that if pure water is placed on one side of an animal membrane (e.g., a bladder wall) and a solution of water containing electrolytes or other molecules is placed on the other side, the water passes through the membrane into the solution. This movement of water down its concentration gradient was called osmosis (from the Greek osmos, "to push"). We don't usually think of "water concentration," but in fact water acts just like any other substance by diffusing down its concentration gradient. It was later found that osmosis produces a hydrostatic pressure gradient. Osmosis is the colligative property of greatest importance to living systems. As can be seen in Figure 4-10, the pressure difference causes a rise in the level of the solution as water diffuses through the semipermeable membrane into the solution. The rise in the level of the solution continues until the net rate of water movement (net flux) across the membrane becomes zero. This occurs when the hydrostatic pressure of the solution in compartment I1 is sufficient to force water molecules back through the membrane to compartment I at the same rate that osmosis causes water molecules to diffuse from I to 11. The hydrostatic back pressure required to cancel the osmotic diffusion of water from compartment I to compartment I1 is called the osmotic pressure of the solution in compartment 11. In 1877, Wilhelm Pfeller made the first quantitative studies of osmotic pressure. He deposited a "membrane" of copper ferrocyanide on the surface of porous clay cups,

6

producing membranes that would allow water molecules to diffuse through them far more freely than sucrose molecules could. These artificial membranes were also strong enough to withstand relatively high pressures without rupturing because of the clay substratum. Using these membranes, Pfeller was able to make the first direct measurements of osmotic pressure. Some of his results are shown in Table 4-1. Note in the table that the osmotic pressure is proportional to the solute concentration. Osmosis is responsible for the net movement of water across cell membranes and epithelia. To understand this, consider a 1.0 M aqueous solution of sucrose carefully layered under a 0.01 M aqueous solution of sucrose. There would be net diffusion of water molecules from the solution of lower sucrose concentration (the 0.01 M solution) into the 1.0 M sucrose solution, and sucrose would show net diffusion in the opposite direction until equilibrium was achieved. If these two solutions were separated by a membrane permeable to water but not to sucrose, the water molecules would still show a net diffusion from the solution in which H,O is more concentrated (the 0.01 M sucrose solution) into the 1.0 M sucrose solution, in which the H 2 0 concentration is lower. Since the sucrose could not cross the membrane, there would be a net diffusion of water (osmoticflow) through the membrane from the solution of lower solute concentration to the solution of higher solute concentration. Osmotic pressure .iris proportional not only to the concentration of the solute, C (moles of solute particles per liter TABLE 4-1 Osmotic pressure of sucrose solutions of various concentrations* Sucrose

Osmotic pressure

Ratio of osmotic pressure

(%I

(atm)

t o percentage of sucrose

1 2 4 6

070 1.34 2 74 4.10

070 067 068 068

*Results were obtarned by Pfeffer (1877) in exper~mentalmeasurements

Semipermeable membrane

Time

Figure 4-10 Water flow produced by osmosis through a semipermeable membrane generates hydrostatic pressures. Compartment I contains pure water; compartment II, water with impermeant solute. Osmotic pressure forces water to enter compartment II from compartment I until the hydrostatic pressure difference equals the opposing osmotic pressure difference. When the pressures are equal, the flux is zero.

MEMBRANES, C H A N N E L S , A N D TRANSPORT 101 .......................................

of solvent = osmolarity), but also to its absolute temperature T: TT

=

KIC

(4-5)

and

where K, and K, are constants of proportionality. Jacobus van't Hoff related these observations to the gas laws and showed that solute molecules in solution behave thermodynamically like gas molecules. Thus, TT

=

RTC

where n is the number of mole equivalents of solute, R is the molar gas constant (0.082 L atm K-l mol-' )," and V is the volume in liters. Like the gas laws, however, this expression for osmotic pressure holds true only for dilute solutions and for completely dissociated electrolytes. Large concentration gradients across cell membranes can generate surprisingly high osmotic pressures-on the order of several atmospheres. Such pressures, if allowed to develop, would be large enough literally to explode a cell. Consequently, mechanisms for regulating osmotic balance have evolved that minimize osmotic pressure gradients across cell membranes and through tissues (see Chapter 14).

-

.

Osmolarity and Tonicity Two solutions that exert the same osmotic pressure through a membrane permeable only to water are said to be isosmotic to each other. If one solution exerts less osmotic pressure than the other, it is hypoosmotic with respect to the other solution; if it exerts greater osmotic pressure, it is hyperosmotic. Osmolarity is thus defined on the basis of an ideal osmometer in which the osmotic membrane allows water to pass but completely prevents the solute from passing. All solutions with the same number of dissolved particles per unit volume have the same osmolarity'and are thus defined as isosmotic. The tonicity of a solution, in contrast to its osmolarity, is defined by the response of cells or tissues immersed in the solution. A solution is considered to be isotonic with a given cell or tissue if the cell or tissue immersed in it neither shrinks nor swells. If the tissue swells, the solution is said to be hypotonic to the tissue; if it shrinks, the solution is said

'R

is the constant of proportionality in the gas equation PV/T = R when referring to 1 mol of a perfect gas, and it has the value of 1.985 cal. molk' .K-'; P is in atmospheres and V is in liters.

to be hypertonic to it. These effects result from movement of water across the cell membrane in response to osmotic pressure differences between the cell interior and the extracellular solution. If cells actually behaved as ideal osmometers, tonicity and osmolarity would be equivalent, but this is not generally true. For example, sea urchin eggs maintain a constant volume in a solution of NaCl that is isosmotic relative to seawater, but they swell if immersed in a solution of CaC1, that is isosmotic relative to seawater. The NaCl solution therefore behaves isotonically relative to the sea urchin egg, whereas the CaCI, solution behaves hypotonically. The tonicity of a solution depends on the rate of intracellular accumulation of the solute in the tissues in question, as well as on the concentration of the solution. The more readily the solute accumulates, the lower the tonicity of a solution of a given concentration or osmolarity. This is because as the cell ,gradually loads up with the solute, water follows according to osmotic principles, causing the cell to swell. Thus, the terms isotonic, hypertonic, and hypotonic are meaningful only in reference to actual experimental determinations on living cells or tissues.

Electrical Influences on Ion Distribution Membrane permeability to charged particles depends both on the membrane permeability constant and on the electrical potential across the membrane. Understanding the interaction of charged particles with membranes is extremely important for understanding how electrically excitable cells function. Neurons are the most highly specialized of this class of cells. Since neurons will be discussed in the next couple of chapters, only a few important observations will be summarized here. Two forces can act on charged atoms and molecules (such as Na+,K+, Clk, Ca2+,amino acids) to produce a net passive diffusion of each species across a membrane:

1.

2.

The chemical gradient arising from differences in the concentration of the substance on the two sides of the membrane The electric field, or difference in electric potential across the membrane

An ion will move away from regions of high concentration, and if that ion is positively charged it will also move toward increasing negative potential. The sum of the combined forces of concentration gradient and electrical gradient determine the net electrochemical gradient acting on that ion. When an ion is at equilibrium with respect to a membrane (that is, when there is no net transmembrane flux of that ion species), there will exist a potential difference just sufficient to balance and counteract the chemical gradient acting on the ion. The potential at which an ion is in electrochemical equilibrium is called the equilibrium potential, measured in volts (or millivolts). Several factors influence the value of the equilibrium potential, but the most prominent is the ratio of the ion concentrations on opposite sides

102


.......................................

of the membrane. For a monovalent ion such as Na+ or K+ at 18"C, the equilibrium potential (in volts) is equal to 0.058 x log,, of the ratio of the extracellular to intracellular concentrations of the ion. Thus, a 58 mV potential difference across the membrane has the same effect on the net diffusion of that ion as a transmembrane concentration ratioof 10:l. An apparently paradoxical situation therefore arises in which an ion species can passively diffuse against its chernical concentration gradient (that is, move "uphill" to an area of higher concentration)if the electrical gradient (i.e.,potential difference) across the membrane is in the opposite direction to and exceeds the concentration gradient. For example, if the interior of a cell has a greater negative charge than the equilibrium potential for K+, potassium ions will diffuse into the cell even though the intracellular concentration of K+ is much higher than the extracellular concentration. The distribution of ions across membranes and the attendant equilibrium potential is described by the Nernst relationship, which is discussed in detail in the following chapter. Electrical forces cannot act directly on uncharged molecules such as sugars. These substances will be influenced primarily by the concentration gradient they experience.

A

Equilibrium

Start

+ Time

B

KA\

Start

Equilibrium

+ Time

Donnan Equilibrium

If diffusible solutes are separated by a membrane that is freely permeable to water and electrolytes but totally impermeable to one species of ion, the diffusible solutes become unequally distributed between the two compartments. This phenomenon was discovered in 1911 by Frederick Donnan, who first described how the solutes would be distributed and hence has been commemorated by having this equilibrium state named for him. To understand a Donnan equilibrium, imagine starting with pure water in two compartments and adding some KC1 to one of them (Figure 4-11). The dissolved salt (K+ and C1-) will diffuse through the membrane until the system is in equilibrium-that is, until the concentrations of K+ and C1- become equal on both sides of the membrane (Figure 4-11A). Now imagine adding the potassium salt of a nondiffusible anion (a macromolecule A-, having multiple negative charges)to the solution in compartment I. The K+ and C1- quickly become redistributed until a new equilibrium is established by movement of some K+ and some C1- from compartment I to compartment I1 (Figure4-11B). Donnan equilibrium is characterized by a reciprocal distribution of the anions and cations so that

Figure 4-1 1 The Donnan equilibrium descr~besion distribution across a semipermeable membrane. (A) When KC1 is added to compartment I of a container divided by a permeable membrane, K+ and CI- diffuse across the membrane until the concentrationsare equal on either side. (B) If the potassium salt of an impermeant anion is added to compartment I, some K+ and C I diffuse into compartment II until electrochemicalequilibrium is reestablished. It should be noted that these chambers (unlikethe living cell) are not distensible.

We can understand this situation by considering the consequences of the following physical principles: within both compart1. There must be electrone~traiit~

2.

3.

At equilibrium, the diffusible cation, K+, is more concentrated in the compartment in which the nondiffusible anion, A-, is confined than in the other, whereas the diffusible anion, C1-, becomes less concentrated in that compartment than in the other.

ments; that is, in each compartment the total number of positive charges must equal the total number of negative charges. Thus, in this example, [K+] = [Cl-] in compartment 11. Considered statistically, the diffusible ions K+ and C1cross the membrane in pairs to maintain electrical neutrality. The probability that they will cross together is proportional to the product [K+]X [Cl-1. At equilibrium the rate of diffusion of KC1 in one direction through the membrane must equal the rate of KC1 diffusion in the opposite direction. It follows, then, that at equilibrium the product [K+] x [Cl-] in one compartment must equal the product in the other compartment. Letting x , y, and z represent the concentrations of the ions in compartments I and 11, as shown in Figure 4-12, we can express the equilibrium

'

Figure 4-12 The Donnan equilibrium can be described algebraically. The equilibrium condition established in Figure 4-1 1 B afterthe salt of an impermeant anion is added to compartment 1 is shown.

condition (i.e., equality of the product [K+] x [Cl-] in the two compartments) algebraically:

meable to a variety of ions and molecules, and there will almost never be a single "nondiffusible anion," which here represents various anionic side groups of proteins and other large molecules. Although the physical and mathematical principles recognized by Donnan play a role in regulating the distribution of electrolytes in living cells, clearly nonequilibrium mechanisms must modify the distribution of many substances across the cell membrane. In particular, the permeability of the cell membrane to particular ions can change over time, changing the conditions dramatically. Thus, cells cannot be considered passive "osmometers," and the distribution of substances across biological membranes cannot be predicted entirely by Donnan equilibrium principles except in certain cases.

OSMOTIC PROPERTIES OF CELLS

This equation also holds, of course, if A- is not present. In that case, K+ and C1- are equally distributed, and z = 0 and x = y. By rearranging equation 4-8, we can see that, at equilibrium, the distributions of the diffusible ions in the two compartments are reciprocal:

From this relation, it is clear that as the concentration of the nondiffusible anion, z, is increased, the concentrations of the diffusible ions (x and y) will become increasingly divergent. This unequal distribution of diffusible ions is the hallmark of Donnan equilibrium. At Donnan equilibrium, the osmotically unequal distribution of solute particles makes water move in the direction of the compartment of higher osmolarity (compartment I in Figure 4-11). This osmotic pressure difference plus any resultant increase in hydrostatic pressure of that compartment is called the oncotic pressure. This concept is important in understanding the balance of hydrostatic and osmotic pressures across certain biological barriers such as capillary walls. The explanation of a Donnan equilibrium depends on an ideal set of conditions for the sake of simplicity. The living cell and its surface membrane are, of course, far more complex. For example, the cell membrane is somewhat per-

[K+], = 140 [Ca2+],< 1 0

- ,.

[CI-1, = 3-4 [A-I, = 140

We can now use the physical principles outlined above to analyze properties of the cell membrane that maintain different concentrations of ions inside and outside the cell (Figure 4-13). Cell membranes ultimately must closely regulate cell volume and thus intracellular osmotic pressure. Ionic Steady State

Every cell maintains concentrations of inorganic solutes inside the cell that are different from those outside the cell (Table 4-2).The most concentrated inorganic ion in the cytosol is K+, which is typically 10-30 times as concentrated there as in the extracellular fluid. Conversely, the internal concentrations of free Na+ and C1- are typically less (approximately one-tenth or less) than the external concentrations. Another important generalization is that the intracellular concentration of Ca2+is maintained several orders of magnitude below the extracellular concentration. This difference is due in part to active transport of Ca2+ out across the cell membrane and in part to the sequestering of this ion within such organelles as the mitochondria and endoplasmic reticulum. As a result, the concentration of Ca2+ M. in the cytosol is generally well below Cell membranes typically are about 30 times more permeable to K+ than to Na+. Membrane permeability to chloride ions varies. In some cells it is similar to that of K+ while in others it is lower. The permeability of the cell membrane to Na' is low, but it is not low enough to prevent Na+ from leaking steadily into the cell.

Figure 4-1 3 Concentrations of common ions areverydifferent inside and outside a vertebrate skeletal muscle cell. The concentrations shown are in millimoles per liter. The concentration given for intracellular Ca2+is for the free, unbbund, and unsequestered ion in the myoplasm. Because the list of ions is incomplete, the totals do not balance out perfectly. [A-l, represents the molar equivalent negative charges carried by various impermeant anions.

104


...............................................................................

TABLE 4-2 Internal and external concentrations o f some electrolytes i n specific nerve and muscle tissues Internal concentrat~ons

External concentrat~ons(rnM)

(mM) Tissue

Na'

KC

CI-

Na'

K+

CI-

Rat~os,~ns~de/outs~de Na'

K+

CI-

Squid nerve

49

410

40-100

440

22

560

1/9

19/1

1/14-1/6

Crab leg nerve

52

410

26

510

12

540

1/10

34/1

1/21

Frog sartorius muscle

10

140

4

120

2.5

120

1/12

56/1

1/30

Certain features of the cell membrane, particularly the differential permeability of the membrane to different ion species, suggest that under some conditions the Donnan equilibrium might apply. To understand when the Donnan equilibrium is useful in determining membrane characteristics of living cells, three related factors are important:

1. Inside the cell, carboxyls and other anionic sites found on nonpermeant peptide and protein molecules contribute most of the net negative charge. These charges must be balanced by positively charged counterions such as Na+, K+, Mg2+,and Ca2+. 2. These anionic sites trapped inside the cell make it similar to the artificial case presented above (see Figure 4-1 1)in which Donnan equilibrium applies. If K+ and C1- were the only diffusible ions, an equilibrium situation similar to that shown in Figure 4-11B would indeed develop in the cell. However, the cell membrane is leaky to Na+ and other inorganic ions, and with time the cell would load up with these ions if they were simply allowed to accumulate. This, in turn, would cause osmotic movement of water into the cell, causing it to swell. 3. Such osmotic disasters are avoided because the cell pumps out Na+, Ca2+, and some other ions at the same rate as they leak in, keeping the intracellular Na+ concentration about an order of magnitude lower than the extracellular concentration. This active pumping, which will be discussed later, is equivalent to an effective impermeability to Na+ and Ca2+.As a result, the concentrations of these ions are not allowed to come into equilibrium, and the cell in fact behaves very much as if it were in a state of Donnan equilibrium. Actually, the unequal distribution of ions represents a steady state requiring the continual expenditure of energy (to pump ions) rather than a true equilibrium. Since K+ and C1- are by far the most concentrated and most permeant ions in the tissue, they distribute themselves in a way similar to that in an ideal Donnan equilibrium. That is, the KC1 product [K+] X [Cl-] of the cell interior will approximately equal the KC1 product of the extracellular solution (Figure 4-14), provided the membrane permeabilities of K+ and C 1 are both high relative to those of other ions present.

Figure 4-14 The KC1 product is governed by the Donnan equilibrium. The distribution of K+ and C I will follow Donnan equilibrium principles, provided the membrane is permeable to both K+ and CI-.

Cell Volume Plant and bacterial cells have rigid walls secreted by the cell membrane. These walls place an upper limit on the size of the cell, allowing the osmotic buildup of turgor pressure in these cells. In contrast, animal cells do not have rigid walls and therefore cannot resist any buildup of large intracellular pressure. As a result, cells will change size when placed in different concentrations of impermeable substances dissolved in water. This shrinkage or swelling is due to osmotic movement of water (Figure 4-15). There are two ways in which the surface membrane might prevent osmotic swelling in the cell. One is to pump water out as fast as it leaks in. There is no evidence that this occurs, although a similar effect is achieved by the contractile vacuole of certain protozoans. The other, which appears to be the major mechanism for regulation of cell volume, is to pump out solutes that leak into the cell (Figure4-16).Thus, at steady state, Na+, the major osmotic constituent outside the cell, is expelled from the cell by active transport as rapidly as it leaks in. In effect, there is no net entry. The situation is osmotically equivalent to complete sodium impermeability, with a relatively fixed concentration of Na+ trapped in the cell. Because Na+ is not allowed to further accumulate in the cell, there is no compensatory osmotic influx of water. The low intracellular (relative to extracellular) sodium concentration is important in balancing the other osmotically active solutes in the cytoplasm. The importance of active transport in maintaining the sodium gradient, and thereby the osmolarity of the cell and the cell volume, is seen when the energy metabolism of the

Na pumped out Passive Na influx

@Na+ Red blood cell

I

N o active transport of Na+

Figure 4-15 Osmotic changes alter the volume of a red blood cell. (A) Isotonic solution: the cell volume.remainsunchanged. (B) Hypotonic solution: water (arrows)enters the cell because of the higher osmoticity of the cytoplasm with respect to the solution, producing swelling. (C) Hypertonic solution: in a more concentrated medium, water leaves the cell, causing shrinkage.

cell is interrupted by metabolic poisons (Figure 4-17). Without ATP to energize uphill extrusion of Naf, the sodium ion, together with its chloride counterion, leaks into the cell, and water follows osmotically, causing the cell to swell.

t

Hyperosmotic solution added

Cell bursts

Figure 4-17 A metabolic inhibitor interferes with Na+ pumping and, therefore, with maintenance of cell volume. Under normal circumstances, levels of Na+ are maintained at equilibrium inside and outside a cell: the ion passively enters the cell and then is pumped out of the cell. With the addition of a metabolic lnhlbitor, however, the cell is rendered unable to pump out the Na+ that steadily leaks into the cell. As a result, [Na+],rises inside the cell and water follows osmotically, increasing cell volume above its initial volume (dashed line). Eventuallythe cell bursts because of massive swelling.

,

Weakly permeant solute

u Time Figure 4-16 Hyperosmotic solutions with impermeant and weakly impermeant solute both cause initial cell shrinkage. If the solute is completely impermeant, it causes maintained cell shrinkage because the solution is basically hypertonic in this situation. If the solute is only weakly impermeant, however, the solution IS hypotonic and enters the cell slowly followed by the osmotic flow of water. This process eventually produces swelling, in spite of the fact that the solution is hyperosmotic.

PASSIVE TRANSMEMBRANE MOVEMENTS Molecules can cross membranes without the direct input of energy, that is, passively, in several different ways. Note that while these processes do not directly require metabolically energized processes, they ultimately depend on a concentration or electrical gradient across a cell membrane that has at some point required energy for its creation and maintenance. The energy stored in such gradients is ultimately responsible for the translocation of molecules across the membrane. Nonetheless, it is useful and appropriate to think of these processes as passive. There are three basic routes for passive transmembrane movements of molecules or ions (Figure4-18).In the first, a

106


...............................................................................

A

Outside

Inside

w .-

"a * m

~II

substrate molecules

C

Extracellular

Carrier molecules

leave the aqueous phase and enter the lipid phase, a solute must first break all its hydrogen bonds with water. This requires about 5 kcal of kinetic energy per hydrogen bond. Moreover, the solute molecule crossing the lipid phase of the membrane must dissolve in the lipid bilayer. So, its lipid solubility will also play a major role in determining whether or not ~twill cross the membrane. Consequently, those molecules having a minimum of hydrogen bonding with water will most readily enter the lipid bilayer, whereas polar molecules such as inorganic ions will almost never dissolve in the bilayer. A number of factors, such as molecular weight and molecular shape, influence the mobility of nonelectrolytes within the membrane, but the empirically measured partition coefficient is the primary predictor of the diffusion of a nonelectrolyte across the lipid bilayer. To measure this property, a test substance is shaken in a closed tube containing-equal amounts of water and olive oil, and the coef. ficient K is determined from the relative solubilities in water and oil at equilibrium, using the equation solute concentration in lipid solute concentration in water

K= molecules occupied

Figure 4-18 Substances cross membranes by three major methods. (A) Dissolving in lipid phase.(B) Diffusion through labile or fixed aqueous channels. (C) Carrier-mediatedtransport (either facilitated transport or active transport).

molecule simply diffuses through the membrane. It leaves the aqueous phase on one side of the membrane, dissolves directly in the lipid layer of the membrane, diffuses across the thickness of the lipid or protein layer, and finally enters the aqueous phase on the opposite side of the membrane. In the second, the solute molecule remains in the aqueous phase and diffuses through aqueous channels, water-filled pores in the membrane. In the third route, the solute molecule combines with a carrier molecule dissclved in the membrane. This carrier "mediates" or "facilitates" the movement of the solute molecule across the membrane. Carriers can "mask" even a polar solute and because of their lipid solubility, allow the solute to diffuse more readily across the membrane, down its concentration or electrochemical gradient. This is called carrier-mediated (or facilitated) transport and may take any of several different forms. Let's now consider each of these three major pathways in turn. Simple Diffusion through the Lipid Bilayer

If a solute molecule comes into contact with the lipid layer of the membrane and its thermal energy is high enough, it may enter and cross the lipid phase and finally emerge into the aqueous phase on the other side of the membrane. To

(4-10)

Is nonelectrolyte membrane permeability related to the lipid-water partition coefficient of the solute? Collander (1937) systematically tested this idea in the giant algal cell Chara by plotting the permeability coefficient (equation 4-4) against the partition coefficient (equation 4-10). Lipid solubility is almost linearly related to permeability of a substance, independent of molecule size (Figure 4-19). Nonelectrolytes exhibit a wide range of partition coefficients. For example, the value for urethane is 1000 times that for glycerol (see Figure 4-19). These differences depend on particular features of the molecular structure, as il-

Water,

Methanol,.

0

urea

Smallest molecule 0

Jrea

Next smallest

o Next largest Largest molecules

0.0001

I

I

I

0.001

0.01

0.1

Olive oil-water partition coefficient Figure 4-19 Membrane permeability of nonelectrolytes is linearly reoil-water partition coefficients.Note that the perlated to their respea~ve meability of nonelectrolytes is independent of molecular size.

MEMBRANES, CHANNELS, A N D TRANSPORT

107

............................................................................... H2C-OH

I

HCH

CHZ-OH

I

I I HO-CH I HC-OH I HC-OH I

HO-CH

HCH

I I

HCH H

H2C-OH

Hexanol

D-mannitol

Figure 4-20 The structure of six-carbon molecules determines their water and lipid solubility. Note the difference between hexanol and mannito1 in the number of hydroxyl groups. Hexanol, with its weak hydrogenbonding capacity, is poorly soluble in water and highly soluble In lipids; mannitol, with its strong hydrogen-bonding capacity, is highly soluble in water and poorly soluble in lipids.

lustrated in Figure 4-20, which compares two molecules with different solubilities. Hexanol and mannitol have similar structures except that hexanol contains only one -OH group while mannitol contains six. These -OH groups facilitate hydrogen bonding to water and therefore decrease lipid solubility. In fact, each additional hydrogen bond results in a fortyfold decrease in the partition coefficient, which is reflected in a decrease in permeability (Figure 4-21). Consequently, hexanol diffuses across membranes much more readily than mannitol does. Water exhibits a much higher permeability across cellular membranes than predicted from its partition coefficient (see Figure 4-19). This is partly because water can pass through selective permanent channels that penetrate the lipid bilayer. Structural evidence of this is seen in certain epithelial cells, where water permeability depends on water channels in the plasma membrane. However, even in channel-free, artificial lipid bilayers, water permeability is still several times higher than that predicted from the solubility of water in long-chain hydrocarbons. A possible explanation is that the small, uncharged water molecules may pass through temporary channels between lipid molecules. Other small, uncharged polar molecules, such as CO, ,NO,

A

Extracellular space

-

Cytoplasm B

Solute

Carrier

Extracellular

Figure 4-21 Hydrogen bonds greatly decrease the l ~ p solublllty ~d and, hence, the permeablllty of a membrane.

and CO, also have relatively high permeabilities across artificial and natural membranes, though it is not known whether this is explained by specialized channels or ones lacking selectivity. Simple diffusion through the lipid bilayer exhibits non-saturation kinetics (see Figure 4-1 8A), meaning that the rate of influx increases in proportion to the concentration of the solute in the extracellular fluid. This is because the net rate of influx is determined only by the difference in the number of solute molecules on the two sides of the cell membrane. This proportionality between external concentration and rate of influx over a large range of concentrations distinguishes simple diffusion from channel permeation or carrier-mediated transport mechanisms (Figure 4-1 8B and C). Diffusion through Membrane Channels

Charged molecules can cross membranes by diffusing through specific water-filled channels. Since inorganic ions such as Na+, Kf, Ca2+,and C1- cannot diffuse through lipid bila~ers,special protein molecules have evolved that extend across the cell membranes and act as pores. When these pores are open, they allow specific solutes to pass through them (Figure 4-22A).

-

Channel protein

Lipid bilayer

Number of hydrogen bonds

Fiqure 4-22 Membrane transport proteins . . act as carriers or form channels in the mernbrane. (A) A channel protein forms a waterfilled pore across the bilayer, through which specific ions can diffuse. (B)In contrast, a carrier protein alternates between two conformations, so that the solute bindinq site is sequentially accessible on one side of the bilayer and then on the other.

"

.. .

SPOTLIGHT 4-2

ARTIFICIAL BILAYERS

The principle of bilayer formation is shown in the figure (part B). The most stable configuration attained consists of two layers of lipid molecules whose hydrophobic, lipophilic hydro-

Many of our ideas of how molecules and ions pass across membranes have grown out of experiments and observations on artificial bilayers that are similar to the bimolecular leaflet that

carbon tails are loosely associated to form a liquid-lipid phase sandwiched between the hydrophilic polar ends of the molecules, which are directed outward toward the aqueous medium. The thickness of the lipid film is easily determined from the in-

forms the basis of the cell membrane. Artificial bilayers are extremely useful in studies of permeation mechanisms because

terference color of light reflected from the two surfaces of the film. Membranes with thicknesses of approximately 7 nm (black

they can be made from chemically defined mixtures of lipids. Se-

interference color) are most commonly used. These membranes

lected substances can be added to test their effects on permeability. Channel-forming substances, such as the antibiotic

have electrical conductances (ion permeabilities) and capacitances consistent with their thickness and lipid composition. Al-

ionophores (moleculesthat facilitate the diffusion of ions across membranes) and membrane channel components of excitable

though their permeability to ions is much lower than that of cell membranes, the addition of certain ionophores increases it to

tissues, have been incorporated into artificial bilayers, allowing

values that are characteristic of cell membranes.

their properties to be studied in isolation under the highly controlled conditions shown in the accompanyingfigure.

Bilayer-filled 1-mm opening

Lipid bilayers can be induced to form across a I-mm opening between two fluid-f~lledchambers. (A) The permeability of the bilayer to electrolytes in the chamber can be measured electrically by placing test sowith different electrolyte concentrations in each of the chambers. lut~ons (B)The bilayer is formed by filling the opening with a small amount ofthe

l i p ~ din a solvent such as hexane. Initially, while the bilayer is forming, its interference color is gray (left).As the membrane assumes the more stable bilayer configuration (right),the interference color changes to black. [From Kotyk and Janacek, 1970.1

The functioning of membrane channels can be demonstrated directly in artificial lipid bilayer membranes that are by themselves highly impermeable to even the smallest of charged molecules (Spotlight 4-2). A dramatic increase in ion permeability occurs upon addition of small amounts of channel proteins extracted from cellular membranes. This increase is measured as discrete pulses of current carried by ions from one side of the membrane to the other, just like those measured in biological membranes. These unitary currents are due to the sudden opening of individual chan-

nels that allow thousands of ions per second to stream down their gradients and across the membrane. Studies of the permeabilities of cell membranes to other polar substances give an estimated 0.7 nm for the equivalent pore size-the pore diameter that would . account for the rate of diffusion across the membrane. Thus, membrane channels presumably have diameters of less than 1.0 nm, close to the practical limits of resolution of contemporary electron microscopes and fixation methods.

As an example, rod-shaped molecules of the antibiotic nystatin applied to both sides of an artificial or a natural membrane aggregate to form channels. These pores permit the passage of water, urea, and chloride, all of which are less than 0.4 nm in diameter. Larger molecules cannot penetrate the channels. Cations also are excluded, presumably because there are fixed positive sites along the channel walls. Incorporation of nystatin into artificial membranes produces a negligible increase in membrane area occupied by fixed channels (0.001%-0.01%), but it produces a 100,000-fold increase in membrane permeability to chloride ions. This means that very little membrane area need be devoted to channels to account for the ion permeabilities of natural membranes. This conclusion is supported by the fact that the electrical capacitance of the cell membrane remains relatively unchanged during large changes in the permeability exhibited during the excitation of some membranes. (This phenomenon is discussed further in Chapter 5.) Facilitated Transport across Membranes

Membranes are permeable to various polar molecules such as sugars, amino acids, nucleotides, and certain cell metabolites that would cross lipid bilayers by diffusion only very slowly. This is because of facilitated transport, the movement of molecules through membranes by the action of membrane transport proteins (see Figure 4-22B). Facilitated transport, unlike active transport discussed later, does not require energy in the form of ATP. Membrane transport proteins, which exist in many forms in all types of membranes, are exquisitely selective about which species of molecules they transport. Carrier proteins that transport a single solute from one side of the membrane to the other are called uniporters, while those that transfer one solute and simultaneously or sequentially transfer a second solute are called coupled transporters. Coupled transporters that transfer two solutes in the same direction are called symporters, while those that transfer solutes in opposite directions are called antiporters (Figure 4-23). These terms can also be applied to active transport systems.

7

Uniporter

Symporter

i

Extracellular

Antiporter

Figure 4-23 Membrane carrier proteins can be configured as uniporters, symporters, or antiporters. Uniporters transport a single type of ion in one direction across the membrane, while symporters simultaneously transport two different ions in the same direction. Antiporters also transport two ions, but create an exchange of ions by moving the two in opposite directions across the membrane. ~

5 Y e,

Facllltated diffusion

m

Passive diffusion

I

External concentration of glucose (mM)

Km

Figure 4-24 The kinetics of simple diffusion differ from those of carriermed~ated(facilitated) diffusion. In this example of glucose movements, the rate of simple diffusion is always proportional to the glucose concentration. However, the rate of carrier-mediated glucose diffusion reaches a maximum (V,,,) when the glucose carrier protein is saturated. The binding constant (K,) of the carrier for glucose, which is analogous to the Kmof an enzyme for its substrate solute, is measured when transport is at half its maximal value. [Adapted from Lodish et al., 1995.1

The existence of such transporters was initially inferred from kinetics studies of molecule transfer across membranes (Figure 4-24). For some solutes, the measured rate of influx reaches a plateau beyond which an increase in solute concentration produces no further increase. This reveals that a rate-limiting step must occur in permeation. Experiments elucidating the kinetics of such permeation led to the conclusion that transport occurs through the formation of a carrier-substrate complex similar in concept to an enzyme-substrate complex. Each carrier protein has a characteristic binding constant for its solute equal to the concentration of solute when the transport rate is half its maximum value (see Figure 4-24). As in enzyme reactions, the solute binding can be blocked by specific competitive inhibitors as well as by noncompetitive inhibitors. The carrier and solute molecule temporarily form a complex based on bonding, stearic specificity, or both. The specificity of these transporters was first established in studies where single gene mutations abolished the ability of bacteria to transport specific sugars across their cell membranes. Similar mutations have now been found in many cases, including human inherited diseases that affect the transport of specific solutes across kidney, intestine, or lungs. For example, in cystic fibrosis, a defect in the chloride transport channel protein (CFTR) appears to be responsible for fluid imbalance in the lungs.

ACTIVE TRANSPORT All channel proteins and most carrier proteins allow solutes to cross the membrane passively at no energetic cost (other than the original cost of generating the

110


........................................

potential energy in the form of different solute concentrations on opposite sides of the membrane, as mentioned earlier). The concentration gradient determines the direction of passive transport. As diffusion proceeds, the solute concentrations in the two compartments approach equilibrium, at which point no further net diffusion will occur. For charged molecules, transport is influenced by both the concentration gradient and the electrical gradient (i.e., the electrochemical gradient) across the membrane. All plasma membranes have an electrical potential difference across them, where the inside is negative relative to the outside of the cell. This favors the entry of positively charged ions and opposes entry of negatively charged ions. In this case, as above, passive processes will continue until the membrane is in equilibrium. The distribution of ions across cell membranes is at true equilibrium only in dead cells. All living cells continually expend chemical energy to maintain the transmembrane concentrations of solutes far away from equilibrium. This energy is typically supplied in the form of ATP. Mechanisms that actively transport substances against a gradient are collectively called membrane pumps. When the source of energy for such pumps is cut off, active uphill transport ceases and passive diffusion governs the distribution of substances. The concentrations of these substances gradually redistribute toward equilibrium. The Na+/K+ Pump a s a Model of Active Transport

Many of the features of active transport are demonstrated in the system that maintains steep concentration gradients for Na+ and KC in the cell. The concentration of K+ is about 10-20 times higher inside cells than outside, while the opposite is true for Na+ (see Figure 4-13). These concentration differences are sustained by a Na+/K+ pump found in the plasma membrane of virtually all animal cells. This pump is an ATPase with binding sites for Na+ and ATP on its cytoplasmic surface and binding sites for K+ on its external surface. In the steady state, the number of Na+ ions pumped, or transported, out of the cell is equal to the number of Na+ ions that leak in. Thus, even though there is a continual turnover of Na+ (and other ion species) across the membrane, the net Na+ flux over any period of time is zero. There are two factors that determine the size of a Na+ concentration gradient that will be built up between the cell interior and cell exterior: the rate of active transport of Na+ and the rate at which Na+ can leak (i.e., diffuse passively) back inro the cell. The rate at which the membrane allows Na+ to leak back into the cell determines, of course, the rate at which the Na+ pump has to work in order to maintain a given ratio of extracellularto-intracellular Na+. There is evidence that an increase in the intracellular concentration of Na+ leads to an increase in the rate of Na+ expulsion by the pump (which may merely be a mass action effect due to the increased availability of intracellular Na+ to the carrier molecules in the membrane).

Several important features of active transport should be noted:

1. Transport can take place against substantial concentration gradients. The most commonly studied membrane pump is the one that transports Na+ from the cell interior to the external fluid against a 10: 1Naf concentration gradient. 2. The active transport system generally exhibits a high degree of selectivity. The Na+ pump, for example, fails to transport lithium ions, which have ionic properties very similar to those of sodium ions. 3. ATP or other sources of chemical energy are required. Metabolic poisons that stop the production of ATP bring active transport to a halt. 4. Certain membrane pumps exchange one kind o f molecule or ion from one side of the membrane for another kind of molecular or ion from the other side. The NaC/K+antiport features active outward transport of Na+ concomitant with the inward transport of K+ by the sodium-potassiumpump. This process involves the obligatory exchange of two potassium ions from outside the cell for three sodium ions from inside the cell (Figure 4-25). When external K+ is absent, the Na+ ions that normally would have been exchanged for K+ ions are no longer pumped out. 5 . Some pumps perform electrical work by producing a net flux of charge. For example, the Naf/K+ exchange pump just mentioned produces a net outward movement of one positive charge per cycle in the form of three Na+ exchanged for only two K+. Ionic pumps that produce net charge movement are said to be rheogenic because they produce a transmembrane electric current. If the current produces a measurable effect on the voltage across the membrane, the pump is also said to be electrogenic. 6. Active transport can be selectively inhibited by specific blocking agents. The cardiac glycoside ouabain, applied to the extracellular surface of the membrane, blocks the potassium-dependent active extrusion of Na+ from the cell. It does this by competing for the K+ binding sites of the Na+/K+pump at the outside surface of the membrane. 7. Energy for active transport is released by the hydrolysis of ATP by enzymes (ATPases) present in the membrane. Active transport exhibits MichaelisMenten kinetics and competitive inhibition by analog molecules. Both behaviors are characteristic of enzymatic reactions. Calcium-activated ATPases have been associated with calcium-pumping membranes. Associated with the Na+/K+ pump are Na+ and K+-activated ATPases isolated from red blood cell membranes and other tissues. These enzymes catalyze the hydrolysis of ATP into ADP and inorganic phosphate only in the presence of Na+ and K+, and they bind the specific Na+ pump inhibitor ouabain. The fact that ouabain binds to the mem-

MEMBRANES,

CHANNELS,

A N D TRANSPORT

111

............................................................................... K+and ouabain

0

Potassium electrochemical gradient

1 ATP

Na+blndlng sites

A D P + Pi

Figure 4-25 The Na+/KCATPase actively pumps Na+ out of the K+ into a cell against their respective electrochemical gradients. (A) For every molecule of ATP hydrolyzed directly to power transmembranetransport, three Na+ ions are pumped out and two K+ ions are pumped in. The specific pump inhibitor ouabain and K+ compete for the same sites on the external side of the ATPase. (B)This schematic model of Na+/K+ATPase shows the movement of Na+ and K+ by a single protein. The binding of Na+ (step 1) and the subsequent phosphorylationof the cytoplasmicface ofthe ATPase by ATP (step 2) cause a conformational change in the protein resulting in a transfer of the Na+ across the membrane (step 3). The Na+ is releasedto rhe cell's exterior and K+ is bound (step 4). Subsequent dephosphorylationofthe protein (step 5) induces a return to the protein's original conformation and consequent transfer of the Kt across the membrane (step 6), where it is released into the cytosol (step 7).

K+ b~ndlng sltes

brane and blocks the Na+/Kt pump is evidence that these ATPases are involved in active transport of Na+ and K+. The operation of the Ka ' / K y ATPasc is thought to depend on a series of co~itormationalchanges in the transport protein that allow the cotransport of K+ and Naf across the cell membrane (see Figure 4-25). The actual process of metabolically energized transport takes place across the cell membrane, pumping molecules either into or out of the cell. However, the organization of cells into an epithelial sheet makes possible the active transport of substances from one side of the epithelial sheet to the other because the cell surfaces at each side are asymmetrical in their transport properties. One side of the cell may tend to import a substance, while the other side tends toward export, thus effecting transfer of the substance to opposite sides of the cell. This characteristic enables the epithelia of amphibian skin and bladder, fish gills, the vertebrate cornea, kidney tubules, the intestine, and many other tissues to move salts and other substances across the tissues.

kinds of transport?

Ion Gradients as a Source of Cell Energy

Electrochemical gradients across biological membranes provide an important energy source immediately available to cells. This energy can be used to drive passive or secondary active transport and is also used to store or conduct information along the surface of cell membranes (see Chapter 5). The amount of free energy stored in an electrochemical gradient depends on the ratio of ion concentrations-or, more accurately, the ratio of the chemical activities of an ion species-on the two sides of the membrane. Energy release occurs when the ions are allowed to flow down their gradient across the membrane. Three important cellular processes utilize the free energy of

112


.........................................

biological gradients: production of electrical signals, chemiosmotic energy transduction, and uphill transport of other molecules. Production of electrical signals Electrochemical energy is stored across the membrane primarily as Na+ and Ca2+gradients. The release of this electrical energy is under the control of "gated" channels. These channels are normally closed, but in response to certain chemical or electrical signals, they switch to an open state in which they exhibit selective permeability to specific ions. These ions then flow passively across the membrane down their electrochemical gradients. Because of the charges it carries, an ion species, as it moves across a membrane, produces an electric current and changes the potential difference that exists across the membrane. This electrical activity is the functional basis of the nervous system (the subject of Chapter 5).

lnner membrane lntermembran

Matrix

space

Translocation of H+during electron transpor

Phosphate transporter

OH-

concentration

Cherniosmotic energy transduction The energy released by the metabolism of foodstuffs culminates in the passage of electrons along the respiratory chain in mitochondria. This, in turn, releases their energy, which is stored as an electrochemical proton gradient across the mitochondrial inner membrane (see Chapter 3). This novel energy storage mechanism, which does not use the conventional high-energy chemical intermediates, puzzled cell biologists for many years until Peter Mitchell proposed the chemiosmotic coupling hypothesis. Chemiosmotic refers to the direct link between chemical ('chemi') and transport ('osmotic') processes. Two ideas are central to the chemiosmotic theory: The redox enzymes are oriented within the inner membrane of the mitochondrion so that the electrontransport system of the respiratory chain pumps hydrogen ions from inside the mitochondrial matrix across the inner membrane into the intermembrane space (Figure 4-26). The inner mitochondrial membrane has a low intrinsic permeability to H+, so that this active pumping produces an excess of OH- (and, therefore, a high pH) within the mitochondrial matrix and an excess of H+ (and low pH) within the intermembrane space. The energy-rich H + gradient set up in this way across the inner membrane provides the free energy that removes HOH from ADP + PI, as required for the production of ATP: ADP

+ Pi

A G O '

=

-

ATP

+ H,O

+7.3 kcal-mol-I

This reaction also requires that an ATPase complex be oriented on the inner mitochondria1 membrane so as to take advantage of the separation of H+ and O H across the membrane. The H+ that is enzymatically removed from

lnner mitochondrial membrane

Figure 4-26 The phosphate and ATP-ADP transport system that generates ATP is located in the inner mitochondrial membrane. The phosphate transporter couples the uptake of one HPOZ- (inorganic phosphate) to the outward movement of one OH- anion. At the same time, the ATPADP antiporter exchanges one incoming ADP3- for one A T P 4 exported from the matrix. The exported O H combines with an H+ translocated As a result, there is a net uptake of one A D P 3 and outward by resp~rat~on. one HPO: in exchange for one ATP4-. This process is powered by the outward translocationof one H+ during electron transport. For every four H+translocated outward, three are used to synthesize one ATP molecule and one 1s used to export ATP in exchange for ADP and P,. [Adapted from Lodish et al., 1995.1

ADP is thought to be "siphoned off" into the OH--rich mitochondrial interior to form HOH (Figure4-27). The OHremoved from the inorganic phosphate molecule is shunted outside the mitochondrion to react with the excess H + to form HOH. Thus, the H + / O H gradient provides the energy needed to remove the water during the phosphorylation. Following the dehydration, phosphate bond formation goes forward on the active site of the ATPase without further need for energy input. ADP

+ Pi

-

ATP

Chemiosmotic energy transduction similar to that proposed for oxidative phosphorylation in mitochondria has been implicated as the mechanism for energy transduction during photosynthesis in chloroplasts and photosynthetic bacteria. In addition, there is evidence that the Na+/K+ pump, which normally utilizes ATP to produce the Na+ gradient, can in special circumstances run in reverse, so that the movement of Na+ down its gradient will cause the pump to synthesize ATP from ADP and P, .

Inner mitochondrial membrane

H+ lntermembrane space

H+

II

Mitochondria1 matrix OH-

Time

HOH

H+ Figure 4-27 The second phase of Mitchell's chemiosmotic theory explains energy transduction in the mitochondrion. With the catalytic aid of F, ATPase located in the inner mitochondrial membrane, ADP and P, have H+ and OH-, respectively, stripped away by high O H levels in the mitochondrial matrix and by the relatively high concentration of H+ in the intermembrane space. This process allows P, to condense with ADP t o form ATP.

Coupled Transport

Movement of some molecules up a concentration gradient is driven by movement of another substance down its concentration gradient. Thus, the ubiquitous Na+ gradient is used to carry certain sugars and amino acids along through the membrane by a symport mechanism and to drive Ca2+ out of the cell by an antiport mechanism. The next section will consider such coupled transport in detail. Symporters Symporters are one form of coupled active transport systems that run on energy stored in ion gradients. An example is the transport of the amino acid alanine, which is coupled to Na+ (Figure 4-28). In the presence of Na+, the amino acid is taken up by the cell until the internal concentration is 7- 10 times that of the external concentration. In the absence of Na+, the intracellular concentration of alanine merely approaches the extracellular concentration. In both cases the rate of influx shows saturation kinetics, indicating a carrier mechanism. The effect of extracellular Na+ is to enhance the activity of the alanine carrier. Increasing the intracellular Na+ concentration by blocking the Na+ pump with ouabain has the same effect as decreasing the extracellular Na+ concentration. Thus, it appears to be the Na+ gradient that is important for inward alanine transport, and not merely presence of Na+ in the extracellular fluid. The transport of amino acids and sugars is coupled to inward Na+ leakage by means of a common carrier. The carrier molecule must bind both Na+ and the organic sub-

l l A l a n i n e concentration Figure 4-28 The cellular uptake of an amino acid such as alanine depends on Na+ concentration. (A) lntracellular concentration of alanine, an amino acid, as a function of time with and without extracellular Na+ present. The dashed line represents the extracellular concentration of alanine. (B) Lineweaver-Burkplots of alanine influx with and without extracellular Na+.The abscissa is the reciprocal of the extracellular alanine concentration. The common intercept indicates that at infinite concentration of alanine, the rate of transport is independent of [Na+]. [From Schultz and Curran, 1969.1

strate molecule before it can transport either (Figure4-29). The tendency for Na+ to diffuse down its concentration gradient drives this carrier system. Anything that reduces the concentration gradient of Na+ (low extracellular Na+ or increased intracellular Na+) reduces the inwardly directed driving force and thereby reduces the coupled transport of amino acids and sugars into the cell. If the direction of the Na+ gradient is experimentally reversed, the direction of transport of these molecules is also reversed. The carrier-mediatedtransport of Na+ in this case also depends on the presence of amino acids and sugars. In the absence of amino acids and sugars, the common carrier will transport Na+ only very weakly, and as a result the inward leakage of Na+ is reduced. The common carrier appears to shuttle between the two sides of the membrane passively, without direct utilization of metabolic energy. The cbupled uphill transport of organic molecules derives its energy from the downhill diffusion of Na+. However, the potential energy stored in the Na+ gradient is ultimately derived from metabolic energy that drives the Na+ pump. The Na+ concentration gradient is thus an intermediate form of energy that can be

114


............................................................................... Na+

Glucose

Extracellular

\

Carrier protein

'1

Cytosol

Figure 4-29 Sugar and amino a c ~ dtransport may be ach~evedby sodium-medlated cotransport The carrier proteln must b ~ n d both the Na+ and the organlc substrate before it will transport elther. Net trans-

port IS Inward because ofthe Na+ gradlent, Indicated by the arrow Note that glucose IS movlng agatnst ~tsgradlent

used to drive several energy-requiring processes in the membrane.

arises from the Na+ concentration gradient, directed from the lumen into the cell. This gradient is maintained by the removal of Na+ from the cell by the Na+/K+pump located in the membrane on the other side of the cell, which faces the plasma and blood.

Antiporters The Na+ concentration gradient also plays a role in the maintenance of a very low intracellular Ca2+concentration in certain cells via the Na+/Ca2+antiport system. In most, if not all cells, the intracellular Ca2+concentration is several orders of magnitude below the extracellular concenM), and many cell functions are trations (less than regulated by changes in the intracellular Ca2+concentration. Efflux of Ca2+from cells is reduced when extracellular Na+ is removed, because Ca2+is expelled from the cell in exchange for Na+ leaking in. The opposing movements of these two ions are coupled to each other by an antiporter. One view is that Ca2+and Na+ both compete for the carrier, but that Ca2+competes more successfully inside the cell than on the outer surface, so that there is a net efflux of Ca2+.Here, again, the immediate source of energy is the Na+ gradient, which ultimately depends on the ATPenergized active transport of Na+. Ca2+is also transported independently of the Na+ gradient by an ATP-energized Ca2+pump, which is the major source of Ca2+extrusion under normal conditions. The Na+/H+ antiporter in the proximal tubule of the mammalian kidney is another example of cotransport in opposite directions (see Chapter 14).Here the extrusion of H+ from inside the cells lining the renal tubule into the urine contained within the tubule is coupled to Na+ uptake into the cell in a 1:1stoichiometry. That is, for each H+ expelled, one Na+ is taken up into the cell. This has the advantages of (1)avoiding the expenditure of energy to perform electrical work, since two equivalent positive charges are exchanged and (2)enabling the kidney to reclaim Na+ from the urine and excrete excess protons. The Na+/H+exchanger, unlike the Na+/K+pump, is oriented so as to move Na+ out of the lumen and into the cell. Also unlike the Na+/K+ pump, this mechanism is not an example of primary active transport, in which ATP is the immediate source of energy. Instead, the Na+/H+exchanger is an example of secondary active transport, in which the source of energy is the electrochemical gradient of one or both exchanged ions. In this case the energy driving the exchange

MEMBRANE SELECTIVITY The utility of cell membranes lies in their selectivity-their ability to allow the passage of only specific types of molecules. This selectivity is important because a nonselective membrane will not protect the contents of the cell from intrusion by unwanted chemicals. Each kind of membrane transport system also displays selectivity, which differs in a given membrane for different transport systems. For example, when the Na+ in a physiological saline solution used to bathe a nerve cell is replaced with lithium ions, the Li+ readily passes through the Na+ channels, which open during electrical excitation of the nerve cell membrane. The other alkali metal cations, K+, Rb+, and Cs+, are essentially impermeant through these channels. On the other hand, the ATPase of the Na+ pump in the same membrane is highly specific for intracellular Na+ and is not activated by Li+. Lithium ions, passing through the Na+ channels, will therefore gradually accumulate in the cell until it comes into electrochemical equilibrium. This is an example of electrolyte selectivity by the transport system, but not by the membrane channels. We will now consider how this selectivity for both electrolytes and nonelectrolytes is achieved. Selectivity for Electrolytes

How do channels discriminate between different ions? Although enzymes recognize substrates via distinct shapes or chemical structures, membranes can distinguish ions of essentially identical shape and size. For example, Na+ and K+ have almost the same shape and size (K+is a little larger), yet the resting nerve cell membrane is about 30 times more permeable to K+ than to Na+. At first glance, we might conclude that these ions are distinguished on the basis of their hydrated size, with K+ passing freely through channels that are too small for Na+. Size can explain how the K+ channel excludes Cs+ or Rb+ (Table 4-3) but not Na+,

...................................... TABLE 4-3

Channel

Ionic radii and hydration energies of the alkali metal cations. Cat~on Ll

+

l o n ~ crad~us(A)

Free energy of hydrat~on (kcal mol-"

OM)

.

-122

Na+

0 95

-98

K+

1 33

- 80

Rb+

1 48

-75

CS+

1.69

-67 Lipid bila

particularly in light of the fact that permeability to Na+ can change dramatically. For example, during the excitation of nerve or muscle membrane, the Na+ permeability of the membrane increases about 300-fold to a value about 10 times greater than the K+ permeability at rest. If, during excitation, the membrane were suddenly to develop channels that pass the Na+ ion on the basis of size alone, there should be a simultaneous increase in permeability to K+ through the same channels, given their comparable sizes. Since this increase does not occur, the membrane's selectivity must rest on properties other than size. Indeed, estimated pore sizes for different membrane channels illustrate that size alone cannot be the agent of membrane selectivity. Two interesting features other than size appear to be important in governing membrane pore selectivity: ease of dehydration and interaction with charges within the pore. For an ion to enter a pore, it must dissociate from water molecules. Ease of dehydration appears to be an important factor in governing selectivity, particularly if the charges within the pore are weak. Since large ions dehydrate more easily than small ones (seeTable 4-3), a pore with weak polar sites along it will admit large ions preferentially over small ones. In channels with strongly charged sites, the interaction of the dehydrated ion with these sites may be more important for conferring specificity than ease of dehydration. Thus, a channel lined with predominantly positively charged residues will selectively repel positively charged ions, but permit negatively charged ions to pass through (Figure4-30). In such cases, smaller ions can approach the polar sites more closely and hence interact more strongly than can large ions, exaggerating the effect. Selectivity for Nonelectrolytes Virtually all nonelectrolytes cross the membrane by dissolving in the lipid bilayer and simply diffusing across it. Since the relationship between permeability and partition coefficient K is essentially linear (see Figure 4-19), selectivity is completely determined by molecular properties responsible for the partition coefficient. Those few nonelectrolytes that deviate from the linear relation between partition coefficient and permeability all have greater than predicted permeability. Some of these substances cross the membrane by carrier-mediated transport. Alternatively,

Figure 4-30 Positive charges lining the membrane channel allow anions t o pass but retard the diffusion of cations through the channel, as shown in this hypothetical cross-section through a highly simplified membrane channel.

small molecules such as ethyl alcohol, methyl alcohol, and urea can cross via both the lipid layer and water-filled channels. All these deviant molecules are small and water-soluble regardless of their relative solubilities in water versus lipid (i.e., their partition coefficients).It is important to note that mechanisms for precise control of nonelectrolyte access through membranes have not evolved, making cells vulnerable to penetration by these molecules. Drugs applied to the human skin, such as anti-nauseants delivered by skin patches placed behind the ear, can use this route to enter the body.

ENDOCYTOSIS AND EXOCYTOSIS The transport processes described above for small polar molecules across membranes cannot transport macromolecules such as proteins, polynucleotides, or polysaccharides. Yet, cells do manage to ingest and secrete macromolecules, using mechanisms very different from those used for small solutes and ions. Transmembrane movement of macromolecules is accomplished through the sequential formation and fusion of membrane-bounded vesicles. The intake of material into the cell is given the general term endocytosis. The prockss is more specifically called pinocytosis if fluid is ingested and phagocytosis if solids are ingested. The secretion from the cell of macromolecules is called exocytosis. In both exocytosis and endocytosis, the fusion of separate regions of the lipid

116


...............................................................................

bilayer occurs in at least two steps: the bilayers come into close apposition and then they fuse. Both processes are thought to be controlled by speciahed proteins. Mechanisms of Endocytosis

and the subsequent budding off of the vesicle from the surface membrane. Once the coated vesicle buds off into the cytoplasm, it is believed to fuse with and deliver its contents to other organelles, such as lysosomes. The clathrin and receptors are recycled into the surface membrane.

Transfer of macromolecules across membranes by endocytosis requires specialized control mechanisms. Receptor-mediated endocytosis depends on the presence of receptor molecules embedded in the cell membrane (Figure4-31A).These bind certain ligand molecules or particles, including plasma proteins, hormones, viruses, toxins, immunoglobulins, and certain other substancesthat cannot pass through membrane channels. The receptors are free to diffuse laterally in the plane of the membrane, but upon binding of ligand, the receptor-ligand complex tends to accumulate within depressions in the membranes called coated pits. The coated pit internalizes the ligand. One theory of how it does this is by the formation of a vesicle that pinches off into the cytoplasm, as shown (Figure4-31B). This is called a coated vesicle because of a layer of the protein clathrin that covers the cytoplasmic surface of the vesicle membrane. The clathrin is organized into pentagonal or hexagonal lattice-like arrays on the membrane surface, and appears to have several functions. These include the binding of ligand-occupied receptor molecules

The release of chemicals from cell membranes through exocytosis plays a crucial role in the endocrine and nervous systems. For example, the presynaptic terminals of nerve cells contain many membrane-delimited internal vesicles about 50 nm in diameter, which contain the neural transmitter substance. These vesicles coalesce with the surface membrane of the nerve terminal and release their contents to the cell exterior, the typical method of exocytosis. This activity occurs with greatly enhanced probability when the terminal is invaded by a nerve impulse, and serves to release the synaptic transmitter that interacts with the postsynaptic membrane. Similar mechanisms are involved in the secretion of hormones. An important feature of exocytosis (as well as endocytosis) is that the secreted or ingested macromolecules are sequestered in vesicles and hence do not mix with macromolecules or organelles in the cell. Since the vesicles can fuse with

A

Plasma membrane

Figure 4-31 Coated vesiclesform during receptor-mediated endocytosis. (A) There are six major steps involved in the process: (1) ligand molecules bind to surface receptor molecules located in coated pits formed by clathrin molecules that are bound to surface membrane; (2)the coated pit is invaginated; (3) the coated vesicle is formed; (4) the coated vesicle fuses with an existing vacuole, shedding the clathrin molecules; (5) the fused complex undergoes further processing, depending on its contents, while (6) clathrin and receptor molecules are recycled for reuse In the plasma membrane. (B) Electron micrographs of coated pit (top) and coated vesicle (bottom).These two stages, taken from a chicken oocyte, show the dense clathr~ncoat on the cytoplasmic surface of the membrane. The surface membrane can be seen pinching offthe vesicle. [Part Afrom Pearse, 1980; part B from Bretscher, 1985.1

Mechanisms of Exocytosis

6

only specific membranes, they assure the directed transfer of their contents in the cell. In exocytosis, once the membrane of the vesicle is incorporated into the surface membrane, the freed contents-hormones, neurotransmitters, and accessory molecules-diffuse away into the interstitial space. Exocytosis requires a method for recovering the relatively large amounts of secretory vesicle membrane that initially surrounds the macromolecules being expelled. In the absence of retrieval of this newly incorporated membrane, the surface area of the plasma membrane would continually grow. However, endocytosis is thought to be responsible for the eventual recovery of this excess membrane through its reformation into new secretory vesicles. Evidence for such membrane recycling through endocytosis comes from experiments in which electron-opaque protein molecules, such as horseradish peroxidase, are introduced into the extracellular fluid and their movement into the cell determined with electron-microscopic methods. In these experiments, horseradish peroxidase shows up inside the cell, but only within vesicles. Since the large size of the horseradish peroxidase molecule prevents its penetration by direct passage across biological membranes, it must have been taken up in bulk during the endocytotic formation of vesicles budding off from the plasma membrane into the cytoplasm. The calcium ion is responsible for the exocytotic secretion of neurotransmitter substances from nerve cells and of hormones from endocrine cells. Although the precise role of Ca2+in initiating secretion is unknown, it appears that an elevation of intracellular Ca2+somehow enhances the probability of exocytotic activity, perhaps by permitting the coalescence of vesicles with the inner surface of the membrane. The membrane regulates exocytotic activity by regulating the intracellular accumulation of Ca2+. As enhanced calcium influx allows Ca2+levels to rise, the rate of exocytotic secretion increases. The vesicle membrane itself may participate actively in the initial steps leading to exocytosis. The secretory granules (or vesicles) of the adrenal medulla have been found to be rich in an unusual phospholipid, lysolecithin, that facilitates the fusion of membranes and thus may help the vesicle membrane fuse with the surface membrane. Before fusion of the two membranes can take place, the secretory granule must come into contact with the plasmalemma. Release of secretory produ%sfrom glandular secretory cells can be blocked by colchicine, an anti-mitotic agent that leads to the disassembly of microtubules, or by cytochalasin, an agent that disrupts microfilaments. This pharmacological evidence has led to the suggestion that microtubules or microfilaments participate in the movement of secretory granules toward sites of exocytotic release on the inner side of the surface membrane.

JUNCTIONS BETWEEN CELLS Cells in animals are organized into cooperative assemblies called tissues. In certain tissues, including epithelium, smooth muscle, cardiac muscle, central nervous tissues, and

many embryonic tissues, neighboring cells are connected by special adaptations of their abutting surfaces. These specialized surfaces are divided into two major groups: gap junctions and tight junctions. Gap junctions enhance cellcell communication through minute, water-filled channels that connect adjacent cells, while tight junctions "sew" cells involved in transepithelial transport into sheets. Gap Junctions

Gap junctions provide communication between cells by allowing inorganic ions and small water-soluble molecules to pass directly from the cytoplasm of one cell to the cytoplasm of the other. These junctions couple cells electrically and metabolically, with important functional consequences for the tissue. The distance between two membranes of a gap junction is only 2 nm (Figure4-32). The two adjoining membranes each contain clusters of hexagonal arrays of six subunits that span the narrow space between the two membranes (Figure 4-33A). The subunit arrays are about 5 nm in diameter and resemble miniature doughnuts whose hollow centers form passageways between the interiors of the neighboring cells (Figure4-33B).The continuity of the cellcell passageways through the gap junction has been demonstrated by injecting fluorescent dyes, such as fluorescein (molecular weight 332) and procion yellow (molecular weight 500), into one cell and following their diffusion into neighboring cells (Figure 4-34). This continuity has been corroborated for direct exchange of ions by the finding that electric current readily passes directly from one cell into another if gap junctions are present. The intercellular channels in these junctions pass molecules with a molecular weight of at least 500, so that small molecules, such as ions,

Gap junctio

,

50nm

,

Figure 4-32 The gap junction interval (2 nm) between neighboring cells is at the lower limit for electron microscopic resolution. The electron micrograph reveals a gap junction between membranes of two neighboring mouse liver cells. [Courtesy of D. Goodenough.]

118


...............................................................................

Figure 4-33 Gap junctions permit passage of molecules between neighboring cells. (A)The two membranes belonging to neighboring coupled cells both contain an array of hexagonal subunits, each of which connects with a matching subunit in the apposed membrane. A central channel penetrates both subunits, providing a path of communication between

the connected cells. (B) Detail of a channel complex. Molecules smaller than about 2 nm can pass between coupled cells through the channel. Molecules larger than 2 nm, such as proteins and nucleic acids, are too large to penetrate the channel. [Part Aadapted from Staehelin, 1974; part B adapted from Bretscher, 1985.1

amino acids, sugars, and nucleotides, are easily exchanged between cells (see Figure 4-33B). This exchange of small molecules is responsible for gap junction-mediated cell-cell communication. Gap junctions are labile and close rapidly (within seconds) in response to any treatment that increases intracellular Ca2+or H+ concentration. Uncoupling of cells from their neighbors can be produced by injecting Ca2+or H+

into a coupled cell, by lowering temperature, or by using poisons that inhibit energy metabolism. The subsequent loss of electrical transmission between cells confirms the uncoupling. Thus, gap junctions are maintained intact only if the metabolic activity of the surface membrane maintains sufficiently low concentrations of intracellular free Ca2+ and H+. The mechanism of closing of the gap junction channel is not clearly understood, but the channel appears to be open or shut depending on the relative positions of the six subunits of the channel.

r

Fluorescein

brate nervous systems, allow the exchange of many types of cytoplasmic materials between adjacent cells. How do assemblages of cells hnked by gap junctions-freely exchanging ions, amino acids, sugars, and nucleotideschallenge the concept of cells? What is the functional difference between a single cell, a series of cells linked by gap junctions, and a tissue?

Distance Figure 4-34 Gap junctions between coupled cells can be demonstrated by following the flow of fluorescent dye injected into one of a group of coupled epithelial cells. Subsequent diffusion of the dye into neighboring cells without loss into extracellular space indicates that there are direct pathways from the cytoplasm of one cell to the cytoplasm of the adjacent cell.

Tight Junctions Tight junctions seal cells together in an epithelial cell sheet so that even small molecules cannot get from one side of the sheet to the other. The two apposing cell membranes make intimate contact, fully occluding the extracellular space in between. Tight junctions are found most commonly in ep-

Zonula occludens

Figure 4-35 Adjacent ep~thelialcells like those that line the mammalian small intestine are connected by intercellularjunctions. The membranes and associated structures are drawn disproportionately large in this reconstruction of the cell-celljunctions.

Zonula adherens (intermediate junction)

Macula adherens

Intercellular space

ithelial tissues as a zonula occludens, a thin band of protein molecules that encircles a cell like a gasket. The zonula occludens is in tight contact with the zonulae of the surrounding cells, forming an impermeable seal that prevents passage of substances from one side of the epithelium to the other via leakage down along the sides of the cells (Figure 4-35). Considered en masse, the zonulae are conceptually like a continuous rubber sheet, penetrated only by the ends of the epithelial cells.3ubstances can pass through the ends of the cells (the transcellular pathway), but not around them (the paracellular pathway). In tissues such as the mammalian small intestine, the gallbladder, and the proximal tubule of the nephron, these zonulae are not fully continuous and thus not really very "tight." These tissues are so leaky, they do not produce a transepithelial potential difference, even though their cells contain ion pumps capable of generating transepithelial ion fluxes. Unlike gap junctions, tight junctions appear to have no special channels for cell-cell communication. Two other types of cell junctions are shown in Figure 4-35: the zonula adherens and the desmosome serve primarily to aid the structural bonding of neighboring cells.

EPITHELIAL TRANSPORT Epithelial cell sheets line the cavities and free surfaces of animal bodies and form barriers to the movement of water, solutes, and cells from one body compartment to another. Each organ or compartment within an animal has such a lining of surface cells. Some of these sheets serve only as passive barriers between compartments and d o

not preferentially transport solutes and water. In other cases they are involved in active transport, performing regulatory functions. For example, osmoregulatory activities of animals are carried out by actively transporting epithelia in a variety of specialized tissues and organs (see e a p t e r 15). Epithelia have several features in common. First, they occur at surfaces that separate the internal space of the organism from the environment. Included are the surfaces lining deep invaginations such as in the lumen of the intestine, which nevertheless comprise external space. Second, the cells forming the outermost layer of the epithelium are generally sealed together by tight junctions, which to varying degrees in different epithelia obliterate paracellular pathways between the serosal (inner) and mucosal (external) sides of the epithelium (Figure 4-36). In epithelia such as that of capillary walls, leaky junctions permit water and solute molecules to cross the epithelial layer by diffusion within the passages that exist between the cells. Such diffusion through paracellular pathways is not coupled to any metabolically energized transport mechanism, so such passages allow only passive movement of water and ions. Substances that are actively transported across an epithelium must follow transcellular pathways, in which the cell membrane participates. Such substances must cross the cell membrane first on one side of the cell and then on the other. As discussed in the next section, the functional properties of the surface membrane of an epithelial cell are dissimilar in some respects on its serosal and mucosal surfaces. This asymmetry is important to epithelial active transport.

120


...............................................................................

-

Current meter

Exterior, lumen, or mucosal side

I,/

Variable current

Transcellular path

Acellular basement membrane Paracellular path

Figure 4-36 Substances cross epithelial layers by two pathways: . para. cellular and transcellular. Active transport takes place only across cell membranes, suggestingthat all actively transported molecules follow the transcellularpathway

Active Salt Transport across an Epithelium

Energy-requiringtransport of ions from one side of an epithelium to the other has been demonstrated in a number of epithelial tissues, including amphibian skin and urinary bladder, the gills of fishes and aquatic invertebrates, insect and vertebrate intestine, and vertebrate kidney tubule and gallbladder. Much of the initial work on epithelial active transport was done on the skin of the frog. In amphibians, the skin acts as a major osmoregulatory organ. Salt is actively transported from the mucosal side (i.e., the side facing the pond water) to the serosal side of the skin to compensate for the salt that leaks out of the skin into the freshwater surrounding the frog. Similar uptake occurs in the gut. Water that enters the skin because of the osmotic gradient between the hypotonic pond water and the more concentrated internal fluid is eliminated in the form of a copious dilute urine that is hypotonic relative to the body fluids (see Chapter 14). Frog skin was first used in the study of epithelial transport in the 1930s acd 1940s by the German physiologist Ernst Huf and the Danish physiologist Hans Ussing. In their procedure, a piece of abdominal skin several square centimeters in area is removed from an anesthetized and decapitated frog and placed between two halves of an Ussing chamber (Figure 4-37). The dissection is very simple, since the skin of the frog lies largely unattached over an extensive lymph space. Once the skin is gently clamped between the two half-chambers, a test solution-for example, frog Ringer's solution (a solution that mimics the ionic composition of frog plasma) -is introduced, with the frog skin acting as a partition between the two compartments. The compartment facing the mucosal side of the skin can be designated as the outside compartment and the one facing the serosal side as the inside compartment. Air is bubbled through the two solutions to keep them well oxygenated.

between two-half-chambers Figure 4-37 Afrog skin separates the two halves of this Ussing chamber. Each half is filled with a physiologicalsaline or other test solution.The current source is adjusted until the potential difference across the skin is zero. Under those conditions, the current flowing through the circuit (and thus through the skin) is equivalent to the rate of charge transferred by the active movement of sodium ions across the skin.

In 1947 Ussing reported the first experiments in which two isotopes of the same ion were used to measure bidirectional fluxes (i.e., the simultaneous movement of that ionic species in opposing directions across the epithelium). The Ringer's solution in the outside compartment was prepared using isotope 12Na+,and the Ringer's in the inside compartment was prepared using 24Na+.The appearance of each of the two isotopes on the opposite side of the skin was tracked over time. The two isotopes were switched around in other experiments of the same type to rule out any effects due to possible (but unlikely) differences in transport rates inherent in the isotopes themselves. In all experiments it was found that Na+ shows a net movement across the skin from the outside compartment to the inside one. That the Na+ flux is the result of active transport is seen in the fact that it Occurs without any concentration gradient, and even against an electrochemical gradient. Is inhibited by general metabolic inhibitors, such as cyanide and iodoacetic acid, and by specific transport inhibitors, such as ouabain. Displays a strong temperature dependence. Exhibits saturation kinetics. Shows chemical specificity. For example, Na+ is transported while the very similarly structured lithium ion is not. How can an active movement of ions be produced across a layer of cells contained in an epithelium? Adjacent cells of transport epithelium are intimately tied together with tight junctions. Assume for the sake of simplicity that this closeness eliminates all extracellular passageways for

MEMBRANES, CHANNELS, AND TRANSPORT 121 .......................................

the diffusion of ions between the two sides of the epithelium. This would force all substances that cross the epithelium to traverse the epithelial cell membrane twice, first crossing the membrane on one side of the cell and then leaving through the membrane on the other side. Active transport by this route requires that the surface membrane of each epithelial cell be differentiated, so that the portion of the cell membrane facing the serosal side of the epithelium differs in functional properties from the portion fating the mucosal side. Experiments on frog skin in Ussing chambers have provided several lines of evidence to support a hypothesis of a differentiated membrane. For example,

.

Ouabain, which blocks the Na+/K+ pump, inhibits transepithelial sodium transport only when applied to the inner (serosal)side of the epithelium. It is ineffective on the outer (mucosal) side. Conversely, the drug amiloride, a powerful inhibitor of passive carrier-facilitated transport, blocks Na+ movements across the skin only when applied to the outer side of the skin. For active Na+ transport to take place, K+ must be present in the solution on the inner side of the chamber, but is not required on the outer side. Transport of Na+ exhibits saturation kinetics as a function of Na+ concentration in the outer solution; it is unaffected by Na+ concentration in the inner solution.

Such evidence led to the model of epithelial Na+ transport shown in Figure 4-38. According to this model, a Na+/K+exchange pump is located in the membrane of the serosal side of the epithelial cell (togetherwith Na+/H+and Na+/NH,+ exchange pumps in the intact animal). This membrane behaves in the manner typical of many cell membranes, pumping Na+ out in exchange for K+, thus maintaining a high intracellular K+ concentration and a low intracellular Na+ concentration. The outward diffusion of K+ across the membrane on this side of the cell produces an insi& negative resting potential. The situation on the mucosal side must be different. The cell membrane on this side of the cell is relatively impermeable to K+. Moreover, a net inward diffusion of Na+ across this membrane (apparently facilitated by carriers or through channels in the membrane) replaces the Na+ pumped out of the cell on the serosal side. This model explains why Na+ pump inhibitors exert an effect only from the serosal side of the epithelium, and why only changes in the concentration of K+ on that side influence the rate of Na+ transport. Thus, there is a net flow of Na+ across the frog skin from the mucosal side to the serosal side as a result of the functional asymmetries of the membranes on the two sides. The driving force is none other than the active transport of Na+ that is common. to cell membranes of all tissues. The frog skin has served as a model system for the general problem of epithelial salt transport. Although details

Mucosal side (toward exterior)

Epithelial cell of froQ skin

Serosal side (toward interior)

Figure 4-38 Transepithelial sodium transport depends on a combination of both diffusion and active transport. In this model of an isolated frog skin that has been bathed in Ringer'ssolution, sodium diffuses passively down its concentration gradient into the cell from the mucosal solution. K+ diffuses out of the cell into the serosal space as it is displaced by Na+ influx. In the face of these leaks, a Na+/K+exchange pump in the serosal membrane of the cell maintainsthe high internal K+ and low internal Na+ concentrations. [From Koefoed-Johnsenand Ussing, 1958.1

may differ from one type of epithelial tissue to another, the major features, listed below, are probably common to all transport epithelia.

I.

2.

3.

4.

To varying degrees, tight junctions obliterate paracellular pathways. As a result, transport through transcellular pathways assumes major importance in epithelial transport. Mucosal and serosal portions of the cell membranes exhibit functional differences, being asymmetrical in both pumping activity and membrane permeabilities. The active transport of cations across an epithelium is typically accompanied by transport (passive or active) of anions in the same direction or by exchange for another species of cation, minimizing the buildup of electric potentials. The converse applies to actively transported anions. Epithelial transport is not limited to the pumping of Na+ and C1- ions. Various epithelia are known to transport H+,HC03-, K+, and other ions.

Transport of Water

To function properly, animal tissues require the right amount of water in the right lace at all times. This is achieved by regulation of water via epithelial sheets. A number of epithelia absorb or secrete fluids. For example, the stomach secretes gastric juice, the choroid plexus secretes cerebrospinal fluid, the gallbladder and intestine transport water, and the kidney tubules of birds and mammals absorb water from the glomerular filtrate. In some of these tissues, water moves across an epithelium in the absence of or against an osmotic gradient existing between the bulk solutions on either side of the epithelium. A number of possible explanations for the uphill movement of

122


. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

water have been given, but all these hypotheses can be placed in one of two major categories:

Mucosal

Serosal

Water is transported by a specific water-carrier mechanism driven by metabolic energy. Water is transported secondarily as the consequence of solute transport. The latter includes classic osmosis, in which water undergoes a net diffusion in one direction owing to concentration gradients built up by solute transport. So far, there has been no convincing evidence to indicate that water is actively transported by a primary water-carrier pump. The osmotic hypothesis of water transport received a boost by Curran, who showed that an osmotic gradient produced by active salt transport from one subcompartment of the epithelium into the other could, in theory, result in a net flow of water across the epithelium (Figure 4-39). Biological correlates of Curran's model were subsequently found in the epithelium of mammalian gallbladder. This finding led to the standing-gradient hypothesis of solute-coupled water transport, presented by Diamond and Bossert (1967).A simplified schematic version is shown in Figure 4-40. Two anatomical features are of major importance. First, the tight junctions near the luminal (mucosal) surface block extracellular pathways through the epithelium. Second, the lateral intercellular spaces, or intercellular clefts, between adjacent cells are restricted at the luminal ends by the tight junctions and are freely open at the basal ends. The basis for the standing-gradient hypothesis is the active transport of salt across the portions of the epithelial cell membranes facing the intercellular clefts. The membranes bordering the lateral clefts have been shown to be especially active in pumping Na+ out of the cell. It is suggested that as salt is transported out of the cell into these long, narrow clefts, the salt concentration will set up an osmotic gradient

Active solute

Water follows

Figure 4-39 Curran's model for solute-linked water transport depends on active transport of a solute across a water-permeable membrane. A solute (e.g., Nat) is pumped through barrier Afrom compartment I to compartment II. Semibarrier B slows diffusion of solute into compartment Ill and thereby keeps the osmolarity high in II. The rlse in osmolarity in compartment II causes waterto be drawn from I to II. In the steady state, both water and solutes diffuse into compartment Ill at the same rate at which they appear in II. Compartment Ill is much larger than II, as indicated by the breaks in the walls of the compartment. [From Curran, 1965.1

Figure 4-40 Curran's model for solute-linked water transport has a biological counterpart. The compartments corresponding to those in F~gure4-39 are numbered I, II, and Ill. Salt transported actively into the intercellular clefts produces a high osmolarity with~nthe clefts. Water flows osmotically into the clefts across the cell, and the bulk solution flows through the freely permeable basement membrane and into the bulk fluid of the interstitium. The barriers A and B are analogous to A and B in Figure 4-39. [From Diamond and Tormey, 1966.1

between the extracellular spaces on either side of the tight junctions that join the epithelial cells. There may also be an osmotic gradient within the cleft, with the salt concentration highest near the closed ends of the clefts, diminishing toward the open ends of the clefts, where it comes into equilibrium with the bulk phase. As a consequence of the high extracellular osmolarity in the clefts, water is osmotically drawn into the cleft across the "not so tight" tight junction, or possibly from within the cell across the cell membrane into the intercellular space. The water leaving the cell would have to be replaced by water drawn osmotically into the cell at the mucosal surface. The water that enters the clefts gradually moves, together with solute, out into the bulk phase. In this way, the steady, active extrusion of salt by one surface membrane of the cell produces an elevated concentration in the narrow intercellular spaces. This, in turn, results in a steady osmotic flow of water from one side of the epithelium to the other. The general applicability of the standing-gradient mechanism of solute-coupled water transport is supported by ultrastructural studies showing that the necessary cellular geometry-namely, narrow intercellular spaces closed off at the luminal end by tight junctions-is present in all the water-transporting epithelia that have been examined. Also important in this regard are deep basolateral clefts and infoldings typical of transporting epithelial cells (see Figure 4-40). These spaces are dilated in epithelia fixed during conditions that produce water transport. In epithelia fixed in the absence of water transport, the intercellular clefts are largely obliterated.

SUMMARY Lipid bilayer membranes are fundamental structures in the formation of various cellular organelles, as well as the surface membrane. Their roles include (1)cellular and subcellular compartmentalization, (2) maintenance of the intracellular milieu using selective permeability and transport mechanisms, (3) regulation of intracellular metabolism by maintaining concentrations of intracellular enzyme cofactors and substrates, (4) metabolic activities carried out by enzyme molecules present in ordered arrays in or on the membrane, (5)sensing and transduction of extracellular chemical signals by means of surface receptor molecules and regulatory molecules located in the membrane, (6)propagation of electrical signals that conduct messages, regulate the transport of substances across the membrane, or both, (7)endo- and exocytosis of bulk material. The foundation structure of membranes is a lipid bilayer in which the hydrophilic heads of the phospholipid molecules face outward and the lipophilic tails face inward, toward the center of the bilayerI The most widely accepted model of membrane structure proposes that a mosaic of globular proteins, including enzymes, penetrates the bilayer. Because of an unequal distribution of solutes between cell interior and exterior, water enters the cell, following its tendency to flow from a region of lower to a region of higher osmotic pressure. Osmotic pressure is equal to the hydrostatic pressure necessary to balance osmotic flow (water movement across a semipermeable membrane) down a concentration gradient at equilibrium. The concept of tonicity describes the osmotic effects that a solution has on a given tissue, whereas osmolarity describes the number of dissolved particles per volume of solvent, as well as the behavior of a solution in an ideal osmometer. Permeability is a measure of the ease with which a substance traverses a membrane. There are several ways in which substances cross the membrane. Nonpolar molecules can diffuse readily through the lipid phase of the membrane. Water and some small polar molecules diffuse through transient aqueous channels created by thermal motion. There is a great deal of evidence for the existence of fixed channels that are more or less specific for certain ions and molecules. Diffusion across the membrane of some substances can occur via carrier molecules that complex with the substance, facilitating its transport across the membrane by shuttling it within the lipid phase of the membrane. In addition to these passive mechanisms, several active transport systems also move substances across the membrane. Active transport of a substance occurs by means of carriers and requires metabolic energy, usually provided by ATP. It is responsible for the movement of a substance across a membrane against a concentration gradient. The most familiar active transport system is the sodium-potassium pump, which maintains the intracellular NaC concentration below that of the cell exterior. The energy stored

in the form of an extracellular-intracellular Na+ concentration gradient is utilized to drive the uphill movement of a number of other substances, such as calcium ions, amino acids, and sugars, by means of exchange diffusion and coupled transport. The Naf and Kt gradients are also important for the production of electrical signals, such as nerve impulses. Another important function of active transport is to compensate for the tendency of certain substances, such as Na+, to leak into cells and thereby cause uncontrolled increases in osmotic pressure and subsequent swelling of the cell. Continual removal of NaC by the Na+/K+ pump is therefore a major factor in controlling cell volume. Transepithelial transport depends on an asymmetry in the permeability and pumping activities of the mucosal and serosal portions of epithelial cell membranes. O n the serosal side of the cell, ions are actively transported across the membrane against an electrochemical gradient; on the mucosal side, ions cross the membrane by diffusion or facilitated transport. Diffusion of ions back through the epithelial layer is slow because the spaces between cells are restricted by tight junctions. Water is transported across some epithelia by being drawn osmotically down a standing salt concentration gradient built up by active salt transport between the epithelial cell interior and the intercellular clefts. There is no evidence for true active transport of water.

REVIEW QUESTIONS What are some of the physiological functions of membranes? What is the evidence for the existence of membranes as real physical barriers? What is the evidence for the lipid bilayer model of the membrane? What is the evidence for a mosaic of globular proteins set into the lipid bilayer of the membrane? Explain the meanings of isotonic and isosmotic. How can a solution be isosmotic but not isotonic to another solution? What factors determine the permeability of the membrane to a given electrolyte? Nonelectrolyte? Describe the probable mechanisms by which water and other small (less than 1 nm in diameter) polar molecules pass through the membrane? Why do nonpolar substances diffuse more easily than polar substances through the membrane? There is no convincing evidence for direct active transport of water. Explain one way in which water is moved by epithelia against a concentration gradient, that is, from a concentrated salt solution to a more dilute salt solution. How does facilitated transport differ from simple diffusion? What factors influence the rate of facilitated transport of ions across a membrane?


.................................. How does active transport differ from facilitated transport? Why can the sodium ion concentration gradient be considered a common cellular energy currency? What are some parameters by which the membrane discriminates between ions of the same charge? Explain the osmotic consequences of poisoning the metabolism of a cell. How does the cell maintain a higher concentration of K+ inside the cell than in the extracellular fluid? What are the morphological and functional distinctions between gap and tight junctions? A given cell is 40 times as permeable to K+ and C1as to any other ions present. If the inside-to-outside ratio of K+ is 25, what would the approximate inside-to-outsideratio of C1- be? Given that cell membranes can transport substances only into or out of a cell, explain how substances are transported through cells. Describe the experiments that first demonstrated active transport of Na+ across an epithelium. What is some of the evidence that active transport of Na+ and K+ occurs only across the serosal membranes of epithelial cells?

SUGGESTED READINGS Bretscher, M. S. 1985. The molecules of the cell membrane. Sci. Am. 253:lOO- 108. (The complex, heterogeneous nature of the cell membrane is explored in this wellillustrated article.) Goodsell, D. S. 1991. Inside a living cell. Trends in Biochem. Sci. 16:203-206. (This article takes the reader on a tour through the amazing structures and processes found in the living cell.) Lodish, H., et al. 1995. Molecular Cell Biology. 3d ed. New York: W. H. Freeman. (This comprehensive textbook describes many of the basic biochemical processes that occur in the cell.) Singer, S. J., and G. L. Nicolson. 1972. The fluid mosaic model of the structure of cell membranes. Science 175720-731. (This is the original paper proposing the fluid mosaic model of the structure of cell membranes.) Verkrnan, A. S. 1992.Water channels in cell membranes. Ann. Rev. Physiol. 54:97- 108. (Thisreview focuses specifically on the channels involved in transmembrane movements.) Yeagle, P. L. 1993. The Membranes of Cells. (Both the morphology and the molecular physiology of plasma and organelle membranes are highlighted in this book.)

126

P H Y S I O L O G I C A L PROCESSES

......................................... At the interface between an animal and its environment are many nerve cells that are specially tuned to receive information; other specialized nerve cells monitor conditions within the body. These cells, whose properties make them particularly suited for gathering information, are called sensory neurons, and they are discussed in Chapter 7. The second major system that contributes to coordinating processes within an animal's body is the endocrine system. The cells of this system are collected into organs called endocrine glands, and their signals are molecules that are released into the blood stream of the animal, a process called secretion. Other glands, the exocrine glands, produce chemicals that are secreted into particular locations. The properties of endocrine and exocrine glands are discussed in Chapter 8, The signaling molecules of endocrine glands, called hormones, can influence widely separate parts of the body simultaneously because they are carried throughout the body in the circulatory system. Hormones act on their target cells through specific receptor molecules, and the effect of a hormone on its targets depends upon both the nature of the receptor molecules and their effect on the internal processes

of the target cell. The mechanisms that control the release of hormones and the mechanisms by which hormones act on their targets is discussed in Chapter 9. The outwardly visible behavior of an animal, as well as much of the activity that goes on inside the body, depends upon contractions of muscle cells. In Chapter 10 we discuss the cellular properties of muscles that allow them to move the body or to change the shape of internal organs. We then turn to how muscular movements can be coordinated to produce effective behavior. Finally, in Chapter 11we consider some examples of how specific behaviors are actually produced. Intensive experimental investigation has elucidated details of how particular behaviors are initiated and shaped, following information from sensory input, through processing in the nervous system, to the production of movements that allow an animal to find food or a mate or to flee a potential predator. One emphasis in Part I1 is on the properties of single cells that allow them to perform their particular tasks and to work together effectively and in harmony. Another emphasis is on the mechanisms that coordinate cellular function into larger levels of organization that enhance an animal's overall fitness.

,

,

:

:, .. . ;. . C H A I ' T E K

,,,"%i'r , , +.,,+ ;

~',U,,+I,,,,~C,!C,, .< '.'YI.," :

, ,4-

, 4 ,:.,

. ,,.*,

,:,:1 ,.,.,, .,i2'9;'4 ,,,,. . , a> R, so A is proportional to k times the square root of r, where kis simply a constant; in other words, the length constant

CONDUCTION VELOCITY The velocity at which an AP is propagated depends in part on the forward distance over which the current arising from the Na+ influx can spread at any instant. This distance depends on the relation between the longitudinal resistance (within the axon) and the transverse resistance (across the axon membrane) encountered by currents flowing in a unit length of axon (equation 6-2). The transverse resistance, R ,,

of a unit length, I,

increases in proportion to the radius of the axon. As the radius increases, h increases. Because the velocity of propagation depends on the rate of depolarization at each point ahead of the AP, membrane capacitance cannot be ignored. Note that the time constant (RmX C ),

of a unit length of axon membrane remains constant

as axon diameter changes, because capacitance (C,)

increases

in direct proportion to surface area, whereas resistance (R,) decreases in proportion to an increase in membrane area. The in-

of axon membrane is inversely proportional to the radius, r, of

creased h that accompanies increased axon diameter therefore

the axon, because the area, As,of a cylinder of unit length is equal to 2rrl. The longitudinal resistance, R,, of a unit length of

occurs without changing the time constant of the membrane. Thus an increase in diameter produces a greater outward rnem-

axoplasm is inversely proportional to the cross-sectional area,

brane current at distance x without an increase in membrane

Ax,of the axon. Because A

r r 2 , resistance R, is inversely pro-

time constant, and the increased rate of depolarization brings

portional to the square ofthe radius. It follows then that, for any

the membrane to threshold sooner at every distance and in-

increase in radius, the drop in R,will be greater than the drop in

creases the conduction velocity.

R.,

=

The length constant A = dR,/(R,

+ R),

(equation 6-2), so

0

B

I Electrode 1f

I

I

I Electrode 27

I

Injured section

Time

myelin layer is very thick. This reduction in C, means that less capacitative current is required to change Vm, so more charge can flow down the axon to depolarize the next segment. The changes in resistance and capacitance greatly increase the length constant, A, of the axonal membrane that is covered by myelin, thus enhancing the efficiency with which longitudinal current spreads. However, this insulation would not have this effect if it completely covered the conducted current would axon, because the electrot~nicall~ eventually decrease to zero as a function of distance. Instead, the length of the myelinated segments are typically about 100 times the external diameter of the axon, ranging from 200 pm to 2 mm long, and the segments are interrupted by short, unmyelinated gaps called nodes of Ranvier, at which about 10 pm of the excitable axon is exposed to the extracellular fluid (Figure 6-8B). The segments of axon that lie under the myelin wrapping are called internodes. In the course of development, myelin is laid down around the axons of peripheral and central tracts in vertebrates by two kinds of glial cells: Schwann cells in periph-

e sthe central nervous sysera1 nerves and ~ l i ~ o d e n d r o c y tin tem. Between nodes of Ranvier, the sheath is so close to the axon membrane that it nearly eliminates the extracellular space surrounding the axon membrane. Moreover, the internodal axon membrane has been found to lack voltagegated Na+ channels. Thus, when a local-circuit current flows in advance of the AP, it exits the axon almost exclusively through the nodes of Ranvier. As noted earlier, very little current is expended in discharging membrane capacitance along the internodes, because of the low capacitance of the thick myelin sheath. An AP that is initiated at one node electrotonicallydepolarizes the membrane at the next node; thus, in myelinated axons, APs do not propagate continuously along the axonal membrane, as they do in nonmyelinated nerve fibers. Instead, APs are produced only in the small areas of the membrane exposed at the nodes of Ranvier. The result is saltatory conduction, a series of discontinuous and regenerative depolarizations that take place only at the nodes of Ranvier, as illustrated in Figure 6-9. The velocity of signal transmission is greatly enhanced because the electrotonic spread of local circuit current occurs

Direction of propagation

-

I

-

1-1

,'

5

\\

I^\

1-1 I \

*

--d * 4

-

4

t -

1-6

+-I\ 4

3

'

*

2

rapidly over internodal segments. The conduction velocity of myelinated fibers varies from a few millimeters per second to more than 100 m . s-l, in contrast with unmyelinated fibers of similar diameter, which conduct at a fraction of a meter per second (see Table 6-1). The evolution of saltatory conduction and the resulting higher speed of AP propagation was probably crucial for the successful coordination of activity in the large muscles of vertebrates. Myelination allows APs to travel rapidly in many axons within a compact nerve trunk. The importance of the specialized insulation provided by myelin for coordinating neuronal information is particularly evident in such demyelinating diseases as multiple sclerosis. In this disease, the myelin sheath is reduced or eliminated along some axons making the velocity of neuronal transmission highly variable among neurons, which severely compromises the control of coordinated movement.

TRANSMISSION OF INFORMATION BETWEEN NEURONS: SYNAPSES All information processing done by neurons depends on the transmission of signals from one neuron to another, which is accomplished at structures called synapses. At electrical synapses, the presynaptic neuron is electrically coupled to the postsynaptic neuron by particular proteins within the membranes. Transmission across electrical synapses proceeds very much like signal transmission along a single axon. However, electrical synapses are relatively rare. Most signaling between neurons takes place at chemical synapses. At a chemical synapse, APs in the presynaptic neuron cause the release of neurotransmitter molecules that diffuse across a narrow space (about 20 nm wide), called the synaptic cleft, that separates the membranes of pre- and postsynaptic neurons. As recently as the 1970s, only a handful of chemicals were known to be synaptic transmitters. All of them were thought to act in a similar fashion, in accord with the results obtained in studies of transmission at the synapses, called neuromuscular junctions (NMJs), between motor neurons and the skeletal muscles that they control. Today, more than 50

4

i

\\

*-,

Figure 6-9 In saltatory conduction, the action ~ o t e n t i ailu m ~ from s node to node. (A) Current spreads longltudlnally between nodes The large red arrows lndlcate Na+ Influx through aalvated Na+ channels that are located at the nodes The smaller red arrow represents the later outfux of Kt through act!vated K+ channels (B) Dots indlcate the value of V , at a single instant at each node shown in part^. ~ t s i t Ie, thernernbrane IS in the falling phase of an AP; at site 2, the membrane is in the rlslng phase At sltes 3,4, and 5, the membrane IS In success~vel~ earher phases of an AP

1

neurotransmitters have been identified in the wide range of animals studied, more are being discovered all the time, and we now know that their modes of action vary greatly. Initially, neurotransmitters were thought to act only by causing the voltage in a postsynaptic cell to change, either by hyperpolarizing the cell or by depolarizing it. Howevel; neurotransmitters can also increase or decrease the number of ion channels inserted into the membrane of the postsynaptic cell, alter the excitability of the postsynaptic cell by changing the rate at which ion channels open and close, or modify their sensitivity to activating signals. The discovery of these varied modes of action has dramatically broadened our understanding of the role that synapses play in neuronal communication. Synaptic transmission was a subject of controversy for a very long time. Early in the twentieth century, the great histologist Santiago Ramon y Cajal used the light microscope and a silver-based staining technique developed by neuroanatomist Camillo Golgi to show that neurons stain as discrete units. In spite of this observation, many anatomists continued to believe that the nervous system was a continuous reticulum, rather than a set of morphologically separate nerve cells. It was not until electron microscopy was developed in the 1940s that unequivocal evidence was obtained supporting the notions that neurons are indeed separate from one another and that particular regions of neurons are specialized for communication between cells. However, in 1897, long before the ultrastructural basis of neuron-neuron interactions was determined, the functional junction between two neurons was given the name synapse (from the Greek, meaning "to clasp") by Sir Charles Sherrington, who is widely regarded as the founder of modern neurophysiology. It was his conclusion that ". . . the neurone itself is visibly a continuum from end to end, but continuity fails to be demonstrable where neurone meets neurone-at the synapse. There a different kind of transmission may occur" (Sherrington, 1906). Although Sherrington had no direct information about the microstructure or microphysiology of these specialized regions of interaction between excitable cells, he had extraordinary insight, the sources of which were his cleverly

designed experiments on the spinal reflexes of animals, most of them mammals. Among other things, he deduced that some synapses are excitatory, increasing the probability that APs will arise in the postsynaptic cell, and that others are inhibitory, reducing the probability of APs in the postsynaptic cell. In this section, we start by considering synaptic transmission across electrical synapses, which is similar to signal conduction along axons. We then turn to the topic of chemical synapses, dealing first with transmission at the neuromuscular junction and then with other, more recently discovered types of chemical synapses.

Synaptic Structure and Function: Electrical Synapses Electrical synapses transfer information between cells by direct ionic coupling. At an electrical synapse, the plasma membranes of the pre- and postsynaptic cells are in close apposition and are coupled by protein structures called gap junctions (Figure 6-10A) through which electrical current can flow directly from one cell into the other (Figure 6-10B; see also Chapter 4).Because current travels across gap junctions, an electrical signal in the presynaptic cell produces a similar, although somewhat attenuated, signal in the postsynaptic cell by simple electrical conduction through the junction (Figure 6-10C).Thus, at an electrical synapse, the transfer of information occurs by purely electrical means, without the intervention of a chemical transmitter, and a key feature of electrical synaptic transmission is its rapidity. As we will soon see, signal transmission across chemical synapses is always slower than purely electrical signal transmission. Electrical transmission can be illustrated experimentally by injecting current into one cell and measuring the effect in a connected cell (see Figure 6-10C). A subthreshold current pulse injected into cell A elicits a transient change in the membrane potential of that cell. If a significant fraction of the current injected into cell A spreads through gap junctions into cell B, it will cause a detectable change in the V,,, of cell B as well. Because there is a potential drop as the current crosses the gap junctions, the potential change recorded across the membrane of cell B will always be less than that recorded in cell A. The gap junctions through which current flows from one cell to another are generally, but not always, symmetrical in resistance to the passage of current-that is, current generally meets the same resistance as it passes in either direction between the two cells. However, at some specialized synapses, the transfer of current between the two coupled cells occurs readily in one direction, but not in the other (Figure 6-10D). Such junctions are said to be rectifying. The transmission of an AP through an electrical synapse is basically no different from propagation within one cell, because both phenomena depend on the passive spread of local circuit current beyond the AP to depolarize and excite the region ahead. Because the safety factor -

-

of an AP (the ratio of the change in V,,, during an AP to the change in Vm required to bring a cell to threshold) is typically about 5, the attenuation of the change in V,,, from one cell to the next must be no greater than the safety factor if the depolarization of the postsynaptic cell is to reach threshold and initiate an impulse. Therefore, a single presynaptic action potential might be unable to provide enough local circuit current across an electrical synapse to elicit an action potential in the postsynaptic cell, which may be one evolutionary reason why electrical synapses are less common than chemical synapses. However, the fact that electrical synapses conduct signals much more rapidly than do chemical synapses gives them definite advantages where rapid signal transmission is important. Electrical transmission between excitable cells was first discovered in 1959 by Edwin J. Furshpan and David D. Potter, who were studying the nervous system of crayfish at the time. They found that an electrical synapse between the crayfish lateral giant nerve fiber and a large motor axon has the unusual property of passing current preferentially in one direction (see Figure 6-10D). Since their early work, electrical transmission has been discovered between cells in the vertebrate central nervous system and in the vertebrate retina, between smooth muscle fibers, between cardiac muscle fibers, between receptor cells, and between axons. The rapidity with which current crosses electrical synapses . makes this means of information transfer particularly effective in the synchronization of electrical activity within a group of cells. It is also effective for rapidly transmitting information across a series of cell-cell junctions-for example, in the giant nerve fibers of the earthworm, which are composed of many segmental axons connected in series along the worm's body, and in the myocardium of the vertebrate heart, in which signals are passed between muscle cells. At some synapses, transmission is both electrical and chemical. Such combined synapses were first identified in cells of the avian ciliary ganglion, and they have also been found in a circuit controlling the fish escape response, in synapses made by some neurons onto spinal interneurons of the lamprey, and in synapses onto frog spinal motor neurons. However, as interesting as they are, combined synapses are unusual phenomena.

Synaptic Structure and Function: Chemical Synapses A common mode of synaptic transmission is known as fast chemical synaptic transmission, which is found at many synapses in the central nervous system and at the neuromuscular junction. (Although this transmission is called "fast," it is in fact considerably slower than transmission across electrical synapses.) At neuromuscular junctions, the neurotransmitter acetylcholine (ACh) is stored in membrane-enclosed vesicles and is secreted by exocytosis into the extracellular fluid separating the neuron and the muscle. The sequence of events at these nerve terminals is

176


......................................................................

A

I

/

Current passes between cells through gap junctions

D

Current source

Pre. 50 mV

\

Current source

Ant~dromic

-

25 mV

Figure 6-10 Pre- and postsynaptic cells are in electrical continuity at electrical synapses, permitting rapid signal transmission between the cells. (A) Electron micrograph of densely packed gap junctions in a membrane. Each "doughnut" in this micrograph is a protein complex that forms a pore, allowing ions and small molecules t o move between the coupled cells. When cells are coupled by gap junctions, the membrane of each cell contains these protein complexes, and the complexes line up with one another, forming continuous channels between the cytosolic compartments of the two cells. (B) Gap junctions connecting pre- and postsynaptic membranes permit ionic currents t o flow between cells. (C) In electrically coupled cells, the injection of current into one cell elicits a potential change in both cells. Usually, the electrical coupling at electrical synapses is symmetric, so the injection of current into either cell changes V, in both cells, although V , changes more in the cell into which current was injected than it does in the cell coupled to it. There are, however, exceptions, such as the one shown in part D. (D) The giant electrical synapse in the crayfish illustrates the relation between pre- and postsynaptic signals in an electrical synapse that has asymmetric electrical coupling. (1eft)An AP in the presynaptic axon is transmitted across the electrical junction, bringing the postsynaptic cell t o threshold and eliciting an AP with only a small delay. This recording is a typical example of signal transmission across an electr~calsynapse. (Right) At this asymmetric electr~calsynapse, however, an AP in the axon that was postsynaptic in the record at the left fails to produce a significant potential change in the neuron that was presynaptic in the record at the left. Injection of current pulses into one cell and then into the other showed that there is preferential flow of current in one direction between these two neurons, an arrangement that is unusual at an electrical synapse. [Part A courtesy o f N. Gilula; part D adapted from Furshpan and Potter, 1959.1

C O M M U N I C A T I O N A L O N G A N D BETWEEN NEURONS

177

...........................................

summarized in Figure 6-11. Briefly, when an AP travels down an axon and spreads to the axon terminals, neurotransmitter molecules that are stored in membranebounded spheres, called synaptic vesicles, within the terminals are released into the synaptic cleft, the fluid-filled space separating the presynaptic and postsynaptic cells. The liberated neurotransmitter molecules bind to specific protein receptor molecules in the postsynaptic membrane, which in Figure 6-11 include ligand-gated ion channels. When neurotransmitter molecules bind to the receptor proteins, the result is a brief ionic current through the membrane of the postsynaptic cell. This mechanism is the basis for synaptic transmission in all animals. The existence of chemical transmission and transmitter substances was the subject of intense scientific debate in the first six decades of the twentieth century. The first direct evidence for a chemical transmitter substance was obtained by Otto Loewi in 1921. In his experiments, he isolated a frog heart with the vagus nerve attached. When he electrically stimulated the vagus nerve, the heart rate slowed down, but he also found that when the stimulated vagus nerve caused the heart to beat more slowly, a substance was released into the surrounding saline solution that could cause a second frog heart to beat more slowly, too. Loewi's finding led to the subsequent discovery that acetylcholine is the transmitter substance released by postganglionic neurons of the parasympathetic nervous system in response to stimulation of the vagus nerve (see Chapter 11)and by motor neurons innervating skeletal muscle in vertebrates.

A Terminal at rest

B AP arr~ves;veslcles

Presynaptic axon

fuse with terminal membrane, producing exocytosis of transmitter

For decades, all synaptic transmission was thought to operate by mechanisms that were very similar to transmission at the neuromuscular junction. However, that view has changed. It is now known that, in addition to fast chemical synapses, most species also have synapses that produce slow chemical synaptic transmission, in which communication between pre- and postsynaptic cells is slower than at the neuromuscular junction and takes place by a different postsynaptic mechanism. In addition, although physiologists believed for decades that each synaptic terminal can contain only a single neurotransmitter, it has recently been discovered that many neurons synthesize and release more than one transmitter substance; in such neurons, one of the substances may produce fast transmission while the other produces slow transmission. In many respects, slow synaptic transmission is similar to rapid chemical transmission (Figure 6-12).The neurotransmitter molecules are packed into vesicles in the presynaptic terminal and are released by exocytosis that is triggered by APs. However, there are significant differences between these two synaptic mechanisms. In slow synaptic transmission, the neurotransmitters are typically synthesized from one or more amino acids and are called biogenic amines, if they contain a single amino acid, or neuropeptides, if they consist of several amino acid residues. As the name implies, the onset of the postsynaptic response is slower (hundreds of milliseconds),and it can last much longer (from seconds to hours). Vesicles used in the fast system are synthesized and packaged within the nerve terminals, whereas vesicles in the slow system are larger and are usually synthesized in the cell body, after

c

Transmitter binds to postsynaptic receptor proteins; ion channels open

D Transrnltter is removed from cleft; fused membrane is recycled

/

Receptor protein

~ostsina~tic membrane

Figure 6-11 In fast chemical synaptic transmission, signals in the preand postsynapticcellsare linked by chemical neurotransmitters. The preand postsynaptic cells are not electrically coupled, and there is no direct flow of current between them. Ionic current flows across the postsynaptic membrane only when ligand-gated ion channels are open in the postsynaptic membrane. (A) At rest, transmitter molecules are packaged into membrane-bounded vesicles contained In the axon terminals. (B) When an AP enters the presynaptic terminal, it causes voltage-gated Ca2+channels in the membrane to open, allowing Ca2+ions to flow into the terminal. The increase in intracellular free Ca2+causes synapticvesicles to fuse with the presynaptic membrane, releasing neurotransmitter into the

synaptic cleft by exocytosis. (C) Neurotransmitter molecules diffuse across the synaptic cleft, driven by their concentration gradient, and bind to receptor proteins in the postsynaptic membrane, opening ligandgated ion channels. In this case, Na' flows through the open channels into the presynaptic cell. The vesicle membrane remains fused with the membrane of the terminal, but it moves to the sides of the terminal. (D) Transmitter molecules are removed from the cleft, the postsynaptic ion channels close, and the membrane that was added to the presynaptic terminal when the synaptic vesicles fused is eventually recycled into the terminal (small arrows) and may be reused for more vesicles.

B

A

Fast chemical transmission

Slow chemical transmission

Large vesicle

Receptor

Figure 6-12 Fast chem~calsynaptic transmlsslon and slow chem~cal synapt~ctransm~sslonact through different postsynaptlcmechan~sms(A) In fast chem~caltransm~ss~on, neurotransm~tters are synthes~zed In the term~nalsand stored In small, clearves~clesThese transmltters are typically small molecules The ves~clesare located near the plasma membrane, and transmltters are released by exocytos~sInto the synapt~ccleft through spec~al~zed sltes on the membrane After they are released, these neurotransmltters act on I~gand-gatedchannels In the postsynapt~cmemthe transmltters are typ~cally brane (B) In slow synaptlc transm~ss~on, larger molecules-for example, pept~desconta~nlngmany ammo ac~ds

These transmitters are stored in large, distinctive vesicles and are released from sites that lack morphological specialization and that are located away from the sites from which the fast neurotransmitters are released. In the postsynaptic cell, these neurotransmitters typically act indirectly through G protein-linked receptors to modify channels and other intracellular processes. Single neurons may produce both kinds of transmission, and a single neurotransmitter may affect postsynaptic neurons both by means of ligand-gated channels and by means of G-protein-coupled receptors.

which they are transported to the nerve terminal. Vesicles that mediate slow synaptic transmission may release their transmitter molecules at many sites in the presynaptic terminal and usually affect the postsynaptic cell, not through ligand-gated channels but by altering the levels of intracellular second messengers through intermediate molecules called G proteins. Physiological and anatomical evidence indicates that single presynaptic neurons may participate in both kinds of neurotransmission. The release of neurotransmitter into the synaptic cleft is controlled by mechanisms that are common to both fast and slow synaptic transmission. When an AP arrives at the axon terminals, it activates voltage-gated Ca2+channels in the membrane of the terminals, allowing Ca2+to enter the terminal (seeFigure 6-11B). The increased concentration of Ca2+inside the terminal initiates the exocytosis of vesicles containing the transmitter substance, dumping neurotransmitter molecules into the synaptic cleft where they diffuse away from the presynaptic terminal. In fast synaptic transmission, neurotransmitter-containing synaptic vesicles fuse with the plasma membrane at specialized sites called active zones. After crossing the synaptic cleft, some neurotransmitter molecules bind to receptor molecules in the postsynaptic membrane. When transmitter molecules bind to these receptor molecules, they modify ionic current through channels that are associated with the receptor molecules, allowing permeant ions to carry a postsynaptic current driven by electrochemical gradients. In slow transmission, the neurotransmitter affects the postsynaptic cell through G-protein intermediates to modify activities of intracellular second messengers that then influence ion channels or other intracellular processes (see Figure 6-12).The

postsynaptic current produced by the neurotransmitter causes a change in the membrane potential of the postsynaptic cell. If the sum of the potential changes caused by many such synaptic events is sufficient to exceed the threshold potential in the postsynaptic cell, an AP will be initiated in the postsynaptic cell. In fact, the currents that are generated in the postsynaptic cell may either increase or decrease the probability that APs will occur in that cell; that is, synaptic effects can be either excitatory or inhibitory. What makes a synaptic signal one or the other is examined later in this chapter.

Fast Chemical Synapses

The most extensive studies of synaptic transmission have been done on fast chemical transmission at the neurbmuscular junctions (also called motor terminals or motor endplates) of vertebrate skeletal muscle, where acetylcholine has been shown to be the neurotransmitter. We will use the neuromuscular junction as our primary example, because it is so well studied. It is a good example because fast chemical synaptic transmission between neurons within the central nervous system closely resembles transmission at the

C O M M U N I C A T I O N ALONG A N D BETWEEN NEURONS

179

.............................................................................. neuromuscular junction, although in many cases the transmitters are different.

Structural features The frog motor endplate (Figure 6-13) includes structural specializations of the presynaptic terminal, of the postsynaptic membrane, and of associated Schwann cells. The axon of the presynaptic motor neuron terminal bifurcates, and the branches, each of which is approximately 2 p m in diameter, lie in a longitudinal depression along the surface of the muscle fiber. The muscle membrane lining the depression is thrown into transverse junctional folds at intervals of 1to 2 pm. Directly above these folds within the nerve terminal are the active zones-transverse regons of slight thickening in the presynaptic membrane above which are clustered many synaptic vesicles. The vesicles are released along the active zones by the process of exocytosis (Figure 6-14).There

are thousands of vesicles, each about 50 nm in diameter, in one presynaptic terminal. For example, the branches of the nerve terminal innervating a single frog muscle fiber typically contain a total of about 10"synaptic vesicles. When the vesicles fuse with the plasma membrane and release transmitter molecules into the synaptic cleft, the transmitter molecules reach the postsynaptic membrane by diffusing down their concentration gradient. The cleft itself is filled with a mucopolysaccharide that "glues" together the pre- and postsynaptic membranes, both of which usually show some degree of thickening at the synapse. The vesicular membrane that fused with the plasma membrane of the terminal is taken up into the terminal and may be recycled (see Figure 6-1lD). When acetylcholine (ACh)is released into the synaptic cleft, it can bind to ACh-specific receptor molecules in the postsynaptic membrane of the endplate, causing ion channels that are selective for Naf and K+ to open briefly.

Mvelinated axons

Nerve terminals

,Synaptic vesicles

,Synaptic cleft

Myelin B

Schwann cell

\

/

fold

Junctional fold

' Figure 6-13 Structural specializations are found in the preand postsynaptic cells of the frog neuromuscular junction. (A) Diagram illustrating the pattern of innervation of frog muscle. Each neuron innervates several muscle fibers. (B) Diagram of the neuromuscularjunction. The nerve terminal lies within a longitudinal depression in the surface of the muscle fiber. The depression contains transverse junctional folds that extend into the muscle fiber. An active zone, which is rich in synaptic vesicles, is located in the neuron over each junctional fold. A Schwann cell covers the terminal. (C) Electron m~crographof the neuromuscularjunction (comparewith part B). The muscle (see cell appears at the bottom and contains striated myofibr~ls Chapter 10).The membrane of the muscle f~beris thrown into numerous junctional folds. The axon terminal is seen in longitudinal section above and contains pale synaptic vesicles grouped in bunches over regionswhere the presynaptic membrane is somewhat thicker than usual, forming the active zones. Denser granules and mitochondria lie above the active zones. The synaptic cleft is filled with an amorphous mucopolysaccharide. [Electron micrograph from McMahan et al., 1972.1

180

PHYSIOLOGICAL PROCESSES

...............................................................................

Figure 6-14 The presynaptic terminal at a neuromuscular junction contains thousands of vesicles. A transverse section in a freeze-etched specimen from the electric organ of the ray Torpedo. Synapticvesicles can be seen in the terminal. Two vesicles (black arrows) had fused with the presynaptic membrane when the t~ssuewas fixed, illustrating the process of exocytosis. Calibration bar = 0.2 p m . [From Nickel and Potter, 1970.1

However, in the synaptic cleft, ACh is subject to hydrolysis by the enzyme acetylcholinesterase (AChE). This enzyme can be detected by histochemical methods and is located in the junctional folds. The removal of neurotransmitter molecules from the synaptic cleft is essential because its effect is to limit the time during which the transmitter can be active. In a cholinergic synapse, hydrolysis of ACh inactivates the transmitter and turns off the synaptic transmission. While many neurotransmitters are inactivated by enzymatic action, others are taken up by the presynaptic terminals, carried by specialized transporter molecules. Synaptic potentials In 1942, Stephen W. Kuffler recorded electrical potentials from single fibers of frog muscle and found depolarizations that were intimately associated with the motor endplate. These depolarizations occurred in response to motor neuron APs and preceded the AP generated in the muscle cell. The potential changes, recorded with extracellular elec-

trodes, were greatest in amplitude at the endplate and gradually became smaller with distance, so they were named endplate potentials (epps),or, more generally, postsynaptic potentials (psps).Kuffler correctly concluded that the arrival of an AP in the presynaptic terminal could cause local depolarization of postsynaptic membrane and thus initiate the propagation of an AP through the muscle. The development of the glass capillary microelectrode in the late 1940s made it possible to record potentials produced within a much smaller tissue volume and, hence, to identify more exactly the source of endplate potentials. Numerous intracellular studies of synaptic transmission at the frog neuromuscular junction, performed largely at the laboratory of Bernard Katz in England, have provided a remarkably complete picture of electrical events at this synapse. Like neurons, muscle fibers have a resting potential across their membranes (see Chapter 10).When a muscle fiber is impaled by a microelectrode at a point several millimeters from the motor endplate, the microelectrode records not only this resting potential, but also all-or-none muscle APs that arise with a delay of several milliseconds after APs arrive in the terminals of the innervating motor axon. Every time the motor axon is stimulated, a muscle AP will be recorded, and the muscle fiber will respond with a twitch. To understand the nature of the nerve-muscle synapse, Katz and others used pharmacological agents to interfere with its biochemical reactions. For example, if the South American poison curare (D-tubocurarine, Spotlight 6-3) is applied to a frog nerve-muscle preparation and the concentration of the curare is increased incrementally, at some particular concentration there is a sudden, allor-none failure of the muscle AP and concomitantly the muscle fails to contract. The APs in the motor axon, however, remain unaffected, as does the ability of the muscle fiber to generate an AP and contract if an electrical stimulus is applied directly to the fiber. Because the presynaptic and postsynaptic APs remain unaffected by the poison, curare must interfere directly with synaptic transmission at the neuromuscular junction. Determining how curare works has been a source of insight into the processes of synaptic transmission. For example, in an experiment designed to reveal how curare affects synaptic transmission, a microelectrode is inserted very close to (i.e., less than 0.1 mm from) the endplate region (Figure6-15), and curare is added to the preparation. What do the following results tell us about the nature of synaptic transmission? As the concentration of curare is gradually increased, the muscle AP is seen to rise from a depolarization that is distinctly slower in time course and lower in amplitude than normal, and the initial slope of the rising phase is not as abrupt as in a normal muscle AP (see Figure 6-15B).This initial slow increase in V,,, is an endplate, or postsynaptic, potential. Raising the concentration of curare further decreases the amplitude of the endplate potentials.


181

...........................................

rnrn

Muscle fiber

0

5

10 T ~ m e(ms)

Figure 6-15 Action potentials in muscle are generated from graded endplate potentials. (A) An all-or-none muscle AP is recorded in the muscle fiber far from the endplate region. (B) Recording closer to the endplate reveals that the AP arises out of an endplate potential. (C) Endplate potentials can be recorded without superimposed APs if the size of the endplate potentials can be decreased to the point that they cannot bring the muscle fiber to threshold. Curare, a drug that blocks receptor channels in the postsynaptic membrane, provides one way to reduce the amplitude of endplate potentials. When a preparation is bathed in saline containing curare, the membranefar away from the endplate (lef?record) remains at its resting potential when the motor neuron fires, while, at the same time, graded endplate potentials are recorded near the endplate.

'

The synaptic potential must reach a minimum level (the threshold potential) to trigger the muscle AP; so, when an increase in the concentration of curare causes the amplitude of the endplate potentials to drop below threshold, there is an abrupt failure of the AP. These results suggest that curare interferes with synaptic transmission by blocking endplate potentials in proportion to its concentration. If the concentration of curare is sufficient to reduce the size of the synaptic potential in the muscle to just below threshold, the AP is eliminated, and the synaptic potential is revealed without a superimposed AP (see Figure 6-15C). If the recording electrode is now reinserted into the muscle fiber a number of times at progressively greater distances from the motor endplate, the amplitude of the measured postsynaptic potential drops approximately exponentially with distance from the endplate (Figure 6-16). In contrast with the AP, which propagates in

Distance from endplate (rnm)

Figure 6-16 The amplitude of an endplate potential decays exponentially with distance from the motor endplate. (A) The endplate potential was recorded with a microelectrode that was sequentially inserted at 0,0.5, I .O, 1.5,2.0,2.5,and 3.0 mm from the endplate in a partly curarized frog muscle fiber. (B)Recordings of endplate potentials at each location. The distance away from the endplate (in millimeters) is given for each recording. (C)A plot of the peak potential of each recording shows that the amplitude of the endplate potential decreases approximatelv exponentially with distance from the endplate. [Adapted from Fatt and Katz, 1951.]

an unattenuated manner because it is regenerative, the synaptic potential spreads passively and thus decays with distance. In experiments like this one, curare enabled physiologists to distinguish among the elements of the synaptic response in vertebrate muscle fibers.

Synaptic currents As described in Chapter 5 , a change in membrane permeability for one or more species of ion (i.e., opening or closing a population of membrane channels that selectively passes those ions) typically shifts the membrane potential toward a new level. This change in V, occurs because when

182


............................................................................... SPOTLIGHT 6-3

texln from the tlger snake also lnhlblts transmitter release, causIng lethal paralysrs.The evolut~onof these toxlns has made them

PHARMACOLOGICAL AGENTS USEFUL IN SYNAPTIC STUDIES

hlgh~yeffectwe for lncapacltatlng a vlctlm (I e , only very small amounts are needed), and they must be handled wlth great caution In the laboratory

Postsynaptic Receptor Toxins

Studies of axonal and synaptic transmission have been greatly aided by the discovery and application of natural toxins-from

Agonists and antagonists for receptor subtypes have contributed importantly to defining the role of these receptors in

animals, plants, or fungi-that selectively interfere with, or partially mimic, certain steps in the process of transmission. Toxins

neuronal processing. y-Aminobutyric acid (GABA), a largely

have been found that interact with ion channels, with receptors, and with enzymes important for nervous system function. Some of the commonly used agents that have been useful in studies of synaptic transmission are described here.

inhibitory neurotransmitter, has been studied extensively with the use of a pair of chemicals, one an agonist of GABA and the other an antagonist. The agonist, muscimol, is derived from the mushroom Amanita muscaria. It specifically acivates GABA, type CI- channels. Bicuculline, produced from the plant Dicentra cucullaria, is a competitive antagonist of the same

Channel Toxins Several toxins are specific for particular types of ion channels.

channel. A huge collection of reagents exists for ACh receptors. Mus-

Tetrodotoxin (TTX) from the puffer fish (Sphoeroidessp.) binds

carine and other agents, including pilocarpine, activate mus-

to a site on voltage-gated Na+ channels and blocks Na+ current

carinic ACh receptors. In vertebrates, muscarinic ACh receptors

across the membrane. Similarly, saxitoxin (STX), derived from di-

are most prevalent in the visceral tissues that are innervated by the cholinergic axons of the parasympathetic system. Atropine

noflagellates, blocks voltage-gated Na+ channels, although by a slightly different mechanism. Potassium ion channels can be blocked by several agents. For example, tetraethylammonium (TEA), a synthetic organic compound, blocks most types of

K+

channels from either inside or outside the membrane, and 4-amino pyridine blocks several types. Calcium ion channels can

(belladonna) is a plant-derived alkaloid that blocks muscarinic synaptic transmission. Nicotine, another plant alkaloid, and certain other agents, such as carbachol, act as agonists of nicotinic ACh receptors. D-Tubocurarineis the active principle of curare, the South American blow-dart poison, made from the plant Chondodendron to-

be blocked by any of several w-conotoxins derived from the piscivorous (fish-eating) cone snail (Conus geographus). The

mentosum. This molecule blocks transmission postsynaptically

various subtypes of this toxin block different classes of Ca2+ channels.

by competing with ACh for the ACh-binding site of the nicotinic receptors. It binds competitively to these sites without opening

Glutamate-gated channel toxins have proved invaluable in

the channels, and it thereby interferes with the generation of a

distinguishing among the variety of channel types. Kainic acid,

postsynaptic current. Similarly, a-Bungarotoxin (a-BuTX) is iso-

from a red alga (Digeneasimplex), is an effective agonist for one

lated from the venom of the krait, a member of the cobra fam-

subtype of glutamate receptors. Quisqualic acid, derived from seeds of the plant Quisqualis indica, is a second potent agonist

ily. This protein molecule binds irreversibly and with very high specificity to nicotinic ACh receptors. With the use of radioac-

that is selective for another subtype. One important antagonist is conatokins, from cone snails, which is a noncompet~tiveantagonist of a third class of glutamate receptors, called NMDAre-

tively labeled a-BuTX, it has been possible to determine the number of ACh receptors present in a membrane as well as to isolate and purify the receptor protein.

ceptors for N-methyl-D-aspartate,which activates them.

Eserine (physostigmine) is an anticholinesterase; that is, it blocks the action of acetylcholinesterase.Use of this alkaloid has

Presynaptic Toxins Several toxins act on presynapticterminals to inhibit transmitter

enabled physiologists to measure the amount of ACh released at a synapse, by preventing the rapid enzymatic destruction of

release. /?-Bungarotoxin, derived from cobra venom, inhibits transmitter release by permeabilizing the nerve terminal. No-

the transmitter molecules. Partial doses accentuate the postsynaptic potential at cholinergic synapses.

channels open they allow a flow of ions that transfers charge from one side of the membrane to the other, which in turn causes the measured transmembrane voltage to change. In chemical synaptic transmission, postsynaptic channels in the membrane open when neurotransmitter binds to receptor proteins, and a synaptic current can then flow through these newly opened postsynaptic channels. (In some cases, postsynaptic channels close, reducing the flow

of ions through the membrane.) The direction and intensity of the synaptic current, which are controlled by the size of the conductance through the open channels and by the electrochemical driving force and charge on the permeant ions, determine the polarity and the amplitude of the postsynaptic potential. Because neurotransmitters activate channels with selective ionic permeabilities, they confer specificity on synaptic signal transmission by allowing only certain ionic


183

............................................................................... species to cross the postsynaptic membrane in response to particular neurotransmitters. The ionic currents that produce postsynaptic potentials can be recorded by voltage-clamping the postsynaptic membrane, thus holding the postsynaptic potential constant (see Spotlight 5-3). In a nerve-muscle preparation, this procedure must be carried out close to the motor endplate (Figure 6-17A). The motor nerve (the presynaptic element) is stimulated while V, of the postsynaptic membrane is voltage-clamped at some predetermined value. The release of transmitter by the presynaptic nerve ending is quickly followed by a synaptic current (Figure 6-17B) that is produced when ions flow down their electrochemical gradients through open channels in the postsynaptic membrane. The ions responsible for carrying the synaptic current at particular synapses have been identified through experiments in which the extracellular concentrations of specific ions were changed and the resulting effect on the synaptic current was measured. Such measurements demonstrated that the depolarizing synaptic current at the vertebrate neu-

A

Stimulating wire

7

-

fiber

Recordlng electrode

- V,,

electrode

Undamped synaptic membrane

B

1,

1

k ...,.Iy n a p t i c current .:=:i

I

50 m s Motor axon stimulated Figure 6-17 Voltage-clamping the postsynaptic membrane allows the synaptic current to be measured. (A) Setup for voltage-clamping the muscle membrane, which holds the postsynaptic potential constant while ionic current flowing across the postsynaptic membrane through channels opened by a neurotransmitteris recorded (see Spotlight 5-3).(6) The upper record shows an endplate potential when the neuron is stimulated and the muscle is not voltage-clamped. The lower record shows a synaptic current when the muscle fiber is voltage-clamped under the same conditions. The synaptic current decays much faster than does the endplate potential.

romuscular junction consists of an influx of Na+ that is partly canceled by a simultaneous, and somewhat smaller; efflux of Kf. At this synapse, both Naf and K+ ions pass through the very same postsynaptic ACh-activated channels, indicating that these channels have a broader ion selectivity than do the highly selective, voltage-gated Na+ and K+ channels that underlie APs (see Figure 5-26). Synaptic currents last a considerably shorter time than d o synaptic potentials (see Figure 6-17B). Acetylcholineactivated channels open only briefly, because the transmitter at the neuromuscular junction is rapidly removed from the cleft by enzymatic destruction, after which the channels close and the synaptic current ceases to flow. A postsynaptic potential lasts longer than the synaptic current because its time course depends on the time constant of the membrane, as well as on the duration of the synaptic current. Reversal potential At every fast chemical synapse; one (or more) species of ions carries current across the postsynaptic membrane, and the change in V, caused by this current determines whether the synapse is excitatory or inhibitory. Measuring the properties of the synaptic current provides an experimenter with clues to the identity of the ions that carry the synaptic current. These measurements are made by injecting current into the postsynaptic cell to set the membrane potential at different values and then observing the sign and amplitude of the postsynaptic potential produced by synaptic inputs (Figure 6-18A and B). The amplitude and sign of the postsynaptic potential depend on the transmembrane voltage and on the species of ion or ions carrying the current. Remember that activation of membrane channels that select for a given ionic species, X, causes V,,, to move closer to the equilibrium potential, Ex, for that ion (see Chapter 5). Consider the experiment that is illustrated in Figure 6-18 for a synapse at which only one ionic species, X, carries the synaptic current. As the membrane potential, V,, is shifted toward the equilibrium potential, Ex, the driving force on X (V, - Ex), will decrease. When V,,, = Ex, no current will flow across the membrane, even though the channels are open, because there is no driving force on the ions. If in the experiment V,,, is set on the other side of Ex,current will once again flow, because V,,, - Ex will again be nonzero, but the sign will have changed, indicating that the driving force is in the opposite direction. As a result, X will flow through the open channels in the direction opposite to that of its previous flow, and the sign of the postsynaptic potential will be opposite to that of its previous value (Figure 6-18B and C). Because the direction of the ionic current and the sign of the postsynaptic potential reverse as V,,, passes through Ex, Ex is called the reversal potential, Ere".When synaptic channels open, the synaptic current causes V,,,to shift toward the Ere"of the current, no matter where V,,,was set experimentally before the synapse was activated. The reversal potential has proved to be a useful property of synaptic currents because it provides a hint about which ions carry the current. In fact, before

184

P H Y S l O L O G l C A L PROCESSES

......................................... Figure 6-18 The synaptic reversal potentlal is measured by changing the membrane potential and recording the postsynaptic potential. (A) Method for determining the reversal potential (E,,) at a synapse. Steady current is injected into the postsynaptic cell with an electrode to set V , at ,, an endplate potential is produced different values. At each value of V by stimulating the presynaptic nerve. The endplate potentials are recorded with a second electrode in the postsynaptic muscle fiber. (B) At , IS set at values that are more negative than the equilibfirst (bottom), V rium potential, Ex, for the ions carrying the synaptic current. When V, is set equal to Ex, no synaptic current flows, and the amplitude of the postsynaptic potential is zero, even though the postsynaptic ion channels are open. When V , is set at values more positive than Ex,the dr~vingforce on the ions carrying the synaptic current is opposite to the direction of the driving force when V , is more negative than Ex.As a result, when V, is more positive than Ex, ionsflowthrough the synaptic channels in the opposite direction from thelr direction when V , is more negative than Ex, and the sign of the endplate potential reverses. (C) The results of this type of experiment can be plotted to show the amplitude of the endplate potentials as a function of the values of Vm The line fitting the experimental points crosses the abscissa at E,". In thls case, E,ev= 0 mV.

Reversal potential -

O

t Nerve s t i m u l a t e d

Time

-

- 100 Membrane

potential (mV)

membrane patch recording was introduced, measuring the reversal potential of a current was the primary method of distinguishing the ionic species that produced a particular postsynaptic potential, although it was-by itself-not conclusive. If a single ion carries the synaptic current, the reversal potential, Ere,, can be calculated by using the Nernst equation for that ionic species (see Nernst Equation in Chap-

ter 5). However, if synaptic channels are permeable to several ionic species, as is the acetylcholine channel, Ere,depends on the concentrations and relative permeabilities of all of the participating ions. If the concentrations and permeabilities of the various ionic species are known, Ere,can be predicted by using the Goldman equation (see Goldman Equation in Chapter S), rather than the Nernst equation. Alternatively, if the current is carried by only two ionic species, Ere, can be calculated from Ohm's law for the two ionic species (Spotlight 6-4). The ACh-activated channels at vertebrate neuromuscular junctions provide an example. When those channels open, they become permeable to both Na+ and Kt. In such a case, the reversal potential, Ere,, of the current will lie between the equilibrium potentials of the two permeant ions (Figure 6-19). In Figure 6-19, V,,, was electronically clamped at several different values and then the synapse was activated. When V, was clamped at ENa (trace a), the driving force on Na+ was zero (V, - ENa= O), but there was a large driving force on K+ (V, - E,). Thus, the synaptic current at ENais carried entirely by an outward flux of K+, which makes V, more negative. In contrast, when Vmis set at E, (trace e), there is no driving force on K+, but there will be a large driving force on Na+. In this case, all of the current through the ACh-activated channel will be carried by an influx of Na+, and Vmwill become more positive. Somewhere between ENa and E,, there must, then, be a value of V, at which the Na+ and K+ currents through this channel will be equal and opposite to one another, so that, although both ions flow through the channel, there will be no net current (trace c). This value of Vm is the reversal potential for the AChactivated current. In the frog endplate channel, the conductances for the two permeant ions, Na+ and K+, are approximately equal. Notice that the synaptic current cannot drive V,,, past Elev,regardless of how many channels become activated. When V, reaches Ere,, the net driving force on the permeating ions drops to zero, and V,,, cannot change further. As a result, E , sets the maximum change


185

............................................................................. K+

A

~n Current out

a

-

Vm= EN,

T~me

c

v m = Ere"

Figure 6-19 The synaptlc current at the vertebrate neuromuscularjunct~onIS carrled by both sod~umand potasslum Ions (A) Sodlum and potasslum currents through activated acetylchollne (ACh) channels at different membrane potentials, beglnnlng at EN, The ACh-act~vated channels are approximately equally permeable to Na' and K+, so the rnagnltudes of ,I and I, depend on the driv~ngforce on each ion The relatlve magn~tudesof the Na' and K+ currents are representedby the lengths of the arrows (B) The arnpl~tudeand tlme course of the net current through the ACh-aa~vatedchannels are shown against tlme At Ere"for the comb~nedcurrents, the net current through the channels IS zero

-t AC h release

in Vmthat can be produced by the activation of synaptic channels (or indeed by the activation of any ion channels). The reversal potential also has a special functional significance at synapses, because the relation between EreVand the threshold for excitation in the postsynaptic cell determines how synaptic events affect the postsynaptic cell. Postsynaptic excitation and inhibition Any synaptic event that increases the probability that an AP will be initiated in the postsynaptic cell is called an excitatory postsynaptic potential (epsp);conversely, any synaptic event that reduces the probability of an AP in the postsynaptic cell is a n inhibitory postsynaptic potential (ipsp). If the reversal po' tential (Ere,)of a synaptic current is more positive than the threshold of the postsynaptic cell, that synapse is excitatory (Figures 6-20A and 6-21A). If Ere"is more negative than threshold, the synapse is inhibitory. At fast chemical synapses, excitatory currents are typically carried through channels that conduct Na+ or Ca2+.These channels may be permeable to K+ as well, as is the ACh channel of the vertebrate neuromuscular junction, but the K+ current itself does not contribute to the excitatory nature of the synapse (see Figure 6-19). Inhibitory synaptic currents are typically carried by channels that are permeable either to K+ or to C1-. The reversal potential, E,, ,for K+ or C 1 typically lies near V,, so it is more negative than the thresh-

old. If E,,, for inhibitory channels is more negative than Vre, in the postsynaptic cell, the synaptic current will cause Vmto become more negative than V, ,hyperpolarizing the cell toward Ere, (see Figure 6-20A). Hyperpolarizing synaptic currents can add to depolarizing synaptic currents, reducing the net amount of depolarization in the postsynaptic cell. Although all excitatory synapses generate depolarizing postsynaptic currents, there are special cases among inhibitory synapses. For example, if Ere"for a synaptic current happens to be identical with Vrest(Vm- Ere" = 0), no net synaptic current will flow even if postsynaptic channels open. The net current will be zero because the driving force on the ion, or ions, that can pass through the channels will be zero. In this case, when the synaptic channels open, Vm will not change. In some cases, Erevis more positive than V,,, but more negative than threshold (Figure 6-21B). In this situation, the postsynaptic potential is depolarizing, but it is, nonetheless, inhibitory because it increases the difficulty of bringing Vmup to threshold. In each of these two special cases, the synapses have an inhibitory action, because activation of these channels can counteract a simultaneous activation of excitatory channels (Figure 6-21C). In effect, the opening of inhibitory postsynaptic channels "short-circuits" excitatory currents, because the positive charge carried into the cell by excitatory currents can leave

186


...............................................................................

'

'1

Figure 6-20 Synaptic currents can be excitatory or inhibitory (A) Transmltter D evokes an excitatory depolarir-

----------

- -

----

- -- - -- - - -

C a, 0

a

-

0

----- ------

- -

-

- -

--- ----

ing postsynaptic potential, because it increases an ionic conductance generating a net inward current that adds positive charge t o the cell's interior For example, transmitter D might increase the permeability t o Na+. Transmitter H produces an inhibitory hyperpolarizing synaptic potential, because ~tIncreases the conductance t o ions that cause a net loss of positive charge from the cell. For example, transmitter H might increase the permeability to K+ or t o CI-. (B) The direction in which positive current flows through channels opened by transmitter D is opposite t o the direction in which ~ o s i t i v ecurrent flows through channels opened by transm~tterH

In

Transmitter D

Transm~tterH

the cell through the inhibitory channels, preventing the positive charges from bringing Vm up to threshold. Note that there is nothing inherently excitatory or inhibitory about any particular transmitter substance. Rathel; the properties of the channels that are opened by the transmitter and the identities of the ions that flow through those channels, determine how a transmitter affects the postsynaptic cell. For example, ACh is an excitatory transmitter at the vertebrate neuromuscular junction, where it opens channels that allow Na+ and K+ to cross the postsynaptic membrane. In contrast, ACh is inhibitory at the terminals of parasympathetic neurons innervating the vertebrate heart, where it affects Kc-selective channels. From this description, it follows that an inhibitory transmitter could be made excitatory if the ionic gradients across the postsynaptic membrane were changed. This experimental manipulation has been accomplished for neurons of the mammalian spinal cord and for neurons of a snail (Figure 6-22). In certain snail neurons, ACh increases g,, of the postsynaptic membrane. In one group of these cells (called H cells, or hyperpolarizing cells), the intracellular C 1 concentration is relatively low, making E,,

more negative than V,, . When ACh is applied to H cells, it opens C1- channels, allowing C 1 to flow into the cell down its electrochemical gradient. The result is to shift Vm toward Ec,, hyperpolarizing the cell (see Figure 6-22A). If all extracellular C 1 is replaced by SO:-, which cannot pass through the chloride channels, application of ACh leads to an efflux of C1-, because it now has an outwardly directed electrochemical gradient. This efflux of negative charge produces both a depolarization and an increase in the frequency of action potentials (see Figure 6-22B). Thus, ACh is normally inhibitory for these cells, but it can produce excitation if the electrochemical gradient for C1- is reversed. In fact, in this species of snail, there are other brain cells (called D cells, or depolarizing cells) that naturally maintain a high intracellular C1- concentration by actively accumulating Clk. Acetylcholine causes an increase in gc, for these cells, as it does in the H cells. However, in the D cells, the net effect is a depolarization, because the electrochemical gradient for C 1 is normally outward. Hence, in this example, excitation and inhibition depend critically on the nature of ionic gradients, and not on the identity of the signaling molecule.

A

n

B

Action potential

-

+ C--

&

Threshold

hE

- - - - - - E r etI,,,,

Threshold

bE

187

COMMUNICATION ALONG A N D BETWEEN NEURONS

..............................................

-------

~nhib

Inhibitory

Rest~ngpotential

Excitatory

- - - - - - - - - - - Ewv, excit

Figure 6-21 Excitatory and inhibitory synaptic signals interact in the postsynapticcell. (A) An action potent~alarises out of an excitatory postsynaptic potential if that postsynaptic potential bringsthe membrane potential V,, above threshold. (B) A postsynaptic potential is inhibitory, even if it depolarizes V,, if its Ere,is more negative than the threshold for impulse generation.(C) An inhibitory transmitter (as in part B) may reduce the depolarizationproduced by an excitatory transmitter (as in part A) sufficiently to keep the postsynaptic potential from reaching threshold.

Threshold

Excitatory and inhibitory

Presynaptic inhibition Experiments performed in the 1960s on neurons of the mammalian spinal cord and on the crustacean neuromuscular junction revealed an additional inhibitory mechanism at some synapses. In this mechanism, called presynaptic inhibition, an inhibitory transmitter is released from a terminal that ends o n the presynaptic terminal of an excitatory axon (Figure 6-23). In this case, the presynaptic terminal of the excitatory axon is itself a postsynaptic element. During presynaptic inhibition, the amount of transmitter released from the excitatory terminal is reduced, which reduces synaptic excitation in the cell that is postsynaptic to the excitatory neuron (see Figure 6-23B). In some cases, the presynaptic inhibitory transmitter increases g, or g,, in the presynaptic terminals of the excitatory axon, which reduces the amplitude of any AP invading the excitatory terminal and hence diminishes the amount of

transmitter released from the terminal. In other examples of presynaptic inhibition, the inhibitory transmitter modifies some property of Ca2+channels in the presynaptic membrane, rendering them less responsive to de~olarization.Because the release of transmitter molecules depends on Ca2+ entry into the terminal (seethe next section of this chapter), reducing Ca2+entry reduces transmitter release. Regardless of the mechanism, the net effect of presynaptic inhibition is that the postsynaptic cell receives less transmitter and thus a smaller postsynaptic potential is generated. Postsynaptic and presynaptic inhibition produce quite different consequences for the postsynaptic cell. Postsynaptic inhibition globally reduces the excitability of the postsynaptic cell, making it is less able to respond to all excitatory inputs. In contrast, presynaptic inhibition acts only on specific inputs to the cell, allowing the cell to remain

In normal Rlnger

C I Rlnger

M

5s

B

SO,?- Ringer

Then change t o chloride-free Ringer

f

ACh ( l o k 5 M)

Figure 6-22 Experimentally changing ionic gradients across the membrane of a postsynapt~ccell can change the sign of a synapse (A)Acetylchol~ne(ACh) appl~edto H-type cells In the snail brain activates CIk channels, produc~nga hyperpolar~zat~on because CIbr~ngsnegatlve charges Into the cell as ~tmoves down its electrochem~calgrad~ent(B) When the extracellular CI ions are entirely replaced by SO:-, leaving CI-

ins~dethe cell, the electrochem~calgradient for CI- IS reversed Revers~ngthe electrochem~calgrad~ent causes the direction of the synaptic current to reverse. As a result, the postsynaptic potential becomes depolarizing, and the synapse becomes excitatory. Electrical activ~tyof the cell before, during, and after synaptlc actlvatlon IS shown at the r~ght[Adapted from Kerkut and Thomas, 1964 1

From this equation, if g, is greater than gNa, then V , must b e

SPOTLIGHT 6-4

, than t o EN,, and vice versa. Solving equation 4 for closer t o E

CALCULATION OF

V,,, = Ere"gives

REVERSAL POTENTIAL The value of the reversal potential of an ionic current elicited by

From equation 5, it is apparent that Ere,will not b e simply the al-

a stimulus or a neurotransmitter depends on the relative con-

gebraic sum of EN, and E, but will lie somewhere between the

ductances of the ions carrying the current as well as on their

,, two, depending on the ratio gN,lgK.Thus, if g

equilibrium potentials. If we assume that only Na+ and K+ carry current in response t o the stimulus, the reversal potential can b e related t o the conductances of these ions by using equation 5-10, with the values g, and gNa representing the respective tran-

and g, become

equal t o each other (e.g., as they may when endplate channels are activated by ACh in frog muscle), the membrane potential will shift toward a reversal potential that lies exactly halfway between g,=, and g,:

sient changes in the two conductances.

1,

=

INa =

g, X (Vm- EK)

(1)

9 a,

(2)

X

(Vm- ENa)

At the reversal potentlal, I, and INa must b e equal and oppos~te regardless of the relat~veconductances, because the net current must b e zero. Thus, when V ,

IS

at the reversal potent~al,Ere,,

(3)

-IK= lNa

Substltut~ngfrom equat~ons1 and 2, atthe reversal potentlal we

For frog muscle, E, is about -100 mV, and EN,is about +60 mV. Hence, we would predict that during synaptic activation of a frog muscle, Ere, = (-100

+ 60) = -20

mV The measured rever-

sal potential of the current at the frog neuromuscular synapse,

- 10 mV, is somewhat more positive than this value, possibly beis actually somewhat greater than g,.

cause g ,

To summarize, the reversal potentials of membrane currents differ according t o the species of ions that participate, the equilibrium potentials of those ions, and the relative conductances

have

t o each of the ionic species that participates in the current.

-gKWm -

EK) =

gNa(Vm - ENa)

(4)

normally responsive to other inputs. Thus presynaptic inhibition provides a mechanism for narrowly targeted and subtle control of synaptic efficacy (the effectiveness of a

presynaptic impulse in producing a postsynaptic potential change) among the many synaptic connections onto a particular neuron.

Inhibitory terminal

Muscle Figure 6-23 Neurons that produce inhibition at the crustacean neuromuscularjunction also inhibit excitatory motor neurons presynaptically. (A) The morphological arrangement of excitatory and inhibitoryterminals, showing the location of an inhib~torysynapse that produces presynaptic inhibition and the arrangement for the experiment illustrated in part B. (B) lntracellular recordingfrom the muscle fiber innervated by excitatory and inhibitory motor neurons. (1) Stimulation of the excitatory axon (labeled E on record) produced a 2 mV excitatory postsynaptic po-

-t20 rns tential (epsp).(2) Stimulation of the inhibitoryaxon(labeled I on record) produced a depolarizing inhibitory postsynapticpotential (ipsp)of about 0.2 mL! (3) If the inhibitory neuron was stimulated afew millisecondsafter the excitatory neuron, the excitatory postsynaptic potential was unaffected. (4) However, if the ~nhibitoryneuron was stimulated a few milliseconds before the excitatory neuron, the excitatory postsynaptic potential was almost abolished. [Adapted from Dudel and Kuffler, 1961.1

PRESYNAPTIC RELEASE OF NEUROTRANSMllTERS The properties

presynaptic terminals determine the effectiveness of synaptic transmission, because the number of transmitter molecules affects the size the postsynaptic Understanding transmitter release is thus of central importance for understanding synaptic transmission and its norma1 role in neuronal communication. Besides its importance for physiology, the history of experimentation on transmitter release provides classic examples of the scientific method and experimental strategies. A particularly striking example is the demonstration, by Sir Bernard Katz and his co-workers, that neurotransmitters are generally released in tiny packets called quanta. More recent experiments have shown that synaptic release is closely related to other forms of exocytosis used by cells, such as glandular cells, to release chemicals (see Chapter 9). The conservation of this mechanism has facilitated experiments designed to understand the details of exocytosis in all cells. Quantal Release of Neurotransmitters

In their investigation of neuromuscular transmission, Paul Fatt and Bernard Katz (1952)discovered that spontaneous "miniature" depolarizations ( a

Indirect Neurotransmission

The biogenic amines constitute an important class of neurotransmitters (Figure 6-33) that act through second messengers to produce slow synaptic transmission. This class of neurotransmitters includes: Epinephrine, norepinephrine, and dopamine, classified as catecholamines on the basis of their chemical structure Serotonin (5-hydroxytryptamine,or 5-HT), an indolamine Histamine, an imidazole These substances can be detected visually in individual neurons because they fluoresce in ultraviolet light after the tissue has been fixed with formaldehyde. They act as neurotransmitters in some invertebrate neurons and in the central and autonomic nervous systems of vertebrates (see Table 6-2). Norepinephrine (also known as noradrenaline) is the primary excitatory transmitter released by postganglionic cells of the vertebrate sympathetic system (see Chapter 11). It is also released by the chromaffin cells of the vertebrate adrenal medulla (see Chapter 8). The chromaffin cells are derived embryologically from postganglionic neurons, and they secrete epinephrine (adrenaline) as well as norepinephrine. Epinephrine and norepinephrine are structurally very similar (see Figure 6-33), and they have similar pharmacological actions. Neurons that use epinephrine or norepinephrine as transmitters are adrenergic neurons. At some synapses, epinephrine is excitatory; at others, it is inhibitory. Its effect depends on the properties of the postsynaptic membrane. Norepinephrine is synthesized from the amino acid phenylalanine ( ~6-34A), i and ~it is inactivated ~ ~ in sev~ of the preinto the ways' It is taken synaptic neuron, where some of it is repackaged into SYnaPtic vesicles for rerelease and some of it is inactivated by

196


...............................................................................

Epinephrine (Adrenaline)

Catecholamines

Norepinephrine (Noradrenaline)

Dopamine

OCH, Mescaline

Seratonin (5-Hydroxytryptarnine)

Histamine

Figure 6-33 Several neurotransmitters are monoamines. These transrnitters are each synthesized from single amino acid molecules and are classified on the basis of their molecular structure. Epinephrine, norepinephrine, and dopamine constitute one group, the catecholamines. Mescaline, a halucinogenic drug, has structural features in common with the catecholamines and appears t o produce its effects by interacting with catecholamine receptors in the central nervous system Serotonin (5-hydroxytryptamine)is an indoamlne, and histamine is an ~midazole. These transmitters are found in the nervous systems of vertebrates, as well as many invertebrates.

monoamine oxidase. In addition, it is deactivated by methylation within the synaptic cleft (Figure 6-34B). Several psychoactive drugs have molecular structures that are similar to the biogenic amines, which allows them to act at synapses that use these transmitters. For example, mesca-

line (see Figure 6-33), a psychoactive drug that is extracted from the peyote cactus, induces hallucinations, apparently by interfering with its analog norepinephrine at synapses in the central nervous system. Both amphetamines and cocaine exert their effects by interacting with adrenergic neurotransmission-amphetamines by mimicking norepinephrine and cocaine by interfering with the inactivation of norepinephrine. In addition to the relatively small, "classic" transmitter molecules, there is a growing list (now more than 40) of peptide molecules that are produced and released in the vertebrate central nervous system. Many of these molecules, or very similar analogs, have also been found in the nervous systems of invertebrates. Some of these peptides act as transmitters; others act as modulators that ~nfluencesynaptic transmission. Interestingly, a number of these neuropeptides are produced in many tissues, not just in neurons. Thus, a single molecular species may be released from intestinal endocrine cells, from autonomic neurons, from various sensory neurons, and in various parts of the central nervous system. In fact, some neuropeptides were initially discovered in visceral tissues and were only later identified in neurons. The gastrointestinal hormones glucagon, gastrin, and cholecystokinin (see Chapter 15)are prime examples. It is not yet clear how many peptide neurotransmitters there are. We know that some neuropeptides act in a neurosecretory fashion; that is, they are liberated into the circulation and are carried by the blood to their targets, rather than being released into the confined space of a synaptic cleft. The pituitary hormone-releasing factors of the hypothalamus operate by neurosecretion (see Chapter 9). There is evidence that a single neuropeptide species may be liberated as a transmitter from some neurons, as a neurosecretory substance from other neurons, and as a hormone from nonneuronal tissue. This multiplicity of function is not really all that novel. It has long been known that norepinephrine (as well as its close relative, epinephrine) acts as a hormone when liberated by the adrenal medulla and as a transmitter when released at synapses. Recently, however, it has become clear-much to the surprise of neurophysiologists-that a neuropeptide can be released as a cotransmitter from nerve terminals that also release a more familiar transmitter such as ACh, serotonin, or norepinephrine. Several combinations of a classic transmitter and a paired cotransmitter have been identified in the mammalian brain (Table 6-3). The first neuropeptide was discovered in 1931 by U. S. von Euler and John H. Gaddum while they were assaying for ACh in extracts of rabbit brain and intestine. The extracts stimulated contraction of the isolated intestine, much as ACh does, but the resulting contractions were not blocked by ACh antagonists. This observation led von Euler and Gaddum to discover that the contraction was produced in response to a polypeptide, which the researchers named substance I? Since then, substance P and a growing list of other neuropeptides have been found in various parts of the central, peripheral, and autonomic ner-


197

.............................................................................

Phenylalanine

OH Tyrosine

neuron Circulation

NH,

OH 3,4-Dihydroxyphenylalanine(dopa)

Figure 6-34 Epinephrine is synthesized from phenylalanine, with dopamine and norepinephrine as intermediates,and is inactivatedby reuptake or by methylation. (A) Biosynthetic pathway leading to epinephrine. Each of the lowerthree molecules is used as a neurotransmitter by some neurons. (B) Norepinephrine is synthesized from the amino acid phenylalanine, through conversion into tyrosine, and stored in synapticvesicles. After it is released into the synaptic cleft, some norepinephrine is taken back up Into the presynapticterminal, and some is deact~vatedby methylat~onand carried away In the blood Cytoplasmic noreplnephrine is either repackaged Into synaptlc ves~clesor degraded by monoamine oxidase (MAO) [Part A adapted from E~duson,1974, part B adapted from Mountcastle and Baldessar~n~, 1968 ]

OH 3,4-Dihydroxyphenylethylarnine (dopamine)

OH Norepinephrine

OH Epinephrine

vous systems of vertebrates and in many invertebrate nervous systems. To explore the localization of these molecules, investigators have typically used immunological labeling with fluorescent antibodies that recognize specific neuropeptides. In histological sections, this labeling can be detected with a fluorescence microscope, and it reveals the distribution of specific peptides in the nervous sys-

tem. Some well known neuropeptides are antidiuretic hormone (see Chapter 14), the hypothalamic-releasing hormones (see Chapter 9), and various gastric hormones (see Chapter 15). Unlike small neurotransmitters, which may be synthesized in the synaptic terminals, neuropeptides are made in the cell body and are transported along the axons to the terminals. Neuropeptides are typically synthesized as a part of larger proteins, called propeptides, which may contain the sequences for many biologically active molecules. Specific enzymes cleave the propeptide into individual peptide molecules. This method of production can limit the amount of peptide neurotransmitter available at a synapse compared with a neurotransmitter synthesized on site. Peptides are, however, more potent than small neurotransmittersfor three reasons. First, they bind to receptors at much lower concenM vertrations than do other neurotransmitters (about sus lo-' M for typical neurotransmitters), so very small amounts of neuroieptide can be effective. Second, they act through intracellular pathways that can provide significant amplification. Thus, even a small amount can produce a large effect. Third, the mechanisms that terminate their actions are slower than are those for other neurotransmitters, so they remain available to their receptors for a longer time. Recent research has focused on two groups of naturally occurring neuropeptides, known as endorphins and enkephalins, that reduce the perception of pain and induce euphoria, much as exogenous opiates such as opium and heroin do. The levels of endorphin and enkephalin

198


.........................................

TABLE 6-3 Examples o f small and large neurotransmitter molecules that have been found together in neurons Small neurotransmitter Acetylcholine

Peptide in same neuron CGRP Enkephalln Galanin GnRH Neurotens~n

Somatostatin Substance P VIP Dopamine

CCK Enke~halin

Epinephrine

Enkephalin Neuropeptide Y Neurotensin Substance P

Norepinephrine

Enkephalln Neuropeptlde Y Neurotens~n Somatostat~n

Vasopressin y-Aminobutyric acid

CCK Enkephalin Somatostatin Neuropeptide Y Substance P VIP

Most of these data are based on immunocytochemistry,and the precise chemical nature of the immunoreactive peptide has not been determined. Abbreviations: CCK, cholecystokinin; CGRP, calcitonln generelated peptide; GnRH, gonadotropin-releasing hormone; VIP, vasoactive intestinal peptide. Source: Adapted from Hall, 1992

molecules have been found to rise in the brain in resDonse to eating, to listening to pleasant music, and to other activities generally pcrccived as pleasurable. Bccause of their properties and beca~~se thcse ncuropeptides bind to the same rcccptors in the nervous s!.stem to which opiates such as o p ~ u m~ n its d deri~ati\.esbind, they are callcd endogenous opioids. Until these endogenous neuropeptides wcre discovered, it was very difficult to understand how alkaloids derived from plants-such as opium, morphine, and heroin-could so powerfully affect the nervous systems of animals. We now know that the surface membranes of many central neurons contain opioid receptors, and this class of receptors normally binds the enkephalins and endorphins that are produced within the central nervous system. Only secondarily, and perhaps coincidentally, do they bind exogenous opioids. However, when opioid molecules bind to the receptors, they elicit such intense feelings of pleasure that people learned to use opiate narcotics to stimulate the receptors. There is, however, a physiological problem associated with this intense pleasure: repeated doses of the exogenous opiates provoke compensatory changes in neuronal metabolism, such that removal of the opiate shifts the nervous system into a state

that is perceived as extreme discomfort until the opiate is readministered. This metabolically induced dependence is termed addiction. The drug naloxone, which acts as a competitive blocker of the opioid receptor, has proved to be a useful tool in studies of opioid receptors. Because naloxone interferes with the ability of either opiates or the opioid peptides to act on their target cells, it has allowed investigators to determine whether a response is mediated by opioid receptors. For example, naloxone has been found to block the analgesic effect that can be produced by a placebo (inert substance given to patients with the suggestion that it will relieve pain). Apparently the very fact that a subject believes a medication or other treatment will relieve pain can induce the release of endogenous opioid peptides, and this observation may have revealed the phy'siological basis for the well known "placebo effect" (i.e., almost anything that you do to research subjects will produce whatever effect that you promise, at least in some of the subjects.) Similarly, naloxone renders acupuncture ineffective in relieving pain, which has led to the suggestion that the stimulation of acupuncture causes the release of natural opioid peptides within the central nervous system. There is some indication that the analgesic properties of the endogenous opioids may depend on the ability of these neuropeptides to block the release of transmitter from certain nerve endings. For example, the sensation of pain may be diminished if neuropeptides interfere with synaptic transmission along afferent pathways that carry information about noxious stimuli. Indeed, enkephalins and endorphins have been found in the dorsal horn of the vertebrate spinal cord, part of the pathway carrying sensory input within the spinal cord.

M o s t vertebrate a n d invertebrate species have b o t h fast a n d slow neurotransmission. What kinds of information processing would b e served best b y fast neurotransmission? W h a t kinds b y slow neurotransmission?

POSTSYNAPTIC MECHANISMS Neurotransmitter molecules act through specific protein receptors in the membranes of postsynaptic cells. The properties of the postsynaptic molecules thus form a crucial link in the chain of events that begin when an action potential arrives at the terminal of a presynaptic neuron and end when the response of the postsynaptic neuron is complete. In this section, we will consider in detail the receptor molecules that mediate the two major classes-fast and slow-of chemical synaptic transmission and the events that take place after a neurotransmitter molecule has bound to these receptors.


199

............................................................................... Receptors and Channels in Fast, Direct Neurotransmission

As we have seen, chemical transmitters act by directly changing the permeability of the postsynaptic membrane to certain ions. (Typically the permeability increases.) This interaction requires two major events:

1. Transmitter molecules must bind to receptor molecules in the postsynaptic membrane. 2. When the transmitter molecules bind to the receptors, closed ion channels must open (or, more rarely, open channels must close) transiently. The receptor site may be located in the same molecular complex that forms the channel or it may be on a molecule that is separate from those that make up the channel. When a synaptic channel opens, a minute ionic current passes through the open channel. Many such single-channel currents normally sum to form the macroscopic synaptic current that produces postsynaptic potentials in response to the release of tens-or even hundreds-of thousands of transmitter molecules from the presynaptic terminal. Most of what we know about these events has been revealed in studies of the ACh-activated channels at the vertebrate neuromuscular junction. The acetylcholine receptor channel The number of postsynaptic channel protein molecules is very small relative to other proteins in a membrane; as a result, the isolation, identification, and characterization of these important proteins was difficult. In early studies, physiologists used a variety of pharmacological agents to distinguish among receptor types, creating a pharmacological taxonomy of receptor types. As a result, various ion channels were named for substances that could modify the activity of the channels. For example, there are two types of acetylcholine receptors. Nicotine, an alkaloid produced by some plants, mimics the action of ACh on the channels found at the vertebrate neuromuscular junction, so these ACh receptors (AChRs) are called nicotinic AChRs. Muscarine, a toxin isolated from certain mushrooms, activates the other type of AChR, which is found in the target cells of parasympathetic neurons in the vertebrate autonomic nervous system. These AChRs are called muscarinic AChRs. Our understanding of nicotinic AChRs was given a huge boost when it was discovered that specialized organs of certain elasmobranch and teleost fishes contain extremely high densities of these receptors. The receptors are found on one side of the elearoplax organ, which consists of many flattened cells that originate from embryonic muscle tissue and produce the very high intensity electrical discharges used by these species to stun prey and to send navigational signals. The unusually high density of nicotinic AChRs in electroplax tissues allowed the nicotinic AChR to be the first ligand-gated channel to be purified chemically and studied electrically. More recently, its molecular struc-

ture has been resolved; we even have images of the form of the receptor channel as it opens. A second important aid to the analysis of the AChR is its sensitivity to a-Bungarotoxin (aBuTX; see Spotlight 6-3),a component of cobra venom that binds irreversibly and with high specificity to nicotinic AChRs. a-Bungarotoxin can be isotopically labeled and used to tag AChR molecules, facilitating chemical isolation and purification. Physiological and biochemical studies have shown that the AChR and the postsynaptic channel that is activated by ACh are identical: the receptor site to which ACh molecules bind is an integral part of the channel protein complex. Each nicotinic AChR consists of five homologous subunits that associate and form a channel at the center of the complex (Figure 6-35). There are two identical a subunits plus one each of three different subunits termed P, y, and 6. Each subunit is a glycoprotein with a molecular mass of approximately 55 kD,giving the entire complex a total molecular mass of about 275 kD. This molecular weight agrees well with the size of channel structures that have been seen, using the electron microscope, to penetrate the surface membrane. The channels protrude from both sides of the membrane, with the funnel-shaped opening bulging outward from the cell surface. Acetylcholine binds to the AChR where the receptor molecule extends into the extracellular space. This location was first deduced because ACh injected into a muscle cell near the endplate produced no electrical effect. Since then, experiments have shown that there are receptor sites on each of the two a-subunits. When both sites are occupied by ligand molecules (i.e., ACh or other agonists, such as carbachol or nicotine, that activate the channel), the channel shows a high probability of shifting from a closed to an open state. The nature of this gating process has been studied most extensively at the neuromuscular junction of frog skeletal muscle. As described earlier, the postsynaptic ion channels of the frog neuromuscular junction become permeable to both K+ and Na+ when they are activated by ACh. The increased permeability permits the flow of an inward current with a reversal potential of about - 10 mV. Normally, these channels and the associated AChRs are confined to the postsynaptic membrane in the endplate region. The density of ACh-activated channels in the postsynaptic membrane of the frog endplate is about lo4per square micrometer. Although this high density of channels proved useful for analyzing the summed activity of many ACh channels, for a long time little was understood about the activity of individual channels. Analysis of single channels was made possible by the invention of patch-clamp recording by Erwin Neher and Bert Sakmann (1976; see Figure 5-24), for which they were awarded a Nobel Prize in 1992. Their work on single AChR channels depended both on the development of the patch-clamp technique (see Chapter 2 and Figure 5-24) and on finding a region of muscle that had a sufficiently sparse distribution of AChR channels that they could isolate and record from a single channel. They

200


....................................................

Neurotransrnitter-

6 nrn

3 nrn

2 nrn

-

Figure 6-35 Nicotinic acetylcholine receptors at the neuromuscular junction consist of five protein subunits associated t o form a transmembrane channel. (A) The channel is inserted through the lipid bilayer, protruding into the extracellular space and into the cytoplasm. The CY subunits contain the sites t o which acetylcholine molecules bind t o activate the channel. The entry t o the channel from outside the cell is a broad funnel that becomes narrower and bears a net negative charge toward the cytoplasm, forming the selectivity filter-the region of the pore that controls which ions will permeate readily. In this diagram, the inside of the channel is darker in color than the surrounding region. The subunit facing you is a ysubunit. (B)Top view showing the five subunits associated t o form the channel. These structural features have been inferred on the basis of electron microscopy and X-ray diffraction analysis. [Adapted from Unwin, 1993.1

2 nrn

generated this sparse distribution by taking advantage of the changes that occur in frog skeletal muscle after the motor nerve controlling the muscle has been cut. When a muscle is denervated (i.e., it loses its neuronal input-experimentally the axons are crushed), the region of the membrane that responds to ACh gradually spreads across the surface. Initially, only the membrane at the endplate region can respond, but eventually most or all of the membrane contains AChRs and can respond to ACh. (The normal suppression of these extrajunctional AChRs is thought to be dependent on two factors: first, trophic action from the motor neuron that innervates each muscle fiber and, second, electrical and contractile activity that takes place in an innervated muscle fiber. If the motor axon is allowed to reinnervate the muscle, the extrajunctional receptors disappear, and sensitivity to ACh is again confined to the endplate.) The broad, but sparse, distribution of extrajunctional ACh-activated channels that develops in denervated frog muscle was exploited by Neher and Sakmann to explore the gating of the channel, using their newly developed patch-clamp method. The muscle membrane was voltageclamped (see Spotlight 5-3) at a hyperpolarized potential to increase the driving force for inward current. They used a

micropipette that had a smoothly polished tip with a tip diameter of 10 pm, which they filled with Ringer solution containing a low concentration of ACh or one of its agonists. They then brought the pipette up to the surface of the muscle fiber, exposing any AChRs under the pipette tip to the ACh. The pipette was connected to a highly sensitive, low-noise amplifier (Figure 6-36A) with which they could record currents flowing in the extracellular pipette. When it was applied snugly to the surface of the denervated muscle fiber, the pipette detected minute (less than 5 x 10-l2 A) and transient inward currents (Figure 6-36B) produced by the transient opening of the ACh-activated channels. With this experiment, Neher and Sakmann produced the first recordings ever made of currents through single ion channels in a biological membrane. Indeed, this effort produced the first direct evidence that ionic currents cross the membrane through discrete, gated channels rather than by some other means, such as carrier molecules. Single-channelcurrents such as those first recorded by Neher and Sakmann in 1976 are more or less rectangular in shape; they turn on abruptly and then turn off abruptly, and they are all-or-none.This observation strongly suggests that the channels can exist only in one of two states, completely shut or completely open. Moreover, the unitary cur-

C O M MU N l C A T l O N A L O N G AND BETWEEN NEURONS

201

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

r-c-

Pipette current

I

Voltage c l a m p

Denervated muscle cell

Figure 6-36 The patch recording technique reveals ionic currents through single acetylcholine receptor channels. (A) The muscle mernbrane is held at a hyperpolarized potential (-120mV) by a voltage-clamp circuit, greatly increasing the driving force on ions through acetylcholine receptor (AChR) channels, while the surface ofthe muscle is explored with a patch pipette filled with Ringer solution containing 2 X lo-' M suberylcholine (an ACh agonist). (B) When the pipette tip is sealed tightly against the membrane, brief, minute, inward currents are recorded. In this experiment, the pipette records current flow through the ion channel of a single AChR protein complex that opens transiently when agonist molecules are bound t o receptor sites. [Adapted from Neher and Sakmann, 1976.1

rents recorded from each nicotinic ACh-activated channel are about the same size as the currents recorded from all other nicotinic ACh-activated channels, provided that the electrochemical driving force is kept constant. Ohm's law indicates that this result must mean that all individual nicotinic ACh channels have similar conductances. When two or more channels in the patch being recorded open with overlapping times, the individual, unitary single-channel currents sum linearly, producing a current two (or three, etc.) times as large as the individual unitary current. These currents are not present unless the pipette contains ACh or an agonist, and the frequency of their occurrence depends on the concentration of the transmitter or agonist in the pipette. From Ohm's law, the conductance of a single open nicotinic AChR channel was calculated to be about 2 x 10-l1 S, which is usually expressed as 20 picosiemens (20 X 10-l2 S; i.e., the channels have a resistance of 5 x lolo a). Since the pioneering patch-clamp experiments of Neher and Sakmann, many ligand-gated postsynaptic ion channels have been studied intensively with this method of recording single-channel currents. Statistical analyses of

these unitary currents indicate that the channels can fluctuate between several closed states and at least one open state. Binding of an agonist molecule to the receptor sites of the closed channel greatly increases the probability that the channel will change to an open state and briefly allow ions to flow through the channel. The channel remains open for only about 1 ms and then closes, even though ACh is still bound to the receptor sites. After a short time, the agonist molecules leave the binding sites, and the channel remains closed until more molecules of ACh bind (Figure 6-37).The macroscopic currents and postsynaptic potentials recorded at a synapse represent the sum of many such single-channel events in the postsynaptic membrane.

Other ligand-gated channels Since the ACh channel proteins were purified from electroplax organs, several types of ligand-gated channels have been isolated from neurons and characterized, including the glycine, GABA,, and neuronal ACh receptors, all of which mediate rapid postsynaptic responses. These receptors have a common pentameric protein structure, and each is composed of two to four different kinds of subunits. As in the muscle ACh channel, only one type of subunit binds the ligand. The remarkable homologies among these different channel proteins have allowed the diversity of subunit types and their distribution in nervous tissue to be characterized at the molecular level. Somewhat surprisingly, for each type of receptor-ACh, glycine, and GABA,-a number of different subunits are found to be assembled in different combinations to produce receptors with slightly different properties. Moreovel; each type of receptor is expressed in a unique and characteristic pattern within the mammalian brain, indicating that expression of receptor subtypes is regulated differently in different regions of the nervous system. Recognizing the large number of permutations that are possible, even within receptors that respond to a single neurotransmitter, has helped us to understand how subtle the mechanisms that allow the brain to achieve its highly differentiated functional states can be. Furthermore, a comparison of the DNA sequences of the receptors for ACh, GABA, and glycine reveals them to be closely related, suggesting that all ligand-gated ion channels may have a common ancestral origin. DNA sequence analysis has revealed that glutamate receptors belong to a separate family having only a slight resemblance to the nicotinic receptors. Currently, there is intense interest in this receptor family, both because glutamate is the most common excitatory neurotransmitter in the mammalian central nervous system and because glutamate receptors participate in modifications of synaptic strength, which may underlie learning and memory. At present, three types of fast-acting glutamate receptors have been identhed and are named for their sensitivity to specific agonists. The agonists that typify the three receptor classes are kainate, quisqualate (a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid), and NMDA (N-methyl-Daspartate). These receptor types are considered further later

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X

Figure 6-37 The nicotinic acetylcholine receptor channel exists In three functional states. The ion channel through the receptor opens when acetylcholine (ACh) or agonist molecules bind to the protein complex. After about 1 ms, the ion channel closes, even though the ACh molecules are still bound. The channel can "flicker" between closed and open states while ACh molecules remain bound. Then the ACh molecules unbind, the channel closes, and it remalns in the closed state until two more ACh molecules bind.

, Closed ## b4

Ion channel

/-

Ion channel

+

4----

Open

in this chapter in the section concerning mechanisms of synaptic modification (see Long-term potentiation). Receptors in Slow, Indirect Neurotransmission

A large family of receptors responds to the family of slow neurotransmitters. Interestingly, these receptors have many features in common with receptors that respond to light, to odor, to hormones, and to other extracellular messengers. Most such receptors act by activating members of a group of proteins, known as G proteins, that are associated with the cell membrane and that bind guanosine triphosphate (GTP). A G protein consists of three subunits, called a, P, and y. The G protein-transmembrane signaling pathway was discovered and described by Alfred Gilman and Martin Rodbell, who studied its role in transduction of nonsteroid hormone signaling (for a more complete treatment of G proteins, see Chapter 9); for this work, they received the Nobel Prize in 1994. When GTP binds to a G-protein molecule, the protein is activated, and it catalyzes the hydrolysis of the bound GTP to GDP, which terminates its activation (Figure 6-38). When a membrane receptor molecule binds to its ligand, this cycle of binding and hydrolyzing GTP is facilitated, because the receptor-ligand complex catalyzes the release of GDP from the G protein, making the binding site more rapidly available to a new GTP molecule. Three separate proteins contribute to G protein-mediated synaptic transmission. The neurotransmitter receptor molecules span the membrane, binding the neurotransmitter on the extracellular face and catalyzing G-protein activation on the cytoplasmic face. The activated G protein can regulate the activity of effector proteins, which can be ion channels or enzymes that control the concentrations of intracellular second messengers or both. We now know of more than 100 receptors that act through G proteins, and these signaling molecules respond to a wide variety of external stimuli ranging from peptides to light and odors. The G proteins themselves constitute a family of at least 20 different proteins. The combinatorial richness of

Closed

this system provides yet another mechanism for producing subtle control within the nervous system. A well studied example of indirect neurotransmission that regulates ion channels is found in heart atrial cells, the system Otto Loewi used more than 75 years ago in the first demonstration that neurons can transfer information by way of chemical signals. Acetylcholine acts on muscarinic receptors in the heart to hold K+-selectivechannels open, prolonging a hyperpolarization. Several different kinds of experiments were needed to establish that this action of ACh depends on a G protein. Some of the experimental results are described here. Acetylcholine has been found to act on heart atrial cells only if GTP is inside the cells, and the muscarinic activation of Kt channels is known to be blocked by pertussis toxin,

....-

Neurotransmitter

Extracellular

Ion channel

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G-protein complex

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\Second messengers, other proteins

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Other cell functions Figure 6-38 lntracellular second messengers modify channel conductances at slow chemical synapses. G proteins participate in signal transmission at many slow chemical synapses. At this kind of synapse, the receptor protein spans the plasma membrane. Neurotransmittermolecules bind to the extracellulardomain of the receptor, which activates a G protein that resides on the cytoplasmic s~deof the membrane. The act~vated G protein regulates the activity of other intracellular proteins, which directly or indirectly changes the conductance through ion channels in the membrane.Activated G proteins can also modify other cellular functions, changing metabolic pathways or the structure of the cytoskeleton.

B

5 pM G,* Control

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Patch of atrial cell membrane

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.Muscarinic r e c e ~ t o r

Figure 6-39 Muscarinic acetylcholine receptors in cardiac cells indirectly cause potassium ion channels in the membrane t o open. (A) Experimental setup for measuring the effect of slow synaptic activation on guinea pig heart atrial cells A nonhydrolyzable analog of GTP, GTPyS, was bound t o asubunits of the G protein t o activate them, and the activated a subunits (the activated state is indicated by an asterisk)were applied t o the intracellular surface of an isolated patch of membrane from atrial cells. The net effect mimics the result of receptor-mediated activation of the endogenous G protein. (B) Typical recordings from an experiment like the one shown in part A. When the concentration of activated asubunits increased, the K+ channels opened more frequently, producing more frequent current steps in the single-channel records. (C) Schematic representation of events at a muscarinic synapse in an intact cell. When ACh binds to muscarinic receptors, G proteins in the membrane are activated, and a subunits of the G proteins bind t o K+ channels, causing them t o open. [Data adapted from Covina et al., 1987.1

which inactivates numerous G proteins. In a direct test of the hypothesis that ACh acts on these cells by means of a G protein, Codina and colleagues (1987)applied G-protein a subunits that had been activated by GTPyS, a nonhydrolyzable analog of GTP, to the inside of membrane patches from cardiac muscle cells (Figure 6-39A). The result mimicked the stable activation of G proteins in the membrane. As the amount of activated a subunit was increased in the bathing solution, the number of open channels increased, as shown by the increased number of singlechannel currents (Figure 6-39B). Similar experiments have identified a large variety of K+, Na+, and Ca2+channels whose activity is regulated similarly by receptor-activated a subunits of G proteins. Neuromodulation

The postsynaptic response to fast synaptic transmitters is immediate, brief, and localized to specialized sites on the postsynaptic cell. In contrast, slow synaptic transmission not only is slow and long lasting, but can also be spatially widespread. In some cases, slow, or indirect, synaptic transmission can interact with and modulate the effects of fast synaptic transmission. The interaction can affect just one postsynaptic neuron, or it may affect many more post-

synaptic neurons, a phenomenon called neuromodulation. Neuromodulation (or, more precisely, modulation of synaptic transmission)refers to transient changes in how effectively a presynaptic neuron can control events in the postsynaptic neuron (i.e., its synaptic efficacy.) Neuromodulatory changes in synaptic efficacy last from seconds to minutes, and this time course distinguishes neuromodulation from synaptic plasticity, described later in this chapter, in which the effects are much longer lasting or even permanent. One of the best understood examples of neuromodulation and its role in normal synaptic excitation is found in cells of sympathetic ganglia of frogs. The system is complex because these cells receive three distinct classes of synaptic

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inputs that are mediated by two different neurotransmitters acting on three distinct types of receptors. Three distinct excitatory postsynaptic responses are produced: a fast epsp, a slow epsp, and a late slow epsp. A typical experimental setup is shown in Figure 6-40A. Both the fast and the slow excitatory postsynaptic potentials are produced by ACh

B

Fast epsp

I

Slow epsp

4 m v L

Late, slow epsp

4 m . L

1 min

1 min

Nerve stimulated

GnRH added 3m v L , 1 min

Figure 6-40 Postsynaptic potentials with very different time courses can be recorded in bullfrog sympathetic ganglion cells. (A) The sympathetic chain ganglia are located on either side of the spinal cord (see Chapter 1 I), and the responses of the large B cells (one class of neurons in the ganglia) can be recorded while nerves that innervate the ganglia are stimulated. Anterior is up in this diagram. (B) Three different kinds of synaptic responses can be recorded in B cells: ( I ) a fast excitatory postsynaptic potential, (with a latency of 30-50 ms) when ACh activates nicotinic receptors in the postsynaptic membrane; (2) a slow epsp (with a latency of 30-60 ms) when ACh binds to muscarinic receptors in the postsynaptic membrane; and (3) a late, slow epsp (with a latency of more than 100 ms) caused by a decapeptide messenger-found in the brains of cold-blooded vertebrates-that is closely related to the hypothalamic releasing factor GnRH. When GnRH binds to postsynaptic receptors, it produces a depolarization in the B cells that lasts many minutes. (Notice the calibration bars below the records.)(C) When exogenous GnRH is applied to B cells, the effect is identical in onset, magnitude, and duration with the late, slow epsp in part B. [Adapted from Jan and Jan, 1982.1

from presynaptic nerve terminals. The postsynaptic cells have both nicotinic receptors (the fast response) and muscarinic receptors (the slow response) in their membranes. In contrast, the late, slow excitatory postsynaptic potential is produced by a neuropeptide that is very similar to the gonadotrophin-releasing hormone (GnRH-see Chapter 9) in mammals and is also released from presynaptic neurons, but not directly onto the postsynaptic neurons. The three postsynaptic potentials depolarize the postsynaptic cell by different amounts and at different times after stimulation, and they act through different, but not entirely independent, mechanisms. When ACh binds to a nicotinic receptor, the ion channel in the receptor complex opens and Na+ and K+ can pass through, producing the fast response (Figure 6-40B). This type of excitatory postsynaptic potential can be elicited by a single stimulus that lasts only a few tens of milliseconds. The slow excitatory postsynaptic potential is produced when ACh binds to muscarinic receptors, and it can be elicited only after several trains of APs have arrived at the presynaptic site and released ACh. The muscarinic receptors act through a G protein to cause a type of K+ channel, called an M channel, to close (Figure 6-41A). When these K+ channels close, the steady state influx of Na+ is no longer balanced by an efflux of K+ and, as a result, the cell depolarizes. The depolarization is small (only about 10 mV; see Figure 6-40B), because it depends on the small steady-state Na+ current. By itself, it cannot produce an AP in the postsynaptic cell, but it can significantly change the response of the cell to fast synaptic signals, particularly when it acts in concert with a late, slow excitatory postsynaptic potential. The late, slow epsp results from the release of a different kind of neurotransmitter, the GnRHlike peptide, that acts through a transmembrane receptor to close the same M channels that are affected by the muscarinic receptors. Adding exogenous GnRH to the postsynaptic neurons produces the same kind of response (see Figure 6-40C). The time course of the response to GnRH is even slower than the muscarinic response; it begins 100 ms after the stimulus and can last for 40 min (see Figure 6-40B). The similarities and differences between these two slower responses are important for understanding how neuromodulation might act in animals. To explore the role of the slow excitatory postsynaptic potential in these sympathetic ganglion cells, the efficacy of an injection of current into the presynaptic cell was evaluated before and during a slow epsp (Figure 6-41B). Before the slow epsp, a presynaptic stimulus caused a single postsynaptic AP; whereas, during the slow epsp, the same stimulus elicited a burst of APs. Clearly, the slow epsp modified signal transmission across this synapse. Normally the KC current through M channels is activated by membrane depolarization and tends to repolarize the cell by shunting depolarizing currents that enter through synaptic channels, thus reducing the effectiveness of any excitatory postsynaptic potentials. When the M channels are kept closed by ACh


205

............................................................................... A

C

ACh

K+ channel

3rd, 4th, or 5th

7th or 8th

GnRH

A

B

GnRH

C cell

B cell

v

ACh

50 mV 9th and 10th Current

chain gangha

Figure 6-41 Both muscarinic acetylcholine receptors and GnRH receptors depolarize a postsynaptic cell by closing M-type potassium ion channels. (A)When acetylcholine (ACh) binds to muscarinic receptors or when the GnRH-like neuropeptide binds to its receptor, M-type channels close, reducing the K+ current across the membrane and depolarizing the neuron. (B) The effect of a fast excitatory postsynaptic potential (epsp) in a postsynapticB cell before, during, and after a slow epsp. During the slow epsp, the decrease in the Kt current through M channels increased the excitability ofthe B cell, producing a train of action potentials in response

t o the fast epsp. (C)Cholinergic neurons from the seventh and eighth spinal nerves Innervate C cells of the ninth and tenth sympathetic ganglia, whereas neurons from the third, fourth, and fifth nerves innervate only B cells in those ganglia. Only C cells receive terminals that are immunoreactive for GnRH, but stimulating the seventh and eighth spinal nerves produces a late, slow epsp in both B and C cells, suggesting that GnRH diffuses from its site of release at the surface of C cells and activates receptorson B cells. [Part B adapted from James and Adams, 1987; part C adapted from Jan and Jan, 1982.1

muscarinic receptors, repolarization of the membrane by the K+ current is prevented and further excitation is potentiated. The late, slow excitatory postsynaptic potential acts similarly, but with a longer latency and for a longer time, and it shares the M channel as a final common pathway. However, there is an additional twist because the peptide neurotransmitter diffuses to nearby neurons, which it can influence identically if the appropriate receptors are present (Figure 6-41C). Only some of the presynaptic neurons can release GnRH, but most postsynaptic cells seem to have GnRH receptors, which strongly suggests that neuromodulation is a normal part of this neuronal circuitry. Taken together, these mechanisms can produce a variety of postsynaptic effects following presynaptic transmitter release. A brief burst of activity in the presynaptic cells would typically generate only the fast excitatory postsynaptic response. More prolonged stimulation might activate the slow pathway in addition, which would effectively amplify the response of the postsynaptic cell to its fast excitatory postsynaptic potentials. With still greater stimulation, the late, slow pathway would additionally increase the effectiveness of fast excitatory postsynaptic potentials and could also potentiate responses in neighboring neurons (see Figure 6-41C), increasing the efficacy of neurotransmission in cells that are not directly postsynaptic to the GnRH-releasing neurons. Moreover, this modulation could be relatively long lived, given the long time constant of the late slow, response.

Within the past few years, studies in the crustacean stomatogastric ganglion have demonstrated the extreme power of neuromodulatory mechanisms. This ganglion contains only 30-40 identified neurons, whose interconnections have been characterized in detail and whose output patterns are well known. When certain neuromodulatory substances, such as proctolin or cholesytokinin, are added to the saline bathing the stomatogastric ganglion, the properties of at least some of the membrane channels change dramatically, effectively rewiring the entire ganglion and generating circuits and outputs that are never seen in the absence of the modulator. Thus, neuromodulators afford a means of remodeling neuronal circuitry, allowing a set of neurons to interact in several distinctly different ways, even though their physical synaptic relations remain unchanged.

INTEGRATION AT SYNAPSES Only rarely are single neurons responsible for producing behavior. Even the simplest behavior requires that several hundred to many thousands of neurons act in a coordinated fashion. This coordination among neurons is called neuronal integration. Used in this sense, "to integrate" means "to combine into a whole." At the level of a single neuron, integration consists of responding to incoming synaptic inputs either by producing an AP or by not producing one, and every neuron integrates the various

excitatory and inhibitory synaptic signals that impinge on it. The process of integration depends heavily on the passive electrical properties of the membrane that lies between the synapses and the spike-initiating zone. In addition, the density and voltage sensitivity of the Na+ and K+ channels determine the threshold and the rate of firing that is produced in response to a given synaptic depolarization. Much of what we know about neuronal integration has been obtained from studies of the large a-motor neurons (Figure 6-42) in the vertebrate spinal cord. These neurons innervate groups of skeletal muscle fibers at neuromuscular junctions. In vertebrates, these are the only neurons that synapse directly onto skeletal muscle fibers, so they play an exceedingly important role in generating overt behavior (see Chapter 10).Thousands of inhibitory and excitatory synaptic terminals contact the dendrites and cell body of each a-motor neuron. The net effect of all synaptic activity is to control the frequency with which APs are generated in the cell. This frequency of firing (typically measured in impulses per second) determines the strength of contraction in the set of muscle fibers innervated by the motor neuron. All of the integrative activity in a neuron is centered on producing APs (i.e., excitation) or suppressing them (i.e., inhibition). Because APs are the only events that can carry information over distances greater than a few millimeters, only synaptic inputs that cause APs in a-motor neurons can generate behavior. Any excitatory input that fails to bring a motor neuron to threshold, either by itself or by summation with other inputs, is lost because no AP is produced in the postsynaptic cell and the signal dies out. In an a-motor neuron, APs are generated in the initial segment of the axon just beyond the axon hillock (see Figure 5-2). This region is more sensitive to depolarization than are the soma and dendrites (perhaps the membrane has a higher density of Na+ channels in this location), so it has a lower threshold for producing APs. If it is to generate APs in the cell, a synaptic current must be able to bring the membrane of the spike-initiating zone up to threshold.

Stimulus

Gray matter

a-Motor neuron

How do the many thousands of individual synaptic inputs onto a motor neuron influence its activity? Synaptic currents spread electrotonically from synapses on dendrites and the soma. How much the current decays over distance is determined by the cable properties of the neuron (Figure 6-43), but in all cases, synaptic potentials become smaller as they spread away from their sites of origin and toward the spike-initiatingzone (see Passive Spread of Electrical Signals earlier in this chapter and Figure 6-16). Because the decrement depends on distance, a synaptic current set up at the end of a long, slender dendrite will decay more than will currents closer to the spike-initiating zone, so distant synapses exert a relatively smaller influence on the activity of the postsynaptic neuron. As a result, the location of synapses, as well as the initial size of synaptic currents, can influence how much control particular synapses have. (Interestingly, recent evidence suggests that in at least some neurons of the mammalian brain, there may be some Na+ channels in dendritic membranes, and these channels can boost synaptic currents, preventing them from decaying as rapidly as they would if they were conducted only electrotonically.) In many cases, the density of inhibitory synapses is highest near the axon hillock, where these synapses can be most effective in preventing excitatory synaptic current from depolarizing the spike-initiating zone to threshold. We have learned many of these concepts from experiments in frogs of the genus Rana. For example, in such an experiment, several segments of the spinal cord of an anesthetized frog are exposed by opening up the vertebral column. Then a microelectrode is lowered into the ventral horn of the gray matter and inserted into the soma of a single a-motor neuron. Small bundles of afferent axons dissected from the dorsal root are placed on silver-wire, stimulating electrodes, providing stimulation to some axons that cause the a-motor neurons to be excited and to others that cause the motor neurons to be inhibited. Initially, the intracellular recording electrode will pick up randomly occurring postsynaptic potentials. These sig-

Figure 6-42 Neurons connected by synapses work together to process information. In t h ~ sdiagram, a spinal a-motor neuron, whose soma is located in the ventral sp~nalcord, is part of a disynaptic reflex arc (called the flexion reflex) in which a noxious stimulus applied to the sk~ncauses excitation of a motor neuron that controls a flexor muscle. The pathway includes one interneuron between the sensory and motor neurons.Activation of the motor neuron causes the muscle fibers that it innervatesto contract.

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.

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Action potential Threshold

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Distance Figure 6-43 Each synaptic input decays with distance as it travels to the spike-initiating zone. An excitatory postsynaptic potential originating in a dendrite spreads electrotonically and gets smaller with distance (top). The density of Na' channels (red dots) in the membrane determines the threshold (blacktrace at bottom) for generating an AP The synaptic potential gets smaller as it spreads toward the axon, and no AP is generated until the current reaches the dense Na+-channeldistribution in the spike-

initiating zone of the axon hillock (or at the first node of Ranvier), where the firing threshold is lowest. The graph shows the relative values of the threshold potential and the synaptic potential along the membrane between the synapse and the spike-initiating zone. The dashed line shows what the amplitude of the excitatory postsynaptic potential would be if the AP were blocked.

nals are caused by synaptic input onto the motor neuron that is not under experimental control. Typically, the activity consists of synaptic potentials with amplitudes of about 1mV, which are similar to those of miniature endplate potentials recorded at the muscle endplate (see Figure 6-24).It has been shown that stimulation of a single neuron that is presynaptic to these motor neurons releases only from one to several quanta of transmitter in response to a presynaptic AP. In this respect, excitatory synapses ending on a motor neuron are quantitatively different from those on a neuromuscular junction, where a single motorneuron terminal releases approximately 100 to 300 quanta in response to a single presynaptic impulse and produces an excitatory postsynaptic potential of 60 mV or more. The transmitter released from a single synaptic ending onto an a-motor neuron depolarizes the neuron by only about 1 mV, far less than the amount required to shift the membrane potential to the firing level. Whereas the vertebrate neuromuscular

junction acts as a single relay synapse, transmitting in a oneto-one manner (i.e., one postsynaptic impulse for each presynaptic impulse), a motor neuron requires the more-orless concurrent activation of numerous excitatory synaptic inputs impinging on it in order for the synaptic potential to reach the firing threshold for the initiation of a postsynaptic AP. Thus, the decision to fire is a response to a collection of presynaptic inputs, and although each small synaptic current is ineffective by itself, the activity at a single ending can contribute significantly to the integrative behavior of the neuron. This rather democratic behavior prevents activation of motor neurons by trivial input or spontaneous activity in their input neurons. More importantly, it provides a means of integrating inputs from various sources, both excitatory and inhibitory, to determine when the neuron will produce APs and how many there will be. As the strength of the stimulus current applied to the presynaptic axons in the dorsal root is increased, more and

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more excitatory axons become active; that is, they are yecruited by the increased stimulus. When these neurons fire in unison, the total amount of transmitter released onto the motor neurons rises, producing more individual synaptic currents that add together to cause a larger excitatory postsynaptic potential. When inputs from several individual synapses add together simultaneously to change Vm in the postsynaptic neuron, the process is called spatial summation. The summed synaptic inputs can lead to greater depolarization if they are all excitatory (Figure 6-44). If inhibitory transmitter is released simultaneously with excitatory transmitter, it also produces synaptic currents that sum with the excitatory currents (Figure 6-45). Open inhibitory synaptic channels can short-circuit the depolarizing current carried by Na+ ions moving through excitatory channels; that is, as depolarizing positive charge is carried into the cell by Na+ ions, some of that charge is promptly removed from the cell when K+ ions move out or CI- ions move in through inhibitory synaptic channels. The activation of inhibitory synapses re. . duces the depolarization at the spike-initiating zone and decreases the probability that an AP will be produced. When a second postsynaptic potential is elicited within a very short time after the first, it can add to, or "ride piggyback" on the first, even if the two synaptic events are caused by the same presynaptic neuron. This effect is called

Synapt~c current

I I

Time

Figure 6-44 Synaptic inputs from several presynaptic neurons produce spatial summation onto a motor neuron. Two excitatory synaptic currents, from two separate neurons a and b, arise at spatially separated synapses. Traces at lower right show synaptic potentials recorded at the spikeinitiating zone when each input acted alone and when the two inputs were active simultaneously, producing spatial summation. Spatial summation of currents from many synapses is required to produce a synaptic potential that exceeds the threshold of a motor neuron. If too few excitatory inputs are active simultaneously, V, at the spike-initiating zone fails to reach threshold, and no APs are produced.

synaptic current

Inhibitory synaptic current

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- - - - - - - a and b

Figure 6-45 Excitatory and inhibitory synapticcurrents sum. Stimulation of separate presynaptic pathways gives rise to excitatory (a) and inhibitory (b) synaptic currents. Traces at lower right show synaptic potentials recorded from the spike-initiating zone when either a o r b was stimulated individually and then when they were stimulated together, illustratingthe effects of summation. The dashed arrows indicate that some of the excitatory synaptic current is diminished by the open inhibitory channels.

temporal summation (Figure 6-46). The shorter the interval between two successive synaptic potentials, the higher the second response rides upon the first and so the bigger the postsynaptic potential can become. Further summation can be achieved if additional stimuli arrive in rapid succession, with the third synaptic potential riding on the second, and so forth. Under natural conditions, spatial and temporal summations often occur together. For example, if different excitatory synapses on one motor neuron are active at slightly different times, the effects will sum both spa'tially and temporally. Both spatial and temporal summations of synaptic potentials depend on the passive electrical properties of the neurons. Spatial summation occurs because synaptic currents that originate at the same time, but at different synapses, each spread electrotonically away from the synapse (see Figure 6-43), so their effects on V, can add together at the spike-initiating zone. Temporal summation, on the other hand, does not require the summation of synaptic currents and can occur even though the individual currents do not overlap (see Figure 6-46C), because the electrical time constant of the membrane is long relative to the time course of synaptic currents. The first synaptic current brings positive charge into the cell, partially discharging the negative resting potential of the cell membrane. The positive charge carried into the neuron by the synaptic current then slowly leaks out (through the resistance-K+


209

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St~mutus

S y n a p t ~ ccurrent

B

Synaptic potentla1

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but randomly, changing V,. Every now and then, excitatory inputs will sum to trigger an AP in the neuron, which in turn leads to an AP and twitch in each of the muscle fibers innervated by the neuron. The result of this activity is a constant background of low-level tension in skeletal muscles as first one and then another motor neuron fires and causes the contraction of the muscle fibers that it innervates. (See Chapter 10 for further discussion of muscle fibers and their control.) The membrane at the splke-initlat~ngzone in motor neurons typically never accommodates completely to maintained depolarization. Therefore, ~f synaptic input is both strong and maintained, it can cause the motor neuron to fire a sustained tram of APs. The frequency of impulses in a train depends on how depolarized the spike-initiating zone becomes (Figure 6-47), which in turn depends on the

Stimulus

Figure 6-46 In temporal summation, presynaptic signals arrive at the synapse in rapid succession. (A) Setup for recording postsynapticevents. (B) Asinglest~mulusevokes a synaptic current (shaded signal) and a more slowly decaying synaptic potential. (C) Summation of synaptic currents is not required for summation of synaptic potentials, because the time constant of the synaptic potential is longer than the time course of the synaptic current. Arrows indicate the time points at which presynapticimpulses arrived at the synapse.

channels-and capacitance of the membrane), and V, gradually returns to rest after the synaptic current has ceased. Thus synaptic potentials outlast synaptic currents by milliseconds and, if a second synaptic current flows before the first synaptic potential has subsided, it will cause a second depolarization that adds to the falling phase of the first, even though the two synaptic currents do not overlap in time. Thus, the membrane's charge-storing capacity allows the voltage effect of synaptic currents to sum over time. The longer the time constant of the membrane is, the slower the decay of postsynaptic potentials will be, and the more effective the temporal summation of asynchronous synaptic inputs can be. The membrane time constant, (T), of vertebrate motor neurons is about 10 ms, and it can range from 1ms to 100 ms in other neurons. Microelectrode recordings reveal that, under normal conditions, motor neurons are almost never electrically silent, but instead always exhibit some synaptic noise (irregular fluctuations in membrane potential) caused by ongoing activity in presynaptic neurons. The result is a constantly,

Time

Depolarization Figure 6-47 The initial frequency of impulses generated in a motor neuron is approximately proportional to the amplitude of the membrane depolarization. (A) Two electrodes, one for passing depolarizing current and one for recording membrane potential, are inserted into a spinal a-motor neuron. (B) Three idealizedtraces show that increased depolarization (top to bottorn)causes an increased rate of flung. (C) Initial firing frequency plotted against amount of depolarization. As the depolarizationis increased, the frequency of APs increases, up to some maximum value.

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amplitude of summed synaptic inputs. Thus the number and frequency of APs produced in the motor neuron carry information about the input to the neuron. In fact, most information transfer in the nervous system depends on this frequency code. To summarize, APs are generated in a neuron when the low-threshold initial segment (often the axon hillock) is depolarized to threshold or beyond. The frequency of APs in the neuron rises as depolarization increases to some maximum firing frequency. The amount of depolarization at the spike-initiating zone depends on the relative timing of excitatory and inhibitory synaptic currents and on where those currents originate.

SYNAPTIC PLASTICITY The nervous system would be much less useful to an animal if it could not be changed by experience. Neuronal plasticity, the modification of neuronal function as a result of experience, is of premier importance for the survival of any organism. Common examples of neuronal plasticity in our lives are learning and the development of motor skills and habits. This plasticity lies behind human intelligence, as well as the ability of all higher animals to respond adaptively to stimuli in ways that allow them to go beyond fixed reflexes programmed into their developing nervous systems by genetic mechanisms. Virtually all animals demonstrate a degree of behavioral plasticity, and the mechanisms that underlie synaptic plasticity are currently the subject of many experiments. Synaptic plasticity also takes place as the result of developmental events over the course of a lifetime. Synaptic connections that are established in embryos are later refined into adult patterns, and even later changes in synaptic strengths are thought to be important mechanisms for learning and memory at mature synapses. Interestingly, shaping the development of mature synapses and modifying them in learning and memory both appear to depend on a retrograde signal that is sent from the postsynaptic neuron to the presynaptic neuron. In fully developed adult organisms, neuronal plasticity requires changes in synaptic efficacy. A change in synaptic efficacy is not the only way in which neuronal function might be modified, but, at present, it is the one for which there is the most experimental support. D. 0. Hebb suggested in 1949 that the effectiveness of an excitatory synapse will increase if activity at this synapse is consistently and positively correlated with activity in the postsynaptic neuron. Since then, one challenge has been to identify the mechanisms that could underlie this kind of change. Two large categories of mechanisms that could fill this role are (1) changes in the presynaptic terminals and (2) changes in the postsynaptic neuron. One example of a presynaptic mechanism would be a change in the amount of transmitter released from presynaptic terminals in response to a presynaptic AP. An example of a postsynaptic mechanism would be a change in the postsynaptic apparatus that altered the amplitude of depolarization produced

when a given amount of transmitter was released from the presynaptic endings. Relatively little is known about the mechanisms of postsynaptic plasticity, although ithas been demonstrated in several tissues. We will consider presynaptic mechanisms of neuronal plasticity. There are two major classes of presynaptic mechanisms that change synaptic efficacy. In one class, activity in the terminal itself causes a use-dependent change in the release of transmitter, so these mechanisms are called homosynaptic modulation. In the other class, changes in presynaptic function are induced by the action of a modulator substance released from another, closely apposed nerve terminal, so these mechanisms are called heterosynaptic modulation. Typically, heterosynaptic modulation lasts longer than homosynaptic modulation. Homosynaptic Modulation: Facilitation

A use-dependent change in synaptic efficacy can be seen in a partly curarized endplate region of a frog skeletal muscle fiber if two stimuli are applied to the motor axon in rapid succession. If the second synaptic potential begins before the first has subsided, they will sum, but the amplitude of the second response will be greater than can be accounted for by summation alone. If the second synaptic potential begins soon after the first has completely subsided, precluding temporal summation, the second postsynaptic potential may still reach a higher amplitude than the first one. This effect, termed synaptic facilitation, lasts from 100 to 200 ms at the frog neuromuscular junction (Figure 6-48). Stimulus

Curare in bath

I (resynaptic

impulses

Time

Figure 6-48 Synaptic facilitation occurs at the frog neuromuscularjunction. In this experiment, curare in the bathing saline blocked some ACh receptors, reducing the amplitude of exc~tatorypostsynaptic potenti& to below the firing threshold. Two st~muliwere delivered to the nerve in rapid succession. The second synaptic potential summed with the falling phase of the first, producing a larger postsynaptic potential; but, in addition, the amplitude of the second response (indicated by the line labeled 2) was greater than could be accounted for by summation alone.


211

............................................................................... A

S t ~ m u l u st o

B

I ?

-

Postsynaptic

Ca2+

tained by Katz and Miledi (1968).They used a carefully positioned micropipette to supply pulses of Ca2+ ions to the external solution near a motor endplate of a frog muscle that was immersed in Ca2+-free Ringer solution (Figure 6-49A). They found that facilitation of the postsynaptic potential evoked by the second stimulus was greatest when a pulse of extracellular Ca2+ions was supplied to coincide with the arrival of the first AP (Figure 6-49B). The first pulse of Ca2+did not significantly enhance facilitation if it was given after the first AP arrived at the terminals (see Figure 6-49B).Thus, if synaptic facilitation is to occur, Ca2+must be available to enter the presynaptic terminal when an AP invades the terminal. If Ca2+ions can enter the terminal from the external fluid, Ca2+ions from the second AP add to any Ca2+ions remaining from the first, leading to the release of more transmitter. Homosynaptic Modulation: Posttetanic Potentiation

Figure 6-49 Synaptic facilitation depends on the presence of calcium ions in the extracellular fluid. (A)The motor neuron innervating the muscle fiber was stimulated, and the resulting postsynaptic potential was recorded. The bathing solution was calcium free, but small pulses of CaCI, were provided by a CaC12-containingpipette positioned just at the endplate region. In this experiment, the relative timing between stimuli to the motor neuron and the delivery of CaCI, was vaned. ( 0 )Recordings of postsynaptic potentials in the muscle fiber. Horizontal black bars show the timing of Ca2+pulses. Thin vertical lines indicate stimuli to the presynaptic neuron.Trace 1 shows the amplitude of a postsynapticpotential in response to a single AP in the motor neuron. In the three other traces, the temporal relation between the first AP and the pulse of CaCI, was varied. In all cases, Ca2+ions were available at the time of the second AP. Facilitation occurred only when Ca2+ions were present at the endplate when both of the APs reached the endplate. [Adapted from Katz and Miledi, 1968.1

The best evidence indicates that synaptic facilitation depends on the amount of free Ca2+within the presynaptic terminal. The concentration of intracellular free Ca2+ions rises in the terminal when the first AP opens voltagedependent Ca2+channels, and this increase in Ca2+ion concentration persists for a short time. When the second impulse arrives at the te5mina1, the Ca2+ concentration is still somewhat elevated,and the ca2+ ions that enter as a of the second add the remaining Ca2+ions, generating an even higher Ca2+ concentration in the terminal. Because the release of transmitter is a Dower function of the intracellular Ca2+ concentration near the presynaptic release sites, this small increase in Ca2+concentration inside the terminal produces a large increase in the amount of transmitter released subsequent to the second impulse. Experimental evidence for this hypothesis was ob-

When a frog motor axon is stimulated tetanically (i.e., at a high frequency for a relatively long time), synaptic transmission at the neuromuscular junction is initially depressed after the stimulation. However, responses to test pulses applied at later times after the stimulation are found to be larger than normal. This increase in the amplitude of the response lasts for as long as several minutes and, during this time, the responses are said to be potentiated. This posttetanic potentiation is another example of a use-dependent change in presynaptic efficacy, and it is found in one form or another at many types of synapses. Figure 6-50 illustrates the results of one such experiment. Initially,

H

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5 min

200

5 +..I

r +g

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0

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50 s-' Figure 6-50 Tetanic stimulation of a frog motor nerve elicits depression and potentiation of excitatory postsynaptic potentials in muscle fibers. Curare was used to reduce the amplitude of synaptic potentials, blocking the produaion of APSand revealing the amplitude of the synaptic POtentials. When the nerve and muscle were bathed in normal frog Ringer's solution, which has a ca2+concentration of about 2 rnM (top), stirnulating the motor nerve at 50stimuli per second for about a minute produced first a depression in subsequent excitatory postsynaptic potentials and When the concentration of extracellularCa2+was rethen duced to 0 225 mM, only potent~at~on was seen after h~gh-frequency st~mulatlon.[Adapted from Rosenthal. 1969 I

212


......................................

excitatory postsynaptic potentials (epsps)were evoked at a frog neuromuscular junction by stimulating the motor nerve at a low control rate (one stimulus every 30 seconds). The rate of stimulation was then increased to 50 per second for a period of 20 seconds, after which a series of test stimuli were administered at the initial rate of one every 30 seconds. In Ringer solution that contained a normal concentration of Ca2+ (see Figure 6-50, top), posttetanic depression of the evoked epsps occurred immediately after the tetanic stimulation. However, within 1minute, the amplitude of the epsps increased; in other words, posttetanic potentiation had occurred. The amplitude of the epsps returned to the control level after about 10 minutes. In Ringer solution that contained a lower-than-normal concentration of Ca2+(see Figure 6-50, bottom), there was no depression, and the posttetanic potentiation subsided more rapidly. These results are thought to depend on events within the terminals. During high-frequency stimulation in a normal concentration of extracellular Ca2+ (1.8 mM), the available synaptic vesicles are released faster than they can be replaced, so the amount of transmitter available for release is depleted and remains low for a time immediately after the high-frequency stimulation. Later in the posttetanic period, quanta of transmitter available for release are restored, and the depression subsides. During tetanic stimulation, Ca2+ ions that have entered the terminals accumulate, load up the available Ca2+-binding sites that ordinarily buffer the intracellular concentration of Ca2+, and linger within the terminals until they are gradually pumped out by active transport across the cell membrane. It is believed that posttetanic potentiation and its slow decay reflect this increase and subsequent decrease in the concentration of Ca2+inside the terminals. In low-Ca2+Ringer solution, fewer Ca2+ions are available to enter the terminals, so fewer synaptic vesicles can bind to the membrane and release transmitter. As a result, there is less depletion of available synaptic vesicles, and there is no posttetanic depression. Posttetanic potentiation is just as pronounced, because repeated stimulation does bring Ca2+ions into the terminals, but the potentiation decays more rapidly, perhaps because the concentration of Ca2+inside the terminals is less elevated or because the presynaptic terminal is able to pump the extra Ca2+out more rapidly because less has accumulated.

but not at, a presynaptic ending, they are said to act heterosynaptically, because transmission through the synapse is altered by an additional, third neuron, which released the modulator. One class of heterosynaptic action that has already been discussed in this chapter is presynaptic inhibition; another, in which the amount of transmitter released is increased by the presence of the modulator, is called heterosynaptic facilitation. In heterosynaptic modulation, the modulator is thought to alter the number of Ca2+ions that enter the terminals subsequent to a presynaptic AP. Synaptic modulators usually do not directly open (or close) ion channels. Instead, they change how ion channels respond to another stimulus; by doing so, they increase or decrease ionic currents carried through channels that are activated by a presynaptic AP. This action by modulators is typically mediated by one or more intracellular messengers that act on the ion channels. In contrast, fast neurotransmitters bind to membrane receptors and open (or close) channels. The most extensively studied example of heterosynaptic modulation at a synapse is found in the sea hareAplysia californica, a sluglike gastropod mollusk that has been widely used in studies of neuronal plasticity. Eric Kandel and his associates have found that excitatory transmission between specific identified neurons in the central nervous system of Aplysia is enhanced during behavioral sensitization. They have found that this enhancement occurs through heterosynaptic facilitation of transmitter release triggered by the release of serotonin near the synapse (Figure 6-51). In this case, serotonin is thought to elevate the concentration of the intracellular messenger 3',5'-cyclic adenosine monophosphate (CAMP),which has been found to influence the opening of a specific type of K+ channel, known as the S channel. Specifically, when CAMP iselevated in the presynaptic neuron, S channels are more likely to be shut at any given Vm. The efflux of K + through S channels contributes to repolarization after an AP, so the closing of S channels will prolong the presynaptic AP and allow more Ca2+ions to enter the terminal through voltage-gated Ca2+channels. An increase in the influx of Ca2+ ions allows more transmitter to be released and increases the amplitude and duration of the postsynaptic potential.

Long-Term Potentiation Heterosynaptic Modulation The release of transmitter from nerve terminals can be influenced at some synapses by the presence of certain neuromodulators. These modulatory agents include serotonin in mollusks and in vertebrates, octopamine in insects, and norepznephrine and GABA in vertebrates. All of these agents are also neurotransmitters (see Table 6-2). In addition, endogenous opioids have been shown to act as modulatory agents in vertebrate neurons. Such agents, released into the circulation or liberated by nerve endings near a synapse, are believed to modify the release of transmitter from presynaptic terminals. When they are liberated near,

In the past few years, intense interest has been focused on long-term changes in synaptic efficacy that have been identified in the mammalian hippocampus, the site of certain memories. High-frequency stimulation of inputs to the hippocampus produces an increase in the amplitude of excitatory postsynaptic potentials recorded in the postsynaptic hippocampal neurons. In an intact animal, the increased amplitude can last for hours-even days or weeks-after the potentiating stimulation. This prolonged facilitation of synaptic transmission, called long-term potentiation, has been shown to occur in many synaptic pathways. At different sites, long-term potentiation may require different


213

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sucrose > glucose) as do the sweetness receptors of the human tongue. Like insects, many vertebrates have taste receptors on the body. The bottom-dwelling sea robin fish, for example has modified pectoral (anterior)fins with taste receptors at the tips of the fin rays, which it uses to probe the muddy bottom for food. In terrestrial vertebrates, taste receptors are found on the tongue and epiglottis, in the back of the mouth, and in the pharynx and upper esophagus. In vertebrates, taste receptor cells are located in taste buds, which have some organizational features in common with olfactory organs (Figure 7-20). The taste receptors are surrounded by support cells and by basal cells, which are progenitor cells that give rise to new taste receptors. Basal cells are derived from epithelial cells, and they regularly generate new sensory receptor cells; taste receptor cells live for only about 10 days. This remarkable turnover of primary sensory cells also occurs in vertebrate olfactory organs and in specialized parts, called outer segments, of photoreceptor cells. All of these cells, or cell parts, that are regularly renewed directly interact with physical stimuli from outside the organism: taste and smell molecules in gustatory and olfactory cells, respectively, and photons in photoreceptor outer segments. The turnover of all sensory cells poses a problem for the maintenance of sensory specificity in an organism because, unless the new cells were precisely integrated into the existing network, specificity would be lost. Just how the integrity of taste and smell sensations is maintained remains an unsolved, but actively studied, mystery. Although our subjective experience would suggest that there is a wonderfully large spectrum of possible tastes, these sensations can be grouped into four distinct qualities: sweet, salt, sour, and bitter. In evolutionary terms, these categories may be related to some basic properties of food. Sweet foods are likely to be rich in calories and thus useful; salt is essential for maintaining water balance (see Chapter 14);a sour taste can signal danger if it is in excess; and many bitter substances are toxic. The discovery that vertebrates respond to only four fundamental categories of tastes suggests that all perceived tastes must depend on various combinations of these fundamental characteristics. In addition, it generated -the hypothesis that there is a separate, identifiable sensory pathway associated with each of the four tastes.

Microvilli of apical membrane

A

\ ia

Vertebrate taste bud

Nasal cavity

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MUCUS layer

to brain

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Vertebrate olfactory receptor

Insect olfactory

receptor

Figure 7-20 Chemosensoryorgans typ~callyconsist of receptor cells surrounded by supporting structures. (A) In vertebrate taste buds, the receptor cells are surrounded by basal cells, which generate new receptor cells, and by supporting cells. Transduction takes place across the apical membrane. The receptor cells do not themselves send axons to the central nervous system, although they can produce APs Instead, they synapto the CNS In tlcally exc~teafferent neurons that carry the ~nforrnat~on contrast, vertebrate (B) and Insect (C) olfactory receptors themselves send primary afferent axons to the CNS. Structures that are analagous between vertebrates and insects are drawn similarly in parts B and C. All three types of receptors extend fine processes into a mucous layer that covers the epithelium. In insects, these fine processes are true dendrites. [Part A adapted from Murray and Murray, 1970; part C adapted from Steinbrecht, 1969.1

How do molecules interact with membranes to produce distinct tastes? In the past few years, the use of patchclamp recording has allowed the mechanisms responsible for each taste modality to be identified (Figure 7-21). Each individual taste receptor cell reacts to particular stimuli, and each class of taste stimuli activates a distinctive cellular pathway in the receptors that respond to it. Salty

Figure 7-21 Each kind oftaste is transduced by a dist~nct~ve mechanism. (A) In the transduction of salty and some sour tastes, Na+ (or H-) ions pass through ion channels in the apical membrane, directly depolar~z~ng the receptor cell (B) In the transduction of other sour tastes and some b~ttertastes, protons (sour) or certain bitter compounds block Ki channels, and residual leakage of cations into the cell depolarizes the recep(Ala)and some other sweet compounds bind to receptor. (C) L-Alan~ne tors (R) and actlvate a G protein (G).The activated G protein activates adenylate cyclase (AC), and the resulting increase in cAMP closes K t channels in the basolateral membrane, depolarizing the cell. (D) L-Arginine (Arg)binds to, and opens, a ligand-gated, nonselective cation channel. (E) Some bitter compounds bind to a receptor and activate a G protein that IS thought to be coupled to phospholipase C (PLC),producing an Increase In intracellular ~nositoltriphosphate (InsP,), which could then release CaZ' from ~ntracellularstores. The net result 1s an increase in transmitter release from the receptor cell, but the mechanism is not yet completely understood. PIP,, phosphoinositol 4.5-biphosphate. [Adapted from Avenet et al., 1993.1

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s t ~ m u l such ~ , as NaCI, read~lyd~ssociateIn water, and the N a + Ions enter receptors through N a + channels In the membrane to depolar~zethe membrane potent~al.These because ve they can be blocked Na+ channels are d ~ s t ~ n c t ~ e, the voltage-gated Na+ chanby the drug a m ~ l o r ~ dunl~ke nels that med~atemost APs. Sour s t ~ m u lw~ h, ~ c hare charr t h ~ ssame acter~zedby excess H+ Ions, act e ~ t h e through channel (observed in the hamster) or by blocking a K+

channel (observed in the salamander Nectuuus). In either case, the membrane is depolarized. Sweet compounds and the amino acid alanine (Ala) bind to receptors coupled to an intracellular cascade that closes K+ channels in the basolateral membrane, depolarizing the receptor. Other sweet substances, including the amino acid arginine (Arg) - and monosodiuin glutamate, activate nonspecific cation-selective channels in taste cells. Some bitter substances, such as Ca2+ and quinine, close K + channels in the apical membrane, allowing the cell to depolarize. Transduction of other bitter substances is less well understood but appears to rely on intracellular second-messenger systems (either the InsP; or cAMP pathways) to excite the cell. It is hypothesized that the sweet and bitter pathways that act through second-messengers are mediated by G proteins, and recent reports have suggested candidate molecules. In all cases, the initial event in the receptor cell eventually causes an increase in the concentration o f intracellular Ca'+ and thus increases the release of neurotransmitter onto the second-order cells in the pathway. Taste receptors generate APs, but they have no axons, so they cannot themselves carry information to the central nervous system. Instead, they synapse onto, and modulate activity in, neurons whose axons run in the facial, glossopharyngeal, and vagus nerves (seventh, ninth, and tenth cranial nerves). The existence of four kinds of taste sensatioils and the specificity of membrane transduction mechanisms for each kind of taste suggest that each receptor subtype might be connected to a particular set of axons. In that arrangement, for example, information about "sweetness" would be carried by some specific subset of axons. Such a pattern is called labeled line coding, but recordings have revealed that taste information is not nearly this neatly organized. Recordings from single neurons show that a receptor will often respond optimally to a particular type of stimulus (Figure 7-22), but many receptors also respond suboptimally to stimuli in other classes. The data thus suggest that a single fiber innervating a taste bud receives information fromseceptors belonging t o different subtypes.

Chorda tympani nerve

Figure 7-22 Each afferent taste neuron is most effectively stimulated by one type of stimulus, but it also responds to other stimuli. The responses to four different taste stimuli were recordedfrom sinqle taste afferent axons in two different nerves in hamsters. Each neuron responded maximally to one of the four taste stimuli; different neurons responded maximally to different stimuli. However, all of the axons responded at least weakly to all four stimuli, indicating that each taste afferent is not restricted to carrying information about only one kind of taste. Abbreviations: S, sucrose (sweet), N, NaCl (salty), H, HCI (sour), Q, qulnlne HCI (b~tter)The number of axons IS Ind~catedfor each group [Adapted from Hanamor~et al ,1988 ]

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Rather than simple labeled line coding, sensory information about taste must depend on the analysis of many gustatory axons in parallel. Mechanisms of Olfactory Reception

In vertebrates, olfactory receptors are located inside the nasal cavity, arranged so that a stream of air or water

flows over them during respiration (Figure 7-23).Animals that are particularly dependent on olfactory cues have complex cavities that are lined by sheets of receptors. These cavities are called turbinates, and the mechanisms guaranteeing air flow through them remains unknown. Each receptor neuron has a long thin dendrite that terminates in a small knob at the surface (Figure 7-24A). Figure 7-23 In vertebrate olfactory organs, air (or water) carrying odorants is moved past olfactory receptors. The olfactory epithelium of human beings covers part of the surface of air passages in the nose. The arrows indicate the route followed by air as it is breathed in through the nose. The dashed portion of each line shows the movement of air in the turbinates (shaded in red) where the olfactory receptors are located. Dashed lines also indicate eddy currents in the air that are created over the olfactory epithelium that lines the dorsal recesses of the nasal cavity.

Figure 7-24 Receptors of the vertebrate olfactory epithelium depolarize in response to oderant molecules. (A) The organization of cells within the mammalian olfactory epithelium. (B) Response of a cultured salamander olfactory receptor neuron to focal pulses of an odorant. (Left) When the stimulating chemical pulse was directed at the receptive membrane on the cilia, it produced a large current (top record). When a solution containing a high concentration of K+ was focused at the same location, the response was small (bottom record). (Right) When the stimulating chemical pulse was directed at the soma, rather than at the cilia, the response was small (top record). However, when the solution containing a high concentration of K+ was directed at the soma, it produced a large response (bottom record). [Part A adapted from Shepherd, 1994; part B adapted from Firestein et al., 1990.1

cilia

,

Solution aimed at cilia

Solution aimed at soma

Odorant

Odorant

Emanating from the knob are several thin cilia (about 0.1 pm in diameter and about 200 pm long) that are covered by a protein solution called mucus. Molecules delivered to the nasal cavity are absorbed into the mucous layer and delivered to the cilia. Two lines of evidence suggest that the cilia are the location of olfactory transduction. First, only ciliated neurons respond to odors, implying that the cilia must be the site of

transduction. The second piece of evidence comes from experiments in which olfactory neurons were grown in culture and were exposed to odorants while the receptor current was recorded by an intracellular electrode in the soma (Figure 7-24B). If a solution of odorant molecules was ejected onto the cilia, the cell responded strongly; whereas, if the same solution was ejected onto the soma, there was only a small response. In contrast, ejecting a solution of

KC1 (which would depolarize the membrane of the receptor) onto the cilia produced a small response, whereas ejecting KC1 onto the soma produced a large response. These data imply that only the cilia were able to respond to the odorant, causing V,,, to change significantly. The olfactory transduction cascade includes an adenylate cyclase that is linked to a G protein. (Seethe discussion of transduction earlier in the chapter.) A very large family of proteins has recently been found to be expressed only in olfactory epithelial cells. The structure of each protein includes seven transmembrane domains, and other features also indicate that these molecules are homologous to proteins that mediate other transduction processes. The large size of this family of proteins suggests that there could be many individual receptor subtypes for distinct odors, in

contrast with the small number of receptor types that code for taste. Olfactory coding in vertebrates has been studied electrically in the olfactory epithelium of the frog (Figure 7-25A) In these experiments, activity of single receptor axons was recorded by one electrode while the summed potential of large numbers of olfactory receptors in the epithelium (the electro-olfactogram, or EOG) were recorded simultaneously by another electrode (Figure 7-25B). Impulses from individual receptors were then superimposed electronically on the electroolfactogram. This technique permits the activity in a single receptor to be compared with the total response of many receptors when a single odorant, or a combination of odorants, is presented. Figure 7-25 Olfactory reception can be studled at the cell and organ level simultaneously in the olfactory epithelium of the frog. (A) Various odorants can be applied to the nasal epithelium while the summed electro-olfactogram (EOG)and spikes from single receptor cells are recorded. The two kinds of records can then be electronically summed to give a composite recording (right). (B) Detail of tissue and electrodes. Electrode 1 records the overall EOG potential, because it is far from any one axon, while electrode 2 records the activity of the single axon to which it is closest. [Adapted from Gesteland, 1966.1

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The results indicate that stimulus coding in the vertebrate nose is far more complex than is coding in the contact chemoreceptors of houseflies. Different receptors respond differently to the same odorant. In some olfactory axons, a particular odorant increased the impulse frequency (Figure 7-26A). Some odorants that smell alike to humans have similar effects on some frog olfactory cells, suggesting that they would smell alike to frogs, too. However, these same odorants have different effects on other cells (see Figure 7-26A, cell a vs. cell b), suggesting that they would not smell alike to the frog. In the olfactory bulb, farther along the chain of olfactory neurons, neurons may respond to an odorant with decreased activity or with increased activity (Figure 7-26B). In fact, it has proved to be impossible to establish a one-to-one relation

Resting discharge Menthone

-

Menthol

Cell a

Cell b

Odorant applied

Suppression of ongoing

Increased

activity

between classes of odorants and types of olfactory cells in the frog. Instead, each olfactory receptor cell appears to express a mosaic of odorant receptor molecules with differing specificities. The response characteristics of a particular olfactory receptor must, then, depend on the proportions of its many types of receptor molecules. This situation implies that the ability of mammals to distinguish among a wide variety of odors must reside in the ability of the higher olfactory centers in the brain to decode a combinatorial signal from a large number of olfactory receptors.

MECHANORECEPTION All animals can sense physical contact on the surface of their bodies. Such stimuli are detected by mechanoreceptors, the simplest of which consist of morphologically undifferentiated nerve endings found in the connective tissue of skin. More complex mechanoreceptors have accessory structures that transfer mechanical energy to the receptive membrane. These accessory structures often also filter the mechanical energy in some way, as described earlier in regard to the mammalian Pacinian corpuscle in which the sensitive ending is covered by a capsule (see Figure 7-14). Other mechanoreceptors include the muscle stretch receptors of various kinds found in arthropods and vertebrates, in which mechanically sensitive sensory endings are associated with specialized muscle fibers (see Figure 7-13), and the hairlike sensilla that extend from the exoskeletons of arthropods (Figure 7-27). The most elaborate accessory structures associated with mechanoreceptive cells are found in the vertebrate middle and inner ear and in the vestibular system, both of which are considered later in this chapter. The stimulus that activates mechanoreceptive membrane is a stretch or distortion of the surface membrane. Indeed, stretch-sensitivechannels are found in all types of organisms from the simplest to the most complex. Patchclamp data indicate that these channels respond to changes in tension in the plane of the membrane, and they can be either activated or inactivated by stretch. Stretch-sensitive channels defy simple classificationwith regard to selectivity, because they show a wide range of conductances and fidelity. Possible transducers of mechanical stress include the cytoskeleton, enzymes, or the ion channels themselves. Mechanically sensitive channels are the only primary mechanotransducers that do not depend on enzymatic activity but instead directly use the free energy stored in the transmembrane electrochemical gradient. Mechanoreceptors can be exquisitely sensitive, responding to mechanical displacements of as little as 0.1 nm. It is a continuing challenge to understand how such small displacements can produce changes in ion permeability through the membrane.

I

Figure 7-26 Olfactory receptors have complicated responses to individual odorants. (A) Recordings from two frog olfactory receptors. Menthone and menthol both slightly suppresssed ongoing activity in cell a, indicating that cell a could not distinguish between the two substances. In contrast, cell b responded differently to the two substances, producing more APs in response to menthol than it did at rest but fewer in response to menthone than it did at rest. Thus, cell b could potentially distinguish between the two substances, whereas cell a could not. Notice that the electro-olfactogram(EOG) has been summed with the individual record for each cell. (B) Recordingsfrom second-order olfactory cells of the tiger salamander. Odorants may reduce or increase ongoing activity in these cells. [PartA adapted from Gesteland, 1966; part B adapted from Kauer, 1987.1

Hair Cells

The hair cells of vertebrates are extraordinarily sensitive mechanoreceptors that are responsible for transducing me-

SENSING T H E E N V I R O N M E N T

239

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Figure 7-27 Hairlike mechanoreceptors extend from the exoskeleton of many insects. (A) Location of jo~nt-positionreceptors. Each hair plate conta~nsa number of sensilla that sense the position of the joint. (B) Anatomical detail of a sensillum at rest. (C) Bending the sensillum stretches and deforms the dendr~te of the receptor cell. [Adapted from Thurm,

Cilium

1965.1

chanical stimuli into electrical signals (Figure 7-28). They are found in several locations. Fish and amphibians have a set of external receptors, called the lateral-line system, that are based on hair cells and that detect motion in the surrounding water (Figure 7-29). The vertebrate organs of hearing and the organs that report the position of the body with respect to gravity (called the organs of equilibrium) also are based on hair cells. Typically, the organs of equilibrium include the semicircular canals and the vestibular apparatus. Hair cells are named for the many cilia that project from the apical end of each cell. These cilia fall into two classes: each hair cell typically has a single kinocilium and 20-300 nonmotile stereocilia. The kinocilium has a "9 + 2" arrangement of internal microtubules (see Figure 7-28A) that are similar to that of other motile cilia. The stereocilia contain many fine longitudinal actin filaments, and they are thought to be both structurally and developmentally distinct from the kinocilium. Although the hair cells of the lateral line and of the organs of equilibrium have both a kinocilium and several stereocilia, some hair cells in the adult mammalian ear lack kinocilia. In addition, the technically remarkable feat of microsurgically removing the kinocilia from hair cells that normally have them does not block transduction. From these two observations, it appears that kinocilia must not be necessary for mechanotransduction. The stereocilia of a hair cell are arranged in order of increasing length from one side of the cell to the other (see Figure 7-28B and C). A plane of symmetry through the kinocilium bisects the

stereocilia, making a hair cell bilaterally symmetric, with the top beveled like a hypodermic needle. In most organs, the hair bundles are coupled to some kind of accessory structure through their kinocilia. Stimuli that affect the accessory structure are transmitted to bundles of stereocilia through bonds that connect the accessory structures and kinocilium to the stereocilia. In addition, if the tip of the bundle of stereocilia is touched with a fine probe, the bundle moves as a unit, regardless of the direction of stimulation. The exact process by which pressure or force from the outside world moves bundles of stereocilia depends on the specific arrangement of hair cells and accessory structures within each sense organ, but ultimately it is the movement of the stereocilia that produces an electrical signal. When the cilia bend toward the tallest cilium, a hair cell depolarizes; whereas, when they bend in the opposite direction, the cell hyperpolarizes (see Figure 7-28D). (If the stereocilia bend to either side, rather than toward or away from the kinocilium, Vm remains the same.) At rest, about 15% of the channels in a hair cell are open, producing a resting potential of about -60 mV. Hair cells do not produce APs. Instead, they form chemical synapses onto afferent neurons and release neurotransmitter in a graded fashion, depending on V, in the receptor neuron; the afferent neurons then carry information into the central nervous system. The amount of transmitter released onto the afferent neurons determines their frequency of discharge. Notice that the input-output relation for hair cells is markedly asymmetrical (see Figure 7-28D); that is,

240


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SENSING THE ENVIRONMENT

241

........................................ Figure 7-28 (At left) The membrane potential of hair cell receptors changes when the cilia are moved away from a rest position. (A) Electron micrograph of a cross sect~onthrough the cilia of a hair cell. The large cilium, containing a typical 9 Zstructure of microtubules, is the kinocilium; the others are stereocilia. (B) Scanning electron micrograph showing the structure of a hair cell from the neuromast of a giant Danio fish. (C)Diagram of a typical hair cell showing the anatomical relations of the stereocilia and the kinocilium The hair cell releases transmitter onto an afferent neuron, which carries the sensory signal t o the central nervous system. It also receives synapses from efferent neurons. Depending on the direction in which the cilia are bent, the hair cell can either increase or decrease the frequency of APs in the afferent fiber. A linear back-and-forth motion applied t o the cilia produces intracellular potential changes that can b e recorded with a microelectrode. Extracellular recording from the afferent axon shows APs associated with changes in Vm in the receptor cell. (D) Input-output relation for a hair cell. Note that the depolarization produced by movementtoward the kinocilium is largerthan the hyperpolarization in response t o movement away from the kinocilium. [PartA from Flock, 1967; part B courtesy of Christopher Braun; part C adapted from Harris and Flock, 1967; part D adapted from Russell, 1980.1

+

the depolarization produced by a given amount of displacement toward the tallest cilium is larger than the hyperpolarization produced by a similar displacement of the cilia in the opposite direction. This asymmetry is important because, when hair cells are subjected to symmetrical vibrations such as sound waves, changes in the membrane potential can faithfully follow the alternating phases of the stimulus only up to frequencies of several hundred hertz (Hz)but sound frequencies are often much higher than this value. At higher frequencies, the response to the vibrations fuses into a steady depolarization; even if the stimulus displaces the cilia in both directions by equal amounts away from zero displacement, the hair cell will depolarize. This steady depolarization in response to high frequency stimuli produces steady, rather than modulated, transmitter release by the hair cell and, hence, high frequency firing of the afferent neurons. The details of transduction by hair cells are presented later (see Excitation of cochlear hair cells).

Organs of Equilibrium

airs embedded

Figure 7-29 The lateral-line sensory system of fish and amphibians is based on hair cells. The drawing shows the location of these receptive organs along the body of an African clawed frog, Xenopus. The lower diagram shows a cross-section through part of the lateral line, illustrating the cupula, an accessory structure that bends cilia when it is displaced by motion of the surrounding water. Compare the structure of this organ with the hair cells shown in Figure 7-28.

The simplest organ that has evolved to detect an animal's position with respect to gravity or its acceleration is the statocyst. Forms of this type of organ are found in a number of animal groups, ranging from jellyfish to vertebrates. (Interestingly, insects lack these sense organs and apparently depend entirely on other senses, such as vision or joint proprioceptors, for orientational information.) A statocyst consists of a hollow cavity lined with ciliated mechanoreceptor cells that make contact with a statolith, which can be sand grains, calcareous concretions, or some other relatively dense material (Figure7-30A). The statolith is either taken up from the animal's surroundings or secreted by the epithelium of the statocyst. For example, a lobster loses its statoliths at every molt and replaces them with new grains of sand. In either case, the statolith must have a higher specific gravity than the surrounding fluid. As the position of the animal changes, the statolith rests on different regions of the statocyst. When a lobster is tilted to the right about its longitudinal axis, the statolith rests on the receptor cells on the right side of the statocyst, stimulating them and causlng a tonic discharge in the sensory fibers from the stimulated receptor cells (Figure 7-30B). Recordings from many different fibers of a statocyst reveal that each cell fires maximally in response to a certain orientation of the lobster (Figure 7-30C). Information from these receptors travels to the central nervous system and sets up reflex movements of the appendages. This pattern of information processing was confirmed in a clever experiment in which molting lobsters were presented with iron filings, rather than with sand. They replaced their statoliths with iron filings, allowing the position of the iron statolith to be manipulated by a magnet. As the magnet was moved through space, pulling on the iron statolith, the lobsterwhose position with respect to gravity had not changedproduced a series of compensatory postural responses.

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nule

C

0

20

40 Degrees

60

Figure 7-30 Statocysts sense accelerat~onand the pos~t~on of an an~rnal w ~ t hrespectto grav~ty(A) Structure of a statocyst In a lobster Astatol~th rests on the receptor part of an array of ha~rcells (B) Act~onpotent~als recorded from ~nd~v~dually d~ssectednerve f~berswh~lethe lobster was tllted Each recording shown here was made from a d~fferentf~berThe

trace below each recording indicates the time course of the tilt and the angle to which the animal was tilted. (C) Frequencies of APs recorded from different fibers plotted as a function of the position of the animal. Each cell responded with a maximum rate of discharge at a different position. [Adapted from Horridge, 1968.1

The Vertebrate Ear

fluid moves in one direction and inhibited when it moves in the opposite direction. The orthogonal arrangement of the three canals allows them to detect any movement of the head in three-dimensional space. Below the semicircular canals, larger bony chambers contain three more patches of hair cells called maculae. Mineralized concretions termed otoliths are associated with the maculae, similar to the statoliths associated with statocysts. The otoliths signal position relative to the direction of gravity; in the lower vertebrates, they can also detect vibrations, such as sound waves, in the surrounding medium. Sensory signals from the semicircular canals are integrated with other sensory input in the brain stem and in the cerebellum for the control of postural and other motor reflexes.

The ears of vertebrates perform two sensory functions, each of which is based on the activity of hair cells. Some structures of the ear, the organs of equilibrium, perform like the statocystsof invertebrates, reporting on the animal's position with respect to gravity and acceleration through space. Other structures, the organs of hearing, provide information about vibrational stimuli in the environmentstimuli that are called sound if they fall within a particular frequency range.

Vertebrateorgans of equilibrium In vertebrates, the organs of equilibrium reside in a membranous labyrinth that develops from the anterior end of the lateral-line system. It consists of two chambers, the sacculus and the utriculus, that are surrounded by bone and filled with endolymph, a specialized fluid. Endolymph differs from most extracellular fluids because it is high in K+ (about 150 mM in human beings) and low in Na+ (about 1 mM in human beings); the significance of this unusual composition is considered in the section titled The mammalian ear. The utriculus gives rise to the three semicircular canals of the inner ear, which lie in three mutually perpendicular planes (Figure 7-31). Hair cells in the three orthogonal semicircular canals detect acceleration of the head. As the head is accelerated in one of the planes of the canals, the inertia of the endolymphatic fluid in the corresponding canal ~roducesa relative motion of the endolymph past a gelatinous projection, the cupula, which moves the cupula. When the cupula moves with respect to the cilia of the hair cells at its base, V, of the hair cells changes. All hair cells in the canal are oriented with the klnocilium on the same side, so all hair cells attached to the cupula are excited when the

The mammalian ear Sound in the environment has led to the evolution of hearing in many phyla. Hearing allows an animal to detect predators or prey and to estimate their location and distance while they are still relatively far away. Sound also plays an important role in intraspecific acoustic communication, which often requires subtle tuning of both production and reception. Sound is a mechanical vibration that propagates through air or water, traveling as waves of alternating high and low pressure, accompanied by a backand-forth movement of the medium in the direction of propagation. The nature of sound, particularly the differences in how it is conducted through air and through water, has set distinct constraints on its detection. The evolution of hearing illustrates many different mechanisms that have evolved to solve the various problems presented by the physical nature of sound. A well-studied example, which we examine here, is the mammalian ear.

243

SENSING THE E N V I R O N M E N T

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Figure 7-31 The human auditory organs and organs of equilibrium are located in the ear. (A) The major parts of the ear. (B) The semicircular canals and cochlea. Thestapes has been removedto reveal the oval window. The pathway taken by auditory signals is shown by black arrows. At the far right, a section has been removed from the cochlea to reveal the inner structure. (Figure 7-33 shows this structure in more detail.) (C) Detailed structure of two parts of the organs of equilibrium. The cilia of re-

ceptors in a semicircular canal are embedded in the gelatinous cupula. When fluid moves in the canal, the cupula bends the cilia (left). Particles called otoconia rest on the cilia of receptors in the sacculus (one of the maculae).Changes in the position of the head cause the otoconia to shift posit~on,changing how much the cilia are bent (right). [Parts A and B adapted from Beck, 1971; part C adapted from Williams et al., 1995.1

External ear, auditory canal, and middle ear The structure of the external ear acts as a funnel that collects sound waves in the air from a large area and concentrates the oscillating air pressure onto a specialized surface, the eardrum or tympanic membrane. The external structures of the ear-the pinna and tragus-facilitate the collection of sound waves. The shell-like outer structures, and in some species the mobility of the shell, can modify the directional sensitivity of the auditory system. In some species, including human beings, the acoustical properties of the external ear amplify sound in particular frequency ranges. In addition, the human ear emphasizes the spatial distribution of stimuli by amplifying sounds coming from some directions more than it does sounds from other directions (Figure 7-32).

To be detected, airborne vibrations must be transmitted to the fluid-filled inner ear, where the receptor hair cells reside. The difficulty of communicating across a air-liquid interface can be appreciated by trying to talk with someone who is under water. Most of the sound energy generated in air is reflected back from the water's surface, so it is difficult to generate sufficient energy with airborne sounds to move the water at the required frequency and displacement. This kind of situation is called acoustical impedance mismatch. In the ear, this mismatch is partially overcome by a series of three small bones connected in series that are attached to the tympanic membrane at one end and to the oval window of the cochlea at the other. These bones, the auditory ossicles (labeled incus, malleus, and stapes in Figure 7-31A),

Figure 7-32 The structure of the human plnna and tragus selectively a m p l ~ f ~ espes clf~cfrequenc~es of sound Thls graph shows the galn In pressure at the eardrum over what the pressure would b e ~f sound Irnplnged on the ear canal w ~ t hall external ear structures removed If there were n o ampllthe graph would be a hor~zontall ~ n e f~cat~on, lntersectlng w ~ t hthe ordinate at an arnpltfic a t ~ o nfactor of 1 Values above 1 lnd~cate

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evolved from the articulation points of the posterlor jaw and are now located in the middle ear. Changes In air pressure produced by sound waves in the external auditory canal cause the eardrum to move, whlch transfers the energy first to the ossicles, and then to the structures of the inner ear. In the inner ear, the first structure to receive the mechanical input is the oval window, which forms the outermost surface of a fluld-filled chamber (the cochlea) that contains the receptor hair cells. At the other end of the fluid-filled compartment is another membrane, the round window. There are two important consequences of this arrangement. First, the properties of the mechanical coupling between the eardrum, ossicles, and oval window amplify the signal by about 1.3 times. Second, the pressure of the signal is greatly amplified between the eardrum and the oval window because the eardrum has an area of about 0.6 cm2, whereas the oval wlndow is smaller, about 0.032 cm2.This ratio of about 17: 1 between the areas of the two membranes means that the sound pressure onto the typanum is concentrated onto the smaller area of the oval wlndow, producing a much greater pressure, whlch IS important because the inertia of the cochlear fluid on the other side of the oval window is greater than that of alr. The increase in pressure helps to efficiently transfer airborne vibrations to the cochlear fluld. As a consequence of these two mechanical features, signals arriving at the eardrum are amplified by at least a factor of 22 by the tlme they reach the cochlea. Structure and function of the cochlea This mechanically amplified sound input is transduced into neuronal signals by the hair cells of the inner ear. The hair cells of the mammalian ear are located in the organ of Corti in the cochlea (Figure 7-33). The movement of fluid in the cochlea causes a vibration of the hair cells, which displaces their stereocilia; the hair cells, in turn, excite the sensory axons of the auditory nerve. The hair cells in the organ of Corti resemble the hair cells of the lateral-line system in lower vertebrates, except that the kinocilium is absent from some cochlear hair cells in adults. The cochlea, a tapered tube encased in the mastoid bone, is coiled somewhat like the shell of a snail (see

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ampl~f~catlon, values below 1 lndlcate suppresslon The galn varles as a funct~onof frequency, and sounds emanating from d~fferent d ~ r e c t ~ o nare s ampl~fiedd~fferent~ally Zero degrees 1s stra~ghtin front o f the face. [Adapted from Shaw, 1974.1

Figure 7-31A and B). It is divided internally into three longitudinal compartments (see Figure 7-33A). The two outer compartments (scala tympani and scala vestibuli) are connected by the helicotrema, an opening located at the apical end of the cochlea (see Figure 7-35B).The scala tympani and scala vestibuli are filled with an aqueous fluid called the perilymph, which resembles other extracellular fluids in having a relatively high concentration of Na+ (about 140 mM) and a low concentration of K+ (about 7 mM). Between these compartments-and bounded by the basilar membrane and Reissner's membrane-is another compartment, the scala media, which is filled with endolymph (high in K+ and low in Na+),the same type of fluid that surrounds the cilia of hair cells in the organs of equilibrium. The unusual ionic composition of endolymph contributes importantly to the process of auditory transduction. The organ of Corti, which bears the hair cells that transduce auditory stimuli into sensory signals, lies within the scala media and sits on the basilar membrane, and signal transduction by the cochlear hair cells depends in part on this anatomical arrangement. Among the vertebrates, only mammals possess a true cochlea, although birds and crocodilians have a nearly straight cochlear duct that contains some of the same features, including the basilar membrane and the organ of Corti. The other vertebrates have no cochlea. Some of the lower vertebrates are able to detect sound waves through the activity of hair cells associated with the otoliths of the utriculus and sacculus and with the lagena, one of the three maculae in the organs of equilibrium. The hair cells of the mammalian cochlea encode both the frequency (i.e., the pitch) and the intensity of sound. The adult human cochlea contains four rows of hair cells, one inner and three outer rows, with about 4000 hair cells in each row (see Figure 7-33B). The stereocilia of the hair cells contact the overlying tectorial membrane. The cilia are bent by shearing forces (i.e., a force perpendicular to the axis of the cilia) that arise when the hairs move through the gelatinous mucus that coats the tectorial membrane. Sound vibrations are transferred by the ossicles to the oval window and then pass through the cochlear fluids and the membranes that separate the cochlear compartments


245

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Reissner's membrane \

Spiral glnnlinn

U

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and efferent nerve fibers

Figure 7-33 Sound stimuli are transduced by hair cells in the cochlea. (A) Cross section through cochlear canal, made at about the location illustrated in Figure 7-31B, showing the two outer chambers (the scala vestibuli and the scala tympani) and the organ of Corti attached to the

basilar membrane in the central canal. (B) Enlargement of the organ of Corti. The cilia of the hair cells are embedded in the gelatinous layer of the tectorial membrane, whereas their cell bodies are fixed with respect to the basilar membrane.

(both Reissner's membrane and the basilar membrane) before their energy is dissipated through the membrane-covered round window. The distensibility of the round and oval windows is an important adaptation because, if the fluid-filledcochlea were encased entirely by solid bone, the displacements of the oval window, the fluid, and the internal tissues would be very small. The distribution of the perturbations within the cochlea depends on the frequencies of vibrations entering the oval window. To visualize this,

imagine a displacement of the eardrum transferred through the ossicles of the middle ear to the oval window. Vibrations displace the incompressible perilymph along the scala vestibuli, through the helicotrema, and back through the scala tympani toward the round window. Excitation of cochlear hair cells Electrical recordings made at various locations in the cochlea show fluctuations in electrical potential that are similar in frequency, phase, and

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amplitude to the sound waves that produced them. These cochlear microphonics.result from the summation of receptor currents from the numerous hair cells that were stimulated by movements of the basilar membrane. The actual transduction event occurs when a perturbation of the basilar membrane forces the tips of the stereocilia to bend laterally, because the basilar membrane has moved with respect to the tectorial membrane (Figure 7-34). This mechanical deflection directly causes ion channels in the tips of the stereocilia to open. In the past few years, our understanding of these events has grown dramatically, although questions still remain about the details of transduction. The perceptual threshold of cochlear hair cells corresponds to a deflection of 0.1-1.0 nm, which is equivalent to a change in membrane current of only about 1 pA through ion channels in the hair cell membrane. These channels have been experimentally shown to be permeable to many small, monovalent cations (e.g., Li+, Na+, K+, Rb+, and Cs+).When they open in uiuo, K+ ions and some Ca2+ ions enter the cell from the endolymph. (The high concentration of K+ in the endolymph produces a net inward driving force on K+, unlike the usual situation in which V, - E, is an outward force. This inward K+ current depolarizes hair cells, because it adds positive charge to the inside of the cells.) On the basis of measurements of current flow, it has been estimated that there are about 30-300 channels per bundle of stereocilia, which implies that as few as one to five channels per stereocilium may be responsible for transduction. The channels are thought to be opened directly by a mechanical stimulus because, when isolated bundles of stereocilia are abruptly deflected in experiments, the transduction current increases with an extremely short latency (about 40 ps). The brevity of this latency period makes it unlikely that an enzymatic or biochemical step is included in this process. This interpretation is reinforced by patchclamp experiments indicating that the channels open faster when the deflection is larger, again suggesting a direct

Hinge point

Tectorial membrane

hair cells

mechanical influence on the conformational states of the channel. Several factors affect the sensitivity of hair cells. Each hair cell in the cochlea appears to be tuned to a particular band of sound frequency as a result of both mechanical and channel properties. Each cell has a resonance frequency that is determined by the length of the stereocilia in the hair bundle. Cells with long hairs are most sensitive to lowfrequency sounds, whereas cells with short hairs are tuned to high-frequency sounds. In addition, each cell responds maximally to a particular frequency of electrical stimulation. This electrical resonance frequency is determined by the balance of currents through voltage-gated Ca2+channels and through Ca2+-sensitiveK+ channels in the basal membrane (which is exposed to perilymph). The outer hair cells of the cochlea may contribute to tuning in the cochlea by modifying the mechanical properties of the organ of Corti. The outer hair cells make-few afferent connections, but they receive a large number of efferent synapses. When these cells are stimulated electrically during experiments, they shorten when depolarized and elongate when hyperpolarized. Thus, it is possible that the outer hair cells could modify the mechanical coupling between the inner hair cells and the tectorial membrane, which would change transduction. It remains to be demonstrated that this mechanism actually affects audition. Hair cells adapt to changes in the position of their stereocilia, a process that has been particularly well studied in the bullfrog sacculus. When the cilia of frog hair cells are deflected by a probe and held at the new position, the operating range of the cell adapts within milliseconds to this new tonic position, causing the hair cell to then respond to small changes in position away from this new set point. Calcium ions have been shown to play a pivotal role in the process, evidently by modifying the tension in the spring that opens the transduction channels. Finally, efferent input onto a hair cell can decrease the cell's response to sound and broaden its frequency selectivity by opening inhibitory

deflected up

membrane

hair cell Figure 7-34 Movement of the bas~larmembrane w ~ t hrespect t o the tector~almembrane produces shear on the stereoc~l~a of cochlear ha~r cells The tector~almembrane sl~desover the organ of Cort~,because the tector~almembrane and the bas~larmembrane p~votabout d~fferent

point polnts when they are d~splacedby waves travel~ngalong the cochlea The movements are greatly exaggerated In thls d~agram[Adapted from Dav~s,1968 ]


247

...................................... K+ channels, which short-circuit the cell's electrical resonance. Taken togetheq the attributes of the hair cells reveal their exquisite tuning. However all of the adaptations that make hair cells so extremely sensitive also make them highly vulnerable to overstimulation, which can cause rupture at the base of the stereocilia. Acoustic trauma can produce permanent hearing loss that is worst at the frequencies of sound that actually damaged the hair cells. Although some cold-blooded vertebrates can recover from such trauma, the loss is permanent in mammals.

The receptor currents of the hair cells faithfully transduce the movements of the basilar membrane over the whole spectrum of audible sound frequencies. The cells transmit their excitation through chemical synapses onto sensory axons of auditory neurons that have cell bodies located in the spiral ganglion. Release of neurotransmitter by the hair cells modulates the firing rate of these axons, which travel in the uestibulocochlear (eighth cranial) nerve and synapse onto neurons in the cochlear nucleus. In fact, the hair cells in the inner row receive about 90% of the contacts made by neurons of the spiral ganglion, suggesting that the inner row of cells is largely responsible for detecting sound. In contrast, hair cells of the outer three rows receive many efferent synapses and may participate in modulating the sensitivity of the cochlea by changing the mechanical relation between the basilar and tectorial membranes. Frequency analysis by the cochlea Pioneering studies of the exposed cochlea, carried out by Georg von Btktsy, greatly enhanced our understanding of how the auditory system can encode information about the frequency of stimuli. His studies showed that:

1. In response to a pure sine wave tone, the perturbations of the basilar membrane have the same frequency as the tone. 2. Low frequency perturbations move as a traveling wave along the whole length of the basilar membrane. 3. The location where the basilar membrane is displaced maximally by a tone is a function of the frequency of the tone. High frequencies displace only the initial parts of the membrane, whereas low frequencies displace more distant parts. Thus, each point along the basilar membrane is displaced most effectively by some unique frequency, and that point

varies in an orderly fashion with higher frequencies displacing the basilar membrane close to the oval window and lower frequencies displacing the basilar membrane farthest from the oval window. For sounds up to about 1kHz, APs in the auditory sensory axons appear to follow the fundamental frequency. Above this level, the time constant of the hair cells and the electrical properties of the axons in the auditory nerve prevent a one-to-one correspondence between sound waves and electrical signals. In this higher frequency range, some other mechanism must inform the central nervous system of the sound frequency. Hermann von Helmholtz noted in 1867 that the basilar membrane consists of many transverse bands that increase gradually in length from the proximal end to the apical end of the basilar membrane (from about 100 pm long at the base to about 500 pm long at the apex), which reminded him of the strings of a piano and led him to propose his resonance theory. He proposed that various locations along the basilar membrane vibrate in resonance with a specific tonal frequency while the other locations remain stationary, just as the appropriate string of a piano resonates in response to a tone from a tuning fork. This theory was later challenged by von Btktsy (1960), who found that the movements of the basilar membrane are not standing waves, as Helmholtz suggested, but consist instead of traveling waves that move from the narrow base of the basilar membrane toward the wider apical end (Figure 7-35). These waves have the same frequency as the sound entering the ear, but they move much more slowly than sound moves through air. A familiar example of a traveling wave can be seen by moving the free end of a rope that is secured at the other end. Unlike a rope, however, the basilar membrane has mechanical properties that change along its length. The compliance of the membrane (the amount that the membrane will stretch in response to a given amount of force) increases from its narrow end to its broad end, which causes the amplitude of a traveling wave to change along the length of the membrane (see Figure 7-35). The position along the cochlea at which the displacement of the basilar membrane is maximal-causing the hair cells at that location to be stimulated maximally-depends on the frequency of the traveling waves and consequently on the frequency of the stimulating sound. The traveling waves produce maximum displacements near the basal end of the cochlea when the stimulating sound has a high frequency. The region of maximal displacement moves along the basilar membrane toward the apex as the frequency of the sound drops. The extent of membrane displacement at any point along the basilar membrane determines how strongly the hair cells are stimulated, so it also determines the rate of discharge in sensory fibers arising from different parts of the basilar membrane. Even at maximum amplitude, all of the movements are very tiny: the loudest sounds produce displacements of the basilar membrane of only about 1pm. The movement of the hair cell cilia is much smaller, and the threshold of stimulus detection is at the lirmt of the thermal noise.

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Distance from oval window Figure 7-35 Sound sets up traveling waves along the basilar membrane. (A) Waves move in the direction shown by the arrow. Lines a and b indicate the shape of the membrane attwo differenttimes. The light dashed lines indicate the envelope generated by the movements, which in this case has the largest amplitude near the apical end. (The amplitudes of the waves are greatly exaggerated in this figure). (B) The cochlea drawn as if it had been straightened out. The locations that respond maximally to sounds of d~fferentfrequency are indicated below. [Part A adapted from Von Bekesy, 1960; part B adapted from Moffett et al., 1993.1

An Insect Ear

Many organisms have ears that operate differently from the mammalian ear, and it is instructive to consider at least one of them to know the variation possible. Crickets find their mates through auditory communication: male crickets produce a song whose pattern is specific to their species, and female crickets are attracted to the song of their species. Cricket ears are located on the first thoracic legs, and they are associated with the respiratory passages, called tracheae (Figure7-36). Each ear contains a tympanum, analogous in function to the tympanic membrane of the mammalian ear, and the changes in air pressure that constitute sound are carried through the tracheae to the tympanum. The tym. panum is exposed to changes in air pressure from both outside the animal and from inside through the tracheae. If a sound arises on the right side of the cricket, it will directly cause the tympanum on the right side to vibrate. In addition, it will be carried through the tracheal system to the left tympanum, causing the left tympanum to vibrate as well.

Figure 7-36 A cricket's ears are located on its anterior thoracic legs. The tympanum receives sound stimuli through the tracheal ducts and vibrates in response either t o sounds from the outside or to sounds carried through the inside of the animal through the tracheae. Nerve cells associated with the tympanum transduce the sound stimuli.

The differences in the time at which the stimulus reached the right and the left tympani can be used to help localize sounds, a principle that is used by vertebrates as well (see Chapter 11).In some species, hair cells are associated with the tympanum, suggesting that excitation within the insect ear may be similar to excitation in the mammalian ear. The insect ear incorporates some of the features of the mammalian ear: a channel conducts sound waves to a movable surface, which vibrates in response to the sound waves. When the tympanum vibrates, it excites receptors either directly or indirectly, sending signals to the central nervous system. However, the tracheal system allows sounds to travel through the body of the animal and to move the tympanum from either the inside or the outside of the animal's body.

ELECTRORECEPTION Hair cells located in the skin of certain species of bony and cartilaginous fishes have lost their cilia and become modified for the detection of electric currents in the water. The sources of these currents are either the fishes themselves or currents that originate in the active tissues of other animals in the vicinity. The weakly electric fishes (such as the Mormyrids) possess specialized electric organs that generate the fields sensed by these receptors; they can use the fields for communicating with one another and for navigating in turbid water. In fact, all electrically active tissue can generate electrical fields, and some sharks are especially adept at locating their prey by sensing the electric currents emanating from the active muscles of the animal. The electroreceptors of fishes are distributed throughout the head and body in the lateral-line system (Figure 7-3712). In weakly electric fishes (as opposed to strongly electric fishes, such as the electric eel), electrical pulses produced by modified muscle or nerve tissue at one end of the body reenter the fish through epithelial pores in the lateral-line system. At the base of each pore, the current encounters an electroreceptor cell (Figure 7-37B), which makes synaptic

Posterior lateral-line nerve

A

orsal branch

Receptor areas

B

Current

I I

Stimulus current

Receptor potential

-

Y

Resting discharge level

Nerve impulses

"

-1

0

+1

Stimulus voltage (mV) Figure 7-37 Electroreceptorcells are specialized hair cells located along the lateral line of many species of fishes. (A) Positions of the electric organ and of the lateral-linenerve trunk and distribution of electroreceptor pores in the weakly electric fish Gnathonernus peters;;. (B) At the base of each electroreceptor pore there is an electroreceptor cell whose apical membrane has a low electrical resistance compared with that of the basal membrane. (C) Receptor cells release transmitter mole-

cules spontaneously (a) Current enter~ngthe cell (b)depolar~zes~ tIn, creasing the rate of release and, hence, the frequency of APs In the sensory f~ber~nnervatlngthe cell Current leav~ngthe cell (c) decreases the rate of release The amount of transm~tterreleased by the receptor cells changes when V,, IS altered by only a few m~crovolts[Adapted from Bennett, 1968 ]

contact with axons of the eighth cranial nerve that innervate the lateral-line system. The cell membrane facing the exterior has a lower electrical resistance than does the basal membrane, so most of the potential drop caused by the current moving across the cell occurs across the basal membrane, depolarizing it. Depolarization of the basal membrane activates Ca2+channels in the membrane, and the resulting influx of Ca2+at the base of the cell increases the release of synaptic transmitter by the receptor cell. This transmitter increases the frequency of APs in the sensory fiber that innervates the receptor. Conversely, a current flowing out of the body of the fish hyperpolarizes the basal membrane of the receptor cell and decreases the release of transmitter below the spontaneous rate. Thus, the firing frequency in the sensory fiber goes up or down, depending on the direction of the current flowing through the electroreceptor cell (see Figure 7-37B and C). The sensitivity of these receptors and their sensory fibers, like that of the hair cells of the vertebrate ear, is truly remarkable. As seen in Figure 7-37C, changes in sensory nerve discharge occur in

response to changes in V, of the receptor cell of as little as several microvolts. The train of current pulses flows through the water from the posterior to the anterior end of the fish (Figure 7-38).Any object whose conductivity differs from that of water will distort the lines of current flow. The lateral-line electroreceptors detect the distribution of current flowing back into the fish through the lateral-line pores on the head and anterior end of the body and can detect the changes in field produced by objects in the water. This sensory information is then processed in the greatly enlarged cerebellum of the fish, enabling it to detect and locate objects in its immediate environment. Electrical signals are produced by other species of fishes and used for quite a different task. In contrast with the weakly electric fishes that use electrical fields for navigation and signaling, some eels, torpedoes, and other fishes produce a powerful discharge of current to stun enemies and prey. These strongly electric fishes produce a continuous series of synchronous, relatively high frequency

250


............................................................................... Figure 7-38 Electroreception allows electric fishes to recognize and to locate objects In their environment. An object that has a conductivity greater than that of water deflects current toward the axls of flow. An object whose conductivity is lower than that of water (inset) diverts the current away from the axis of flow. [From "Electric Location by Fishes," by H. W. Lissman. Copyright 0 1963 by Scientific American, Inc. All rights resewed.]

Lateral-line system

depolarizations with their electric organs, and the way in which the electrical discharges of these fishes are generated and used resembles the way in which muscles are controlled to produce movement (see Chapter 10).

THERMORECEPTION Temperature is an important environmental variable, and many organisms acquire sensory information about temperature from the action of specialized nerve endings, or thermoreceptors, in the skin. Higher-order neurons receive input from thermoreceptors and contribute to the mechanisms that regulate the temperature of the body (see Chapter 16).In addition, some of the neurons in the hypothalamus of vertebrates are able to detect changes in body temperature. Temperature receptors can be remarkably sensitive. The infrared (radiant heat) detectors in the facial pits of rattlesnakes provide an example (Figure7-3912).The receptor membrane consists of the branched endings of sensory nerve fibers, with no readily apparent structural specializa-

tions, and the endings appear to detect changes in tissue temperature, rather than the radiant energy itself. The mechanisms by which temperature changes can alter receptor output are not known. The sensory axons from the pit organs of the rattlesnake increase their firing rate transiently when the temperature inside the pit increases as little as 0.002"C, and this change in receptor firing rate can modify behavior. For example, a rattlesnake can detect the radiant heat emitted by a mouse that is 40 cm away if the body temperature of the mouse is at least 10°C above the ambient temperature. Furthermore, the temperature receptors lie deep within the facial pits, and this arrangement allows the snake to detect the direction of a source of radiant heat (Figure 7-39B). Both the external skin and the upper surface of the tongue of mammals contain two kinds of thermoreceptors: those that increase their firing when the skin is warmed ("warmth" receptors) and those that increase their firing when the skin is cooled ("cold" receptors). These receptors, too, are quite sensitive. Human beings can detect a change in skin temperature of as little as O.Ol°C. The two cate- -


251

...................................... A 0

Outer chamber

Cold

Membrane

\ Temperature

1n6er chamber

B

(OC)

~rdnnchof trigeminal nerve

Object in front stimulates both pits

Time (s) Figure 7-40 The frequency of APs in mammalian thermoreceptorsvaries with temperature at the surface of the body. (A) The steady-state firing rate of cold and warmth receptors that arborize in the surface of a mammal's tongue. (B) Time course of a cold receptor's response when the tongue was f~rstcooled and then rewarmed as shown In black. [After Zotterman, 1959.1

30"-3S°C, this pattern changes for both kinds of receptors, and the frequency of APs drops (see Figure 7-40A). The response of the thermoreceptors consists of a large transient change in firing rate, followed by a longer-lasting, steadystate phase. The transient phase is an accurate response to any change in temperature (Figure 7-40B),even though the steady-state phase behaves as shown in Figure 7-40A.

VISION Figure 7-39 The facial pits of rattlesnakes contain extremely sensitive thermoreceptors. (A) Structure of a facial pit in the rattlesnake Crotalus viridis. (B) The position of the facial pits makes thermoreception by the pit organs directionally sensitive. [Adapted from Bullock and Diecke, 1956.1

gories of thermoreceptors are distinguished from one another in accord with how they respond to temperature changes near the normal temperature of the human body (about 37°C). Both warmth and cold receptors increase their firing rate as the temperature becomes increasingly different from 30"-35°C (Figure 7-40A): warmth receptors fire faster as the temperature gets warmer; cold receptors fire faster as the temperature gets colder. However, when the temperature becomes sufficiently different from

Since the Earth formed more than 5 billion years ago, sunlight has been an extremely potent selective force in the evolution of living organisms, and most organisms are able to respond to light in some way. Photoreception consists of transducing photons of light into electrical signals that can be interpreted by the nervous system, and photoreceptive organs-typically called eyes-have evolved into many shapes and sizes and with many distinct designs. Interestingly, although the physical structure of eyes varies greatly among species, visual transduction is based upon a very highly conserved set of protein molecules that provide an optical pathway leading light to the photoreceptive surface and that capture photons within the photoreceptors. This conservation of visual molecules suggests that, when suitable biochemical means had evolved to solve the

252


............................................................................

problem of capturing light's energy, the sequences were conserved even though they were packaged into organs with highly diverse structural properties. For example, the opsins are protein visual pigment molecules. Each molecule includes seven transmembrane domains. Opsins are coupled to photopigment molecules, which are structurally altered by the absorption of photons and which in turn modify the properties of the opsin protein (see Figure 7-3). Opsins are found widely in the animal kingdom, even in photoreceptive structures that are extremely simple and that lack the features that would constitute an eye. In many organisms, the structure of the eye has evolved to collect and focus incident light rays before they arrive at the site of transduction. Eyes refract light through highly concentrated soluble proteins that are formed into lenses, and these refractive structures also have an interesting evolutionary history. We first consider how eyes collect and focus light.

The physics of light tightly constrains the structure of an eye that will produce a usable image. Most of the possible designs have been "discovered" in the course of evolution, giving rise to similar structures in unrelated animals. One of the most well known examples of convergent evolution is the similarity of eyes in the phylogenetically unrelated squid and fishes. These eyes are similar in many details because optical laws have dictated convergent solutions to the problem of seeing under water. In contrast, the eyes of human beings and fishes are similar because they share com-

mon evolutionary descent, although they differ to some extent because the two species live in different optical media. The evolution of eyes has proceeded in two stages. Virtually all major animal groups have evolved simple eyespots consisting of a few receptors in an open cup of screening pigment cells (Figure 7-41A). Some biologists estimate that such photon detectors have evolved independently between 40 and 65 times. Eyespots provide information about the surrounding distribution oflight and dark, but they do not provide enough information to allow detection of either predators or prey. For pattern recognition or for controlling locomotion, animals need an eye with an optical system that can restrict the acceptance angle of individual receptors and form some kind of image. This stage of optical evolution happened less frequently, occurring in only 6 of the 33 metazoan phyla (Cnidaria, Mollusca, Annelida, Onychophora, Arthopoda, and Chordata). Because these phyla contain about 96% of all extant species, it is tempting to speculate that having eyes confers significant selective benefits. Ten optically distinct designs for image-formingeyes have been discovered to date. They include nearly all the possibilities known from physical optics except Fresnel and zoom lenses. In addition, there are some variations, such as array optics, that have not been used by physicists studying optics. Simple eyespots are typically less than 100 pm in diameter and contain between 1 and 100 receptors. Even simple eyespots allow some visually guided behavior. In protozoa and flatworms, the direction of a light source is detected with the help of a screening pigment that casts a shadow on the photoreceptors. Some flagellates, for exam-

A

E

Optic Mechanisms: Evolution and Function

C

-

+

Shallow pit

Vertebrate eye

I' B

slble evolutionary relations among types of eyes [Adapted from Land and Fernald, 1992.1

D

Pinhole eye

6

Figure 7-41 The structures of eyes Incorporate many different optlcal prlnc~ples(A) The slmplest eye conslsts of a shallow open plt llned wlth photoreceptor cells (B) In sllghtly more complicated eyes, the aperture of the eye IS small In proportion to the slze of the eye, and the eye operates slmllarly to a pinhole camera (C)An alternative Improvement allowlng Image format~onIS the addltlon of a refracting element between the aperture and the layer of photoreceptors (D)Three lenses arranged In serles Improve the optlcal propertles of the eye In Pontella, a copepod (E) The vertebrate eye IS an elaborat~on of a slmple eye to wh~cha small aperture and posa lens have been added Arrows ~nd~cate

Many lenses in series


253

...................................... ple, have a light-sensitive organelle, near the base of the flagellum, that is shielded on one side by a pigmented eyespot. This shielded organelle provides a crude, but effective, indication of directionality. As the flagellate swims along, it rotates about its longitudinal axis roughly once per second. If it enters a beam of light shining from one side and perpendicular to its path of locomotion, the eyespot is shaded each time the shielding pigment passes between the source of the light and the photosensitive part of the base of the flagellum. Each time this happens, the flagellum moves just enough to turn the flagellate slightly toward the side bearing the shielding pigment. The net effect is to, turn the flagellate toward the source of the light. The simplest eyes are improvements on eyespots that have been achieved by reducing the size of the aperture to produce a pinhole eye (Figure 7-41B) or by adding a refracting structure (Figure 7-41C). The evolutionarily ancient cephalopod mollusc Nautilis has a pinhole eye that, except for the absence of a lens, is quite advanced. It is nearly 1 cm in diameter and the aperture is variable, expanding from 0.4 to 2.8 mm. In addition, extraocular muscles compensate for the rocking motion produced when the animal swims, stabilizing the eye. Most aquatic animals have a single-chambered eye with a spherical lens (see Figure 7-41C). This type of lens provides the high refractive power needed to focus images under water, but it poses the problem of spherical aberration. The lenses found in fishes and cephalopods avoid this

Compound

eye

Vertebrate eye

difficulty because the material of the lens is not homogeneous. Instead, it is dense with a high refractive index in the center and has a gradient of decreasing density and refractive index toward the periphery. This pattern was first noted in 1877 by Matthiessen, who showed that a consequence of the density gradient is a short focal length, about 2.5 times the radius (known as Matthiessen's ratio). This remarkable gradient in density has evolved eight times among aquatic animals, suggesting that it is a very good solution and perhaps the simplest. Other aquatic species have eyes with multiple lenses. For example, the eye of the copepod Pontella (Figure 7- 4 ID) contains three lenses in series that together correct for spherical aberration. The vertebrate eye (Figure 7-41E) combines a relatively small aperture with a refractile lens. These two features together provide a very high quality image that is focused on the layer of photoreceptors in the retina, located at the back of the eye. Compound Eyes

The compound eyes of arthropods are image-forming eyes composed of many units, each of which has the features of the eye shown in Figure 7-41C. Each optic unit, called an ommatidium, is aimed at a different part of the visual field (Figure 7-42A), and each samples an angular cone that takes in about 2-3 degrees of the visual field. In contrast, in the vertebrate eye, each receptor may sample as little as 0.02 degree of the visual field. Because the receptive field of

Figure 7-42 Compound eyes produce mosaic images. (A) In a compound eye, each ommatidium samples a different part of the visual field through a separate lens. The image at the right illustrates a mosaic image of a butterfly as it would be perceived by a dragonfly at a distance of 10 cm. (B) In a simple eye, each receptor cell samples part of the field through a lens that is shared by all receptor cells. For comparison, the image at the right illustrates the same butterfly as it would be perceived by a simple vertebrate eye. Arrows indicate that the optics of the vertebrate eye invert the imaqe - on the retina, whereas the optics of the compound eye do not. [Adapted from Kirschfeld, 1971, and Mazokhin-Porshnyakov,1969.1

254


.......................................

each unit in a compound eye is relatively large, compound eyes have lower visual acuity than do vertebrate eyes. However, although the mosaic image formed by this type of eye is coarser than the image produced by a vertebrate eye (Figure 7-42B), it is certainly recognizable.

tally accessible and its activity could be monitored with simple electrical recording techniques. The visual receptor cells of the Limulus compound eye are located at the base of each ommatidium (Figure 7-43B and C). Each ommatidium lies beneath a hexagonal section of an outer transparent layer, the corneal lens. The primary photoreceptors are 12 retinular cells, which surround the dendrite of another neuron, the eccentric cell. Each retinular cell has a rhabdomere, in which the surface membrane of the cell is thrown into a dense profusion of rniaovilli, which are miniature tubular evaginations of the surface membrane (see Figure 7-43D). The microvilli greatly increase the surface area of the cell membrane in the rhabdomere. Light enters through the lens and is absorbed by molecules of the photopigment rhodopsin that are located in the receptor membrane within the rhabdomere. Transient, random depolarizations of the membrane po-

The eyes of Limulus Each ommatidium of a compound eye contains several photoreceptors. The most intensively studied invertebrate photoreceptors are those in the lateral eyes and the ventral eye of the horseshoe crab Limulus polyphemus (Figure 7-43). The two lateral eyes of Limulus are typical compound eyes, similar to those in Figure 7-42A, whereas the unpaired ventral eye is simpler in structure and more like the eyespot shown in Figure 7-41A. Most of the early electrical recordings made from single visual units were done with this lateral eye, because the eye was experimenC

Light

eccentric cell

t

L~ght

on

Figure 7-43 Early studies of the compound eyes of Limuluspolyphemus provided Insights into visual transduction. (A) The lateral eyes of the horseshoe crab Limulus are located on the dorsal carapace. (B) A cross section through a lateral eye, which is made up of ommatidia. (C) The structure of a s~ngleommatidium (outlined in red in part B). Light enters through the lens and is intercepted by visual pigment in the rhabdomeres of the retinular cells. The cells are arranged like the segments of an orange around the dendrite of the eccentric cell. The eccentric cell depolarizes and generates APs when light shines on the rhabdomeres.(D)An electron micrograph of a cross section through the microvilli of a rhabdomere. [Part C from "How Cells Receive Stimuli," by W. H. Miller, F. Ratliff, and H. K. Hartline. Copyright 01961 by Scientific American, Inc. All r~ghtsresewed. Part D courtesy of A. Lasansky.]

1

Light

off

0.1 pm

SENSING T H E E N V I R O N M E N T

255

...................................... tential occur in the retinular cells when the eye is exposed to very dim, steady illumination. These "quantum bumps" in the recording increase in frequency when the light intensity is gradually increased, which causes more photons to impinge on the receptors. The transient depolarizations are electrical signals generated by the absorption of individual quanta of light by single photopigment molecules. A single photon captured by a single visual pigment molecule in Limulus produces a receptor current of A. This transduction event amplifies the energy of the absorbed photon between lo5 and lo6times. How can capture of a single photon lead to the rapid release of so much energy? In this case, the amplification occurs through a cascade of chemical reactions inside the cell that include G-protein activation (see From Transduction to Neuronal Output earlier in this chapter). The net effect is to open ion channels, allowing cations to enter the cell. In Limulus, the receptor current through the lightactivated channels is carried by Na+ and KC. This current causes a depolarizing receptor potential, by a mechanism similar to the depolarizing postsynaptic potential that is generated when acetylcholine activates the motor endplate channels in muscle (see Chapter 6).When the light goes off, these channels close again, and the membrane repolarizes. The sensitivity of individual photoreceptors drops with exposure to light, and this adaptation is thought to be mediated by Ca2+ions, which enter the cells when light opens ion channels and which then reduce the current through light-activated channels. Although retinular cells have axons, they apparently do not produce APs. Instead, the receptor current arising in

retinular cells spreads through low-resistancegap junctions into the dendrite of the eccentric cell, and from there the depolarization spreads to the eccentric cell axon where it generates APs. The APs are conducted in the optic nerve to the central nervous system. Although the organization of the Limulus eye is simple in comparison with that of vertebrate eyes, the Limulus visual system is capable of generating electrical activity that parallels some of the more sophisticated features of human visual perception (Spotlight 7-1). Perceiving the plane of polarized light The arrangement of cells within the ommatidia of compound eyes confers special abilities on some arthropods. For example, some insects and crustaceans can orient behaviorally with respect to the sun, even when the sun itself is blocked from their view. This ability depends on the polarization of sunlight, which is different in different parts of the sky. It has been found that many arthropods can detect the plane of the electric vector of polarized light entering the eye, and some use this information for orientation and navigation. Measurements of the birefringence (the ability of a substance to absorb light polarized in various planes) of the retinular cells in the crayfish show that the absorption of polarized light is maximal when the plane of the electric vector of light is parallel to the longitudinal axis of the microvilli of the rhabdomeres. Each crayfish ommatidium consists of seven cells, and the rhabdomeres of the seven retinular cells interdigitate, forming the rhabdome. Within the rhabdome, the microvilli of some receptors are oriented at 90 degrees to the microvilli of a second group of receptors (Figure 7-44). If the photopigment molecules

Retinular cell Microvilli of rhabdornere

Figure 7 4 4 The structure of ommatidia allows some arthropods to perceive the plane of polarized light. (A)The interdigitating rhabdomeresof separate retinular cells produce two sets of mutually perpendicular microvilli. (B) Electron micrograph of a section through the rhabdome

formed by two sets of microvilli.The upper microvilli were sectioned parallel to their longitudinal axis, and those in the lower set were sectioned perpendicular to their longitudinal axis. [Part A adapted from Horridge, 1968; part B courtesy of Waterman et al., 1969.1

256


............................................................................... SPOTLIGHT 7 - 1

judgments made by a human subject who

IS asked t o

com-

pare the lntensltles of different l ~ g hstlmuli t

SUBJECTIVE CORRELATES OF PRIMARY

2. A receptor's response t o flashes of 11ghtthat are less than 1 second In duratlon IS proportional t o the total number of photons In the flash, regardless of the actual duratlon That

PHOTORESPONSES

IS,

photoplgment molecules lsomerlzed by photons lmplnglng

tivlty in the photoreceptors and the parameters ofthe stimuli Al-

on the receptor For short flashes, a human observer cannot

though these receptors dlffer In some featuresfrom human pho-

tell the dlfference when lntens~tyand duration of the flash

toreceptors, they are slmllar In fundamental ways, such as the

are changed reciprocally

chemlcal Identity of thew vlsual plgment and some electrlcal Hartllne's work is that many features of human vlsual perceptlon, measured In psychophysical experlments, parallel the electrlcal behav~orof slngle Llmulus vlsual cells Thls relat~on suggests that some propertles of vlsual perceptlon or~glnateIn the behavior of

relatively un-

modlfled as lnformatlon undergoes further processing by the

kept con-

should be determined, wlthin some Ilmlts, by the number of

h ~ associates s In the 1930s revealed correlations between the ac-

propertles of the cells One of the most lnterestlng results of

IS

stant Thls result mlght b e expected, because the response

Studles on the L~muluseye carrled out by H Keffer Hartllne and

the photoreceptor cells themselves and remaln

the number of lmpulses generated remalns constant as

long as the product of Intensty and duration

3.

If a photoreceptor

IS

stimulated with a fllckerlng Ilght, V, will

follow the frequency of the flashes up t o nearly 10 Hz (part B ofthe adjoining flgure) Beyond this frequency, the receptor potentlal can no longer follow the flashes, Instead, the rlpples In V , fuse Into a steady level of depolarizatlon (see Flgure 7-55 also) Actlon

potentials In sensory flbers no longer

follow the patternlng of the flashes but, Instead, are generated at a steady rate When the patternlng of APs no longer

nervous system For example.

conforms t o the frequency of fllcker, the message sent t o the

1. The frequency of APs recorded from the axons of slngle ommatldla is proportional t o the

logarithm of the intensity of

the stlmulatlng llght (shown at the rlght in part A of the adjolnlng flgure) Thls logarithmic relatlon 1s also typlcal of the

central nervous system lndlcates that the light

IS

constant,

even though the actual stlmulus IS not In fact, human belngs cannot tell the dlfference between a steady llght and one that fllckers at a rate higher than the frequency that can no longer b e encoded by receptors The lowest frequency at wh~chfhckerlng l~ghtsproduce constant stlmulatlon of visual sensory flbers

Duration (s)

A

IS

called the c r ~ t ~ cfusion al frequency For ex-

ample, a standard Incandescent llght bulb fllckers at 60 Hz,

0 0001

0 001

"dlulk

0 01

01

10

but t o us ~tappears as a constant light source Thls charac-

1 0 teristlc of photoreceptors IS very important t o the film and televlslon lndustrles

+

-c

-I"""'lillL 0.001

(A) When l~ghtflashes are shorter than 1 second, the product of intensity and duration determines the number of APs produced by a Limulus photoreceptor. Short, bright flashes can produce a response that is indistinguishablefrom the response to a dimmer, but longer, stimulus. (B) Flickering lights above a certain frequency cannot be distinguished from constant illumination. The on-off pattern of the stimulus is shown under the response to the stimulus recorded from a Limulus photoreceptor.At 10 Hz, the photoreceptorfollows the flicker faithfully; at 12 Hz, the photoreceptor becomes less accurate in reporting the flicker; and, at 16 Hz, the response in the photoreceptor is continuous. [Part A adapted from Hartline, 1934; part B from "How Cells Rece~veStimuli," by W. H. Miller, F. Ratliff, and H. K. Hartline. Copyright O 1961 by Scientific American, Inc. All rights reserved.]


257

............................................................................... planes of polarized light by arthropods. In electrical recordings from single retinular cells in crayfish, the response to a given intensity of light did indeed vary with the plane of polarization in the stimulating light, consistent with this hypothesis (Figure 7-45).

R e t ~ n u l a cell r

a

Cell a

cd b

w-

-

I

I

I

I

400

500

600

700

1

,

I

500

600

J

' 0 °

Figure 7-45 The response of crayflsh photoreceptors to polarized light varies with the plane of polarization. Two cells, a and b, were presented with a series of equal-energy flashes of polarized light at various wavelengths. The color of light in each flash (indicated by its wavelength in nanometers) is indicated along the lower axis. Cell a responded maxirnally to light with a wavelength of about 600nrn; cell b responded maximally to light of 450 nm. When the plane of ~olarization(red arrow) was perpendicular to the microvilli, the responses in both cells were small (left).Responses of both cells were enhanced when the plane of polarization (red arrow) was rotated so as to lie parallel to t6e microvilli (right). [Adapted from Waterman and Fernandez, 1970.1

were oriented systematically in the microvilli and each preferentially absorbed light with its electric vector parallel to the microvilli, the anatomical arrangement within the rhabdome could provide a physical basis for the detection of

The Vertebrate Eye Vertebrate eyes (see Figure 7-41E) have certain structural features similar to those of a camera. In a camera, the image is focused on the film by moving the lens forward or backward along the optic axis. For example, to bring objects that are close to the camera into focus, the lens must be positioned relatively far away from the film. To focus on distant objects, the lens is moved forward. In the vertebrate eye, incident light is focused in two stages. In the initial stage, incident light rays are bent as they pass through the clear outer surface of the eye, called the cornea (Figure 7-46). They are further bent. or refmcted, as they, Dass through a second structure, the lens, and finally form an inverted image on the rear internal surface of the eye, the retina. In fact, most of the that Occurs in the eye 85% the tal) takes place at the air-cornea interface, and the rest depends on the effect of the lens. Like a camera, certain bony fishes focus images on the retina by moving the lens of the eye with respect to the retina. (This principle of changing the distance between the lens and the light-receptive surface has also been adapted by some invertebrates. For example, in the eyes of jumping spiders, the position of the lens is fixed, and focusing depends on moving the retina.) In contrast, neither the lens nor the retina can be moved in the eyes of higher vertebrates. Rather, the image is

-

Rectus tendon

Figure 7-46 In the mammalian eye, incident light is refracted by the cornea and the lens and is focused on the photosensitiveretina. In this diagram, the refraction of light has been simplified; refraction at the aircornea interface is omitted. The image focused on the retina is inverted

by the lens. The lens is held in place by the zonularfibers. When ciliary muscle fibers contract, tension on the zonular fibers is reduced, and the elastic properties of the lens cause it to become more rounded, shortening the focal length.

258


...............................................................................

focused by changing the curvature and thickness of the lens. Changing the curvature of the lens surfaces changes the distance at which an image passed through the lens comes into focus, called the focal length of the lens. The shape of the lens is changed by modification of the tension exerted on the perimeter of the lens. The lens is held in place within the eye by the radially oriented fibers of the zonula (seeFigure 7-46).The fibers of the zonula exert outwardly directed tension around the perimeter of the lens. Radially arranged ciliary muscles adjust the amount of tension exerted on the lens. When the ciliary muscles relax, the lens is flattened by elastic tension exerted by the fibers of the zonula, which pull the perimeter of the lens outward. In this state, objects far from the eye are focused on the retina, but objects close by would be fuzzy. Objects close to the eye are brought into focus on the retina when the ciliary muscles contract, relieving some of the tension on the lens and allowing the lens to become more rounded. This process is called accommodation to close objects. The ability to accommodate decreases with age in human beings as the lens becomes less elastic, producing a type of "far-sightedness," called presbyopia. Perhaps the most remarkable thing about accommodation is not the mechanical mechanisms for altering the focal length of the lens, but the neuronal mechanisms by which a "selected" image-out of all the complexity in the visual environment-is correctly focused on the retina as a result of nerve impulses to the ciliary muscles. A related neuronal mechanism produces binocular convergence, in which the left and right eyes are positioned by the ocular muscles in such a way that the images received by the two eyes fall on analogous parts of the two retinas, regardless of the distance between the object and the two eyes. When an object is close, each of the two eyes must rotate toward the middle of the nose; when an object is far away, the two eyes rotate outward from the midline. Responses to changes in light intensity In a camera, the intensity of light admitted to the film is controlled by adjusting the aperture of a mechanical diaphragm through which light is admitted when the shut-

Gamma rays

ter opens. The vertebrate eye has an opaque iris with a variable aperture called the pupil, which is analogous to the mechanical diaphragm of a camera. When circular smooth muscle fibers in the iris contract, the pupillary diameter decreases, and the proportion of incident light that is allowed to enter the eye is reduced. Contraction of radially oriented muscle fibers enlarges the pupil. The contraction of these muscles-and, hence, the diameter of the pupil-is controlled by a central neuronal reflex that originates in the retina. This pupillary reflex can be demonstrated in a dimly lit room by suddenly illuminating a subject's eye with a flashlight. Changes in pupillary diameter are transient. After a response to a sudden change in illumination, the pupil gradually returns after several minutes to its average size. Moreover, the area of the pupil can change only about fivefold, making it no match for the changes in the intensity of illumination normally encountered by the eye, which equal six or more orders of magnitude. Thus, although the pupil can produce rapid adjustments to moderate changes in light intensity, other mechanisms must be available. The eye adapts to extremes of illumination by changes in the state of visual pigments and by the processes of neuronal adaptation (see Mechanisms of Adaptation earlier in this chapter). Pupillary constriction provides an additional advantage: the quality of the image on the retina improves. The edges of the lens are optically less perfect than is the center; so, when the pupil is constricted, light is prevented from passing through the perimeter of the lens and optical aberrations are reduced. The depth of focus (the distances through which objects are in focus when the lens is in one fixed shape) increases with decreased pupillary diameter, just as it does in a camera when the aperture is reduced. Visual receptor cells of vertebrates The stimulus to all visual receptor cells is electromag~ietic radiation that falls within a particular range of energy, called visible light (Figure 7-47). The energy of electromagnetic radiation varies inversely with its wavelength, and we perceive this variation in energy as variation in color. Violet light, the highest energy of light to which the human

X rays

Ultraviolet

Infrared

Short radio waves

Log of wavelength

Figure 7-47 The spectrum of electromagnet~crad~at~on encompasses a broad range of energy that IS detected by vanous sensory modal~t~es Most v~sualreceptors detect energy In the v~s~ble range, but some can detect Into the ultrav~olet as well The p ~organs t of some snakes can detect Infraredradlatlon [Adapted from Lehn~nger,1993 ]

400

500

600 Wavelength (nm)

700

SENSING T H E ENVIRONMENT

259

............................................................................... eye responds, has a wavelength of approximately 400 nm. Red light, at the low-energy end of the visible spectrum, has wavelengths between 650 and 700 nm. Bright light delivers more energy per unit of time than does dim light. The photoreceptor cells that capture the energy of light and transduce it into neuronal signals are located in the retina of the vertebrate eye. In mammals, birds, and other vertebrates, the retina contains several types of cells that are interconnected into a network. The visual receptor cells themselves fall into two classes, rods and cones, which were named for the shapes of the cells as observed under a microscope (Figure 7-48). All neurons within the retina, as well as the epithelial cells, contribute to the light response of the vertebrate eye, but rods and cones have different physiological characteristics. For example, cones function best in bright light and pro-

vide high resolution, whereas rods function best in dim light. These different capabilities are used by different animals to provide particular visual capabilities. For example, animals that live in flat, open environments (e.g., cheetahs and rabbits) usually have horizontal visual streaks, regions within the retina that contain unusually high densities of cone receptors. Such a region corresponds to the horizon in the visual world and is thought to confer maximal resolution in this part of the scene. The visual streak also contains a high-density population of ganglion cells-the cells that transmit visual information to the brain. In contrast, arboreal species (and human beings) typically have a radially symmetric density gradient of photoreceptors. An important feature of this kind of retina is the fovea, or area centralis. It is a small (about 1 mm2) central part of many mammalian retinas, and it provides very detailed

Pigment epithelium

cilium

Figure 7-48 Vertebrate photoreceptors are classified as rods or cones on the basis of their morphological and physiological properties. The outer segments of rods and cones, where light is captured, face away

from the source of light. The pigmentthat absorbs the light energy is contained in membrane lamellae, and the ends of the outer segments lie against the pigment epithelium.

260


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information about the visual world, a characteristic called high visual acuity. In human beings and in certain other mammals, the fovea contains only cones, whereas the remainder of the retina contains a mixture of rods and cones, with the rods significantly outnumbering the cones. In mammals, cones mediate color vision, and rods, which are more light-sensitive, mediate only achromatic vision. This distinction between rods and cones does not, however, pertain to all vertebrates. In fact, the retina in some species contains only rods, based on morphology, but may nonetheless be capable of color vision. Rods and cones are structurally and functionally more similar to one another than are the wide variety of photoreceptors found in the invertebrates. Each vertebrate receptor cell contains a segment with an internal structure similar to that of a cilium. This rudimentary cilium connects the outer segment, which contains the photoreceptive membranes, to the inner segment, which contains the nucleus, mitochondria, synaptic contacts, and so forth (see Figure 7-48).The receptor membranes of vertebrate visual cells consist of flattened lamellae derived from the surface membrane near the origin of the outer segment. In the

cones of mammals and some other vertebrates, the lumen of each lamella is open to the cell exterior. In rods, the lamellae pinch off completely from the surface membrane of the outer segment so as to form flattened sacs, or disks, that are stacked like pita bread on top of one another within the rod outer segment. The stack of disks is completely contained within the surface membrane of the visual cell. The photopigment molecules are embedded in the. membranes of the disks. Because the photopigment lies in the disk membranes of the rod outer segment but not in the surface membrane, the primary step in photochemical transduction must take place in the d s k membranes, rather than in the surface membrane. The eyes of many invertebrateslack the ciliary structure that connects the inner and outer segments of vertebrate rods and cones (Figure 7-49).In these invertebrate eyes, the photopigment is located in microvilli formed by the cell membrane, and these pigment-containing microvilli make up the rhabdomeres. Because many invertebrate species have simple eyes in which the photoreceptors are of the rhabdomeric type, it might be tempting to conclude that rhabdomeric photoreceptors are found only in simple eyes. Arthropods

+

Ciliary line Vertebrata

. Echinodermata

Rhabdomeric line

Figure 7-49 Vertebrate photoreceptors contain a typical 9 2 ciliary structure that connects the inner and outer segments, but many invertebrate photoreceptors lack this ciliary structure and instead contain many rnicrovilli. This diagram illustrates the phylogenetic distribution of ciliary and rhabdorneric eyes. There are, however, exceptions. Both the scallop, Pecten, and the surf clam, Lima, have complicated eyes with two layers of photoreceptors. One layer contains ciliary photoreceptors and the other contains rhabdomeric receptors. [Adapted from Eakin, 1965.1

However, the eyes of the octopus are optically very complex and the photoreceptors are rhabdomeric. In addition, some bivalve mollusks (e.g., the scallop, Pecten, and the clam, Lima) have eyes with two separate layers of photoreceptors. One layer contains ciliary receptors, and the other contains rhabdomeric receptors.

In all photoreceptor cells, the transduction of light energy produces a change in the membrane potential; however, the effect of the transduction is different in vertebrate and invertebrate photoreceptors. Invertebrate photoreceptors depolarize in response to light (Figure 7-50A; see also Figure 7-45), but vertebrate rods and cones hyperpolarize in response to a light stimulus (Figure 7-50B). Membrane conductance measurements before and during illumination have shown that the effect of light on vertebrate photoreceptors is to decrease the conductance for sodium, gNa,of the outer segment membrane. In the dark, the surface membrane of the vertebrate rod outer segment is nearly equally permeable to Na+ and K+, and V, lies about halfway between EKand EN,. In this state, Na+ ions leak into the outer segment through channels that are steadily open in the dark (Figure 7-51A). The Na+ ions that carry this inward current, which is called the dark current because

A Invertebrate

t

Increased g,

B Vertebrate

Time

t

Decreased g,

nn Light o n

Light on

Figure 7-50 Most invertebrate photoreceptors depolarize in response to a stimulus, whereas vertebrate photoreceptors hyperpolarize. (A) Transduction of light energy into chemical energy w ~ t h ~most n invertebrate photoreceptors causes an increase in the permeability of the surface membrane to Na+ and K+, depolarizing the cell. (B)Vertebrate photoreceptors respond to light with a decrease in gNaof the surface membrane, leaving a residual low g,and shifting V, toward EK.As a result, the cell hyperpolarizes.

it is maximal in the dark, are kept from accumulating in the cell by the steady action of the metabolically driven Na+,K+ATPase. A dark current is found only in vertebrate photoreceptor cells and not in invertebrate photoreceptors. After light absorption by the photopigment, the conductance to sodium, gNa,of the outer segment decreases, causing the dark current to decrease and V,,, to hyperpolarize toward EK(see Figures 7-50B and 7-51B). When the light stimulus stops, gNaof the membrane returns to its high resting level, and V,,, becomes more positive, returning to its resting level between ENaand EK.The change in V,,, at the onset of light is carried electrotonically (see Passive Spread of Electrical Signals in Chapter 6) into the inner segment of the photoreceptor. In the inner segment, changes in V,, modulate the steady release of neurotransmitter from the presynaptic sites located in the basal part of the inner segment. Like vertebrate auditory receptors, vertebrate photoreceptors lack axons. They synapse onto other neurons, which carry the visual signal toward the central nervous system. The neuronal signal is passed along by other neuronal cells of the retina, ultimately influencing the activity of axons that project to the brain within the optic nerve. It is interesting that a hyperpolarization, rather than a depolarization, is produced when a vertebrate photoreceptor absorbs light, because in most sensory systems reception of a stimulus depolarizes the receptor cell. In vertebrate photoreceptors, the inner segment steadily secretes a transmitter while it is being partially depolarized by the dark current. The hyperpolarizationthat occurs in response to illumination decreases the amount of transmitter released onto the next neuron in line, modifying the activity of that second-order neuron. The change in membrane potential that is produced in a group of photoreceptors when they are illuminated can be recorded by extracellular electrodes, as can action potentials traveling down the axons of a nerve. Many photoreceptors are tiny cells making intracellular recording difficult, so this method of recording-called an electroretinogram-has been extremely useful in the study of vision (Spotlight 7-2). Photoreception: Converting Photons into Neuronal Signals When photons strike the photosensitive pigment molecules of a photoreceptor, the cell must generate APs, either by itself (in invertebrate photoreceptors) or in higher-order neurons (in vertebrate photoreceptors) if the signal is to be carried to the central nervous system. The process of visual transduction has received an enormous amount of research attention, and the features of the visual process have provided clues to physiologists studying sensory transduction in other sensory modalities. Studies of photoreception have been carried out in many different species spanning several phyla. Many similarities between vertebrate and invertebrate photoreception have been found, although it is now thought that invertebrate photoreception may be more complex because it relies on two light-activated pathways,

In

Figure 7-51 Illumination reduces the dark current in vertebrate rods. The g,, of the rod outer segment is high in the dark(A) and becomes reduced in the light (B). Forthis reason, the dark current, which is carried by Na+ ions that leak into the outer segment, drops during illumination. In the equivalent circuit (top left), the battery is the Na+, K+ ATPase, and the light-activated variable resistor (R,,) represents the gNaof the outer segment. [Adapted from Hag~ns,1972.1

4 Out

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rather than the single pathway found in vertebrates. There are other, perhaps related, differences as well. For example, capture of a single photon by a photoreceptor in Limulus produces a peak current of -1 nA, whereas capture of a single photon by a vertebrate rod photoreceptor changes the current by -1 pA, three orders of magnitude smaller. Moreover, invertebrate photoreceptors may respond to light intensities spanning seven orders of magnitude, whereas vertebrate rods respond to intensities only within four orders of magnitude. Despite these differences in detail, all types of photoreceptors have been shaped by evolution to convert the energy of photons into neuronal energy, and studies of all types of eyes have contributed to our understanding of the process.

Visual pigments The spectrum of electromagnetic radiation extends from gamma rays, with wavelengths as short as 10-l2 cm, to radio waves, with wavelengths greater than lo6 cm (see Figure 7-47). The segment of the electromagnetic spectrum

cm and lo-' cm is termed with wavelengths between light. Only a small part of this segment of the spectrumranging from about 400 nm to about 740 nm-is visible to human beings. Below this range is the ultraviolet (W)part of the spectrum, and above it the infrared (IR),neither of which is visible to human beings and other mammals. There is nothing qualitatively special about those parts of the spectrum that renders them invisible to us. Rather, what we see depends on which wavelengths are absorbed by our visual pigments. For example, there is a condition called cataract, in which the lens becomes opaque. Treatment of the condition consists of surgically removing the lens; after this surgery, patients can see light into the W range because it is absorption of UV light by the lens that prevents people from seeing those wavelengths. The compound eyes of many insects can detect light into the UV range, causing some flowers containing W-reflecting pigments to look much less plain to insects than they do to mammals, but all animals are sensitive to only a part of the spectrum of electromagnetic radiation that is available in

S P O T L I G H T 7-2

THE

activity of the photoreceptor cells and other neurons in the retina. It took several years t o sort out the source of each of the components of the ERG, but we now believe that the a wave is

ELECTRORETINOGRAM

due t o the receptor current produced by the visual receptor cells. The b wave follows the a wave, and it is produced by elec-

In a teaching laboratory, it is sometimes useful t o record the summed electrical activity of the eye, which is technically much less complicated than recording from single cells with microelectrodes. The recording electrode (which can be a thread or a

trical activity of the second-order retinal neurons that receive inp u t from the receptor cells. The c wave is found only in vertebrates and appears t o b e produced by the pigment epithelial cells against which the outer segments of the visual cells abut. In

wick that is saturated with saline) is placed on the cornea, and the

the developing eyes of tadpoles, the ERG consists of only an a wave before synaptic contacts are established. Similarly, in the

ground electrode is attached t o another part of the body. When

eye of an adult frog, if synaptic transmission between the photo-

a light is flashed on the eye, a complex waveform is recorded by the electrode (as shown in the adjoining figure). This recording is called an electroretinogram (ERG), and it records the summed

receptors and the second-order neurons is blocked pharmacologically, the ERG consists of only an a wave.

1

A vertebrate electroretlnogram conslsts of several components, each from a different source The tlmlng of the st~mulusIS shown under the recording [Adapted from Brown, 1974 ] Off

sunlight. Perhaps the visual pigments of vertebrates absorb only a limited range of the electromagnetic spectrum of sunlight because vertebrate life evolved in water, which heavily filters electromagnetic radiation. The range of the spectrum to which vertebrate photopigments-including those of terrestrial mammals such as human beings-are sensitive matches rather closely the spectrum of light that is admitted through water. All known organic pigments owe their ability to absorb light selectively to the presence of a carbon chain, or ring, that contains alternating single and double bonds. When a photon is captured by one of these molecules, the energy state of the molecule is changed. The energy contained in a quantum of radiation is equal to Planck's constant di,vided by the wavelength, A (in centimeters):

Thus, the energy in a photon increases as the wavelength of radiation decreases. Quanta with wavelengths less than 1 nm contain so much energy that they break chemical bonds or even atomic nuclei; quanta with wavelengths greater than 1000 nm lack sufficient energy to affect molecular structure. The visual pigments absorb maximally between these two limits. When a quantum of radiation is absorbed by a photopigment molecule, it raises the energy state of the molecule by increasing the orbital diameter of the electrons associated with a conjugated double bond, the

same process that is used in the photosynthetic conversion of radiant energy into chemical energy in plants. Photochemistry of visual pigments The energy content of visible light is just low enough to be absorbed by molecules without breaking them up. The concept that a pigment is essential for the process of absorbing light and transducing its electromagnetic energy into chemical energy originated with John W. Draper, who concluded in 1872 that, to be detected, light must be absorbed by molecules in the visual system. R. Boll found soon thereafter that the characteristic reddish purple color of the frog's retina fades (bleaches)when the retina is exposed to light. The light-sensitivesubstance, rhodopsin, that is responsible for the purple color, was extracted in 1878 by W. Kiihne, who also found that, after the pigment has been bleached by light, its reddish purple color can be restored by keeping the retina in the dark, provided that the receptor cells remain in contact with the pigment epithelium at the back of the eye. Since then, much has been learned about the chemical nature and physiological effects of rhodopsin. It absorbs light maximally at wavelengths of about 500 nm. It is found in the outer segments of rods in many vertebrate species and in the photoreceptors of many invertebrates. Rhodopsin molecules are packed at high density into receptor membranes; there may be as many as 5 x 1012molecules per square centimeter, which is equivalent to an intermolecular spacing of about 5 nm.

All known visual pigments consist of two major components: a protein (opsin) and a light-absorbing molecule. In all instances, the light-absorbing molecule is either retinal or 3-dehydroretinal (Figure 7-52). Retinal is the aldehyde of vitamin A,, a carotenoid. Vitamin Al is an alcohol and is also called retinol; 3-dehydroretinal is the aldehyde of vitamin A,, which is also called 3-dehydroretinol. In addition to its major components, rhodopsin includes a sixsugar polysaccharide chain and a variable number (as many as 30 or more) of phospholipid molecules. The lipoprotein opsin, which binds the phospholipids and the polysaccharide chain, appears to be an integral part of the photoreceptor membrane. Carotenoid molecules move back and forth between the photoreceptor membrane and the pigment epithelium at the back of the retina during bleaching and regeneration of the visual pigment. (Incidentally, the pigment that confers a dark color on the pigment epithelium is photochemicallyinactive and is unrelated to the visual pigment. Instead, it keeps light from scattering and reflecting diffusely back toward the retina.) The retinal molecule assumes two sterically distinct states in the retina. In the absence of light, the opsin and the retinal are linked covalently by a Schiff's base bond, and retinal is in the 11-cisconfiguration (seeFigure 7-3). When the 11-cis retinal captures a photon, it isomerizes into the all-trans configuration (see Figure 7-52). This cis-trans isomerization is light's only direct effect on the visual pigment. The conversion from 11-cis to all-trans retinal initiates a series of changes in the relation between the retinal and the opsin protein, including changes in the conformation of the opsin itself.

When light hits the photopigment, an intermediate, metarhodopsin II, is formed. Metarhodopsin I1 activates another protein that is associated with the membrane and that binds GTP in exchange for GDP. This protein, which we now know belongs to the family of G proteins, is called transducin in recognition of its key role in the transduction of light. The activated subunit of transducin diffuses in the plane of the membrane, encountering many phosphodiesterase molecules, which hydrolyze cGMP to 5'-GMP. In vertebrate photoreceptors, the dark current Na+ channels are open only in the presence of cGMP; so, when cGMP is hydrolyzed, these channels close (Figure 7-53). The membrane of the rod outer segment contains a class of channels that are permeable to three cations: Na+, Mg2+and Ca2+. When the level of cGMP drops, the conductance through these channels drops. Most importantly, the inward I,= drops, and the residual K+ current through other channels causes the cell to hyperpolarize. When the light stimulus ends, cGMP is regenerated by the action of another enzyme, guanylate cyclase. As the level of cGMP rises, the dark current channels open, and the receptor current returns to its full value in the dark. Activated transducin collides with, and activates, phosphodiesterase molecules at a rate of about lo6 molecules per second, allowing the capture of a single photon to affect the conductance through an enormous number of ion channels. This numerical relation generates an impressive amplification between the capture of a single photon and the effect that the event produces on V,. After the cis-trans isomerization of retinal has occurred, further changes in the molecule appear to be irrel-

l

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Figure 7-52 The carotenoid pigment retinal changes its steric conformation when it absorbs a photon. (A) In the dark, the bonds of carbon 11 are arranged in the cis configuration. (B) When a photon is captured, these bonds are converted into the straight, all-trans configuration. Both

space-f~ll~ng and llne d~agramsof the molecular structure are shown [Part B from "Molecular Isomers In Vls~on,"by R Hubbard and A Kropf Copyr~ght0 1967 by Scient~flcAmer~can,Inc All r~ghtsresewed.]

SENSING T H E ENVIRONMENT

265

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evant to the excitation of visual receptor cells, but the subsequent reactions (Figure 7-54)are necessary for regenerating active rhodopsin. Activated rhodopsin is hydrolyzed spontaneously to retinal and opsin, which are both reused repeatedly. Free retinal is reisomerized back into the 11-cis form and reassembled with an opsin to form rhodopsin. Any retinal that is lost or chemically degraded in the process is replenished from vitamin A, (retinol) stored in the cells of the pigment epithelium, which actively take up the vitamin from the blood. A nutritional deficiency of vitamin A, decreases the amount of retinal that can be synthesized and, hence, decreases the amount of available rhodopsin. The result is reduced photosensitivity of the eyes, a condition that is commonly known as night blindness. Rod photoreceptors can respond to the absorption of a single photon, partly because rhodopsin is so densely packed into their disc membranes. There are about 20,000 rhodopsin molecules per square micrometer in the rod outer segment, which is much closer packing than, for example, the density of acetylcholine receptors at the neuromuscular junction. By recording from a single rod, Denis Baylor, of Stanford University, has measured the response to the capture of a single photon (Figure 7-55).In these ex-

Rhodopsin

all-trans Retinal 4 Isomerase

Opsin Il-cis Retinal

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periments, rods are teased apart and one of them is drawn into a recording pipette where it is stimulated by a small beam of light. When the stimulating light is very dim, it is possible to record small current fluctuations, each of which occurs when a single rhodopsin molecule has been photoisomerized by a single photon. The properties of the current recorded under these conditions are similar to the properties of the current measured through a single acetylcholine receptor channel at the neuromuscular junction. (The change in current that is associated with the capture of one photon is about 1 PA.) Because photoreceptors can respond to a single photon, or quantum of energy, the sensitivity of photoreception is limited by the quanta1 nature of light; there is no smaller amount of light than one photon. Elucidating the process of visual transduction has demonstrated the power of a comparative approach. Although vertebrate and invertebrate photoreceptors seem quite different from each other at the electrophysiological level, there are many similarities between them at the molecular level. The molecular genetics available in Drosophila and the physiological accessibility of the vertebrate retina have been combined to provide an array of enormously powerful experimental approaches to the question of how visual information is acquired and processed by

t all-trans Retinol (Vitamin A)

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Figure 7-53 When light is absorbed by retinal, a series of reactions causes the Na+ channels that carry the dark current to close. (A) Activated rhodopsin increases the activity of a G protein, transducin. The activated G protein then activates many phosphodiesterase (PDE) moiecules, reducing the intracellular concentration of cyclic guanosine monophosphate (cGMP),which leads to the closing of Na+ channels that carry the dark current. The receptor cell then hyperpolarizes. (B) Recordings of currents in single rod photoreceptors isolatedfrom toad retina. (Left) A flash of light causes the inward dark current of 10 pA to drop to zero (R~ght) The photoreceptor has been broken open, and the external sal~nehas been changed to rnlrnlc Intracellular lonlc concentrat~onsWhen cGMP IS added to the external sallne (exposing the Inside of the outer segment to a hlgh concentratlon of cGMP), a very large Inward current develops [Part B adapted from Yau and

!

Pigment epithelium

Figure 7-54 When rhodopsin is activated, the all-trans retinal separates from the opsin. Rhodopsin is reconstitutedafter an isomerase returns the retinal to the II-cis configuration. Retinol (vitamin A) is stored in the pigment eplthellum and can be deltveredto the photo receptors for generating new rhodopsln molecules.

760 photons. pm-Z.s-'

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light

'outer segment

of rod

Time (s) Figure 7-55 Rods can respond electrically to the capture of a single photon. (A) A single rod outer segment is sucked into a smooth glass pipette electrode and is illuminated by a narrow bar of light while the ionic current across the membrane is recorded by the pipette. (B) The recorded membrane current changes in response to illumination. In very dim light (bottom record), small individual changes in the current ac-

company the capture of single photons. As the light intensity is increased, (intensity is indicated above each recording), the responses become larger and smoother. The duration of the illumination is indicated by the bar under each recording. Membrane currents are in PA. [Adapted from Baylor et al., 1979.1

photoreceptors. If the molecular identities of the players had not been so strongly conserved through phylogeny, teasing out the details of visual transduction would probably have taken much longer.

action spectrum of a photoreceptor depends upon the absorption properties of its visual pigment. In addition, results of such experiments confirm that each photoreceptor synthesizes only one of the visual pigments. Light that contains different wavelengths generates photochemical reactions in a particular photoreceptor cell in proportion to the amount of each wavelength absorbed; thus, a photoreceptor cell is excited by different wavelengths in proportion to the efficiency with which its pigment absorbs each wavelength. Any photon that is not absorbed can have no effect on the pigment molecule; any photon that is absorbed transfers part of its energy to the molecule as described in Photochemistry of Visual Pigments in this chapter. Thus, it is possible to restate Young's trichromacy theory (see Spotlight 7-3) in relation to cone photoreceptors and their photopigments: there are in the human retina three classes of cones, each of which contains one visual pigment that is maximally sensitive to blue, to green, or to orange light. The electrical output of each class depends on the number of quanta that are absorbed by the pigment and can thus contribute to the events of transduction. The sensation of color arises when higher-order neurons integrate signals received from the three classes of cones. Knowledge about the molecular basis of color reception has grown enormously since 1984, when Jeremy Nathans described the molecular structure of human opsins and thus provided an explanation for hereditary color blindness. For example, point mutations in individual pigment genes cause defects in sensitivity to particular wavelengths. Indeed, the molecular basis for differential spectral sensitivity among the opsins has been characterized by using naturally occurring variants in visual pigments.

Cones and rods The ability to distinguish color, rather than just perceiving the visual world in shades of gray, is correlated with the possession of multiple visual pigments, each of which absorbs maximally at a different wavelength (Spotlight 7-3). In vertebrate species that have color vision, it has been found that different groups of photoreceptors contain spectrally identifiable visual pigments, and each class of photoreceptor has a distinctive action spectrum. That is, the electrical response of each photoreceptor, when it is illuminated, is maximal at a particular wavelength and falls off when the wavelength of incident light is either raised or lowered. In many species for which action spectra have been recorded, three classes of photoreceptors have been found. The action spectra for some species have then been compared with the absorption spectra of individual photoreceptors. Absorption spectra for single photoreceptors have been measured by a process, called microspectrophotometry, in which a tiny beam of light is focused on one photoreceptor at a time and the absorption properties of that cell are determined. Photoreceptors studied in this way fall into distinct classes for each species; there are no intermediate spectra, which implies that each photoreceptor synthesizes a single visual pigment (Figure 7-56). Both the action spectra and absorption spectra of photoreceptors have been determined in many species, and the two kinds of spectra match one another closely, confirming that the


267

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I

Figure 7-56 Each class of cones in the carp retina has a distinctive action spectrum. (A) Absorption spectra of individual cones in the retina of a goldfish indicate that there are three separate visual pigments, each with a distinctive absorbance peak. These measurements were made by microspectrophotometry,which allows the absorption spectrum of a single photoreceptorto be measured. In human beings, the class of cones that is equivalent to the red-absorbingcone in the goldfish absorbs maximally closer to 560 nm, which is in the yellow part of the spectrum. (B) Electrical responses of three single cones in the retina of a carp to flashes of d ~ f ferent wavelengths, as shown by the scale at the top. The wavelength that produces a maximal response is different for each of the three cones. (C)When the amplitude of activity in each cell shown in part B was plotted as a function of wavelength, three classes of cones were revealed, each with an action spectrum approximating one of the absorption spectra in part A. [Part A adapted from Marks, 1965; parts B and C adapted from Tomita et al., 1967.1

It appears that 11-cis-retinal(or 11-cis-3-dehydroretinal) is the light-absorbing molecule in all visual pigments, and this prosthetic group is combined with different opsin molecules to produce visual pigments with different absorption maxima. Differences in the amino acid composition of opsins-rather than variation in the light-absorbing prosthetic group-produce rhodopsins with different absorption maxima. Nathans and his co-workers discovered three genes that encode for the opsins in human cones. The gene encoding the protein part of the blue-absorbing pigment is located on an autosomal chromosome, whereas the two genes for the "red"-absorbing and green-absorbing proteins are closely linked on the X chromosome. The "red" and green opsins differ at only 15 of 348 amino acids, and each shares about half its amino acids with rhodopsin in rods (Figure 7-57).On the basis of sequence similarity, we can surmise that the genes for these pigments probably arose from a common ancestral gene that underwent duplication and divergence. A comparison of the amino acid sequences suggests that, of the cone pigments, the blue-sensitive pigment arose first, followed by the red and the green. Color blindness is caused by an absence of,

or a defect in, one of the cone opsin genes. With the use of these molecular markers in conjunction with visual tests, it is now possible to define the molecular basis of this perceptual problem. For example, the high incidence of

Cvtosol

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Figure 7-57 The two opsin proteins in the human rhodopsins that absorb maximally in the red and the green parts of the visible spectrum differ by only 15 amino acids, most of which are thought to be in membranespannlng helices. In this diagram, these variable amino acids are red. [Adapted from Nathans et al., 19861

SPOTLIGHT 7 - 3

LIGHT, PAINT, AND

ceptors would be stimulated maximally by separate monochromatic "red" and "yellow" wavelengths, respectively, and both would be stimulated to a lesser degree by monochromatic or-

COLOR VISION

ange light. Young proposed, in other words, that the sensation for "orange" results from the simultaneous excitation of "red"

In 1666, Sir Isaac Newton demonstrated that white light is separated into a number of colors when it is passed through a prism. Each spectral color is monochromatic; that is, it cannot be separated into yet more colors. At that time, however, it was already known that a painter could match any spectral color (e.g., orange) by mixing two pure pigments (e.g., red and yellow), each of which reflects a wavelength different from that of the color produced. Thus, there seemed to be a paradox between Newton's demonstration that there is an infinite number of colors In light and the growing awareness of Renaissance painters that all colors could be produced by combinations of three primary pigments-red,

yellow, and blue. This paradox appeared

to be resolved by Thomas Young's suggestion, in 1802, that the receptors in the eye are selective for the three primary colors: red, yellow, and blue. Young reconciled the infinite variety of spectral colors that can be duplicated with the small number of painter's pigments required to produce all colors by proposing that each class of color receptor is excited to a greater or lesser

receptors and "yellow" receptors. Young had no notion about the physiology of photoreceptors, making his insight truly remarkable. Extensive psychophysical investigations carried out in the nineteenth century by James Maxwell and Hermann von Helmholtz supported Young's trichromacy theory, and additional support came from later investigations by William Rushton. However, direct evidence for the existence of three classes of color-receiving photoreceptors was missing. Then, in

1965, W. B. Marks and E. MacNichol measured the color absorption of single cone photoreceptors in the goldfish retina (see Figure 7-56A). They found three classes of cones, each of which absorbed maximally at a unique wavelength. Subsequent measurements of the absorption spectra of cones in retinas from human beings, monkeys, and other species of fishes reproduced these results. It thus appears that the retinas of species that can percelve and respond to color contaln photoreceptor cells w ~ t h d~fferentabsorption spectra and that, In many of these species, there are three d~sttnctclasses of receptors.

degree by any wavelength of light: The "red" and "yellow" re-

red-green color blindness arises from the recombination of these closely linked red and green opsin genes. Color vision has been demonstrated in some members of all classes of vertebrates. In general, retinas that include cone photoreceptors are associated with color vision, but examples of different color classes among rods have been found. For example, frogs have two kinds of rods-red (containing rhodopsin, which absorbs in the blue green) and green (containing a pigment that absorbs in the blue)-in addition to cones. If color vision is mediated primarily by the cones, what do rods contribute? Rods are more sensitive to light than are cones (the records in Figure 7-55 were made from rods), and their connections to the next neurons in line are characterized by greater convergence than are the connections of cones (see Visual Processing in the Vertebrate Retina, Chapter 1I),producing greater summation of weak stimuli. Thus, rods are most effective in dim light. Because the cones produce color vision, when only our rods are stimulated by dim light, we see in shades of black and gray, rather than in color. In the human retina, most images are prefentially focused onto the fovea, which contains only tightly packed cones. Rods are found only outside the fovea. The differential distribution of rods and cones in the retina causes us to be most sensitive to dim light when an image is focused outside the fovea, onto parts of the retina where the rod population is higher. For example, a dim star will appear brighter if you adjust your gaze to make its image fall outside the fovea. If you s M your gaze to make the

image fall on the fovea, the star will fade or even disappear. This increased sensitivity comes at a price: the broader connections among rods reduces the acuity of rod-based vision. Our visual sensitivity is greatest when an image is focused onto the rods outside the fovea; our visual acuity is greatest when we focus an image onto the cones of the fovea. When visual pigments are explored throughout phylogeny, some interesting patterns emerge. For example, all visual pigments for which retinal is the prosthetic group are called rhodopsins. All human visual pigments-the rod pigment and the three cone pigments-are rhodopsins. Visual pigments in which 3-dehydroretinal is the prosthetic group are called porphyropsins, and the distribution of the rhodopsins and porphyropsins among species shows an interesting correlation with environment. All visual pigments of terrestrial vertebrates are rhodopsins. In addition, rhodopsins are found among invertebrates, including Limulus, insects, and crustaceans. In contrast, porphyropsins are found in the retinas of freshwater fishes, euryhaline fishes (see Chapter 14),and some amphibians. This distribution suggests that some feature of the porphyropsins makes them particularly well adapted to conditions found in freshwater. In fact, anadromous fishes, which migrate from freshwater to saltwater-or vice versa-during their life cycle, change their visual pigment between porphyropsin and rhodopsin during the migration. They synthesize porphyropsin during their stay in freshwater and rhodopsin while they are in saltwater. The absorption maxima of the porphyropsins is shlfted toward the longer-

wavelength, red end of the visual spectrum, whereas rhodopsins absorb maximally at shorter wavelengths. Perhaps the freshwater environment makes sensitivity to the red end of the spectrum important. Tracing the transduction of information from the absorption of a photon to the production of neuronal signals leaves the question of how all this information about incident radiation is molded into a coherent view of the world unanswered. The collected information is passed on to higher neuronal centers, where it is integrated and can be used to shape behavior-a topic that is explored in Chapter 11.

LIMITATIONS O N SENSORY RECEPTION To be most useful, a sensory receptor should be very sensitive to stimulation from the environment and should encode the information with perfect accuracy. In fact, no receptor meets these requirements, because of the physical properties of stimuli and of receptors; all receptors represent compromises in how they receive and encode sensory information. Some physical principles that apply to receptors of many sensory modalities necessarily limit the fidelity with which sensory information is received and transmitted by cells. In some cases, the accuracy of sensory reception is limited by the relative magnitudes of the signals and the background noise. This signal-to-noise ratio limits the performance of all systems that receive and transmit information, whether or not they are living. In other cases, the performance and sensitivity of a sensory system are limited by the form of energy to which the receptors are tuned. For example, light is by its nature quantized into photons. No receptor can receive less than one quantum of light, because light does not exist in fractions of quanta. A major source of background noise arises from a corollary of the Third Law of Thermodynamics. That is, at all temperatures above O°K, molecules have kinetic energy and are in motion. Thermal energy is given by

where k is Boltzmann's constant (1.3805 X 10-l6 erg K-l) and T is the absolute temperature. This equation gives the energy that is associated with the movement of molecules (i.e., Brownian motion) at an animal's body temperature. It sets a lower limit on the sensitivity of receptors in detecting signals because thermal energy provides a constant noise level against which stimulation occurs. To detect an external signal, a receptor must be able to distinguish the signal from this baseline thermal noise. How easily can receptor cells accomplish this task? Photoreceptors provide an example. At a body temperature of 2S°C, the thermal energy is about 0.58 kcal-mol-', or 4 x 10-l4 erg. We must compare this energy to the energy of a typical sensory stimulus. The stimulus for a vertebrate photoreceptor is light in the visible

part of the electromagnetic spectrum (seeFigure 7-47). The energy of a single photon of light is given by the Einstein relation:

where h is Planck's constant and v, c, and A are the frequency, speed, and wavelength of light, respectively. Substituting the values for a photon of blue light (A = 500 nm), the energy is calculated to be about 57 kcal-mol-lalmost 100-fold greater than the thermal energy. In vision, detection is definitely not limited by thermal energy within the detector. Instead, it has been found to be limited by the quanta1 nature of light itself. In audition, the energy is given by the Einstein relation for single phonons, which are quantum units of sound energy analogous to photons of light. Animals hear sounds across a remarkably broad range of frequencies, from 10 to lo5Hz. The energy of phonons at these frequencies ranges to 7 x erg. In the middle of this from 7 x range, the energy of a single phonon is 10 orders of magnitude (101°)below the limit of detection set by thermal energy. This result indicates that the detection of acoustic stimuli is fundamentally limited by thermal noise, and there must be special mechanisms that permit auditory sensory reception. Indeed, some advantage is gained by tuning the detectors to limit their range, a common feature found in the hair cells of auditory systems. Numerous mechanisms have evolved to combat the limitations of thermal noise, but direct measurements have also shown that sensory cells in auditory systems faithfully reproduce the thermal noise at their inputs. As discussed earlier in this chapter, most chemical stimuli (olfaction, taste, chemotaxis) bind to specific receptors, rather than directly change ionic currents through membrane channels. In this case, the relation between binding energy and thermal energy determines the limits of detection. The binding energies that have been measured in chemical sensory systems are typically about 1kcal mol-I . This energy is sufficiently greater than thermal energy that chemoreceptors could theoretically count single molecules. There is, however, an important constraint dictated by the physics of receptor binding. The greater the binding energy, the longer the molecule remains associated with its receptor. For a binding constant of lop6M, the association time seconds; whereas, for a binding constant is about 3 X of lo-'' M (giving very high specificity), the association would last for more than 5 minutes. Because the performance of a receptor system depends at least in part on comparisons across many receptors, long binding times would require that comparisons be made over very long time periods, and evolution seems to have shunned such a mechanism. Instead, the binding constants between chemical stimuli and their receptor molecules are moderately high, reducing the binding energy but also the time required to transduce and interpret chemical signals.

-

270


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Signal-to-noise properties can be predicted for stimuli that activate electroreceptors and thermoreceptors. Electroreception is relatively widespread in aquatic organisms, in which it is used for navigation, communication, and predation. The energy in the electric fields, carried through water at the frequencies used, is about 10 orders of magnitude below the thermal energy. Thus, the process of electroreception, like that of auditory reception, must be dominated by thermal noise in the detector. Thermoreception depends on the detection of photons in the infrared region of the electromagneticspectrum (which has lower frequencies and longer wavelengths than those in the visible spectrum), and, by definition, it is limited by the temperature difference between the measured object and the measuring organ. Some animals, particularly beetles, have been found to perform at or near the theoretical limits; others have apparent adaptations that keep their detectors cooler than the rest of the body, decreasing the background thermal noise. As scientists have explored the limits of detection achieved by animal senses, it has become clear that many modalities operate at or near the theoretical limits imposed by physical laws. To accomplish this prodigious task, many types of receptors have evolved similar molecular mechanisms, and the mechanisms that are available to be used in each sensory modality depend, at least to some extent, on whether sensory reception is limited by thermal energy or by the quanta1 nature of the stimulus.

SUMMARY Receptor cells are highly sensitive to specific kinds of stimulus energy and relatively insensitive to other kinds of stimulation, and they transduce the stimulus into an electrical signal, usually (but not always) a depolarization. The lower limit to sensation often depends on how much energy is carried in the signal compared with the energy in the thermal noise within the organism. The transduction process is most sensitive to weak stimuli, producing receptor signals that contain several orders of magnitude more energy than the stimulus itself. This sensitivity drops off with increasing stimulus strength. In most receptor cells, the primary sites of reception and transduction are receptor molecules located in the cell membrane or in intracellular membranes. Activation of receptor molecules causes the conductance of particular classes of ion channels in the membrane to change; typically, the change in conductance permits the flow of a receptor current, producing a receptor potential. In many sensory modalities, the receptor cells do not themselves produce APs. Instead, receptor potentials modulate the amount of neurotransmitter that the receptor cells release onto second-order neurons, which in turn initiates or modulates the number of APs in second-order neurons. Stimulus intensity is typically encoded in the frequency of impulses, which in many sensory fibers is roughly proportional to the logarithm of the intensity, up to a maximum frequency. The logarithmic relation between stimulus and response magnitudes permits reception over a large

dynamic range while retaining high sensitivity to weak stimuli. Parallel inputs from receptors that cover different parts of the intensity range increase the range of stimulus intensities that can be perceived. The time-dependent loss of sensitivity to a maintained stimulus, termed sensory adaptation, is a common property of receptor cells; some receptors adapt rapidly and others adapt slowly. The mechanisms responsible for sensory adaptation vary. Some take place in the receptor cell, others in the network of neurons that carry the sensory information. In at least one case (the Limulus photoreceptor), adaptation results in part from intracellular elevation of Ca2+, which blocks the lightdependent activation of Na+- K+-selectiveion channels. Some receptor cells exist individually, but others are organized into sensory tissues and organs, such as the vertebrate nasal epithelium or the retina of the eye. Anatomical organization affects how a sensory organ functions. For example, the quality of the image formed by the vertebrate visual system depends on the presence of a lens and a huge population of photoreceptor cells in the retina. Several sensory systems have features in common. In particular, many receptor molecules contain seven transmemt>ranedomains, a feature that is also found in some neurotransmitter and hormone receptors. Many sensory systems also have common elements in the cascade of biochemical events that immediately follow signal detection and that amplify the signal. Mechanoreception is a result of distortion or stretching of the receptor membrane, directly producing changes in ion conductances. The deflection of the stereocilia of hair cells provides directional information by modulating, upward or downward, the frequency of spontaneously occurring impulses of axons in the eighth cranial nerve. This function is the basis of reception in several sensory organs-the lateral-line system of fishes and amphibians, vertebrate audition, and the organs of equilibrium in both vertebrates and invertebrates. The mammalian cochlea analyzes sound frequencies according to their effectiveness in displacing different parts of the basilar membrane, which bears hair cells. Mechanical waves travel along the basilar membrane, set up by sound-driven movements of the eardrum and auditory ossicles; they stimulate the hair cells, which in turn modulate their synaptic activity onto auditory nerve fibers. Certain sound frequencies stimulate each location along the basilar membrane more strongly than do other frequencies, which is the basis for frequency discrimination in mammals. Electroreceptors of fishes are modified hair cells that have lost their cilia. Exogenous currents flowing through electroreceptor cells produce changes in the transmembrane potential that modulate the release of transmitter at the base of the receptor cell, thus determining the rate of APs in sensory fibers. Visual receptors employ pigment molecules, in specialized membranes, that undergo a conformational change after absorbing a photon. The change in the conformation of

the photoreceptive molecules initiates a cascade of reactions that leads to achange in the conductance of the receptor cell membrane. All visual pigments consist of a protein molecule (an opsin) combined with a carotenoid chromophore, either retinal (in rhodopsins) or 3-dehydroretinal (in porphyropsins). The amino acid sequence of the opsin determines the absorption spectrum of each visual pigment. A cis-trans isomerization of the carotenoid initiates all visual responses. Absorption of photons is coupled to the opening (in invertebrates) or closing (in vertebrates) of ion channels by intracellular second-messengers. In vertebrate rods, photon capture by rhodopsin molecules leads to the activation of associated G-protein molecules located in the receptor membrane. Each G protein then activates many phosphodiesterase molecules, each of which hydrolyzes many molecules of the internal messenger cGMP. In the dark, cGMP continually activates Na+ channels that carry the dark current. The light-dependent hydrolysis of cGMP reduces the dark current, and a residual K+ current hyperpolarizes the photoreceptor cell, reducing the steady release of neurotransmitter at the inner segment. The reduced rate of transmitter release causes a change in activity in the next higher-order neuron. Some vertebrates have three types of cones in the fovea, each containing a visual pigment that is maximally sensitive to a different part of the spectrum. The integration of activity from all of these cones produces color vision. Rods, which in human beings all contain only one type of photopigment, are present in great densities in the periphery of the retina outside the fovea, are more sensitive than cones, and show much greater synaptic convergence. As a result, they exhlbit low acuity and high sensitivity.

REVIEW QUESTIONS 1.

2.

3. 4.

5.

6.

Visual receptor cells can be stimulated by pressure, heat, and electricity, as well as by light, as long as the intensity of these other stimuli is sufficiently great. How can this fact be reconciled with the concept of receptor specificity? Choose one sensory modality and outline the steps from energy absorption by a receptor cell to the initiation of action potentials (APs) that will travel to the central nervous system. Why must receptor potentials be converted into APs to be effective? All sensory information enters the central nervous system in the form of APs having similar properties. How can we differentiate among various stimulus modalities? What is the difference between sensory transduction and sensory amplification? Choose one sensory modality and describe how these two processes are related in that modality. Discuss the relation between the intensity of a stimulus and the magnitude of the signal sent to the central nervous system by receptor cells. How is stimulus in-

tensity encoded? How can a sensory system respond to stimuli whose intensity varies over many orders of magnitude? 7. Discuss three mechanisms that contribute to sensory adaptation. 8. Discuss one example in which efferent activity can regulate the sensitivity of receptor cells. 9. How are movements of the basilar membrane converted into auditory nerve impulses? 10. Discuss the function of inner and outer hair cells in the cochlea. How does spontaneous firing enhance the sensitivity of certain receptor systems-for example, lateralline electroreceptors? How is the presence of an object perceived by electroreceptors of the weakly electric fishes? What is the major difference between vertebrate and invertebrate photoreceptor cells in their electrical responses to illumination? Compare the mechanisms that allow the auditory system to distinguish the frequency of incident sounds and the visual system to distinguish the frequency of incident light. Outline the steps, as currently understood, in the transduction of light energy in vertebrate visual receptors. How does our current understanding of the physiology of color vision corroborate Young's trichromacy theory? Compare and contrast the morphological and functional properties of vertebrate rods and cones. What allows some arthropods to respond to the orientation of polarized light? Human beings cannot do it. Why? Compare the ways in which mammalian and teleost lenses focus images.

SUGGESTED READING Corey, D. P., and S. D. Roper. 1992. Sensory Transduction. 45th Annual Symposium of the Society of General Physiologists. New York: Rockefeller University Press. A series of papers that discuss recent data from studies of transduction in many different sensory modalities. Dowling, J. 1987. The Retina. Cambridge, Mass.: Belknap Press of Harvard University Press. A very readable compendium of information on the vertebrate retina written by a major contributor to our understanding of this sensory organ. Finger, T. E., and W. L. Silver. 1987. Neurobiology of Taste and Smell. New York: Wiley. A collection of papers considering the chemical senses in a broad range of animals. Hudspeth, A. J. 1989. How the ear's works work. Nature 341:397-404. A beautifully written account of auditory transduction by hair cells, written by a man who has played a major role in exploring the subject.

272


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Kandel, E. R., J. H. Schwartz, and T. M. Jessell. 1991. Principles of Neural Science. 3d ed. New York: Elsevier. An enormous and authoritative compendium of information about the function of the nervous system, from the biophysics of membrane channels to the physiological basis of memory and learning. Several chapters consider sensory mechanisms, with some emphasis on vertebrates.

Land, M., and R. Fernald. 1992. The evolution of eyes. Ann. Rev. Neurosci., 15:l-29. A consideration of the physical and optical properties of visual organs across all of animal phylogeny. Shepherd, G. M. 1994. Neurobiology. 3d ed. New York: Oxford University Press. A concise text that considers several sensory modalities in both vertebrates and invertebrates.

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Mammal Figure 11-32 Visual information is transmitted from the retina to the brain through layers of cells. (A) In an amphibian, the left and right sides of the optic tectum each receive projections from the entire field of view of the contralateral eye. (B) In a mammal, each side of the visual field is projected to the opposite side of the visual cortex. For example, the temporal half ofthe left retina and the nasal half of the right retina project to the left visual cortex. (C)The neurons that initially process visual inforrna-

tion are organized in layers. The retina contains the firstthree layers, and the remainder are in the brain, in the lateral geniculate nuclei and in the cortex. Information converges and diverges between the layers, and it flows in both directions between the layers. [Part A from "Retinal Processing of Visual Images" by C. R. Michael. Copyright O 1969 by Scientific American, Inc. All rights reserved. Part B adapted from Noback and Demarest, 1972.1

BEHAVIOR: I N I T I A T I O N , PATTERNS, A N D CONTROL

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Figure 11-33 The function of the vertebrate retina is based on five major types of neurons. Photoreceptors receive light stimuli and transduce them into neuronal signals. Bipolar cells carry signals from photoreceptors to the ganglion cells, which send their axons into the central nervous

system through the optic nerves. Horizontal and amacrine cells, which are located in the outer and inner plexiform layers, respectively,carry signals laterally. [From "Visual Cells," by R. W. Young. Copyright O 1970 by Scientific American, Inc. All rights reserved.]

(Figure 11-34). Vertebrate photoreceptor cells hyperpolarize when they are illuminated (see Chapter 7). They release synaptic transmitter continuously in the dark, and transmitter release is reduced when they hyperpolarize in response to illumination. Similarly, horizontal cells produce only graded hyperpolarizations in response to light (see Figure 11-34). Bipolar cells can produce graded potential changes of either polarity. A ganglion cell responds with the same polarity as do bipolar cells that innervate it. It becomes depolarized and fires APs when the bipolar cells synapsing on it depolarize, and it becomes hyperpolarized and ceases spontaneous firing when its bipolar inputs hyperpolarize. Amacrine cells respond transiently at the onset and offset of light in response to input from bipolar cells. Bipolar cells typically connect more than one receptor to each ganglion cell, and they may also connect each receptor cell to several ganglion cells. Thus, convergence and divergence already exist between the first- and third-order cells of the visual system, but the amount depends on retinal location. In mammals, both convergence and divergence are minimal in the fovea, or area centralis, (the area

in the center of the retina on which visual images are sharply focused). This lack of convergence and divergence produces very high visual acuity based on one-to-one-toone connections between cone photoreceptors, bipolar cells, and ganglion cells. (Cones are the majority of photoreceptors in the fovea.) Outside the fovea, each ganglion cell receives input from many receptor cells-primarily rods-conferring on these ganglion cells a greater sensitivity to dim illumination but a lower degree of visual acuity. Structurally, the output of the retina is carried in the optic nerve by axons of ganglion cells, but how is the output organized? Understanding the information exported by ganglion cells hinges on the concept of a receptive field, an idea that was first proposed by Sherrington and was applied to visual processing by Hartline in the 1940s. A cell's receptive field is the area on the retina in which light stimuli affect the cell's activity. The receptive field of a ganglion cell is roughly centered on the cell and varies in size, depending on the degree to which photoreceptor and bipolar cells converge in the pathway to each ganglion cell. At the center of the fovea, a ganglion cell's

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Figure 11-34 Each type of retinal neuron has a distinctive electrical response to light. Activity was recorded in each type of cell in response to a spot of light focused direaly on the receptors in the region (left) and in response to an annulus of light surrounding the photoreceptors (right). The duration of the stimulus is indicated in the lower trace on each record. In this example, the ganglion cell is activated by a light that shines on the center of its receptive field. Note that the bipolar and ganglion cells produce responses of opposite polarities to the spot and the annulus. This effect is believed to be due to lateral inhibition similar to that in Limulus. Notice that the off bipolar cell and the on-center ganglion cell shown in this figure would not be synaptically connected. See Figure 11-36 for a detailed depiction of how ganglion cell responses are related to signals in bipolar cells. [Adapted from Werblin and Dowling, 1969.1

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receptive field extends over only one or a few photoreceptors; at the periphery of the retina, where convergence is great, the receptive field of a ganglion cell can be as large as 2 mm in diameter. Each ganglion cell is spontaneously active in the dark, and the level of activity changes when a spot of light falls within its receptive field. Depending on which receptor cells are illuminated, the frequency of APs in a ganglion cell may increase if a small spot of light enters the cell's receptive field-an on response. Alternatively the frequency of APs may drop in response to a light-an off response. The receptive field of a ganglion cell is typically divided into a center and a surround, and the response of the cell depends on whether the center or the surround is being stimulated or both are (Figure 11-35). In an on-center ganglion cell, the frequency of APs increases when the center of its receptive field is illuminated (see Figure 11-35A). If a ring of light shines on the entire receptive field, with the center of the ring over the center of the field, activity in the cell drops. A

weaker off response is elicited by a spot of light that illuminates only a part of the field. The ring surrounding the center of the receptive field is called the inhibitory surround of the receptive field. An off-center cell exhibits the converse behavior, ceasing or reducing its activity when the center of its receptive field is illuminated and increasing its firing when the surround is illuminated. The center-surround organization of receptive fields depends on lateral inhibition similar to that found in the compound eye of Limulus. Lateral interaction in the vertebrate retina takes place primarily through the activity of the horizontal cells in the outer plexiform layer (see Figure 11-33). The horizontal cells have extensive lateral processes and are interconnected with neighboring horizontal cells through electrotonic junctions. In addition, they make chemical synapses on bipolar cells and receive synaptic inputs from receptor cells. Light that falls in the surround of a ganglion cell's receptive field exerts its effects through lateral connections made by horizontal cells. Because hori-

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Figure 11-35 Retinal ganglion cells have either on-center responses or off-center responses to light stimuli. (A) Four recordings from a typical on-center retinal ganglion cell. Each record shows activity in the ganglion cell during a 2.5second interval. Stimuli are shown in the middle of the figure. In the dark, APs in the cell are slow and more or less random.The lower three records show responses to a small spot, to a large spot that includes the center of the receptive field plus a surround, and to a ring that covers only the surround (B) Responses of an off-center ret~nalgangl~oncell [Adapted from to the same set of st~mul~ Hubel, 19951

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Stimulus

zontal cells form an extensive syncytial network, communicating with one another through low-resistancegap junctions, input from any receptor onto a horizontal cell produces a hyperpolarizing signal that spreads electrotonically in all directions away from the receptor. Every bipolar cell receives input from surrounding receptor cells by means of horizontal cells, and this input falls off with distance because the graded, hyperpolarizing potentials in horizontal cells decay as they spread electrotonically. The indirect input that a bipolar cell receives from outlying receptors through the horizontal cell network opposes the direct input that it receives from photoreceptors, providing the basis of the center-surround organization in retinal receptive fields. The local, direct-line pathway from photoreceptor through bipolar cell to ganglion cell produces the center response. The indirect pathway from photoreceptors through horizontal cells to bipolar cells and, thence, to ganglion cells mediates the response to the surround. These two pathways show how particular features of a stimulus can be extracted by even a relatively simple neuronal network. The distinctive responses of on-center and off-center ganglion cells arise from their connectionswith two classes of bipolar cells: on bipolar cells and off bipolar cells. These two types of bipolar cells respond oppositely to synaptic * input, both from receptors and from horizontal cells (Figure 11-36).The off bipolar cells become hyperpolarized by illumination of receptors, whereas the on bipolar cells become depolarized. In both types of bipolar cells, a light flashed onto the surround produces a response, mediated by horizontal cells, that is of the opposite electrical sign to that produced by illumination of the center. Each bipolar cell causes potential changes in its ganglion cell or cells that are of the same sign as the potential change occurring in the bipolar. Thus, ganglion cells innervated by on bipolars will have on-center receptive fields, whereas those innervated by off bipolars will have off-center fields. An on-center ganglion cell is excited by light in the center of its

receptive field because it receives direct synaptic input from on bipolar cells. It is inhibited by light in the surround of its receptive field, because horizontal cells that receive input from surrounding photoreceptors inhibit the on bipolar cells in the direct pathway from photoreceptors to the ganglion cell. The responses of on and off bipolar cells depend on how the cells respond to the neurotransmitter released by photoreceptor cells and the different neurotransmitter released by horizontal cells. On bipolar cells are steadily hyperpolarized in the dark by transmitter that is steadily secreted from the partially depolarized receptor cells. When a light stimulus causes photoreceptors to hyperpolarize, their release of transmitter drops and on bipolar cells are allowed to depolarize. This depolarization causes the on bipolar cells to release an excitatory transmitter that depolarizes ganglion cells, increasing the frequency of APs in the ganglion cells. In contrast, the off bipolars have a different class of postsynaptic channels with different ionic selectivity and are steadily depolarized in the dark by neurotransmitter released by the photoreceptors. When light falls on the photoreceptors and they hyperpolarize, reduction in their release of neurotransmitter causes the off bipolar cells to hyperpolarize. This hyperpolarization is accompanied by a drop in transmitter release by the off bipolar cells, producing a hyperpolarization of postsynaptic ganglion cells. In summary, the receptive field organization of the vertebrate retina depends on three basic features:

1. Two kinds of ganglion cells receive input from two corresponding kinds of bipolar cells. The connections produce on-center and off-center ganglion cell responses. 2. Receptors in the surround of the receptive field exert their effects through a network of electrically interconnected horizontal cells that synapse onto the two kinds of bipolar cells.

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Optic nerve Off center

On surround

Figure 11-36 Connections within the retina produce the responses characteristic of on-center and off-center ganglion cells. Two kinds of bipolar cells, EON,and BoFF,respond oppositely to direct input from the receptors, R, and to indirect input carried laterally by the horizontal cells, H. The on bipolar cells become depolarized during activation of the overlying receptor cells and are weakly hyperpolarizedby lateral input from horizontal cells. The offbipolars behave oppositely. (A) Responses of bipo-

lar cells and ganglion cells to a spot of light. (B) Responses of bipolar cells and ganglion cells to an annulus of light. Amacrine cells have been omitted from the diagram for simplicity. The direct pathway from photoreceptors to ganglion cells, G, is shown in color. The indirect, lateral pathway through horizontal cells is shown in gray. The plus and minus signs indicate synaptic transfer that conserves (+) or inverts (-)the polarity of the signal.

3.

polarization of the presynaptic cell. The postsynaptic response depends on the ionic currents produced in the postsynaptic cell as a result of the modulated release of transmitter by the presynaptic neuron.

Direct input to bipolar cells from overlying receptors and indirect input to the cells through the horizontal cell network oppose each other and thereby produce the contrasting center-surround organization seen in both the on-center and the off-center ganglion cells.

The organization of the retina reveals several general principles that apply to the other parts of the central nervous system. First, nerve cells can signal each other electrotonically without APs if the distances are small. Nonspiking neurons can in fact convey more information more accurately than can all-or-none signals. Electrotonic signals are attenuated with distance, which limits the range of effects such as lateral inhibition. Second, reception of stimuli is not necessarily synonymous with depolarization. In some nerve cells (e.g., photoreceptors and some horizontal cells), hyperpolarization is the normal response to stimulation; it modulates synaptic transmission by causing a drop in the steady release of transmitter. Third, the postsynaptic response in a neuron cannot be predicted from the sign of the potential change in the presynaptic neuron. A cell can be either depolarized or hyperpolarized in response to hyper-

Information processing in the visual cortex What happens to a retinal image after it has been transformed into an array of receptive field responses within the retina? Physically, the information is carried by axons to visual areas within the brain. The details of this pathway vary among species. In mammals and birds, the axons of retinal ganglion cells are routed either to the ipsilateral or to the contralateral side of the brain at the optic chiasm, the site where some axons cross the midline (see Figure 11-32B); whereas, in vertebrates more primitive than birds, all optic fibers are routed to the contralateral side at the chiasm (see Figure 11-32A).To some extent, the degree of crossing at the optic chiasm depends on how much overlap there is between the visual fields of the two eyes. In animals in which the visual field of one eye is entirely different from the visual field of the other eye, all retinal ganglion cell axons cross the midline. In mammals, ganglion cell axons

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............................................................................... synapse with fourth-order cells in the lateral geniculate nucleus of the thalamus. Lateral geniculate neurons send axons to synapse on fifth-order cortical neurons in the occipital cortex (see Figure 11-11)in an area called Area 17, also called primary visual cortex because it is the first region of the cortex in the pathway to receive visual information. The pattern of synaptic relations within the lateral geniculate is based on the source and the nature of information carried by retinal ganglion cells, and it constitutes another step in the processing of visual input. Each lateral geniculate nucleus, or body, is composed of six layers of cells, stacked like a club sandwich that has been folded (Figure 11-37). The top four layers contain neurons with small somata, which are called parvocellular neurons, and the bottom two layers contain neurons with large somata, called magnocellular neurons. Input onto these neurons is tightly organized. Each lateral geniculate body receives information from only one half of the visual field (i.e., one of the two visual fields illustrated in Figure 11-32B), and cells in each layer receive input from only one retina. Each neuron in the lateral geniculate receives information from only one eye. Neurons in a given layer receive information from the same eye, and the layers alternate from one eye to the other, with the pattern of alternation changing between the fourth and fifth layers (see Figure 11-37).Across all layers, the topography of the corresponding retinal surface is preserved exactly, and the topography is kept in register among the layers. If we pass an electrode along the path indicated by a dashed line in Figure 11-37, we will encounter cells that respond to a light stimulus in precisely the same

point in the visual field, but the eye of origin will switch from left to right as o w electrode moves from one layer to the next. Are there functional differences among the layers that receive information from each eye? Yes, the cells in each layer respond to particular properties of a stimulus, and the response varies from layer to layer. For example, in the monkey, cells within the four dorsal layers respond to the color of a stimulus, whereas cells of the two deepest layers do not. In contrast, the two deepest layers respond to movement, whereas the outer four layers do not. This spatial sorting of outputs from ganglion cells illustrates another principle of brain organization: information about a single stimulus is divided among parallel pathways. This pattern, called parallel processing, is a major theme of research into higher brain function. The receptive fields of neurons in the geniculate do not differ substantially from those of retinal ganglion cells. That is, they have a concentrically arranged center-surround organization of either the off-center or on-center type. The difficult question of how the visual world is organized in the next visual projection area, Area 17, was extensively and insightfully analyzed by David Hubel and Torsten Wiesel in the 1960s, and they received the Nobel Prize in 1981 in recognition of the importance of their work. In their experiments, they recorded the activity of individual neurons in the brain of an anesthetized cat while a simple visual stimulus-such as a dot, circle, bar, or edge-was projected onto a screen positioned to cover the entire visual field of the cat (Figure 11-38A). The Figure 11-37 Cells of the mammalian lateral geniculate nucleus are organized into layers, each of which receives information from only one eye. Histological section of the left lateral geniculate body of a macaque monkey; section is parallel t o the face. The cells of the outer four layers have small somata and are called pa~ocellular.Cells in the deeper layers are magnocellular. In the left lateral geniculate, all cells receive information about the right visual field. In addition, the outermost layer receives input from only the left eye, whereas cells in the next layer receive input from only the right eye, and so forth. A recording electrode passed from one layer t o the next would reveal that cells along the path indicated by the dashed line respond t o precisely the same location in visual space, but the eye that received the information alternates. [Adapted from Hubel, 1995.1

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B

Stimulus 1 \

Ret~na

Stimulus 2

I

Light

cortex

C

Stimulus 1

Stimulus 2

Stimulus 3

responses that they recorded from cortical neurons were correlated with the position, shape, and movement of the projected images. In retrospect, Hubel, Wiesel, and their collaborators made two important decisions in their experiments that allowed them to uncover order and regularity amid the enormous complexity of the visual brain. First, they chose to use more complex stimuli than just simple spots, and they asked which of these stimuli was most effective in eliciting a response in each neuron. Second, they recorded from many cells with each electrode penetration, allowing them to learn what neighboring cells had in common and how cells were grouped in the brain. These strategies allowed them to discover several different kinds of order among the interconnections of the visual cortex, and their discoveries have provided a model for examining other sensory systems. Hubel and Wiesel's major discovery about the responses of cells in the visual cortex was that they responded to entirely different properties of stimuli than did retinal ganglion cells. Cortical cells responded most strongly to bars projected in different orientations. They called the two major classes of cells that they found simple cells and complex cells, based on the nature of their optimal stimulus.

Figure 11-38 Neurons in Area 17 of a cat have very different receptive fields from those of retinal ganglion cells or lateral geniculate cells. (A) Experimental setup used to study the responses of cells in the visual cortex. An electrode is advanced through the cortex while light stimuli are projected onto a screen. (B)The receptive field of cortical simple cells is bar shaped. Aspot of light anywhere along the on part of this receptive field (stimulus 1) produces a small excitation of the simple cell. A spot of light adjacent to the bar-shaped on region (stimulus2) causes inhibition of APs in these tonically active cells. (C)Rotating a bar of light (red bar) across the receptive field of a simple cell produces maximum activity in the cell when the bar coincides completely with the on region of the cell's receptive field (stimulus 3) and partial excitation at other orientations (e.g., stimulus 2). [Part A from "Cellular Communication" by G. S. Stent. Copyright 0 1972 by Scientific American, Inc. All rights reserved. Parts Band Cfrom "Thevisual Cortexof the Bra~n"by D. H. Hubel. Copyright0 1963 by Scientific American, Inc. All rights reserved.]

They found that cells of each type were arranged systematically in space according to their optimal stimuli. The receptive fields of simple cells are long and bar shaped, and the on region of the field has a straight border separating it from the off region (Figure 11-38B), rather than the circular border found for cells in the retina and in the lateral geniculate. As for retinal ganglion and geniculate cells, the receptive field of a simple cell lies in a fixed position on the retina and, hence, represents a particular part of the total visual field. There is some variation in the receptive fields of simple cells: some have a bar-shaped on region surrounded by an off region; for others, the receptive field consists of an off bar surrounded by an on region; and, for still others, it consists of a straight edge with an off region on one side and an on region on the other side. A stimulus bar elicits maximal activity in a simple cell when it overlaps completely with the cell's on receptive field (Figure 11-38C). When the bar is rotated so it no longer aligns with the orientation of the receptive field, either it has no effect on the spontaneous activity of the simple cell or it inhibits the cell's activity. If the bar of light is displaced so that it falls just outside the on region, the cell is maximally inhibited. The orientation and the on-off boundaries

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................................................................................ differ from one simple cell to another; so, when a bar of light moves horizontally or vertically across the retina, it activates one simple cell after another as it enters one receptive field after another. What makes simple cells respond specifically to straight bars or to borders of precise orientation and location? Hubel and Wiesel suggested-and recent experiments have confirmed-that each simple cell receives excitatory connections from lateral geniculate cells whose on centers are arranged linearly on the retina (Figure 11-39A). Simple cells that respond to borders, rather than to bars, are thought to receive inputs as shown in Figure 11-39B. A simple cell would receive maximal input when light fell on all of the receptors that activate the on-center fields of the ganglion cells and the geniculate cells on the pathway to that cell. Any additional illumination would fall on the inhibitory surround of the ganglion cells and could only reduce the response of the cortical cell. Complex cells constitute the next level of abstraction in the processing of visual information. Complex cells are believed to be innervated by simple cells, which would make complex cells sixth-order cells in the hierarchy of visual information processing. Like simple cells, complex cells respond best to straight borders of specific angular orientation on the retina. Unlike the simple cells, however, complex cells do not have topographically fixed receptive fields. Appropriate stimuli presented within relatively large areas of the retina are equally effective at activating complex cells; as for simple cells, general illumination over the whole retina is not an effective stimulus. Some complex cells respond to bars of light of specific orientation (Figure 11-40A).Others give an on response to a straight border when the light is on one side and an off response when the light is on the other side. Still other complex cells respond optimally to a moving border that progresses in only one direction (Figure 11-40B).For these cells, movement in the other direction evokes either a weak response or no response at all. These receptive fields can be explained by a combination of synaptic inputs from simple cells. As a light-dark border moves through the receptive fields of the simple cells that synapse onto a complex cell, each simple cell excites the complex cell in turn as the light-dark border passes the in the the simple cells. This arrangement could produce directional sensitivityto movement of the on-off boundary (see ~i~~~~1 1 - 4 0 ~ )1f. the boundary moved so that sequential were One after would be excited, exciting the complex cell. When each simple cell became inhibited by the dark side of the moving edge, the next one in line would be excited. In contrast, if the boundary moved so that simple cells were sequentially exposed first to inhibition and only later to stimulation, one simple cell after another would inhibit the complex cell, counteracting any tendency for excitation caused by the bright side of the edge. The properties of individual cortical cells suggest that they abstract features of the visual scene, such as edges, as

On-center retinal receptive fields I

I I I

Lateral geniculate cells

I

I Receptive field of simple cell I

L- - +

L!?

Simple cell w i t h bar-shaped fi.eld

Off

Receptive fields of lateral geniculate cells

r--

I I I I I

I I

I I

Recept~ve field of simple cell I

I I

I

44

Lateral geniculate cells

I

I I

L---

on-off edge field

Figure 11-39 The responses of simple cells in the visual cortex arise from the pattern of their synaptlc inputs. (A) The fixed, bar-shaped receptive field of a s~mplecell arises from convergence of outputs from ganglion and lateral geniculate cells whose circular on-center receptive fields are linearly aligned. (B)An on-off straight-border receptive field results from the convergence of off-center and on-center geniculate cells onto the simple cell.

a first step toward analysis and recognition. The spatial relations among visual cortical cells are correlated in an orderly fashion with their functional properties. In their experiments, Hubel and Wiesel discovered that cells adjacent to one another responded to similar features of a stimulus. When they penetrated the visual cortex with an electrode that was perpendicular to the cortical surface and recorded

446


............................... A

Rece~tivefields

of

response

response

Off On "EdgeH-receptive fields projected from retina to simple cells

cells

Figure 11-40 Responses in complexcells could be based on their pattern of input from simple cells. (A) Some complex cells respond to bars of light that have a specific angular orientation, but their location can be anywhere within a large receptivefield. This pattern of response could be evoked by the convergence of many simple cells, each having similarly oriented bar-shaped receptive fields. In this example, the vertical bar of light stimulates one simple cell to fire because it falls on a row of ganglion-cell receptive fields that produce the bar-shapedreceptive field of a simple cell. If the bar were moved to the right, it would excite another simple cell that synapses onto the same complex cell, producing activ-

ity in the complex cell. In contrast, a horizontal bar of light produces only a subthreshold response in the simple cells, and hence no signal is sent to the complex cell. (B) Some complex cells respond to edges of light moving in only one direction. This response pattern could be produced by the convergence of a population of simple cells, all of which are sensitive to light-dark edges of the same orientation. Excitation of the complexcell would occur ifthe edge were moved so that it illuminated the on side of the simple-cell receptive fields before it illuminated the off side. Movement in the opposite direction would produce only inhibition.

responses from cells that were located along that pathway, they discovered that the cells along each pathway responded to bars having the same orientation. When they moved the electrode laterally and made another penetration, they found a column of cells that responded optimally to a stimulus having a different orientation from that of the optimal stimulus for the neighboring column of cells. Each such set of cells is called a cortical column. In contrast, recording from cells along a track parallel to the surface revealed an amazingly regular change in the optimal stimulus orientation, with the preferred orientation shifting about 10 degrees each time the electrode advanced 50 pm. This result implies that the cells of the visual cortex are organized in columns according to a feature of their optimal stimulus and that this difference changes in an orderly fashion across the cortex (Figure 11-41A). The columnar organization of cells with similar response properties had been seen earlier in the somatosensory cortex, where adjacent columns contained cells re-

sponding to touch or to the bending of a particular joint. However, the orderly array of orientation columns was only the first of the functionally based subdivisions found in the visual cortex. The next to be discovered related to the eye from which the visual signal came. By injecting one eye with a radioactive tracer molecule that is transported to the visual cortex, Hubel and his colleagues identified the projection pattern of each eye onto the cortical surface. These experiments revealed a second columnar system in which alternating columns represent one or the other eye (Figure 11-41B, but see Spotlight 11-3).Three-dimensional reconstructions of these columns, called ocular dominance columns, show their distribution across the cortical surface (Figure 11-41C). These experiments revealed that the visual cortex is subdivided into small functional units that analyze the stimulus into its constituent features before passing it on to higher levels for further analysis. This modular organization is superimposed on a fundamental spatial map, which

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447

............................................................................... SPOTLIGHT 11-3

eyes from precisely the same small region of the visual field These cort~calcells recelve extremely accurate neuronal projec-

SPECIFICITY OF

tions from ganglion cells "seelngn the same part of the f~eld,but

NEURONAL

of Johannes Muller, made more than a century ago, that Infor-

located In the two retinas These f ~ n d ~ n g conf~rm s the suggest~on

CONNECTIONS AND

mation originating from analogous receptors (i.e., those that

INTERACTIONS

nas converges on specific neurons in the brain. This high degree

"see" the same part of the visual field) on both right and left retiof morphological specificity underscores the precision with

Notice in Figure 11-32B that the half of a visual image that falls

which synaptic contacts are established within the central ner-

on the temporal part (the side toward the ear) of one retina falls

vous system.

on the nasal part (the side toward the nose) of the other retina

The neurons of the visual cortex are arranged in a remark-

and vice versa. In human beings, the ganglion cells on the right

ably orderly manner.When a recording electrode is gradually ad-

side of each retina send their axons t o the right side of the brain,

vanced through the cortex in a path perpendicular t o its surface,

and those on the left side send their axons t o the left side of the

and encounters neurons in successively deeper layers, all of the

brain. Thus the nasal half of the right retina and the temporal half

neurons along the track have receptive field properties in common. For example, all of the cells may b e simple cells with the

of the left retina project t o the left side of the brain. David Hubel and Torsten Wiesel, in their studies of visual

same orientation.

processing in the brain, found that some neurons in the right and

The precise and orderly arrangements of neuronal connec-

left visual cortices have receptive fields in both retinas, and that

tivity present one of the greatest challenges t o neurobiology: t o

these receptive fields are optically in register. That is, cortical cells

discover the mechanisms that guide neurons t o find functionally

that receive input from both retinas get information from both

appropriate synaptic partners during embryonic development.

1

Into cortex

Figure 11-41 Neurons of the visual cortex are arranged in columns that are perpendicular to the cortical surface. (A) D~agramillustratingthe organization of columns of cells that respond to orientation of stimuli. Columns are collections of cells for which the optimal stimulus orientation is the same for all cells in the column. Cells in adjacent columns have different optimal stimulus orientations, and this orientation varies systematically from column to column. (B)The eye that excites cortical neurons also alternates between neighboring columns. Red columns are excited by the left eye; gray columns by the right eye. (C) Simon LeVay's reconstruction of the ocular-dominance columns in part of Area 17. [Adaptedfrom Hubel, 1995.1

persists through the layers of the visual cells. To understand the nature of the spatial map at the cortical level, experiments have been done to plot the visual field onto the cortex directly, with the use of a radioactive labeling technique (Figure 11-42).Radioactive 2-deoxyglucose was injected into an anesthetized monkey and then a complex, target-shaped stimulus was projected onto its retina. Active neurons take up more 2-deoxyglucose than do resting neurons, so cortical neurons that were activated by the stimulus would be expected to contain more radioactivity than their inactive neighbors. The pattern of radioactivity observed in the visual cortex revealed that, although the twodimensional surface of the retina was completely represented on the cortical surface, the cortical pattern was not an exact replica of the spatial features of the stimulus on the retina. Instead, retinal regions representing the center of gaze (the fovea) were greatly magnified relative to those representing the peripheral view. This pattern corresponds to the difference in visual acuity across the retinal surface, as well as to differences in the convergence of primary photoreceptors onto subsequent layers of neurons. This distortion of the map in accord with the needs and habits of the animal is characteristic of all animals with well developed visual systems. For example, animals, such as rabbits, that live on large open plains have an elongated, horizontal region of specialization, called a retinal streak, that provides the greatest number of photoreceptors, and the least convergence, for receiving stimuli along the visual horizon.

All of the various levels of cortical organization must be combined to provide the next set of cortical cells with a complete picture of visual stimuli, and the manner in which this synthesis is accomplished is still the subject of intense research. For example, it now appears possible that some high-order visual neurons may be active only if a specific object (e.g., a face) enters their receptive field. The visual cortex has taught physiologists several important principles about the organization of sensory networks. First, the visual system is organized hierarchically. At each level, cells require more complicated stimuli to excite them optimally, and this complexity arises from the convergence of cells having simpler receptive fields onto cells having more complicated receptive fields. Second, although convergence is apparent as we follow a stimulus into the system, the parallel analysis of distinct features of a stimulus requires divergence of information as well. The simultaneous analysis of different features of a stimulus, which occurs along parallel pathways, appears to be an important principle of functional organization. Third, the activity of cortical neurons in Area 1 7 results in abstractions of some features of the visual stimuli. Fourth, the visual cortex does not receive a simple one-toone projection from the retina in either space or time. Instead, some regions in the visual field are expanded dramatically in their cortical representation, whereas others are compressed.

Figure 11-42 Visual space is represented on the surface of the visual cortex, but in a somewhat distorted form. This target-shaped stimulus with radial lines was centered on the visual fields of an anesthetized macaque monkey for 45 minutes after radioactive 2-deoxyglucose had been injected into the monkey's bloodstream. One eye was held closed. The cortexwas removed, flattened, frozen, and sectioned. The lower picture shows a section parallel to the cortical surface. The roughly vertical lines of label represent the curved lines of the stimulus; the horizontal lines of label represent the radial lines in the right visual field. The lines are broken because only one eye was stimulated. This dotted pattern displays the ocular dominance columns. [Adapted from Tootell et al., 1982.1


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'

The auditory map of an owl brain The retinotopic and somatotopic maps described previously are found in many levels of the brain as sensory information is transmitted through the nervous system. We can recognize these maps because, even in a distorted form, they mimic the spatial organization of objects in the outside world. The two-dimensional array of cells on the retinal surface produces a two-dimensional map of the surroundings, and the spatial relations in the surroundings are preserved as the image is projected onto the cells of the lateral geniculate and into the cortex. For other sensory systems, the nature of the possible central maps is not so obvious. In the auditory system, for example, the arrangement of hair cells along the cochlea is correlated with their sensitivity to particular frequencies of sound (see Chapter 7).If the spatial order of these hair cells were preserved in the projection of their axons to the brain, a brain map of sound frequency, a tonotopic map, would be the result. Indeed, tonotopic maps have been found in some auditory regions of the brain. However, it is not obvious how sorting sounds by frequency would help an animal acquire information about its environment. We know that human beings can locate the source of a sound in space, but knowing just the frequency of the sound would not help much in solving this problem. How does an animal locate a sound in space? Information about where the source of a sound lies relative to a listener is encoded in the intensity of the sound and in the relation between the times at which the sound reaches the two ears. If a source is to the left of the animal, sounds reach the left ear first and arrive somewhat later at the right ear. The time separating the arrival of the sound at first one ear and then the other can be computed by the nervous system as an indication of where the sound originated. To understand how this is achieved, Eric Knudsen and Mark Konishi studied barn owls, birds that depend critically on locating the sources of sounds in darkness. Barn owls have several characteristics that make them excellent animals in which to study the neuronal mechanisms that underlie sound localization. First, if light is available, owls use both vision and hearing to guide hunting, but they can capture mice in complete darkness, finding their prey only by listening to sounds (Figure 11-43). In addition, an owl cannot move its eyes within the orbits; instead it must move its whole head whether it is orienting to a sound or to a visible object, and this orienting response is quite accurate. Owls can point their heads to-

Figure 11-43 Barn owls can capture mlce in total darkness. These images are from afilm thatwas made by using only infrared illuminationthat the owl cannot see. The owl successfully captured the mouse in total darkness. [Courtesy of M. Konishi.]

ward the source of a sound with an accuracy of 1 to 2 degrees in both azimuth (lateral distance away from a point straight in front of the owl's head) and elevation (vertical distance away from a point straight in front of the owl's head). To test its orienting ability, an owl was placed on a perch and sounds were generated by a speaker whose location could be varied over a hemisphere in space while remaining at a fixed distance from the bird (Figure 11-44A). The orientation of the owl's head was monitored as the owl oriented toward the sounds produced by the speaker. The orientation of the head in response to each sound was expressed in degrees of elevation and azimuth (Figure 11-44B).Careful behavioral observations indicated that the owl was using two kinds of cues in its orienting response: the intensity of sounds was used to determine the elevation of the target and their relative times of arrival at the two ears were used to determine the azimuth of the target. To examine the role played by intensity cues, either the right or the left ear was plugged to attenuate the sounds, using plugs that either weakly or strongly reduced the sound intensity. The results of this experiment revealed that an owl consistently misdirected its gaze when one of its ears

A

along track; track can be raised and lowered

B

Figure 11-44 Owls move thew heads to orlent toward a sound, a behavlorthat IS read~lyobservable (A) Experimental setup forstudylng the ab~l~ty of an owl t o locate the source of a sound The target speaker can be moved to any locatlon In a hemisphere surrounding the front of the owl (6)Coordinate systems for determ~nlngthe locatlon of a sound Elevatlon lnd~catesthe angle along a vertlcal axls and az~muthlndlcates the angle In the horizontal plane Dlstance 1s malntatned constant [Adapted from "The hear~ngof the barn owl" by E I Knudsen Copyr~ght0 1981 by Sclent~ficArnerlcan, Inc. All rlghts resewed.]

was plugged (Figure 11-4SA). With the right ear plugged, the owl oriented below the actual source and slightly to the left. With its left ear plugged, it oriented above the source and slightly to the right. In other words, when the sound was louder in the right ear, it seemed to the owl to be corning from above; whereas, when the sound was louder in the left ear, it seemed to be coming from below. The slight difference in the azimuth angle of orientation suggests that some information about the horizontal location is available from the intensity, but intensity cannot entirely account for orientation along that dimension. How can interaural intensity differences allow an owl to discriminate the elevation of a sound source?The answer

lies-at least in part-in anatomy. The region around the openings of an owl's ears is made of stiff feathers, called the facial ruff,which form a surface that very effectively directs sounds into the ear canals like the fleshy pinna of the mammalian ear. When these feathers are removed, the external auditory canals of the owl are seen to be asymmetric (Figure 11-4SB).The opening of the right ear is directed upward, whereas the opening of the left ear is directed downward. This arrangement could provide a basis for discriminating elevation from intensity cues. The importance of the facial ruff was revealed by removing these feathers. If it lacked its facial ruff, an owl was no longer able to identify the elevation of sound sources, although its estimates along

-

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Preaural flap

/

Facial ruff

'

Figure 11-45 Plugging one of its ears caused an owl to make errors in locating the source of a sound. (A)A target was presented directly in front of the owl, which had either a hard (high attenuation, solid symbols) or a soft (less attenuation, open symbols) plug in one ear. Note that with the left ear plugged (circles) the owl judged the sound to be above ~tsreal location. With the right ear plugged, the owl made mistakes in the oppo-

site direction. (B) Facial ruff showing the asymmetry in the auditoryopenings. The right ear canal points slightly upward, whereas the left ear canal points slightly downward. This small difference is amplified by the position of the feathers in the facial ruff. [Adapted from "The hearing of the barn owl" by E. I. Knudsen. Copyright 01981 by Scientific American, Inc. All rights resewed.]

the horizontal axis remained as accurate without the facial ruff as with it. Thus, the facial ruff must amplify the directional asymmetry of the ears and is essential for discriminating differences in elevation among sound sources. How does an owl locate sounds along the horizontal or azimuthal meridian? From behavioral experiments, it was clear that differences in the time at which sounds arrived at each of the ears were important for this discrimination. However, the relevant cue could have been either disparity in the onset (or offset) of the sound or ongoing disparity that occurred during the duration of sound (Figure 11-46). Disparity of onset (or offset) refers to the difference in the time at which a given signal first reaches each ear; the ear nearest the source receives the signal first. Disparity can also occur between the signals that are received at the two ears as a sound continues; just as the onset of a sound reaches the two ears at different times, identifiable features of the sound reach first one ear and then the other. These two types of disparity were independently varied by implanting small speakers in an owl's ears. In response to disparity of onset, the owls failed to make "correct" head movements; whereas, in response to ongoing disparities ranging from 10 to 80 ps, owls made rapid head orientations to the correct place in the azimuth corresponding to that time difference (Figure 11-46B). These experiments show that owls orient to sounds in space with remarkable accuracy. Elevation is judged from differences in the inten-

sity of sounds arriving at each ear, and azimuth is judged from the ongoing temporal disparity between sounds arriving at each ear. How is information about a sound's location in space represented in the nervous system? The ears cannot directly provide the brain with a representation of external space. Instead, as we have seen, an owl must compute the difference in intensity between sound signals sensed by its two ears to determine the elevation of a sound, and it must compute an ongoing evaluation of disparity between sound signals reaching its ears to determine the position of the sound in the azimuthal plane. How and where these comparisons are made and how the output is represented in the brain were discovered by Knudsen and Konishi in the late 1970s. Knudsen and Konishi identified a collection of spacespecific neurons in a midbrain nucleus. Each of these cells responds best to sound signals that are located at a particular point in space, and each cell has a receptive field with an on-center, off-surround organization similar to that found in retinal ganglion cells (Figure 11-47A). Sounds that are located within the center region of the cell's receptive field (mean diameter = 25") excite the cell, whereas sounds in the surround of the receptive field inhibit its response. The neurons are arrayed in the nucleus to form a spatial map (Figure 11-47B) analogous to the retinotopic map derived from the retina and the somatotopic map derived

452


............................................................................... Figure 11-46 Owls judge azimuthal location from the disparity between ongoing sounds that arrive at the ears. (A) Onset disparity occurs when a sound reaches one ear before the other. Ongoing disparity refers t o the continued difference in sound waves as perceived by both ears. (B) Owls use the ongoing disparity between the sounds reaching the two ears t o localize a signal accurately in space. The linear relation between azimuth and ongoing disparity between signals at the two ears suggests that this type of disparity is the relevant cue. [Adapted from "The hearing of the barn owl" by E. I. Knudsen. Copyright O 1981 by Scientific American, Inc. All rights re-

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Disparity at onset

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from the body surface. Cells at each point on the surface of the nucleus respond by firing APs in response to sounds at a particular point in space. Adjacent points in the nucleus respond to stimuli that are adjacent in space.

Another feature common to this map and other brain maps is that the size of the receptive field for cells that receive information from directly in front of the animal is smaller than it is for cells that receive information from the sides of the animal. The area directly in front of the animal projects to a larger part of the nucleus and is therefore magnified compared with the area to the sides of the animal. This representation is reminiscent of the exaggerated representation in the visual cortex of the retinal fovea and of the large representation of the hands and face on the somatosensory cortex. In barn owls, the nucleus where these spatial fields are recorded is the rnesencephalicus lateralis dorsalis (MLD),which is the avian homolog of the mammalian inferior colliculus. (The inferior colliculus is a major auditory center that lies just beneath the superior colliculus-the mammalian homolog of the optic tectum). The MLD nucleus passes a map of sound location in space to higher centers. Disparity between signals is sensed by neurons in nuclei that lie below the MLD in the midbrain. These neurons, which are called coincidence detectors, receive input from both ears, and their activity changes, depending on whether signals from the two ears arrive simultaneously or sequentially. The mechanisms by which


453

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Fiaure 11-47 Aud~torvneurons in part of an " owl's bra~nhave spatially organized receptive fields. (A) Receptive field of a single cell showing the on-center (red) and off-surround(gray) plotted on a hemisphere. This cell responds most strongly to sounds at 0 degrees of elevation and 10 degrees to the right of center. Sounds that are 20 degrees away from this location stimulate the cell only weakly, and sounds that are 40 degrees away inhibit it. (B) Spatial auditory map in a barn owl's mesencephalicus lateralis dorsalis nucleus. Data for three electrode penetrations into the nucleus are shown. The location and orientation of each electrode track is shown on the bottom diagram, which depicts the nucleus as if it had been sectioned in a horizontal plane (orientation is indicated below the diagram). Neurons encountered along one track are numbered sequentially, and the receptive field of each neuron is shown. Neurons along one track respond to contiguous locations in space; and, as the electrode moved from one track to another, the az~muthalangle of the receptive fields (indicated on the diagram of the nucleus) changed smoothly. [Adapted from Knudsen and Konishi, 1978.1

Sound azimuth

sponse properties of neurons. Since then, similar computed maps have been found in the brains of bats, which, like owls, hunt by using auditory information. The spatial representation of sound in an owl's brain ultimately projects to the tectum, where it meets-and is congruent with-a map of space generated by the visual system. Adjacent layers of the tectum, then, are topographically correlated, with one processing information about sounds and the other processing information about visual input. This arrangement suggests that behavior can be organized more effectively if all of the sensory information about an object in space is first assembled at one location. The next problem in understanding the production of behavior is a consideration of where and how sensory information leads to a decision to act. Motor Networks Optic tectum

Left MLD

Lateral

Posterior

3-

Anterior

Medial

differences in sound intensity are computed by the owl's brain are still being investigated. The barn owl's map of acoustic space was the &st example of a brain map that is generated de nouo from the re-

The sensory side of the nervous system acquires and analyzes information about the outside world, which is essential for producing behavior that is matched to an animal's current circumstances. This information must then be passed on to neurons that are responsible for generating coordinated movement. Relatively little detail is known about the interface between the sensory and motor sides of this process, in part because investigators have worked independently at understanding either sensory or motor systems. However, in a few cases, this sensory-to-motor connection has been successfully explored, either in very simple reflexes of vertebrates or in more complex behavior of invertebrates. We will consider motor control systems of increasing complexity, from those that produce simple reflex responses through networks controlling repetitive actions to

454


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complex networks that reveal general principles of central neuromotor organization. Motor patterns of different complexity display various amounts of flexibility. Fixed action patterns are relatively inflexible; they occur repeatedly with little variation, but many behaviors are extremely plastic. The animal can shape them to fit each new set of circumstances. One of the challenges for studies of motor control is to understand the neuronal activity that allows an organism to produce behavior that changes from moment to moment, as the situation changes. Levels of motor control The study of how neurons control muscle activity has mostly been focused either on animals with simple nervous systems or on repetitive actions by more complicated animals. The neuronal control of fixed action patterns has been a major topic of this work, because the all-or-none property of these behaviors suggests that a single neuronal decision must generate the behavior. This concept of a decision does not imply a conscious process, but rather that activation of a neuronal "switch" in the central nervous system is sufficient to initiate the behavioral pattern. Conceptually, this idea can be formalized as a hierarchical motor control system in which sensory input is used to select specific motor output. The lowest level of control is the motor neuron that connects to a muscle; activity in motor neurons is regulated by integrated neuronal input (Figure 11-48). Initially, some physiologists believed that a short feedback loop between stretch receptors in leg muscles and the spinal motor neurons controlling those muscles might account for the motions of walking made by vertebrates. However, it has become clear that repetitive motor output-such as walking, swimming or flying-depends on activity in a central network that generates essential features of the motor pattern. The pattern of walking, swimming, or flying can be modified in response to sensory feedback and varies with features of the terrain or with water or wind currents in the environment. Finally, control is exerted from centers higher in the nervous system, whose decisions or commands are also influenced by sensory input. Notice that, in this control hierarchy, no strict chain of command is followed identically in every case. A wide variety of distinct environmental inputs can lead to related kinds of motor output, and feedback control operates at all levels of the system. Simple reflexes The simplest circuitry that controls the activity of skeletal muscles is the reflex arc. It takes only two kinds of neurons-muscle stretch receptors (also called la-afferent neurons) and spinal a motor neurons-connected together to produce the myotatic reflex, or muscle stretch reflex (Figure 11-49A). Because the basic form of this reflex requires only the synapse between afferent and efferent neurons, with no interposed interneurons, it is a monosynaptic reflex.

Central feedback

I

1

I

Sensory feedback from proprioceptors and sense organs

Figure 11-48 Motor control systems are arranged hierarchically. Neurons in the brain and nerve cord exert control over the entire motor side of the nervous system, generating decisions concerning motor output. These decisions modulate activity within sets of neurons, called central pattern generators, that activate motor neurons accordingto more or less preset patterns. The motor neurons provide the only pathway between the nervous system and the muscles, which in the final analysis cause behavior. Feedback occurs at all levels of the hierarchy, potentially shaping the output.

The sensory endings of stretch receptor neurons are located within each muscle, associated with sensory structures called muscle spindle organs. Each spindle organ contains a small bundle of specialized muscle fibers called intrafusal fibers to distinguish them from the majority of contractile fibers, which are called extrafusal fibers. Extrafusal fibers are the skeletal muscle fibers discussed in Chapter 10, and they are innervated by a motor neurons. The intrafusal fibers are small in mass and in number, and they do not contribute to the production of tension by the muscle. Instead, they participate in a feedback loop that regulates how sensitive the spindle organs are to stretch. Muscle spindles lie parallel to the extrafusal fibers; so, if something happens to stretch the muscle (e.g., a weight is added to an isolated muscle or a joint bends, stretching the muscle that runs over the joint), the muscle spindles are stretched too. Stretching the central region of the muscle spindles increases the frequency of APs in the la-afferent axons. These afferent axons make excitatory synapses directly on the a motor neurons that control the muscle that contains their spindle organs; so, when the activity in the la-afferent axons increases, it tends to excite the motor -

- -

455 BEHAVIOR: I N I T I A T I O N , PATTERNS, A N D CONTROL ............................................................................... Figure 11-49 Only two klnds of neurons are requ~red to produce the muscle stretch reflex (A) The steady we~ght(B) state of a muscle that 1s hold~ngup a l~ght If a heav~erwe~ght1s added to the muscle, ~tstretches the muscle, actlvatlng stretch receptors, whlch synapse onto or motor neurons In the same sp~nalsegment and cause the muscle to contract more forcefully If the sensory axons were cut, there would be no feedback onto the motor neurons and the we~ght would cause the muscle to elongate (dashed Ihnes) (C) Sequence of events that lead to produalon of the stretch reflex.

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neurons, causing reflex contraction in the muscle that was stretched (Figure 11-49B, C). The stretch receptors provide negative feedback because stretch of the muscle initiates neuronal activity that causes the muscle to contract, opposing the stretch. A familiar example of a stretch reflex is the knee-jerk reflex evoked when the tendon that crosses the knee cap (also called the patella) is tapped. Tapping the tendon produces a sudden stretch of the quadriceps muscle on the ventral surface of the thigh, activating the muscle and causing the knee joint to extend. The arclike nature of the reflex is revealed when the dorsal root into the appropriate segment of spinal cord is cut. Severing the dorsal root leaves all of the motor innervation intact but removes sensory input to the spinal segment. When the dorsal root is cut, the muscles innervated by the spinal segment go limp, even though their motor input is intact.

Notice that, when a muscle contracts under the influence of the stretch reflex, tension is removed from the muscle spindles. If nothing else happened, the la-afferents would then become silent; and, if the muscle were to be stretched a little more, the muscle spindles would be unable to respond unless their own length could be regulated. The intrafusal fibers-under the control of another set of motor neurons, the y-efferents-regulate the length of the stretch receptors. When a muscle shortens, driven by its a motor neurons, activity in the y-efferents also causes the intrafusal fibers to shorten, maintaining a constant tension in the spindle fibers. In this way, the y-efferents allow the spindle fibers to maintain their sensitivity to muscle stretch through a wide range of muscle length. Centrally generated motor rhythms Locomotion and respiration typically consist of rhythmic movements produced by repetitive patterns of muscle contraction. Each phase of such a neuromotor cycle is both preceded and followed by characteristic activity in motor neurons. Bursts of activity are consistently related to one another in time. Logically, these repetitive acts could depend on moment-to-moment sensory input to the nervous system or on the autonomous motor output of patterngenerating networks that happens entirely independently of sensory input or on some combination of these two mechanisms (Figure 11-50).Regulation of repetitive motor output has been examined in many animal systems, and it appears that both mechanisms play a role. These experiments are typically carried out in semi-intact preparations-that is, in animals in which the nervous system has been exposed for recording but that can still carry out recognizable behaviors. In some behaviors, isolated nerve cords can

456


........................................... Figure 11-50 Motor output from the nervous system depends on a comblnatlon of sensory lnput and central pattern generation Sensory Input arlses In part from the environment and In part from sensory receptors ~nthe body of the anlmal Central pattern generators-represented by a tape recorder In thls dlagram because these neurons produce the same pattern of output over and overagaln-play an Important role In shaplng behavlor, but they provlde only part of the Input onto the motor neurons.

I

Ongoing sensory input

Pattern qenerator

Sensory filter network

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BEHAVIOR

produce all features of a motor output pattern; and, although the concept of an isolated nerve cord behaving may seem strange, these behaviors can be studied either in semiintact preparations or in isolated nerve cords, depending on which is most convenient. Central motor patterns have been most clearly demonstrated in the nervous systems of some invertebrates-for example, in the neuromotor control of rhythmic locomotory movements. Grasshopper flight is controlled by mus-

cles that cause alternate up-and-down movements of the two pairs of wings, and these muscles receive the appropriate sequence of nerve impulses carried by several motor axons. (See Chapter 10 for more information on insect flight.) The patterns of activity in these motor neurons continue to occur with appropriate phase relations, even if sensory input from the muscles or joints of the wings is eliminated by cutting the sensory nerves (Figure 11-SlA).This persistence suggests that the motor pattern may be largely generated

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Figure 11-51 Both a central pattern generator and sensory feedback contribute to the production of grasshopper flight. (A) Experimental arrangement.An eviscerated grasshopper or locust is mounted so it can flap its wings when it is stimulated by air blowing on facial receptor hairs. Electrodes for recording motor output and for stimulating receptor nerves are fixed in place. (B) When sensory receptor neurons at the base of the wings are destroyed, the central pattern generator produces a lowfrequency pattern. Electr~callystimulating the receptor axons increases the frequency of the endogenous motor output. The time during which the receptor nerve was being stimulated is indicated by the black line. After the stimulation ceases, the rhythm returns t o a low frequency. (C)Cyclic organization of flight motor output. Externalsensory input (e.g., a puff of air on hair receptors) stimulates the flight motor output. Wing movements activate stretch receptors that provlde Input stlmulatlng the fllght motor Notlce that thls loop resembles the posltlve feedback loop

illustrated In Flgure 11-26 [Adapted from Wllson, 1964,1971.]


457

............................................................................. within the central nervous system by a network of neurons that interact to coordinate the timing between contraction of different muscles. Does sensory input play any role in the control of grasshopper flight, which seems to be driven by centrally programmed motor output? Sensory feedback from stretch receptors at the base of each wing is stimulated by the movement of the wings and can modify motor output, increasing the frequency, intensity, and precision of the rhythm. If these receptors are destroyed, the neuronal output to the wing muscles slows down to about half its normal frequency, although the phase relations among impulses in the different motor neurons are retained. The original frequency of the rhythm can be restored if the nerve roots containing the axons of the wing-joint receptors are stimulated electrically (Figure 11-SIB). Interestingly, although the motor rhythm increases in frequency when it is ~rovidedwith sensory input, the timing of the motor output is not closely related to the timing of impulses in the sensory nerves. Random stimulation of the wingjoint receptor axons can speed up the motor output, although sensory input is most effective if it occurs during a particular phase of the wingbeat cycle. Thus, proprioceptive feedback is not required for proper phasing of motor impulses to the flight muscles; but, when the central flight pattern generator becomes activated, sensory feedback reinforces its output (Figure 11-SIC). What turns the flight motor on and off? When a grasshopper jumps off the substrate to begin flight, hair receptors on its head are stimulated by the passing air. This specific sensory input initiates the output of the flight motor. When the insect alights, the flight central pattern generator is turned off by signals originating in mechanoreceptors of the foot (the foot is called the tarsus in insects). Endogenous pattern-generating networks have been shown to exist in a number of invertebrate nervous systems. For example, the cyclic motor output to the abdominal swimmerets of the crayfish persists not only in an isolated nerve cord, but even in single, isolated, abdominal ganglia. This intrinsic rhythm is initiated and maintained by activity of "command" interneurons, whose somata are located in the supraesophageal ganglion of the brain. Although the bursting pattern in each abdominal ganglion requires maintained activity in one, or perhaps several, of the interneurons, there is no simple one-to-one relation between the firing pattern of these interneurons and the pattern of motor output to the swimmerets. The crucial interneurons appear to be providing a general level of excitation, which keeps the central pattern generator active. One of the best studied rhythmic patterns is escape swimming in the nudibranch mollusk Tritonia (Figure 11-52A). This sea slug swims away from noxious stimuli by making alternating dorsal and ventral flexions of its body, which are produced by alternating contractions of dorsal and ventral flexor muscles. The central pattern is generated by interconnections of three types of neuronsa cerebral neuron (C2),the dorsal swim interneurons, and

the ventral swim interneurons-which synapse onto the flexion neurons (Figure 11-52B).The cerebral neuron (C2), the dorsal swim interneurons, and the ventral swim interneurons are linked by reciprocal connections many of which are a mixture of excitatory and inhibitory synapses. Reciprocal inhibitory synapses between neurons have been found in many central pattern generators that produce rhythmic outputs; the reciprocal inhibitory synapses in the central pattern generator for swimming in Tritonia have been shown to be necessary for generating swimming in this species. After the initial stimulus, the dorsal and ventral swim interneurons produce alternating bursts of neuronal activity, which activate the flexion neurons responsible for motor output. Intracellular recordings of activity in all five neuron types indicate that the swimming rhythm depends on both the membrane properties of individual neurons and their synaptic connections. Thus, the rhythm is neurogenic, produced by interactions between neurons. Recently, it has been demonstrated that synaptic strengths among the neurons of this network can be modulated during an episode of swimming to change the properties of the network, even while it is producing the swimming output. Autonomous central neuronal control also exists to various degrees in vertebrates. Respiratory movements, which are driven by cells in the brain stem, persist in mammals when sensory input from the thoracic muscles is eliminated by cutting the appropriate sensory nerve roots. Toads in which all sensory roots, except those of the cranial nerves, have been cut still produce simple coordinated walking movements, although these movements are hard to discern, because loss of the myotatic reflex arc causes muscles to become flaccid. Motor output to the swimming muscles of sharks and lampreys continues in a normal, alternating pattern when segmental sensory input is eliminated. However, the intersegmental sequencing of motor output, which normally travels from the anterior to the posterior may be disrupted. Walking movements have been investigated in cats that are supported on a treadmill after the brain stem has been transected above the medulla oblongata (called a spinal cat preparation). Such studies reveal that the walking sequence can occur without input from the brain. Moreover, a rudimentary walking rhythm has been seen to continue even after the dorsal roots have been transected, eliminating sensory input. Thus, even in vertebrates, some aspects of rhythmic movements are programmed into intrinsic connections between neurons within the spinal cord and hind brain, and they can continue even if sensory feedback and other sensory inputs are disrupted. Central command systems The stimulation of appropriate neurons in the central nervous system can elicit coordinated movements of various degrees of complexity. Electrical stimulation of one such command system, in the nerve cord of the crayfish, causes the animal to assume the defense posture, with open claws held high and body arched upward on extended forelegs.

CPG

Flexible

neurons

C2 DSI

DFN

Figure 11-52 Swimming in the nudibranch mollusk Tritonia is controlled by a central pattern generator consisting of three types of neurons. (A) If a Tritonia is threatened (such as by a nudibranch-eating starfish), it rises off the substrate and swims by rhythmically contracting dorsal and ventral flexor muscles. (B) Three interconnected types of neurons act together to generate the swim motor pattern. An excitatory synapse is represented by a bar; an inhibitory synapse by a solid circle; a combination of the two symbols represents a multifunctional synapse. Membrane

properties and synaptic interactions determine the swim motor pattern, Recordings of activity in which changes if these parameters change. (C) swim central pattern generating (CPG) neurons in an isolated brain after electrical stimulation of the pedal nerve.Abbreviations: C2, cerebral neuron; DSI, dorsal swim interneurons; VSI, ventral swim interneurons; DFN, dorsal flexion neurons; VFN, ventral flexion neurons. [Part A courtesy of P. Katz; parts B and C adapted from Katz et al., 1994.1

Appropriate sensory input excites this system through one specific interneuron, and this interneuron diverges broadly, producing excitation in some motor neurons and inhibition in others. Command systems in arthropods characteristically activate many muscles in a coordinated manner and produce reciprocal actions in a given body segment; that is, antagonists are inhibited while synergists are excited. Perhaps not surprisingly, the command interneurons that are most effective in eliciting a coordinated motor response are generally least easily activated by simple sensory input. The discovery of this command neuron in the crayfish initially caused physiologists to hypothesize that a lot of an animal's behavior might be controlled by a small population of command neurons, each of which was responsible for producing and shaping a particular behavior. In this case, "choosing" among behaviors would depend on which command neurons were most active. However, further study of the neuronal basis of behavior suggests that most command functions arise within networks of neurons, in which all contributing neurons play an important role. To determine experimentally whether a neuron fills a command function, it is necessary to show that the activity of

the neuron is both necessary and suficient for causing the particular motor output. That is, removing the neuron from the network must block, or greatly modify, the behavior (necessity) and activating only that neuron must produce the behavior (sufficiency). When the necessity and sufficiency tests are carried out to determine the neuronal basis of many behaviors, three observations appear again and again. First, many neurons are multifunctional, functioning differently under different conditions. For example, some retinal bipolar cells have been found to carry signals from rod photoreceptors in dim light and from cone photoreceptors in bright light. There must be a shift in their connectivity pattern as the level of ambient light changes. Second, one neuron may belong to different levels of a hierarchical control system (see Figure 11-48). For example, one neuron in the Tritonia swim control network acts both in the central pattern generator for swimming and in the command system for escape. Third, because networks can be reconfigured, depending on the situation, there must be mechanisms that can modify neuronal connectivity. Anatomical connections may constrain the range of possible outputs for a set of neu-


459

............................................................................ rons, but functional connections define their output at any given time. One of the best understood mechanisms for shifting neuronal networks among possible functional configurations is neuromodulation (see Chapter 6 ) . Neuromodulators can cause changes in synaptic efficacy that dynamically reconfigure a collection of neurons into a new functional unit. Recognition that "anatomy is not destiny" in the nervous system has changed the way systems are analyzed. In this section, we consider two systems that have been analyzed in sufficient detail to provide examples

of these three principles in the organization of command systems. Many invertebrates escape from potential predators by using stereotypical movements. One well studied example is the crayfish, Procambarus clarkii, which has two types of escape response, depending on the location of the stimulus (Figure 11-53A).In each behavior, at least one giant axon is part of the control circuitry, a typical pattern in the neuronal control of many escape responses. Large axons carry signals rapidly, allowing an animal to escape faster. In the

A Abdomen

30

40

60

Lateral giant interneuron

Motor giant

Sensory receptor Sensory interneuron

Muscle Abdominal segment 1, 2, o r 3

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Figure 11-53 Tactile stimulation of giant interneurons cause crayfish to change their posture. (A) Stimulation of the abdomen (upper left) evokes an abdominal flexion that moves the crayfish upward and forward. This behavior is mediated by the lateral giant interneurons. Anterior stimulation at the antennae (upper right) evokes an abdominal flexion of a different form that propels the animal backward. This response is mediated by the medial g~antInterneurons In both cases, the behavlor moves the an~malaway from the st~mulus In the d~agrams,the tlme after st~mulat~on IS ~nd~cated In m~ll~seconds and time proceeds from top to bottom (B) S ~ m p l ~ f ~ d~agram ed of the clrcult that med~atesthe crayf~shescape response to touch on the abdomen Sensory Input IS carr~edby chem~cal(tr~angles)and electr~cal(res~storsymbols) synapses to the lateral glant neuron, wh~chmakes a rap~d, electroton~csynapse onto the motor glant neuron The motor g~antneuron synapses onto the abdom~nalflexor muscles The large size of the giant axons produces high conduction velocities, and the electrotonic synapses provide rapid communication between neurons. Electrically stimulating the lateral giant interneuron produces flexion only in abdominal segments 1 through 3. Compare this effect with the final posture of a crayfish that has been touched on the abdomen-flexion is pronounced in the anterior abdomen. [Adapted from Wine and Krasne, 1972,1982.1

1

crayfish, there are two giant fibers: the medial giant interneuron, which controls flexion to propel the animal backward; and the lateral giant interneuron, which plays a key role in propelling the animal up and forward. The basic circuitry surrounding the lateral giant interneuron is shown in Figure 11-53B. The neuronal network surrounding the medial giant is quite different-on both the input and the output sides of the network-which explains why the behavior is so different when the crayfish is touched on its antenna. The escape responses of crayfish illustrate some other features of motor control systems. First, if a crayfish is stimulated repeatedly, it fails to respond after about 10 minutes of stimulation; the response is said to habituate. Although habituation can occur at many different points in the network, it has been found that this behavior habituates because less neurotransmitter is released from the terminals of the sensory afferent neurons when stimuli are repeated for long periods. Second, the overall control of the crayfish tail includes a second, parallel pathway that can also elicit the tail-flip response. The fast flexor motor neurons, which are not giant motor neurons, produce more precise control of the tail flip, although it is neither as rapid nor as vigorous as the flip produced by the giant neurons. When the tail flip is initiated by the motor giant neurons, the second pathway is activated, too, although the slow pathway can operate by itself. Third, if the level of serotonin, a neuromodulator, changes in the crayfish, the response of the crayfish to a particular stimulus can change dramatically. An aggressive crayfish can become submissive, and vice versa. Thus neuromodulation must modify the connections between sensory and motor neurons. The crayfish escape response is a typical fixed action pattern, and the neurons that control it illustrate several features of command systems outlined earlier. Perhaps the most important feature is the existence of multiple control points within the network, which offer several ways to initiate or to slightly alter the performance of the behavior. This flexibility within the constraints of a fixed action pattern has been a source of insight into the organization of behavior.

circumstances. The premier example of dynamic network assembly can be found in a collection of 30 large neurons that make up the stomatogastric ganglion (STG) of crustaceans. The esophagus and stomach of lobsters and crabs is a complex structure that is responsible for ingesting, storing, chewing, grinding, and filtering food (Figure 11-54). There are four functional regions of the stomatogastric system: the esophagus, the cardiac sac, the gastric mill, and the pylorus. The neurons of the STG control all of the muscular chambers responsible for ingestion and peristaltic movement of food. They also control the bony teeth that are responsible for chewing and grinding it. Because most neurons in the STG are motor neurons that innervate the muscles in the stomatogastric system, their intrinsic properties have been of direct interest in efforts to discover the functional architecture of each subnetwork that can be formed from this small set of neurons. The ganglion can be divided into three networks of neurons, which control muscles in the esophageal, gastric mill, and pyloric regions of the stomatogastric system. The esophageal, gastric, and pyloric networks can each generate patterns of rhythmic output that are independent of the other two (Figure 11-55A). The frequency of output from each of the networks is a characteristic of the network. Input from modulatory neurons changes the behavior of these neurons drastically. For example, two electrically coupled neurons, called PS neurons, reconfigure the networks. When PS neurons fire, a valve between the esophagus and the stomach opens, and swallowing behavior is initiated. Then an entirely new rhythm begins, coordinating

dilator

The recognition that synaptic neuromodulators can change the properties of a network has opened new avenues of thought. Central command systems, each of which was once believed to drive a single behavioral pattern to completion, must now be viewed as being plastic, with neurons taking on different synaptic relations, depending on

muscle

Figure 11-54 The stomatogastric nervous system controls aaivity in the esophagus, gastric mill, and pylorus of the lobster. The stornatogastric ganglion (STG, one of four ganglia in the system) contains only 30 neurons, most of which are motor neurons and all of which have been identified and characterized. The output of these neurons controls contraction of muscles that cause food to be swallowed, chewed, and moved to the rest of the dlgest~vesystem. (Muscles that control the pylorus are shown. Constrictor muscles close the pylorus, preventing food from moving out. Dilator muscles open the pylorus, allowing food to move into the next segment of the digestive system. These muscles receive input from STG neurons.) [Adapted from Hall, 1992.1

........................... PS silent

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Pyloric network

Es Neuronal activity

Gast PYl PS

I all three parts of the STG system to produce a set of peristaltic waves that travel from the esophagus to the pylorus (Figure 11-55B).All other rhythms are inhibited during this behavior. When activity in the PS neurons ceases, yet another rhythm transiently appears, but eventually all of the neurons in the swallowing network return to their original activity patterns. The neurons that control swallowing behavior are called the swallowing network, which includes neurons that, in the absence of PS neuron activity, are active in the esophageal, gastric, or pyloric networks. Some of the neuromodulators that control the activity of STG neurons have been identified. The biogenic amine serotonin and the neuropeptides proctolin and cholecystokinin all change the output pattern of at least some neurons in the STG. The reconfiguration of a small pool of neurons into several functional networks suggests a new view of the neurons responsible for controlling motor output. Previous work has shown that a single anatomically defined network can produce different forms of output in response to neuromodulatory agents, but the stomatogastric system suggests that the composition of the network, too, can be plastic. Dynamic specification of many functional networks within a defined set of neurons offers a large increase in the number of possible ways that motor output can be controlled. Clearly, one challenge is to discover where the control of this mechanism resides and how it is regulated.

SUMMARY All behavior is controlled by the motor output of the nervous system. Motor neurons are organized into &verse net-

Figure 11-55 Modulatory inputs to the stomatogastric gangl~onchange the neuronal outputs dramat~cally,reconfiguring subnetworks in the ganglion. (A) When the modulatory PS neurons are silent. neurons in the stornatogastric ganglion are organized into three separate subnetworks, the esophageal, gastric, and pyloric networks. Each of these subnetworks ~roducesa rhvthmic o u t ~ u t but: , the outputs are not temporally coord~natedw ~ t h one another In th~sstate, food IS chewed and moved about wlthln the cardlac sac and the pylorlc cav~ty(~ndlcatedby red arrows), no food enters or leaves this part of the digestive tract. (B) When PS neurons are active, neurons of all three subnetworks are recruited into a new network in which their activity is coordinated to produce "swallowing" (indicated by the red arrow). Abbreviations Es, Gast, Pyl, and PS indicate activity of neurons In the esophageal, gastric, and pyloric subnetworks and of the PS neurons. [Adapted from

enters

works that may be somewhat plastic, allowing flexibility in behavioral responses. Understanding behavior at the neuronal level requires an understanding of how neurons interact to produce behavioral output. In the course of evolution, the primitive, distributed, anatomically diffuse "nerve nets" characteristic of coelenterates became condensed into nerve cords and ganglia, which are seen even in some jellyfish. In segmented animals, the anterior end, initially specialized as the location of many sensory organs, became differentiated to contain a superganglion, or brain. The most complicated nervous systems are found in vertebrates. These systems can be divided into the central and the peripheral nervous systems. All neurons in the nervous system are afferent neurons, efferent neurons, or interneurons, and most of the neurons in complex neuronal networks are interneurons. To a large extent, the connections in central networks appear to be preprogrammed genetically; but, during development and thereafter, they are sustained by and can be modified by use. The integration of input and the production of subsequent activity in each neuron of a network depends primarily on two major sets of factors: (1)the organization of circuits and synapses formed by interacting neurons, and (2) the way in which individual neurons process or integrate incoming information to produce their own APs. The integrative properties of a neuron depend on the anatomy of the neuron, on its connections, and on the properties of its cell membrane and ion channels. The study of the neuronal control of behavior has been aided by the identification of specialized behaviors called fixed action patterns. These highly stereotyped motor

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patterns are typically elicited by a particular stimulus, or key stimulus. Understanding the behavioral capacities of animals at the neuronal level is the goal of neuroethology. Sensory neuronal networks sort and refine information available to the animal. They magnify, amplify, filter, and reconfigure the original sensory input. The mammalian visual system has taught us a great deal about how sensory systems function. Electrical recording from cells in the visual cortex indicates that individual central neurons are activated by, and extract, certain features of a stimulus, rather than generating a point-to-point representation of the peripheral input. In addition, studies of the visual system indicate that there is a heirarchical arrangement of neurons and that the specificity of sensory features evoking activity in the neurons increases with each level, until only specific features of the visual stimulus evoke responses at the higher levels. Some cells may be activated only by such complicated stimuli as a face. Studies in the barn owl have shown that a map of auditory space is computed from intensity and timing differences between sounds as they are received at the two ears. Such computed spatial maps are in register anatomically with other sensory maps, such as the retinotopic map from the visual system. The simplest neuronal networks are monosynaptic reflex arcs, the most familiar of which is the stretch reflex of vertebrates. More complex behaviors include locomotory movements that are based in part on central "motor programs" that determine, for example, the sequence of muscle contractions that produce coordinated locomotory movements. Feedback from proprioceptive sensory neurons can exert an influence on the strength and the frequency of the motor output and, in most rhythmic motor activity, it also contributes to fine-tuning its coordination. Muscle output is controlled by a hierarchiacal system. An example of the lowest level of control is the monosynaptic stretch reflex arc that is responsible for maintaining postural tone. At the next level are rhythmic motor patterns characteristic of walking, swimming, and crawling. Finally, the control of complex fixed action patterns resides at the top of the hierarchy. Attempts to understand the relations between levels of control have been most successful in the relatively simple motor systems of invertebrates. In these model systems, it is clear that a particular neuron may participate in several motor networks that function at different levels. Moreover, in some systems, neuromodulatory substances dynamically control the configuration of networks.

REVIEW QUESTIONS 1. Action potentials in all neurons are fundamentally alike; so how is the modality of input from the various sense organs recognized by the central nervous system? 2. Describe the general organization of the vertebrate brain and spinal cord. 3. Compare and contrast the sympathetic and the parasympathetic divisions of the autonomic nervous

system. How do they differ anatomically? How do they differ functionally and biochemically? It has been said that all sensation takes place in the brain. Explain what is meant by this statement. How can an increased rate of firing in an inhibitory interneuron produce increased activity in other neurons? What is the source of the continuous low-level synaptic input and slow tonic firing in vertebrate spinal a motor neurons? Describe the organization of the vertebrate retina. Each eye of a primate sees about the same field, but the right hemisphere of the brain "sees" the left half of the visual field, whereas the left hemisphere "sees" the right half of the field. How does this occur? Why does the evening sky appear to have a lighter band outlining the silhouette of a mountain range? What is meant by the "receptive field" of a cortical neuron? How can the receptive field of a simple cell in the visual cortex be a bar or a straight edge, when cells of the lateral geniculate have circular receptive fields? What would happen to your posture if all of your muscle spindles suddenly ceased to function? How do y-efferent fibers change the sensitivity of muscle spindles? Discuss some of the general insights into neuronal organization that have resulted from studies of the retina and visual cortex. The nervous system is sometimes compared to a telephone system or a computer. Discuss some properties of the nervous system that make this a good analogy and others that make it a poor analogy. What evidence indicates that some complex behavior patterns are inherited and cannot be ascribed entirely to learning? What supports the statement that the major factor responsible for differences in the functioning of different nervous systems is neuronal circuitry and not the properties of single nerve cells? What are releasing stimuh and fixed action patterns? Give at least one example of each. What is a central pattern generator? What are some of the properties of central pattern generators, and what roles do they play in the control of behavior? Describe some examples of central pattern generators. What is a command neuron? What is a command system? Describe some examples of command systems. How can one neuron play different roles in several central pattern generators?

SUGGESTED READINGS Camhi, J. 1984. Neuroethology. Sunderland, MA: Sinauer. (An excellent textbook summarizing many subfields of this rapidly growing discipline.)

Carew, T. J., and C. L. Sahley. 1986. Invertebrate learning and memory: From behavior to molecule. Ann. Rev. Neurosci. 9:435-487. (A review of progress toward understanding this important form of plasticity in the nervous system.) Dowling, J. 1987. The Retina: An Approachable Part of the Brain. Cambridge, MA: Belknap Press. (A description of the structural and functional organization of the vertebrate retina, written by a major contributor to our current knowledge of this remarkable organ.) Ewert, J.-P. 1980. Neuroethology. Berlin: Springer-Verlag. (An introduction to a relatively new field: the study of the neuronal basis of behavior.) Grillnel; S., and P. Wallen. 1985. Central pattern generators for locomotion, with special reference to vertebrates. Ann. Rev. Neurosci. 8:233-261. (Areview of the properties of central pattern generators, with emphasis on the CPG for swimming in lampreys.) Gwinner, E. 1986. Internal rhythms in bird migration. Scientific American 25494 - 92. (A biological approach to this otherwise apparently mysterious navigational ability.) Hubel, D. 1995. Eye, Brain, and Vision.New York: Scientific American Library Paperbacks. (Anexceedingly readable review of information processing in the visual system written by one of the most prolhc and creative researchers in the field.)

Kandel, E., J. Schwartz, and T. Jessell. 1991. Principles of Neural Science, 3d ed. New York: Elsevier. (A giant compendium of information about the nervous system, with some emphasis on vertebrate-particularly mammalian-species.) Knudsen, E. I. 1981. The hearing of the barn owl. Scientific American 245:113 - 125. (A very readable discussion of the remarkable auditory nervous system of this bird, including a description of some very creative physiological experimentation.) Konishi, M. 1985. Birdsong: From behavior to neuron. Ann. Rev. Neurosci. 8:125-170. (A review of the neuronal basis of the production of bird songs, written by one of the most eminent experts on the avian brain.) McFarland, D. 1993. Animal Behaviour: Psychobzology, Ethology, and Evolution. New York: Wiley. (A classic text covering the study o f animal behavior.) Nicholls, J. G., A. R. Martin, and B. G. Wallace. 1992. From Neuron to Brain: A Cellular and Molecular Approach the Nervous System, 3d ed. Sunderland, MA: Sinauer. (A very readable text describing both singlecell properties and circuitry.) Nauta, W. J. H., and M. Feirtag. 1986. Fundamental Neuroanatomy. New York: W. H. Freeman and Company. (A comprehensive description of mammalian neuroanatomy.)

P A R T

INTEGRATION OF PHYSIOLOGICAL SYSTEMS

T

o this point we have discussed the basic principles of animal physiology (Chapters 1-4), followed by a discussion of the nervous, muscular, and endocrine systems and the processes by which they regulate physiological function (Chapters 5 - 11). What remains to be discussed in Part I11 (Chapters 12-16) are the various regulated physiological systems that are involved in the day-to-day efforts of animals to acquire and store nutrients and energy, to expel wastes, to respond to changing environments, and to reproduce. Textbooks in animal physiology historically have treated each of the regulated physiological systems of animals more or less separately, with relatively little focus on their mutual functional and structural interdependencies. This approach persists both for convenience of discussion and because, to some extent, it reflects the patterns of interests of biologists in particular animal systems. Physiologists usually identify themselves as, for example, "cardiovascular physiologists;" few would stress the more integrated aspects of their field by identifying themselves as, for exam-

ple, "energy transfer physiologists" studying the coordinated transport of nutrients, wastes, and heat between the environment and an animal's interior. Further, because there are similarities between the circulatory systems of all animals, it is convenient to discuss individual systems in a single chapter. The division of physiological systems into units, useful in organizing a course or a book, however, has yielded generations of students with the misimpression that animals function as a series of loosely linked physiological systems that happen to be enclosed in a single organism. For that reason we want to stress that animals operate as integrated systems that are responsive to, and constrained by, their surrounding environment. These interrelated systems act in a highly coordinated fashion when faced with stresses that are either environmental (temperature, pressure, etc.) or biological (predation, disease, etc.). The actual design and function of an individual physiological system is modified by constraints placed on it because it is part of a larger physiological network. Because these systems are highly mutually

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dependent upon each other, environmental stresses may present conflicting demands upon individual systems. It is important to think about these interactions in terms of both space and time. Examples abound. Lung vital capacity in some snakes is initially reduced following ingestion of a large prey item because of space limitations in the visceral cavity. However, full lung capacity slowly returns as the meal is digested (interactions between respiration and digestion in space and time). A similar condition exists in humans after a large meal or during pregnancy. As another example, muscle power responds to physical training over time but this is not simply related to increased muscle mass. There must also be increased blood flow to the muscle which may require changes in the heart and respiration (interactions between locomotor and cardio-respiratory systems over time). In addition, the skeletal frame must be strengthened to withstand the increased stress placed on the body by this training. Although we wish to emphasize the importance of taking an integrated view of the physiology of an animal, we realize that, at the same time, it is not practical to ask a student to learn simultaneously everything about all regulated physiological systems. Thus, we have divided the regulated systems into several different chapters. While each of these chapters focuses

on a particular system and its functions, examples are used throughout that will emphasize the interactions between physiological systems and the way they respond in a coordinated manner to environmental change. Chapters 12 through 14 of Part I11 discuss truly multi-function systems. The circulation (Chapter 12) is a means of distributing material between tissues, in particular oxygen, carbon dioxide, and various nutrients and excretory products. Acquisition of oxygen and the elimination of carbon dioxide is the subject of Chapter 13. The circulation and respiratory systems of animals both function together in homeostasis, for example, by regulating acid-base status and, in some systems, ionic and osmotic conditions within the animal (Chapter 14).Animals use a variety of mechanisms to acquire energy, ranging from filter feeding to predation, as described in Chapter 15. This chapter discusses the mechanics, control, and chemistry of food acquisition, digestion, and assimilation. The concluding chapter (Chapter 16) is, in many ways, a summary of the themes of the book, delving into the energetics of animals. Energy use in movement, reproduction, growth, and maintaining homeostasis is explored and placed in the overall objective of surviving to reproduce.

I

n animals 1 mm or less in diameter, materials are transported within the body by diffusion. In larger animals, however, adequate rates of material transport within the body can no longer be achieved by diffusion alone. In these animals, circulatory systems have evolved to transport respiratory gases, nutrients, waste products, hormones, antibodies, salts, and other materials among various regions of the body. Blood, the medium for transport of such materials, is a complex tissue containing many special cell types. It acts as a vehicle for most homeostatic processes and plays some role in nearly all physiological functions. This chapter reviews the circulation of blood and how it is controlled to meet the requirements of the tissues. Most attention is given to the mammalian circulatory system, because it is the best known. Mammals are very active, predominantly aerobic, terrestrial animals, and their circulatory system evolved to meet their particular requirements. The mammalian system is only one of several types of circulation. All circulatory systems, however, comprise the following basic parts, which have similar functions in different animals:

1. A main propulsive organ, usually a heart, which forces blood around in the body 2. An arterial system, which can act both to distribute blood and as a pressure reservoir 3. Capillaries, in which transfer of materials occurs between blood and tissues 4. A venous system, which acts as a blood (volume)reservoir and as a system for returning blood to the heart The arteries, capillaries, and veins constitute the peripheral circulation.

GENERAL PLAN OF THE ClRCULATORY SYSTEM The movement of blood through the body results from any or all of the following mechanisms:

Forces imparted by rhythmic contractions of the heart Elastic recoil of arteries following filling by cardiac contraction Squeezing of blood vessels during body movements Peristaltic contractions of smooth muscle surrounding blood vessels The relative importance of each of these mechanisms in generating flow varies among animals. In vertebrates, the heart plays the major role in blood circulation; in arthropods, movements of the limbs and contractions of the dorsal heart are equally important in generating blood flow; in the giant earthworm, Megascolides australis, peristaltic contractions of the dorsal vessel are responsible for moving blood in an anterior direction and fillingthe lateral hearts, which pump blood into the ventral vessel for distribution to the body (Figure12-1A).This worm, which can be up to 6 m in length, is divided into segments separated by membranous structures (septa).Tracer studies have shown that the anterior 13 segments, each of which contains two lateral hearts, have a rapid circulation, but the remaining segments, which lack lateral hearts, have a very sluggish circulation. Because of the peristaltic contractions of the dorsal vessel, the blood pressure is considerably higher in the dorsal vessel than in the ventral vessel (Figure 12-1B). In all animals, valves andlor septa determine the direction of flow, and smooth muscle surrounding blood vessels alters vessel diameter, thereby regulating the amount of blood that flows through a particular pathway and controlling the distribution of blood within the body. Open Circulations

Many invertebrates have an open circulation, that is, a system in which blood pumped by the heart empties via an artery into an open fluid space, the hemocoel, which lies between the ectoderm and endoderm. The fluid contained within the hemocoel, referred to as hemolymph, or blood, is not circulated through capillaries but bathes the tissues directly. Figure 12-2 (Aand B) illustrates the organization of the main vessels in the

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Time (s) Figure 12-1 In the giant earthworm, Megascolides australis, peristaltic contractions of the dorsal vessel and pumping by the lateral hearts are both important in moving blood. (A) Blood flows from the dorsal vessel into the lateral hearts, present in the 13 anterior segments, and then is

pumped into the ventral vessel. (B)Peak blood pressure is about twice as high in the dorsal vessel, because of its peristaltic contractions, than in the ventral vessel. [Adapted from Jones et al., 1994.1

open circulation of two groups of invertebrates. The hemocoel is often large and may constitute 20%-40% of body volume. In some crabs, for instance, blood volume is about 30% of body volume. Open circulatory systems have low pressures, with arterial pressures seldom exceeding 0.6- 1.3 kilopascals (kPa),or 4.5-9.7 rnm Hg (1kPa = 7.5 rnrn Hg). Higher pressures have been recorded in portions of the open circulation of the terrestrial snail Helix, but these are exceptional. In snails, these high pressures are generated by contractions of the heart, whereas in some bivalve mollusks high pressures in the foot are generated by contractions of surrounding muscles rather than of the heart.

Animals with an open circulation generally have a limited ability to alter the velocity and distribution of blood flow. As a result, in bivalve mollusks and other animals that have an open circulation and use blood for gas transport, changes in oxygen uptake are usually slow and maximal rates of oxygen transfer low per unit weight. Nonetheless, such animals exert some control over both the flow and distribution of hemolymph; moreover, the blood is distributed throughout the tissues in many small channels in animals with an open circulation. In the absence of such features, even moderate rates of oxygen consumption would be impossible because of the large diffu-

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oxygen transfer. They have evolved a tracheal system in which direct gas transport to tissues occurs through airfilled tubes that bypass the blood, which plays a negligible role in oxygen transport. Consequently, although insects have an open circulation, they have a large capacity for aerobic metabolism. The insect tracheal system is described in Chapter 13. Closed Circulations

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12-2 Most invertebrates have an open circulation, but cephalopods have a closed circulation. The main blood vessels in the open circulationof crayfish (A) and bivalve mollusks (B) empty into a large surrounding space, the hemocoel, which makes up about 30% of total body volume. Compared with an open circulation, the closed circulation of cephalopods (C) is characterized by a higher blood pressure and more efficient delivery of oxygen. In all diagrams only the main blood vessels are shown. The arrows indicate blood flow.

sion distances for oxygen between the hemolymph and the active tissue. Insects have an open circulation but do not depend on it for oxygen transport and thus can achieve high rates of

In a closed circulation, blood flows in a continuous circuit of tubes from arteries to veins through capillaries. All vertebrates and some invertebrates, such as cephalopods (octopuses, squids), have this type of circulation (Figure 12-2C). In general, there is a more complete separation of functions in closed circulatory systems than in open ones. The blood volume in the closed circulation of vertebrates typically is about 5% - 10% of body volume, much smaller than that of open-circulation invertebrates. The extracellular volume in vertebrates, expressed as a percentage of body volume is similar to the hemocoel volume in invertebrates. The closed circulatory system of vertebrates is a specialized portion of their extracellular space. In a closed circulation, the heart is the main propulsive organ, pumping blood into the arterial system and maintaining a high blood pressure in the arteries. The arterial system, in turn, acts as a pressure reservoir forcing blood through the capillaries. The capillary walls are thin, thus allowing high rates of transfer of material between blood and tissues by diffusion, transport, or filtration. Each tissue has many capillaries, so that each cell is no more than two or three cells away from a capillary. Capillary networks are in parallel, allowing fine control of blood distribution and, therefore, oxygen delivery to tissues. Animals with a closed circulation can increase oxygen delivery to a tissue very rapidly. For this reason, squid, unlike many other invertebrates, can swim rapidly and maintain high rates of oxygen uptake; that is, their closed circulation permits sufficient flow and efficient distribution of hemolymph to the muscles to support short bursts of highlevel activity. The blood is under sufficiently high pressure in a closed circulation to permit the ultrafiltration of blood in the tissues, especially the kidneys. Ultrafiltration refers to the separation of an ultrafiltrate, devoid of colloidal particles, from plasma by filtration though a semipermeable membrane (capillary wall) using pressure (blood pressure) to force the fluid through the membrane. Ultrafiltration occurs in most vertebrate kidneys, resulting in the net movement of a protein-free plasma from the blood into the kidney tubule. In general, all capillary walls are permeable and as pressures are high, so fluid slowly filters across the walls and into the space between cells. A lymphatic system has evolved in conjunction with the high-pressure, closed circulatory system of vertebrates to recover fluid lost to tissues from the blood. The extent of filtration depends largely on the blood pressure and the permeability of the capillary wall. Filtration across capillary walls can be decreased either by a reduction

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in the permeability of the capillary walls or in the pressure of the blood. For example, the vessels in some tissues have less permeable walls than those found in other tissues. And in the liver and lung, where permeability is high for other reasons, pressures are lower than those in the rest of the body. Different pressures can be maintained in the systemic (body)and pulmonary (lung)circulations of mammals because the mammalian circulatory system is equipped with a completely divided heart (Figure 12-3).The right side of the heart pumps blood in the pulmonary circulation, and the left side pumps blood in the systemic circulation. This means, however, that the flows in the pulmonary and systemic circuits must be equal because blood returning from the lung is pumped around the body. In other vertebrates the heart is not completely divided and flow to the lung can be varied independently of body blood flow. The venous system collects blood from the capillaries and delivers it to the heart via the veins, which are typically low-pressure, compliant structures in which large changes in volume have little effect on venous pressure. Thus, the venous system contains most of the blood and acts as a large-volume reservoir. Blood donors give blood from this

reservoir, and since there is little change in pressure as the venous volume decreases, the volumes and flows in other regions of the circulation are not markedly altered.

THE HEART Hearts are valved, muscular pumps that propel blood around the body. Hearts consist of one or more muscular chambers connected in series and guarded by valves or, in a few cases, sphincters (e.g., in some molluscan hearts), which allow blood to flow in only one direction. The mammalian heart has four chambers, two atria and two ventricles. Contractions of the heart result in the ejection of blood into the circulatory system. Multiple heart chambers permit stepwise increases in pressure as blood passes from the venous to the arterial side of the circulation (Figure 12-4).

Vertebrate cardiac and skeletal muscle fibers are similar in many respects, except that the T-tubule system is less extensive in cardiac muscle cells of lower vertebrates and cardiac muscle cells are electrically coupled (see Chapter 10). Except for differences in the uptake and release of Ca2+,the mechanisms of contraction of vertebrate skeletal and cardiac muscle are generally considered to be alike. The myocardium (i.e., heart muscle) consists of three types of muscle fiber, which differ in size and functional properties: The myocardial cells in the sinus node (or sinoatrial node) and in the atrioventricular node are often smaller than others, are only weakly contractile, are autorhythmic, and exhibit very slow conduction between cells. The largest myocardial cells, found in the inner surface of the ventricular wall, are also weakly contractile, but are specialized for fast conduction and constitute the system for spreading the excitation over the heart. The intermediate-sized myocardial cells are strongly contractile and constitute the bulk of the heart. Electrical Activity of the Heart Arterioles, c a ~ i l l a r i e s(5- 7%)

Figure 12-3 The closed circulation in mammals includes a fully divided heart, which permits different pressures in the pulmonary and systemic portions. This diagram illustrates the main components of the mammalian circulation, with oxygenated blood in the systemic system and the pulmonary system shown in red, and deoxygenated blood shown in blue. The associated lymphatic system (yellow)returns fluid from the extracellular space to the bloodstream via the thoracic dud. The percentages indicate the relat~veproportion of blood in different parts of the circulation. The lymphatic system and associated lymph nodes also play a key role in the immune response.

A heartbeat consists of a rhythmic contraction (systole)and relaxation (diastole)of the whole muscle mass. Contraction of each cell is associated with an action potential (AP) in that cell. Electrical activity, initiated in the pacemaker region of the heart, spreads over the heart from one cell to another because the cells are electrically coupled via membrane junctions (see Chapter 4). The nature and extent of coupling determines the pattern by which the electrical wave of excitation spreads over the heart and also influences the rate of conduction.

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Neurogenic and myogenic pacemakers In vertebrate hearts, the pacemaker is situated in the sinus venosus or in a remnant of it called the sinoatrial node (see Figure 12-4).The pacemaker consists of small, weakly contrade, specialized muscle cells that are capable of spontaneous activity.These cells may be either neurons, as in the neurogenic pacemaker in many invertebrate hearts, or muscle cells, as in the myogenic pacemaker in vertebrate and some invertebrate hearts. Hearts are often categorized by the type of pacemaker and hence are called either neurogenic or myogenic hearts. In many invertebrates, it is not clear whether the pacemaker is neurogenic or myogenic. Decapod crustaceans (shrimps, lobsters, crabs), however, do have neurogenic hearts. In these animals, the cardiac ganglion, situated on the heart, acts as a pacemaker. If the cardiac ganglion is removed, the heart stops beating, although the ganglion continues to be active and shows intrinsic rhythmicity The cardiac ganglion consists of nine or more neurons (depending on the species),divided into small and large cells. The small cells act as pacemakers and are connected to large follower cells, which are all electrically coupled. Activity from the small pacemaker cells is fed into and integrated by the large follower cells and then distributed to the heart muscle. The crustacean cardiac ganglion is innervated by excitatory and inhibitory nerves originating in the central nervous system (CNS);these nerves can alter the rate of firing of the ganglion and, therefore, the heart rate (beats per minute). Vertebrate, molluscan, and many other invertebrate hearts are driven by myogenic pacemakers. These tissues

Figure 12-4 The multi-chambered mammalian heart permits the pressure to increase as blood moves from the venous to the arterial side. This cutaway view depicts the rear portion of the human heart with the impulse pathways shown in color. Impulses originate in the pacemaker, located in the sinoatrial node, spread to the atrioventricular node, from which they are transmitted to the ventricles. Pacemaker cells in some invertebrates are modified nerve cells; in other invertebrates and all vertebrates, they are usually described as modified muscle cells. [Adapted from E. F. Adolph, 1967. Copyright0 1967 by Scientific American, Inc. All rights reserved.]

have been studied extensively in a variety of species. A myogenic heart may contain many cells capable of pacemaker activity, but because all cardiac cells are electricallycoupled, the cell (or group of cells) with the fastest intrinsic activity is the one that stimulates the whole heart to contract and determines the heart rate. These pacemaker cells will normally overshadow those with slower pacemaker activity; however, if the normal pacemaker stops for some reason, the other pacemaker cells take ovel; producing a new, lower heart rate. Thus, cells with the capacity for spontaneous electrical activity may be categorized as pacemakers and latent pacemakers. In the event that a latent pacemaker becomes uncoupled electrically from the pacemaker, it may beat and control a portion of cardiac muscle, generally an entire chamber, at a rate different from that of the normal pacemaker. Such an ectopic pacemaker is dangerous because it will desynchronize the pumping action of the heart chambers. Cardiac pacemaker potentials An important characteristic of pacemaker cells is the absence of a stable resting potential. Consequently, the membranes of cells in pacemaker tissue undergo a steady depolarization, termed a pacemaker potential, during each diastole (Figure 12-5).As the pacemaker potential brings the membrane to the threshold potential, it gives rise to an all-or-none cardiac action potential. The interval between cardiac APs, which of course determines the heart rate, depends on the rate of depolarization of the pacemaker potential, as well as the extent of repolarization and the threshold potential for

472


............................................................................ Figure 12-5 Pacemaker cells undergo spontaneous depolarlzat~onof the membrane, referred to as the pacemaker potent~al,trlggertng cardlac actlon potent~alsautorhymlcally (curve A) A more rap~ddepolar~zat~on tncreases the f ~ r ~ nrate g (curve B ) and thus the heart rate, whereas a slower depolar~zat~on slows the flrlng rate (curve C) and heart rate.

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the cardiac Al? A faster depolarization brings the membrane to a firing levd sooner and thus increases the frequency of firing, leading to a faster heart rate, whereas a slower depolarization does the opposite (see Figure 12-5). Pacemaker activity has its origin in time-dependent changes in membrane conductance. In the frog sinus venosus, the pacemaker depolarization begins immediately after the previous AP,when the potassium conductance of the membrane is very high. The potassium conductance then gradually drops, and the membrane shows a corresponding depolarization owing to the intracellular accumulation of potassium ions and a moderately high, steady conductance for sodium. The pacemaker depolarization continues until it activates the sodium conductance. The Hodgkin cycle then predominates to produce the rapid regenerative upstroke of the cardiac AP (see Chapter 5). Acetylcholine, which is released from parasympathetic terminals of the vagus nerve (tenth cranial nerve), slows the heart by increasing potassium conductance of the pacemaker cells. This increased conductance keeps the membrane potential near the potassium equilibrium potential for a longer time, thereby slowing the pacemaker depolarization and delaying the onset of the next upstroke (Figure 12-6A). On the

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other hand, norepinephrine released from sympathetic nerves accelerates the pacemaker depolarization potential, thus increasing the heart rate (Figure 12-6B). Although norepinephrine increases sodium and calcium conductance, this probably is not the main mechanism involved in speeding up the pacemaker rhythm. It is possible that norepinephrine decreases the time-dependent potassium efflux during diastole and thereby increases the rate of pacemaker depolarization. Cardiac action potentials The action potentials that precede contraction in all vertebrate cardiac muscle cells are of longer duration than those in skeletal muscle. The AP in skeletal muscle is completed and the membrane is in a nonrefractory stage before the onset of contraction; hence, repetitive stimulation and tetanic contraction are possible (Figure 12-7A).In cardiac muscle, by contrast, the action potential plateaus and the membrane remains in a refractory state until the heart has returned to a relaxed state (Figure 12-7B).Thus, summation of contractions cannot occur in cardiac muscle. Cardiac APs begin with a rapid depolarization that results from a large and rapid increase in sodium conductance. This differs from the slow depolarization of the

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Seconds

St~mulation Pacemaker potential

Figure 12-6 Parasympathet~cst~mulat~on vla the vagus nerve and sympathet~cst~mulat~on have opposite effects on the pacemaker potent~al and heart rate (A) Vagus st~mulat~on produces a rlse In dlastol~c(rest~ng) transmembranepotent~al,a decrease In the rate of depolarlzat~on,and a

decrease In the durat~onand frequency of the actlon potentlal (B) Symproduces an Increase ~nthe frequency of flr~ngofthe pathet~cst~mulat~on pacemaker cells [Hutter and Trautweln, 19561

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........................................ A Skeletal muscle

B Cardiac muscle

I

Plateau phase

Muscle contraction Muscle contraction

0

100

200

300

Time (rns)

0

100

200

300

Time (rns)

Figure 12-7 Action potentials in skeletal muscle are of very short duration (A), whereas cardiac action potentials exhibit a prolonged repolarization, or plateau phase, during which cardiac muscle is refractory to

st~mulation(0). For this reason, repetltlve stlmulatlon during a contraction and summation of contractions can occur in the skeletal muscle but not In heart muscle

pacemaker potential, which is marked by a stable sodium conductance and decreasing potassium conductance. Repolarization of the cell membrane is delayed while the membrane remains depolarized in a so-called plateau phase for hundreds of milliseconds (see Figure 12-7B).The long duration of the cardiac AP produces a prolonged contraction, so that an entire chamber can fully contract before any portion begins to relax-a process that is essential for efficient pumping of blood. The prolonged plateau of the cardiac AP results from maintenance of a high calcium conductance and a delay in the subsequent increase in potassium conductance (unlike the situation in skeletal muscle). The high calcium conductance during the plateau phase allows Ca2+ions to flow into the cell, because the equilibrium potential for calcium is directed strongly inwards. This influx is especially important in lower vertebrates, in which a considerable proportion of the calcium essential for activation of contraction enters through the surface membrane. In birds and mammals, the surface-to-volumeratio of the larger cardiac cells is too small to allow sufficient entry of calcium to fully activate contraction. Therefore, most of the calcium is released-by depolarization of the T tubules (see Chapter 10)-from the extensive sarcoplasmic reticulum characteristic of the hearts of higher vertebrates. A rapid repolarization terminates the plateau phase, due to a fall in calcium conductance and an increase in potassium conductance. The duration of the plateau and the rates of depolarization and repolarization vary in different cells of the same heart. The summation of these changes are recorded as the electrocardiogram (Figure 12-8).Atrial cells generally have an AP of shorter duration than ventricular cells. The duration of the AP in atrial or ventricular fibers from hearts of different species also varies. The duration of the AP is one factor correlated with the maximum frequency of the heartbeat; in smaller mammals, the duration of the ven-

tricular AP is shorter, thus heart rates generally are higheq than in larger mammals. Because of the great diversity among the hearts of different invertebrate phyla, few generalizations can be made about the ionic mechanisms generating the cardiac AP of invertebrate hearts. The one widespread characteristic is participation of calcium. For instance, bivalve mollusk hearts have a calcium AP. Transmission of excitation over the heart Electrical activity initiated in the pacemaker region is conducted over the entire heart, depolarization in one cell resulting in the depolarization of neighboring cells by virtue of current flow through gap junctions (see Figure 4-35). These junctions between cells are located in regions of close apposition between neighboring myocardial cells, termed the intercalated disk. Adhesion of cells at intercalated disks is strengthened by the presence of desmosomes. The area of contact is increased by folding and interdigitation of membranes (Figure 12-9).The extent of infolding and interdigitation increases during development of the heart and also varies among species. Gap junctions are regions of low resistance between cells and allow current flow from one cell to the next across intercalated disks. Although the junctions between myocardial cells can conduct in both directions, transmission is usually unidirectional because the impulse is initiated in and spreads only from the pacemaker region. There are usually several pathways for excitation of any single cardiac muscle fiber, since intercellular connections are numerous. If a portion of the heart becomes nonfunctional, the wave of excitation can easily flow around that portion, so that the remainder of the heart can still be excited. The prolonged nature of cardiac APs ensures that multiple connections do not result in multiple stimulation and a reverberation of activity in cardiac muscle. An AP initiated in the pacemaker region

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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Electrocardiogram

Nucleus

I

h

Figure 12-9 Electrical activitycan spread throughout the heart because of the close apposition of myocardial cells at intercalated disks, which are densely packed with gap junctions. Shown here is a schematic diagram of myocardial cells from a mammalIan heart. The folding and the interdigitation of membranes are characteristicof intercalateddisks. Although desmosomes are present in these reglons, thereby strengtheningthe adhesion between cells, they are not easily distinguished.

1\

I 0

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\

intercalated disk;

B Cardiac action potentials Pacemaker

Myocardial cells

/

plateau

I

I

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I 500

Time (ms) Figure 12-8 The electrocardiogram represents the summation of the electrical activity in various parts of the heart. (A) The major components of the electrocardiogram (ECG) reflect atrial depolarization (P), ventricular depolarization (QRS), and ventricular repolarization (T). (6) The amplitude, configuration, and duration of cardiac action potentials differ at various sites. The potential changes were recorded from the following sites: ( I ) sinoatrial node, (2) atrium, (3) atrioventricular node, (4) bundle of His, (5) Purkinje fiber in a false tendon, (6) terminal Purkinje fiber, and (7) ventricular muscle fiber. The numbers indicate the sequence in which the various s~tesfire.[Part B from Hoffman and Cranefield, 1960.1

results in a single AP being conducted through all the other myocardial cells, and another AP from the pacemaker region is required for the next wave of excitation. In the mammalian heart, the wave of excitation spreads from the sinoatrial node over both atria in a concentric fashion at a velocity of about 0.8 ma s-'. The atria are connected electrically to the ventricles only through the atrioventricular (AV) node; in other regions the atria and ventricles are joined by connective tissue that does not conduct the wave of excitation from the atria to the ventricles (see Figure 12-4). Excitation spreads to the ventricle through small junctional fibers, in which the velocity of the wave of excitation is slowed to about 0.05 m . s-'. The junctional fibers are connected to nodal fibers, which in turn are connected via transitional fibers to the bundle of His; this struc-

ture branches into right and left bundles, which subdivide into Purkinje fibers that extend into the myocardium of the two ventricles. Conduction is slow through the nodal fibers (about 0.1 m.s-') but rapid through the bundle of His (4-5 m .s-l). The bundle of His and the Purkinje fibers deliver the wave of excitation to all regions of the ventricular myocardium very rapidly, causing all the ventricular muscle fibers to contract together. As each wave of excitation arrives, the ventricular myocardial cells contract almost immediately, with the wave of excitation passing at a velocity of 0.5 m . s-' from the internal lining of the heart wall (endocardium) to the external lining (epicardium). The functional significance of the electrical organization of the myocardium is its ability to generate separate, synchronous contractions of the atria and the ventricles. Thus, slow conduction through the atrioventricularnode allows atrial contractions to precede ventricular contractions and also allows time for blood to move from the atria into the ventricles. Because of the large number of cells involved, the currents that flow during the synchronous activity of cardiac cells can be detected as small changes in potential from points all over the body. These potential changesrecorded as the electrocardiogram-are a reflection of electrical activity in the heart and can be easily monitored and then analyzed. A P-wave is associated with depolarization of the atrium, a QRS complex with depolarization of the ventricle, and a T-wave with repolarization of the ventricle (see Figure 12-8A). The electrical activity associated with atrial repolarization is obscured by the much larger QRS complex. The exact form of the electrocardiogram varies with the species in question and is affected by the nature and position of recording electrodes, as well as by the nature of cardiac contraction. The electrocardiogram is valuable medically because it can be used to diagnose cardiac abnormalities. As noted earlier, acetylcholine (ACh), released from cholinergic nerve fibers, increases the interval between APs in pacemaker cells and thus slows the heart rate (see

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....................................... Figure 12-6A). This decrease in heart rate is sometimes referred to as a negative chronotropic effect. Parasympathetic cholinergic fibers in the vagus nerve innervate the sinus node and atrioventricular node of the vertebrate heart. As the heart rate slows, acetylcholine also reduces the velocity of conduction from the atria to the ventricles through the atrioventricular node. High levels of acetylcholine block transmission through the atrioventricular node, so that only every second or third wave of excitation is transmitted to the ventricle. Under these unusual conditions, the atrial rate will be two or three times that in the ventricle. Alternatively, high levels of acetylcholine may completely block conduction through the atrioventricular node (atrioventricular block), giving rise to an ectopic pacemaker in the ventricle. The result is that the atria ana ventricles are controlled by different pacemakers and contract at quite different rates, the two beats being uncoordinated. This would be devastating for a fish in which atrial contraction is very important for ventricular filling. It is not quite so devastating in mammals because atrial contraction only tops up the ventricles, they are filled mainly by the direct inflow of blood from the venous system through the relaxed atria. The catecholamines epinephrine and norepinephrine have three distinct positive effects on heart function:

Because the heart primarily uses aerobic pathways to generate energy, it is very dependent on a continual oxygen supply. Thus a continual coronary flow is required to maintain cardiac performance. An increase in cardiac activity depends on increased metabolism, which in turn requires increased coronary flow. Adenosine is probably a key metabolite in maintaining the relationship between coronary flow and cardiac activity. Adenosine, which is formed from adenosine triphosphate (ATP) during cardiac metabolism, and other local metabolic factors cause dilation of , coronary vessels and therefore increase coronary flow. Formation and release of adenosine increases with increased metabolism or during myocardial hypoxia (drop in oxygen level), leading to higher coronary flow. Sympathetic stimulation is a second, but less important, mechanism of increasing coronary flow. Circulating catecholamines increase cardiac contractility and cause coronary vasodilation mediated via PI-adrenoreceptors.

Increased rate of myocardium contraction, or heart rate (positive chronotropic effect) Increased force of contraction of the myocardium (positive inotropic effect) Increased speed of conduction of the wave of excitation over the heart (positive dromotropic effect) The effect of these catecholamines on the rate of contraction is mediated via the pacemaker, whereas the increased strength of contraction is a general effect on all myocardial cells. Norepinephrine also increases conduction velocity through the atrioventricular node. It is released from adrenergic nerve fibers that innervate the sinus node, atria, atrioventricular node, and ventricle, so that sympathetic adrenergic stimulation has a direct effect on all portions of the heart.

'

Coronary Circulation

The coronary circulation supplies nutrients and oxygen to the heart. The coronary supply to the heart is extensive and cardiac muscle has a much higher capillary density and more mitochondria than most skeletal muscles. There is also a high myoglobin content resulting in the typical red color of the heart. The blood pumped by the heart supplies nutrients to the inner spongy layer of the heart in many fish and amphibia as it flows through the heart, but even in these animals the coronary supply is necessary to deliver oxygen and other substrates to the outer, more dense, regions of the heart wall. In general hearts can use a wide variety of nutrients, including fatty acids, glucose, and lactate; the particular substrate used is determined largely by availability.

Mechanical Properties of the Heart

The mechanical aspects of heart function relate to the changes in cardiac pressure and volume that lead to ejection of blood during each heartbeat. Now we'll examine these properties and determination of the work done by the heart. Cardiac output, stroke volume, and heart rate Cardiac output is the volume of blood pumped per unit time from a ventricle. In mammals it is defined as the volume ejected from the right or left ventricle, not the combined volume from both ventricles. The volume of blood ejected by each beat of the heart is termed the stroke volume. The mean stroke volume can be determined by dividing cardiac output by heart rate. Stroke volume is the difference between the volume of the ventricle just before contraction (end-diastolic volume) and the volume of the ventricle at the end of a contraction (end-systolicvolume). Changes in stroke volume may result from changes in either end-diastolic or end-systolicvolume. The end-diastolicvolume is determined by four parameters: Venous filling pressure Pressures generated during atrial contraction Distensibility of the ventricular wall The time available for filling the ventricle

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..................................... SPOTLIGHT 12-1

No single Starling curve, however, describes the relationship between venous filling pressure and work output from the ven-

THE FRANK-STARLING

tricle. The mechanical properties of the heart are affected by a number of factors, including the level of activity in nerves inner-

MECHANISM Otto Frank observed that the more the frog heart was filled, the greater the stroke volume. That is, increased venous return results in increased stroke volume. Frank derived a length-tension relationship for the frog myocardium and demonstrated that contractile tension increases with stretch up to a maximum and

vating the heart and the composition of blood perfusing the myocardium. For instance, the relationship between ventricular work output and venous filling pressure is markedly affected by stimulation of sympathetic nerves innervating the heart. Starling was a versatile researcher who along with William Bayliss discovered the hormone secretin. He coined the term

then decreases with further stretch. Ernest Starling, a dominant

hormone and defined their basic properties (see Chapter 9). Starling also made many contributions to our understanding of

figure in many areas of physiology during the early 1900s, had come to similar conclusions as Frank. Although neither Starling

the circulation. In addition to the observations described by the

nor Frank considered mechanical work, the increase in mechanical work from the ventricle caused by an increase in enddiastolic volume (or venous filling pressure) is termed the Frank-

Starling mechanism (plot A). The curves derived from measur-

Frank-Starling mechanism, he proposed the Starling hypothesis that the exchange of fluid between blood and tissues is due to the difference in the filtration and colloid osmotic pressures across the capillary wall. This hypothesiswas subsequently verified largely by the work of E. Landis.

ing work output from the ventricle at different venous filling pressures are known as Starling curves (plot B).

Starling curves (measured in a mammalian heart)

Frank-Starling mechanism in frog heart

/ Filling pressure

The end-systolic volume is determined by two parameters: The pressures generated during ventricular systole The pressure in the outflow channel from the heart (aortic or pulmonary artery pressure) Increasing the venous filling pressure causes an increase in end-diastolicvolume and results in an increased stroke volume from an isolated mammalian heart (Spotlight 12-1). End-systolic volume also increases, but not as much as enddiastolic volume. Thus, cardiac muscle behaves in a way similar to that of skeletal muscle in that stretch of the relaxed muscle within a certain range of lengths results in the development of increased tension during a contraction. Increases in arterial pressure also cause a rise in both enddiastolic and end-systolic volume with little change in stroke volume. In this instance, the increased mechanical work required to maintain stroke volume in the face of an

Increasing catecholamine stimulation Left atrial mean pressure -+

elevated arterial pressure results from the increased stretch of cardiac muscle during diastole. As noted above, epinephrine and norepinephrine released from sympathetic nerves or circulating in the blood increase the force of contraction of the ventricle; hence both the rate and the extent of ventricular emptying are increased by these catecholamines. The effects of cholinergic (i.e., vagus) nerve activity on the rate and force of ventricular power output during each beat are much less marked than the effects of adrenergic sympathetic nerves. This difference stems from the much more extensive innervation of the ventricles by adrenergic nerves than by cholinergic nerves. The effects of sympathetic nerve stimulation andlor increased circulating levels of catecholamines represent a series of integrated actions. Stimulation of pacemaker cells leads to an increase in heart rate. Conduction velocity over the heart is increased to produce a more nearly synchro-

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...................................... nous beat of the ventricle. Both the rate of production of ATP and the rate of conversion of chemical energy to mechanical energy in ventricular cells increase, leading to an increase in ventricular work: the rate of ventricular emptying increases during systole, so that the same or a larger stroke volume is ejected in a much shorter time. This increased force of contraction is mediated by the action of catecholamines on both a- and p-adrenoreceptors (see Chapter 8 for more details). Thus, when adrenergic nerve stimulation increases the heart rate, the same stroke volume is ejected from the heart in a shorter time. So, although the time available for emptying and filling the heart is reduced as heart rate increases, stroke volume remains quite stable over a wide range of heart rates. For example, exercise in many mammals is associated with a large increase in heart rate with little change in stroke volume; only at the highest heart rates does the stroke volume fall (Figure 12-10). This situation occurs because, over a wide range of heart rates, increased sympathetic activity ensures more rapid ventricular emptying, and elevated venous pressures result in more rapid filling as heart rate increases. There are limits, however, to which diastole can be shortened, determined by the maximum possible rate at which the ventricles can be filled and emptied as well as the nature of the coronary circulation. During contractions of the myocardium, coronary capillaries are occluded, so flow is very reduced during systole. Flow rises dramatically during diastole, but a decrease in the diastolic period tends to reduce the period of coronary blood flow. Catecholamines also cause coronary vasodilation and increase coronary flow. As we have noted, increases in cardiac output with exercise often are associated with large changes in heart rate and small changes in stroke volume in mammals (see Figure 12-10).Following sympathetic denervation of the heart, exercise results in similar increases in cardiac output, Maximum O2 consumption I I I

Cardiac

I

I

Oxygen consumption ___)

Figure 12-10 In humans and many other mammals, increased cellular oxygen needs during exercise are met in part by increasing heart rate rather than stroke volume, leading to higher cardiac output. At high levels of oxygen consumption, heart rate levels off and stroke volume increases and then decreases. In addition, extraction of oxygen from the blood in the capillaries increases during exercise, as indicated by the increase in the arterial-venous(A-V)0, difference. [Adapted from Rushmer, 1965b.l

but in this instance there are large changes in stroke volume rather than in heart rate. The increases in cardiac output are probably caused by an increase in venous return. The sympathetic nerves are not involved in increasing cardiac output per se but rather in raising heart rate and maintaining stroke volume, avoiding the large pressure oscillations associated with large stroke volumes and keeping the heart operating at or near its optimal stroke volume for efficiency of contraction. The sympathetic nerves thus play an important role in determining the relation between heart rate and stroke volume, but additional factors are involved in mediating the increase in cardiac output with exercise. Changes in pressure and flow during a single heartbeat Contractions of the heart cause fluctuations in cardiac pressure and volume as illustrated by the tracings in Figure 12-11A. The following sequence of events occurs during contraction of a mammalian heart (Figure 12-11B):

1. During diastole closed aortic valves maintain large pressure differences between the relaxed ventricles and the systemic and pulmonary aortas. The atrioventricular valves are open, and blood flows directly from the venous system into the ventricles. 2. When the atria contract, pressures rise in them and blood is ejected from them into the ventricles. 3. As the ventricles begin to contract, pressures rise in them and exceed those in the atria. At this point, the atrioventricular valves close, thus preventing backflow of blood into the atria, and ventricular contraction proceeds. During this phase, both the atrioventricular and the aortic valves are closed, so that the ventricles form sealed chambers and there is no volume change. That is, the ventricular contraction is isometrzc. 4. Pressures within the ventricles increase rapidly and eventually exceed those in the systemic and pulmonary aortas. The aortic valves then open, and blood is ejected into the aortas, resulting in a decrease in ventricular volume. 5. As the ventricles begin to relax, intraventricular pressures fall below the pressures in the aortas, the aortic valves close, and there is an isometric relaxation of the ventricle. Once the ventricular pressures fall below those in the atria, the atrioventricular valves open, ventricular filling starts again, and the cycle is repeated. In the mammalian heart, the volume of blood forced into the ventricle by atrial contraction is about 30% of the volume of blood ejected into the aorta by ventricular contraction. Thus, ventricular filling is largely determined by the venous filling pressure, which forces blood from the veins directly through the atria into the ventricles. Atrial contraction simply tops off the nearly full ventricles with blood; but maximal cardiac output may be compromised if atrial contraction is impaired. Contraction of cardiac muscle can be divided into two phases. The first is an isometric contraction during which

....................................... A Changes in pressure and volume during heartbeat Left side of heart

B Sequence of events in heartbeat

Right side of heart

(1) Mid-diastole

(3) Isometric ventricular contraction

(2) Atrial contraction

(4) Ventricular ejection

(5) Isometric ventricular relaxation

Diastole Diastole Diastole Diastole Systole Systole Figure 12-11 During a single cardiac cycle, sequential contraction of the atria and ventricles and the opening and closing of valves produce characteristic changes in pressure and volume. (A) Changes in pressure and volume in the ventricles and aorta (left) and pulmonary artery (rightjdur-

ing a single cardiac cycle. (B) Sequence of events in contraction of mammalian heart. Black indicates contracted muscle; gray, relaxed muscle. See text for discussion. [Part A adapted from Vander et al., 1975.1

tension in the muscle and pressure in the ventricle increase rapidly. The second phase is essentially isotonic; tension does not change very much, for as soon as the aortic valves open, blood is ejected rapidly from the ventricles into the arterial system with little increase in ventricular pressure. Thus, tension is generated first with almost no change in length; then the muscle shortens with little change in tension. In other words, during each contraction, cardiac muscle switches from an isometric to an isotonic contraction.

ventricles eject equal volumes of blood, but the pressures generated in the pulmonary circuit (right ventricle) are much lower; consequently, the external work done by the right ventricle is much less than that done by the left ventricle. As described in the previous section, blood is ejected from the ventricle when intraventricular pressures exceed the arterial pressure. If the arterial pressure is elevated, more external work must be done by the heart to raise the intraventricular pressure enough to maintain stroke volume at the original level. This, of course, means that there is an extra strain on the heart if blood pressure is high. Not all energy expended by the heart will appear as changes in pressure and flow; some energy is expended to overcome frictional forces within the myocardium, and more is dissipated as heat. The external work done by the heart, expressed as a fraction of the total energy expended, is termed the efliciency of contraction. The external work done can be determined from measurements of pressure and flow and converted into milliliters of 0, consumed. This, in turn, can be expressed as a fraction of the total 0, uptake by the heart in order to measure the efficiency of contraction. In fact, not more than 10% -15% of the total energy expended by the heart appears as mechanical work. Energy is expended to increase wall tension and raise blood pressure within the heart. According to Laplace's law, the relationship between wall tension and pressure in

Work done by the heart It is a simple principle of physics that external work done is the product of mass times distance moved. In the present context, work can be calculated as the change in pressure times flow. Flow is directly related to the change in volume with each beat of the ventricle. With the pressure given in grams per square centimeter and the volume in cubic centimeters, pressure times volume equals grams times cubic centimeters divided by square centimeters, which equals grams times centimeters-the equivalent of mass times distance moved, or work. Thus, a plot of pressure times volume for a single contraction of a ventricle yields a pressurevolume loop whose area is proportional to the external work done by that ventricle. Figure 12-12 illustrates pressure-volume loops for the right and left ventricles of a mammalian heart. The two

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...............................................................................

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Filling

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Volume (ml) Figure 12-12 The area of a ventricular pressure-volume loop is proportional tothe external work done by a ventricle in one cardiac cycle. Shown here are loops for the right and left ventricles of the mammalian heart. Once around a loop in a counterclockwise direction is equivalent to one heartbeat. Ventricular filling occurs at low pressure; pressure increases sharply only when theventricles contract (the sharp upswing on the righthand side of each loop). Ventricularvolumedecreases as blood flows into the arterial system, and ventricular pressure falls rapidly as the ventricle relaxes. Filling then begins again. Note that although the volume changes in both ventricles are similar, the pressure changes are much larger in the left ventricle than in the right one. Therefore, the left ventricle has a larger loop and hence does more external work than the right ventricle.

pericardial sac depend on the rigidity of the pericardium and on the magnitude and rate of change of the heart volume. The membrane may be thin and flexible (compliant), in which case pressure changes within the pericardial cavity during each heartbeat are negligible. Or the pericardium may be quite rigid (noncompliant), in which case the intrapericardial pressure oscillates during each heartbeat. The compliant pericardium enveloping the mammalian heart is formed of two layers, an outer fibrous layer and an inner serous layer. The serous layer is double, forming the inner lining of the pericardial space and the outer layer (epicardium) of the heart itself. In mammals, the serous layer secretes a fluid that acts as a lubricant, facilitating movement of the heart. Crustaceans and bivalve mollusks have a noncompliant pericardium. In these animals, contractions of the ventricle reduce pressure in the pericardial cavity and enhance flow into the atria from the venous system (Figure 12-13).Thus, tension generated in the ventricular wall is utilized both to eject blood into the arterial system and to draw blood into the atria from the venous system. The peficardium of elasmobranchs (sharks)and lungfishes also is noncompliant, whereas that of teleosts is compliant. The elasmobranch heart consists of four chambers-sinus venosum, atrium, ventricle, and conus-all contained within a rigid pericardium (Figure 12-14).The reduction in intrapericardial pressure that occurs during ventricular contraction in elasmobranchs produces a suction that helps expand the atrium and thereby increases venous return to the heart. If the pericardial cavity is opened, cardiac output is reduced; hence the increased venous return to the atrium caused by reduced pericardial pressure is

a hollow structure is related to the radius of curvature of the wall. If the structure is a sphere, then

where P is the transmural pressure (the pressure difference across the wall of the sphere), y is the wall tension, and R is the radius of the sphere. According to this relation, a large heart must generate twice the wall tension of a heart half its size to develop a similar pressure. Thus, more energy must be expended by larger hearts in developing pressure, and we might expect a larger ratio of muscle mass to total heart volume in these hearts. Hearts are not, of course, perfect spheres, but have a complex gross and microscopic morphology; nevertheless, Laplace's law applies in general. The energy expended in ejecting a given quantity of blood from the heart will depend on the efficiency of contraction, the pressures developed, and the size and shape of the heart.

The Pericardium The heart is contained in a pericardial cavity and is surrounded by a connective-tissue membrane called the pericardium. The magnitude of the pressure changes within the

r(

PAV

Diastole

\

PAV

Systole

Figure 12-13 In the heart of the bivalve mollusk Anodonta, ventricular contraction not only ejects blood but also reduces pressure in the pericardial cavity, thus enhancing atrial filling. This occurs because of the noncompliant pericard~um.Numbers are pressures in centimeters of seawater, which are expressed relative to ambient pressures. Large black arrows indicate the movements of the walls of contracting chambers; small arrows indicate movements of walls of relaxing chambers. The red arrows indicate the direction of blood flow. AAV, anterior aortic valve; PAY posterior aortic valve; AVV atrioventricular valve. [Adapted from Brand, 1972.1

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Atrial contraction

Pericardium Ventricle

Ventral

Conus

\

/

Atrium

\

venosus

Pericardial cavity

Ventricular contraction

Conal contraction

Figure 12-14 Because the elasmobranch heart is contained in a noncompliant pericardium, contractions of the ventricle reduce pressures in the pericardial cavity and assist atrial filling. In some elasmobranchs,fluid loss via the pericardioperitoneal canal during exercise, feeding, and coughing leads to an increase in heart size and stroke volume. Black arrows indicate direction of wall movement during muscle contraction or relaxation. Red arrows indicate direction of blood flow.

important in augmenting cardiac output. In some elasmobranchs, a pericardioperitoneal canal exists between the pericardial and peritoneal cavity. There is little or no fluid flow through this canal in resting fish, but during exercise, coughing, or feeding, the loss of fluid from the pericardial cavity via the canal causes an increase in heart size and stroke volume. This fluid is slowly replaced by plasma ultrafiltrate. Thus the thin, flexible pericardium of mammals, although protective, has little effect on cardiac output, whereas the more rigid pericardium of sharks, and the possible variations in pericardial fluid volume, can have a marked effect on cardiac output. Vertebrate Hearts: Comparative Functional Morphology The structure of the heart varies in different vertebrates, and a comparative analysis of vertebrate circulatory systems produces insights into the relationships between heart structure and function. Numerous cardiovascular differ-

ences distinguish air-breathing vertebrates from those that do not breathe air. Air-breathing vertebrates differ in the extent to which the systemic (body)and pulmonary (respiratory) circulations are separated. The pulmonary circulation of birds and mammals is maintained at much lower pressures than is the systemic circulation. This is possible because they have two series of heart chambers in parallel. The left side of the heart ejects blood into the systemic circulation, and the right side ejects blood into the pulmonary circulation (see Figure 12-3).The advantage of a high blood pressure is that rapid transit times and sudden changes in flow can be readily achieved for blood passing through small-diameter capillaries. However, when the difference in pressure across a vessel wall (i.e., the transmural pressure) is high, fluid filters across the. capillary wall; as a result, extensive lymphatic drainage of the tissues is necessary. In the mammalian lung, capillary flow can be maintained by relatively low input pressures, reducing the requirements for lymphatic drainage and avoiding the formation of large extracellular fluid spaces that could increase diffusion distances between blood and air and impair the gas transfer capacity of the lung. The advantage of a divided heart, like that of mammals, is that blood flow to the body and the lungs can be maintained by different input pressures. The disadvantage of a completely divided heart is that in order to avoid shifts in blood volume from the systemic to the pulmonary circuit, or vice versa, cardiac output must be the same in both sides of the heart, independent of the requirements in the two circuits. In contrast, lungfishes, amphibians, reptiles, bird embryos, and fetal mammals have either an undivided ventricle or some other mechanism that allows the shunting of blood from one circulation to the other. These shunts usually result in the movement of blood from the right (respiratory, pulmonary) to the left (systemic) side of the heart during periods of reduced gas transfer in the lung. At such times, blood returning from the body, instead of being pumped to the lung, is shunted from the right to the left side of the heart and once again ejected into the systemic circuit, bypassing the lungs. In lungfishes, amphibians, and reptiles, flow to the lungs commonly is reduced during prolonged dives when gas transfer occurs across the skin andlor oxygen stores in the body are being used. Blood flow to the lungs is also reduced during development within the mother (mammals)or egg (birds),before the lungs become fully functional in gas exchange. Although a single undivided ventricle permits variations in the ratio of flows to the pulmonary and systemic circuits, the same pressures must be developed on both sides of the heart, Water-breathingfishes The heart of water-breathing fishes, including elasmobranchs and some bony fishes (teleosts), consists of four chambers in series. All chambers are contractile except the elastic bulbus of bony fish. A unidirectional flow of blood through the heart is maintained by valves at the sinoatrial and atrioventricular junctions and at the exit of the ventricle.

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............................................................................... In elasmobranchs, the exit from the ventricle to the conus is guarded by a pair of flap valves, and there are from two to seven pairs of valves along the length of the conus depending on the species (see Figure 12-14).Conus length is variable among species; in general, more valves are found in those species with a longer conus. Just before ventricular contraction, all valves except the set most distal to the ventricle are open; that is, the conus and the ventricle are interconnected, but a closed valve at the exit of the conus maintains a pressure difference between the conus and the ventral aorta. During atrial contraction, both the ventricle and the conus are filled with blood. Ventricular contraction in elasmobranchs does not have an isovolumic phase, as in mammals, because at the onset of contraction blood is moved from the ventricle into the conus. Pressure rises in the ventricle and conus and eventually exceeds that in the ventral aorta. The distal valves open, and blood is ejected into the aorta. During conal contraction, which begins after the onset of ventricular contraction, the proximal valves close, preventing reflux of blood into the ventricle as it relaxes. Conal contraction proceeds relatively slowly away from the heart toward the aorta; each set of valves closes, in turn, to prevent backflow of blood. As illustrated in Figure 12-15, blood pumped by the heart in typical water-breathing fish passes first through the gill (respiratory) circulation and then into a dorsal aorta that supplies the rest of the body (systemic circulation). Thus, unlike mammals, the respiratory and systemic circulations of fish are in series rather than in parallel, and the gill circulation is under higher pressures than the systemic circulation. The gills of fish are involved in ionic regulation as well as gas transfer and many of the functions of the

Efferent

Afferent branchial artery

mammalian kidney are located in the gills. The consequences of a high blood pressure in the fish gill on ionic and gas transfer is not clear.

Air-breathing fishes Air-breathing has evolved in vertebrates many times, generally in response to hypoxic conditions, high water temperatures, or both. In general, air-breathing fish remain in water but rise to the surface to take in an air bubble to supplement oxygen supplies. Because the gill filaments usually collapse and stick together when exposed to air, they cannot be used for gas transfer in air. Hence, fish that have the ability to breathe air generally use structures other than the gills for this purpose, such as a portion of the gut or mouth, the swimbladder, or even the general skin surface. Although the gills in air-breathing fish are not used for oxygen uptake from air, they are used for carbon dioxide excretion, as well as ionic and acid-base regulation. In many air-breathing fish, however, the gills are reduced in size, presumably to ameliorate oxygen loss from blood to water. The gills of the air-breathing teleost Arapaima, which is found in the Amazon River are so small that only a fifth of oxygen uptake occurs across the gills even in water with normal oxygen levels. The bulk of oxygen uptake by this fish occurs via the swimbladder, which is highly vascularized and has many septa to increase surface area for exchange. In fact the gills of Arapaima are too small for the animal's oxygen requirements, and these fish die if denied access to air; that is, Arapaima is an obligate air-breathing fish. Air-breathing fish have evolved a variety of blood shunts to permit changes in the distribution of blood to the

Arterio-arterial

Ventral aorta

Primary circulation

Tissues

Ventricle

Figure 12-15 In a "typical" water-breathingteleost such as the trout, the respiratory circulation through the gills and the systemic circulation are in series. In the four-chamber, undivided heart, the pacemaker is in the sinus venosus. The ventricle ejects blood into the compliant bulbus and short ventral aorta. Blood flows through the gills into a stiff, long dorsal

aorta. Most teleosts contain a low-hematocrit secondary circulation, which supplies nutrients but not much oxygen to the skin and gut. Black arrows indicate flow of deoxygenated blood; red arrows, flow of oxygenated blood. BV, body volume.

482


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gills and the air-breathing organ. In the tropical freshwater teleost Hoplerythrinus, the posterior gill arches give rise to the coeliac artery, which perfuses the swimbladder and connects to the dorsal aorta by a narrow ductus. When the animal is breathing water, most of the cardiac output is directed to the first two gill arches and flows to the body. Following intake of air, the proportion of the blood flow to the posterior gill arches and therefore to the swimbladder increases, providing increased opportunity for oxygen uptake from the swimbladder.

There are many more species of alr-breathing flsh in tropical than in temperate reglons. Why?

The air-breathing fish Channa argus uses several mechanisms for achieving some separation of oxygenated and deoxygenated blood in the circulation. The most important mechanism is a division of the ventral aorta into two vessels, a posterior and an anterior ventral aorta. The anterior vessel supplies the first two gill arches and the air-breathing organ, whereas the posterior vessel supplies the posterior arches (Figure 12-16).The posterior arches are reduced in size, and the fourth arch is modifikd so that the afferent and efferent branchial arteries are in direct connection. Oxygenated blood is preferentially directed to the posterior arches, and the deoxygenated blood to the first two arches. This is achieved without division of the heart. The ventricle, however, is spongy (trabeculate), which may serve to prevent the mixing of blood in the ventricle, as has been

suggested for the spongy heart of amphibians. In addition, the absence of sinoatrial valves in the Channa heart and the arrangement of the veins probably play an important role in preventing mixing of oxygenated and deoxygenated blood as these flows return to the heart in common vessels. Finally, muscular ridges on the wall of bulbus may prevent mixing of the oxygenated and deoxygenated flows when they are ejected from the heart. Once again this situation is similar to that seen in amphibians. The division of the heart is more complete in the lungfishes (Dipnoi),which possess gills, lungs, and a pulmonary circulation. The African lungfish, Protol)terus, has a partial septum In the atrium and ventr~clcand 5piral folds in the bulh~lsiordis (Figure 12-17).This arrangement mantains d blood in the srpdrdtlon of oxygenated ~ n deoxygenated the heart. The anterior gill arches lack lamellae and oxygenated blood can flow from the left side of the heart directly to the tissues. Within the lamellae present in the posterior gill arches is a basal arterio-arterial connection that allows blood to bypass the lamellae when only the lung is in operation (e.g., during estivation, a state of torpor occurring in the summer). Blood from the posterior gill arches flows to the lungs or enters the dorsal aorta via a ductus. The ductus is richly innervated and is undoubtedly involved in controlling blood flow between the pulmonary artery and the systemic circulation. The initial segment of the pulmonary artery is muscular and is referred to as the pulmonary vasomotor segment. This vasomotor segment and the ductus probably act in a reciprocal fashion: when one constricts, the other dilates. The ductus in lungfish is analogous to the ductus arteriosus of fetal mammals, acting as a lung bypass when the lung is not functioning.

Anterior cardinal vein

Efferent

branchial artery

Afferent branchial artery ~ulbus Figure 12-16 Even though the heart of the air-breathingteleost Channa argus is undivided, the flows of oxygenated and deoxygenated blood are partially separated. Deoxygenated blood (black arrows) preferentially flows through the first two gill arches and a~r-breathingorgan, whereas oxygenated blood (red arrows) flows through posterior arches into the

Ventricle

dorsal aorta. The fourth gill arch is modified so that the afferent and efferent branchial arteries are connected. Compare with Figure 12-15, which illustrates the circulation of more typical water-breathing teleosts. . [Adaptedfrom lshirnatzu and Itazawa, 1993.1

Tissues A

Dorsal aorta

~ u l b u scordis '

~enkicle~ t r i u m

Figure 12-17 The circulation of the African lungfish, Protopterus, is marked by nearly complete separation of oxygenated blood (red arrows) and deoxygenated blood (black arrows). This separation is achieved by a septum dividing the atrial and ventricular chambers and a long spiral fold in the bulbus cordis. This fish possesses a lung and dis-

EJOxygenated blood

tinct pulmonary circulation. The absence of lamellae in the anterior gill arches permits blood to flow directly to the systemic circulation via the dorsal aorta. The ductus and pulmonary vasomotor segment act reciprocally to direct blood to the dorsal aorta or lungs depending on whether the fish is breathing in water or air. [Adapted from Randall, 1994.1

Sub-

lrnocutaneous

Amphibians Amphibia have two completely separated atria, but a single ventricle. In the frog heart, the oxygenated and deoxygenated blood is separated even though the ventricle is undivided. Oxygenated blood from the lungs and skin is preferentially directed toward the body via the systemic arch, whereas deoxygenated blood from the body is directed toward the pulmocutaneous arch. This separation of oxygenated and deoxygenated blood is aided by a spiral fold within the conus arteriosus of the heart (Figure 12-18). Deoxygenated blood leaves the ventricle first during systole and enters the lung circulation. Pressures rise in the pulmocutaneous arch and become similar to those in the systemic arch. Blood then begins flowing into both arches, with the spiral fold partially dividing the systemic and pulmocutaneous flows within the conus arteriosus. The volume of blood going to the lungs or body is inversely related to the resistance of the two circuits to flow. Immediately following a breath, the resistance to blood flow through the lung is low and blood flow is high; between breaths, resistance gradually increases and is associated with a fall in blood flow. These oscillations in pulmonary blood flow are possible because of the partial division of the amphibian heart. Although deoxygenated blood is directed toward the pulmocutaneous arch, the -

-

Figure 12-18 Even though the frog heart has a single ventricle, deoxygenated blood is directed to the lungs via the pulmocutaneous arch and oxygenated blood to the tissues via the systemic arch. This ventral view of the internal structure of the frog heart shows the position of the spiral fold, which aids in separating the two blood flows. [Adapted from Goodrich, 1958.1

ratio of pulmonary to systemic blood flow can be adjusted. That is, when the animal is not breathing, blood flow to the lungs can be reduced, so that most of the blood pumped by the ventricle is directed toward the body. When the animal is breathing, a more even distribution of flow to the lungs and body can be maintained. This distribution is possible only if the ventricle is not completely divided into right and left chambers (as it is in mammals).

.

Noncrocodilian reptiles Most noncrocodilian reptiles, including turtles, snakes, and some lizards have a partially divided ventricle and right and left systemic arches. In these animals, the ventricle is partially subdivided by an incomplete muscular septum referred to as the horizontal septum, Muskelleiste, or muscular ridge. This horizontal septum separates the cavum pulmonale from the cavum venosum and cavum arteriosum; the latter two are partially separated by the vertical septum (Figure 12-19).The right atrium contracts slightly before the left atrium does and ejects deoxygenated blood into the cavum pulmonale across the free edge of the horizontal septum; ventricular contraction ejects this blood into the pulmonary artery. Oxygenated blood from the left atrium fills the cavum venosum and cavum arteriosum; from here the blood empties into the systemic arteries. Measurements in turtles support the view that oxygenated blood from the left atrium passes into the systemic circuit, whereas deoxygenated blood from the right atrium passes into the pulmonary artery. Pulmonary artery diastolic pressure is often lower than systemic diastolic pressure; as a result, the pulmonary valves open first when the ventricle contracts. Thus, flow occurs earlier in the pulmonary artery than in the systemic arches during each cardiac cycle. In turtles, there may be some recirculation of arterial blood in the lung circuit; that is, there is a left-to-right shunt within the heart. The ventricle remains functionally undivided throughout the cardiac cycle, and the relative flow to the lungs and systemic circuits is determined by the resistance to flow in each part of the circulatory system. When the turtle breathes, resistance to flow through the Right atrium

\

\

Right a o r t a l

Right pulmonary artery

/

Brachiocephalic artery

artery

'~ertlcal

Horizontal septum

Y

septum

Cavum venosum

pulmonary circulation is low, and blood flow is high. When it does not breathe, as during a dive, pulmonary vascular resistance increases, but systemic vascular resistance decreases, resulting in a right-to-left shunt and a decrease in pulmonary blood flow. As in many other animals, there is a reduction in cardiac output associated with a marked slowing of the heart (bradycardia) during a dive. The similarity in the pressures in the pulmonary and systemic outflow tracts in turtles, snakes, and some lizards indicates that their heart has a single ventricular chamber partially divided into subchambers even during systole (Figure 12-20A).In monitor lizards and related varanid lizards, however, the pulmonary outflows are at much lower pressures than the systemic outflows during systole (Figure 12-20B).The pressure in the cavum pulmonale, for instance, can be only a third of that in the cavum venosum during systole in Varanus. This pressure differential in veranid lizards is achieved by a pressure-tight contact between the muscular ridge (horizontal septum) and the wall of the heart during systole (Figure 12-21). Crocodilian reptiles Unlike other reptiles, crocodilian reptiles have a heart with a completely divided ventricle. The left systemic arch arises from the right ventricle; the right systemic arch, from the left ventricle. Close to the ventricles, the systemic arches are connected via the foramen of Panizzae (Figure 12-22A). The systemic arches also are joined by a short anastomosis caudal to the heart. When a crocodilian reptile is breathing normally, the resistance to blood flow through the lungs is low, and pressures generated by the right ventricle are lower than those generated by the left ventricle during all phases of the cardiac cycle. In this case, blood is pumped by the left ventricle into the right systemic arch during systole, with the open aortic valve closing off the foramen of Panizzae (Figure 12-22B).There is a small reflux of blood into the left aorta from the right aorta via the anastomosis during sysiole. Because of this connection, pressures in the left systemic arch remain higher than the pressure in the right venFigure 12-19 In noncrocodilian(chelonian)hearts, the ventricle is partially divided by the horizontal septum into the cavum venosum and ventral cavum pulmonale. The common pulmonary artery arises from the cavum pulmonale, whereas all of the systemic arteries arise from the cavum venosum. In this ventral view of the turtle heart, the arrows schematically indicate movement of oxygenated blood (red) and deoxygenated blood (black) but are not intended to represent the flow of separate bloodstreamsthrough the heart. [Adapted from Shelton and Burggren, 1976.1

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Cavum venosum Common pulmonary artery Cavum venosum and cavum pulmonale

I

I

I

..

I

I

I

I

Time (s)

I

I

I I I I I Time (100 ms)

I

I

I

I

Figure 12-20 In turtles, the pressures in the systemic and pulmonary outflows are nearly identical during systole, whereas in veranid lizards they differ considerably. Shown are blood pressures measuredsimultaneously

at the indicated sites during a single heartbeat in (A) a turtle, Chrysemys scripta, and (B) a monitor lizard, Varanus exanthematicus. [Part Afrom Shelton and Burggren, 1976; part B from Burggren and Johansen, 1982.1

tricle; consequently, the valves at the base of the left systemic arch remain closed throughout the cardiac cycle (Figure 12-22C).All blood ejected from the right ventricle passes into the pulmonary artery and flows to the lungs. Thus, the crocodilian reptile is functionally the same as the mammal in that there is complete separation of systemic and pulmonary blood flow. Crocodilian reptiles, however, have the added capacity to shunt blood from the pulmonary to the systemic circuit. S shunt is achieved by active closure of a valve This P at the base of the pulmonary outflow tract towards the end

of systole. Under some experimental circumstances peak right ventricular pressure becomes equal to left ventricular pressure and exceeds left systemic pressure. As a result, the valves at the base of the left systemic arch open, and blood from the right ventricle is ejected into the systemic circulation during late systole (Figure 12-22D,E). In this case, a portion of the deoxygenated blood returning to the heart from the body via the right atrium is recirculated in the sysS shunt operates temic circuit. Exactly when the P normally in the animal is not clear. The role of the foramen of Panizzae also remains enigmatic; it is only open during

A

Diastole

Sinus Pulmonary venosus arch

-

Systole

B

Left and

Sinus venosus

Right

I

atrium

Left atrium Atrioventricular

Figure 12-21 In veranid lizards, a pressure-tightseparation between the cavum pulmonale and cavum venosum occurs during systole. (A) During diastole, the muscular ridge only partially separates the cavum venosum and cavum pulmonale. Thus, oxygenated blood (red arrows) remaining in the cavum venosum from the preceding systole is washed into the cavum pulmonale by deoxygenated blood (black arrows). The cavum arteriosum is filled with oxygenated blood. Separation between the cavum arteriosum and the cavum venosum is provided by at least one atrioventricular valve. (B) During systole, the muscular ridge is pressed tight

against the outer heart wall, forming a pressure-tight barrier. Deoxygenated blood remaining in the cavum venosum from the preceding diastole is mixed with oxygenated blood from the cavum arteriosum and flushed into the aortic arches. Deoxygenated blood with an admixture of oxygenated blood is expelled from the cavum pulmonale into the pulmonary arch. With no connection between the cavum venosum and cavum pulmonale, different pressures can develop in the outflow tracts. [Adapted from Heisler et al., 1983.1

486

I N T E G R A T I O N O F P H Y S I O L O G I C A L SYSTEMS

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . A

-

Diastole

oramen of panizza

B Late systole-no

shunt

D

c No P+ S shunt

E

Towards heart

Figure 12-22 Under some condltlons, a P S shunt operates durlng late systole in crocodiles These schemat~cdlagrams and pressure and flowtraclngs illustrate what happens durlng the cardlac cycle with and wtthout the shunt See text for d~scusslon[Adapted from Jones, 1995 ]

Late systole-shunt

P+ Sshunt

Towards heart

I Late Diastole systole Time (s)

diastole, allowing flow between the aortic arches as the heart relaxes.

Mammals and birds The heart in both mammals and birds, which consists of four chambers, is in fact two hearts beating as one. The heart originates as two separate tubes that join together dur-

Time (s)

ing development to form the multi-chambered heart of the postnatal animal. The right side pumps blood to the lung, the left side pumps blood around the body. Blood returning from the lungs enters the left atrium, passes into the left ventricle, and is then ejected into the body circulation. Blood from the body collects in the right atrium, passes into the right ventricle, and is pumped to the lungs (seeFigure 12-3).

Valves prevent backflow of blood from the aorta to the ventricle, the atrium, and the veins. These valves are passive and are opened and closed by pressure differences between the heart chambers. The atrioventricular valves (bicuspid and tricuspid valves) are connected to the ventricular wall by fibrous strands (see Figure 12-4).These strands prevent the valves from being everted into the atria when the ventricles contract and intraventricular pressures are much higher than those in the atria. The walls of the ventricle, especially the left chamber, are thick and muscular.'The inner surface of the ventricular muscle, or myocardium, is lined by an endothelial membrane, the endocardium. The ventricular myocardium is covered by the epicardium. Mammalian fetus At birth, mammals shift from a placental to a pulmonary circulation, a process that involves several central cardiovascular readjustments. The lungs of the mammalian fetus are collapsed, presenting a high resistance to blood flow. In the fetus, the pulmonary artery is joined to the systemic arch via a short, but large-diameter, blood vessel, the ductus arteriosus (Figure 12-23).Heart function in the mammalian fetus exhibits three important features:

Figure 12-23 In the mammalian fetal heart, most of the blood ejected from the right ventricle returns to the systemic circulation via the duaus arteriosus. Oxygenated blood returning from the placenta is shunted from the right to the left atrium through the foramen ovale and then pumped into the aorta. After birth, the ductus arteriosus normallycloses, so systemic and pulmonary circulations become separated. The numbers refer to the percentage of the combined cardiac output from the right and left ventricles that flows to and from different regions of the body.

Most of the blood ejected by the right ventricle is returned to the systemic circuit via the ductus arteriosus. Blood flow through the pulmonary circulation is greatly reduced. A marked right-to-left (P +S) shunt operates; that is, blood flows from the pulmonary to the systemic circuit. At birth, the lungs are inflated, reducing the resistance to flow in the pulmonary circuit. Blood ejected from the right ventricle passes into the pulmonary vessels, resulting in an increased venous return to the left side of the heart. At the same time, the placental circulation disappears, and the resistance to flow increases markedly in the systemic circuit. Pressures in the systemic circuit rise above those in the pulmonary circulation; if the ductus arteriosus fails to close after birth, this pressure difference results in a left-to-right (S P) shunt with blood flowing from the systemic to the pulmonary circuit. Generally, however, the ductus arteriosus becomes occluded, and blood flow through the ductus does not persist. If the ductus arteriosus remains open after birth, blood flow to the lungs exceeds systemic flow, because a portion of the left ventricular output passes via the ductus into the pulmonary artery and to the lung. In these circumstances, systemic flow is often normal, but pulmonary flow may be twice the systemic flow, and cardiac output from the left ventricle may be twice that from the right ventricle. The result is a marked hypertrophy of the left ventricle. The work done by the left ventricle during exercise is also much greater than normal, and the capacity to increase output is limited. As a result, the maximum level of exercise is much reduced if the ductus arteriosus remains open after birth. Furthermore, this condition increases the blood pressure in the lungs, leading to a greater fluid loss across lung capillary walls and to possible pulmonary congestion. These problems only become detrimental when the left ventricle has become enlarged. An open ductus arteriosus is readily and easily correctable by surgery. Fetal blood is oxygenated in the placenta and mixed with the blood returning from the lower body via the inferior vena cava, a vein that in turn empties into the right atrium (see Figure 12-23). In the interatrial septum is a hole, the foramen ovale, that is covered by a flap valve; oxygenated blood returning via the inferior vena cava is directed into the left atrium through the foramen ovale. Oxygenated blood is then pumped from the left atrium into the left ventricle and ejected into the aorta, whence it flows to the head and upper limbs. Deoxygenated blood returning to the right atrium via the superior vena cava is preferentially directed toward the right ventricle, whence it flows into the systemic circuit via the ductus arteriosus. At birth, pressures in the left atrium exceed the pressure in the right atrium; as a result, the foramen ovale closes, but its position is later indicated by a permanent depression.

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Bird embryo A network of blood vessels, forming the chorioallantois, lies just under the shell of bird eggs. Oxygen diffusing across the eggshell is taken up by blood passing through the chorioallantois. The oxygenated blood leaving the chorioallantois and deoxygenated blood from the head and body enter the right atrium of the bird embryo heart. Oxygenated blood from the chorioallantoic circulation passes from the right into the left atrium through several large and numerous small holes in the interatrial septum. The oxygenated blood is then pumped into the left ventricle and ejected into the aorta, whence it flows to the head and body. After the young bird hatches, the holes in the interatrial septum close, completely separating the pulmonary and systemic circulations.

Capillaries

5000 r

8

8

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Arterioles I

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I Venules I

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HEMODYNAMICS As we have noted, contractions of the heart generate blood flow through the vessels-arteries, capillaries, and veinsthat form the circulatory system. Before examining the properties of these vessels in detail, it is necessary to discuss the general patterns of blood flow in these vessels and the relationship between pressure and flow in the circulatory system. The laws describing the relationships between pressure and flow apply to both open and closed circulatory systems. In vertebrates and other animals with a closed circulation, the blood flows in a continuous circuit. Since fluids are incompressible, blood pumped by the heart must cause flow of an equivalent volume in every other part of the circulation. That is, at any one time the same number of liters per minute flows through the arteries, the capillaries, and the veins. Furthermore, unless there is a change in total blood volume, a reduction in volume in one part of the circulation must lead to an increase in volume in another part. The velocity of flow at any point is related not to the proximity of the heart but to the total cross-sectional area of that part of the circulation-that is, to the sum of the cross sections of all capillaries or arteries at that point in the circulation. Just as the velocity of water flow increases where a river narrows, so in the circulation the highest velocities of blood flow occur where the total cross-sectional area is smallest (and the lowest velocities occur where the cross-sectional area is largest). The arteries have the smallest total cross-sectional area, whereas the capillaries have by far the largest. Thus, the highest velocities occur in the aorta and pulmonary artery in mammals; then velocity falls markedly as blood flows through the capillaries, but it rises again as blood flows through the veins (Figure 12-24).The slow flow of blood in capillaries is of functional significance, because it is in capillaries that the time-consuming exchange of substances between blood and tissues takes place. Laminar and Turbulent Flow

In many smaller vessels of the circulation, blood flow is streamlined. Such continuous laminar flow is characterized by a parabolic velocity profile across the vessel (Figure 12-25A). Flow is zero at the wall and maximal at

Figure 12-24 Blood velocity is inversely proportional to the crosssectional area of the circulation at any given point. Blood velocity is highest in the arteries and veins and lowest in the cap~llaries;the converse is true for the cross-sectional area. [Adapted from Feigl, 1974.1

A Continuous laminar flow

B Pulsatile laminar flow

Figure 12-25 Blood flow through smaller vessels approximates continuous laminarflow, but in large elastic arteries pulsatile larninarflow is observed. As shown in these velocity profiles,the flow rate is higher towards the center of the vessel. (A) The presence of red blood cells flattens the profile of blood compared with that of plasma. (6) Pulsatile flow is marked by a flat profile and reversal of flow during each heartbeat.

CIRCULATION

489

...................................... the center along the axis of the vessel. A thin layer of blood adjacent to the vessel wall does not move, but the next layer of fluid slides over this layer, and so on, each successive layer moving at an increasingly higher velocity, with the maximum at the center of the vessel. A pressure difference supplies the force required to slide adjacent layers past each other, and viscosity is a measure of the resistance to sliding between adjacent layers of fluid. An increase in viscosity will require a larger pressure difference to maintain the same rate of flow, as discussed later. The pulsatile laminar flow characteristic of large arteries has a more complex velocity profile than the continuous laminar flow characteristic of smaller vessels. In large arteries, blood is first accelerated and then slowed with each heartbeat; in addition, since the vessel walls are elastic, they expand and then relax as pressure oscillates with each heartbeat. Close to the heart, the direction of flow reverses each time the aortic valves shut. The end result is that the velocity across large arteries has a much flatter profile than the velocity across more peripheral blood vessels and the direction of flow oscillates (Figure 12-25B). In turbulent flow fluid moves in directions not aligned with the axis of the flow, thus increasing the energy needed to move fluid through a vessel. Laminar flow is silent; turbulent flow noisy. In the bloodstream, turbulence causes vibrations that produce the sounds of the circulation. Detection of these sounds with a stethoscope can localize the points of turbulence. Blood pressure measurement with a sphygmomanometer depends on hearing the sounds associated with blood escaping past the pressure cuff as blood pressure rises during systole. Sounds can be heard in vessels when blood velocity exceeds a certain critical value and in heart valves when they open and close. Although turbulent flow is uncommon in the peripheral circulation, it does occur in some situations. The Reynolds number (Re) is an empirically derived value that indicates whether flow will be laminar or turbulent under a particular set of conditions. A high Reynolds number indicates flow will be turbulent, whereas a low number indicates flow will be laminar. The Re is directly proportional to the flow rate, Q (in milliliters per second), and density, p, of the blood, and inversely proportional to the inside radius of the vessel, r (in centimeters), and viscosity, q, of the blood:

The ratio of viscosity to density (qlp)is the kinematic viscosity. The larger the kinematic viscosity, the less the likelihood that turbulence will occur. The relative viscosity, and therefore the kinematic viscosity, increases with hematocrit (volume of red blood cells per unit volume of blood), so that the presence of red blood cells decreases the occurrence of turbulence in the bloodstream. In genera', 's high enough ate turbulence in undivided vessels with smooth walls, except during the very high blood flows associated with

strenuous exercise. The highest flow rates in the mammalian circulation are in the proximal portions of the aorta and pulmonary artery, and turbulence may occur distal to the aortic and pulmonary valves at the peak of ventricular ejection or during backflow of blood as these valves close. In general, flow will be turbulent in portions of the circulation where vessel walls are smooth and the vessels are undivided only if Re is greater than about 1000, a value seldom observed. Small back eddies may form at arterial branches and, like the back eddies in rivers, can become detached from the main flow regime, being carried downstream as small discrete regions of turbulence. These eddies can form in the circulation when the Re is as low as 200. Relationship between Pressure and Flow

Flow will occur between two sites where there is a difference in potential energy, which can be measured as a difference in pressure. Thus differences in pressure between two points in a flow path establish a pressure gradient and therefore the direction of flow- from high to low pressure. (An exception is a fluid at rest under gravity, where pressure increases uniformly with depth but flow does not occur.) When the heart contracts, the potential energy (pressure)in the ventricle increases. Pressures generated by heart contractions are dissipated by the flow of blood, because energy is used to overcome the resistance to flow through the vessels. For this reason, blood pressure falls as the blood passes from the arterial to the venous side of the circulation (Figure 12-26). Capillaries I I Arterioles! ! Venules

Figure 12-26 The pressure (potentla1energy) generated durlng each cardlac contraction IS dlsslpated In overcoming the reslstance to flow provlded by the vessels Because reslstance IS highest In the arter~oles,the major pressure decrease occurs In this reglon of the c~rculat~on [Adapted from Frelgl, 1974.1

,

Role of kinetic energy Energy is expended in setting the blood into motion, but once in motion flowing blood has inertia; that is, fluids in motion possess kinetic energy. In static fluids, potential energy is measured in terms of pressure; in fluids in motion, potential energy is measured in terms of both pressure and kinetic energy. As we'll see, however kinetic energy generally makes a negligible contribution to the flow rate of blood. The kinetic energy per milliliter of fluid is given by t(pv2),where p is the density of the fluid and u is the velocity of flow. If the velocity is measured in centimeters per second and density in grams per millimeter, then kinetic energy has the units of dynes per square centimeter, the same as pressure. The maximum velocity of blood flow occurs at the base of the aorta in mammals and is about 50 cmss-' at the peak of ventricular ejection, and the density of blood is about 1.055 g-ml-l. Thus the kinetic energy of the blood in the aorta during peak ejection is calculated as f x 1.055 X 502, or 1mm Hg. This is small compared with peak systolic transmural pressures of around 120 mm Hg. Blood velocity is low in the ventricle but accelerates as blood is ejected into the aorta; that is, blood gains kinetic energy as it leaves the ventricle. Pressure is converted into kinetic energy as blood is ejected from the heart, and this conversion accounts for most of the small drop in pressure that occurs between the ventricle and the aorta. Kinetic energy is highest in the aorta. In the capillaries the velocity is about 1rnm s-I and kinetic energy, therefore, is negligible.

.

Poiseuille's law The relationship between pressure and continuous laminar flow of fluid in a rigid tube is described by Poiseuille's law, which states that the flow rate of a fluid, Q, is directly proportional to the pressure difference, PI - P,, along the length of the tube and the fourth power of the radius of the tube, r, and inversely proportional to the tube length, L, and fluid viscosity, 77:

As Q is proportional to r4, very small changes in r will have a profound effect on Q. A doubling of vessel diameter, for instance, will lead to a 16-fold increase in flow if the pressure difference (PI - P2) along the vessel remains unchanged. Although Poiseuille's equation applies to steady flows in straight rigid tubes, it has been used, with some limitations discussed later, to analyze the relationship between pressure and flow in small arteries (arterioles),capillaries, and veins, even though these are not "rigid" tubes. Blood pressure and flow are pulsatile, and blood is a complex fluid consisting of plasma and cells. Since the blood vessel walls are not rigid, the oscillations in the pressure and flow of blood are not in phase; consequently, the relatian-

ship between the two is no longer accurately described by Poiseuille's law. The extent of the deviation of the relationship between pressure and flow from that predicted by Poiseuille's law is indicated by the value of a nondimensional constant a:

where p and 77 are the density and viscosity of the fluid, respectively; f is the frequency of oscillation; n is the order of the harmonic component, and r is the radius of the vessel. If a is 0.5 or less, the relationship between pressure and flow is described by Poiseuille's equation. Because the value for a in the small terminal arteries and veins is about 0.5, this equation can be used to analyze the relationship between pressure and flow in this portion of the circulation. In contrast, the values of cu for the arterial systems of mammals and birds range from 1.3 to 16.7, depending on the species and the physiological state of the animal. Thus, Poiseuille's law is not applicable to this portion of the circulation. There have been only a few studies of the in vivo microcirculation due to the difficulty of measuring blood flow and pressure in capillaries. In those tissues where the relationship between pressure and flow in the microcirculation has been measured, it has been found to be nonlinear, indicating that Poiseuille's equation does not accurately describe the microcirculation. There are two reasons for this: the capillaries branch with collateral pathways that may open and close, and they are so small that red blood cells are deformed as they squeeze through the capillaries. Resistance to flow Because it is often difficult or impossible to measure the radii of all vessels in a vascular bed, we designate 8Lrl17rr4, the inverse of the term in Poiseuille's law (equation 12-3), as the resistance to flow, R, which is equal to the pressure difference (PI - P2) across a vascular bed divided by the flow rate, Q:

The resistance to flow in the peripheral circulation is sometimes expressed in peripheral resistance units (PRUs),with 1PRU being equal to the resistance in a vascular bed when a pressure difference of 1 mm Hg results in a flow of 1m1.s-'. Blood flow through a vessel increases with increased pressure difference along a vessel and decreased resistance to flow, which is inversely proportional to the fourth power of the radius of the vessel. As pressure increases in an elastic vessel, so does the radius; as a result, flow increases as well. Let us consider a blood vessel with a constant pressure differential along its length but operating at two pressure levels:

...................................... Example 1: input pressure 100 mm Hg and outflow pressure 90 mm Hg; A = 10 mm Hg Example 2: input pressure 20 mm Hg and outflow pressure 10 mm Hg; A = 10 mm Hg The flow rate in this vessel will be much greater at the higher pressure (example 1)if the vessel is distensible, simply because the radius will be increased and the resistance to flow reduced. Viscosity of blood According to Poiseuille's law, the flow of blood is inversely related to its viscosity. Plasma has a viscosity relative to water of about 1.8; the addition of red blood cells increases the relative viscosity, so that mammalian and bird blood at 37°C have a relative viscosity between 3 and 4. Thus, ow.. ing largely to the presence of red blood cells, blood behaves as though it were three or four times more viscous than water. This characteristic means that larger pressure gradients are required to maintain the flow of blood through a vascular bed than would be needed if the vascular bed were perfused by plasma alone. However, blood flowing through small vessels behaves as if its relative viscosity were much reduced. In fact, in vessels less than 0.3 mrn in diameter, the relative viscosity of blood decreases with diameter and approaches the viscosity of plasma. This phenomenon, called the Fahraeus-Lindqvist effect, is explained later. As we saw earlier, the velocity profile across a vessel with continuous laminar fluid flow is parabolic, as is seen with plasma (see Figure 12-25A ). The maximum velocity is twice the mean velocity, which can be determined by dividing the flow rate by the cross-sectional area of the tube. The rate of change in velocity is maximal near the walls and decreases toward the center of the vessel. In flowing blood, red cells tend to accumulate in the center of the vessel, where the velocity is highest but the rate of change in velocity between adjacent layers smallest. This accumulation leaves the walls relatively free of cells, so that fluid flowing from this area into small side vessels will have a low level of red blood cells and consist almost entirely of plasma. Such a process is referred to as plasma skimming. The accumulation of red blood cells in the center of a bloodstream means that blood viscosity is highest in the center and decreases toward the walls. This differential in viscosity between the center and the walls of the bloodstream will alter the velocity profile of blood compared with that of plasma. The effect of this viscosity difference is a slight increase in blood flow at the walls and a slight reduction in flow at the center; that is, the parabolic shape of the velocity profile is flattened somewhat (see Figure 12-25A). The hematocrit (percent volume of rbc's in blood) in small vessels is smaller than that in larger ones. In small vessels the boundary layer of plasma occupies a larger portion of the vessel lumen at a given flow than in larger vessels. This axial flow of red blood cells in small vessels means that the greatest change in velocity occurs in the plasma layers

close to the walls and explains why the apparent viscosity of blood flowing in these small vessels approaches that of plasma. Thus the Fahraeus-Lindqvist effect can be explained in terms of the reduced hematocrit seen in small vessels. This decrease in the apparent viscosity of blood, which occurs in arterioles, reduces the energy required to drive blood through the microcirculation. In very small vessels-those with a diameter of approximately 5 to 7 pm-further decreases in diameter lead to an inversion of the Fahraeus-Lindqvist effect, namely, an increase in the apparent viscosity of blood. In such vessels, the red blood cell completely fills the lumen and is distorted as it passes through. Because the red blood cell membrane is not firmly anchored to underlying structures, it can move over its own cell contents, acting somewhat like a tank tread as it moves along the walls of the vessel. Deformation of red blood cells in small vessels leads to a complex flow of erythrocyte membrane and surrounding fluid as the cells squeeze through the narrow lumen. If flow is laminar but pulsatile, as in arteries, the velocity profile is flattened even more than with continuous laminar flow (see Figure 12-25B).Thus blood velocity is constant across much of the vessel and drops sharply near the walls. In turbulent flow, blood moves in various directions in relation to the axis of flow, so there is little accumulation of red blood cells in the center of the vessel. As a result, the blood viscosity and velocity of flow changes little across the vessel.

these fish? What modifications may have evolved to compensate for these low temperatures?

Compliance in the circulatory system A further consideration in analyzing the relationship between pressure and flow in the circulation is that blood vessels contain elastic fibers that enable them to distend. Vessels are not, in fact, the straight, rigid tubes to which Poiseuille's law applies. Rather, as pressure in a vessel increases, the walls are stretched and the volume of the vessel increases. The ratio of change in volume to change in pressure is termed the compliance of the system. The compliance of a system is related to its size and the elasticity of its walls. The greater the initial volume and elasticity of the walls, the greater will be compliance of the system. The venous system is very compliant; that is, small changes in pressure produce large changes in volume. For this reason, the venous system can act as a volume reservoir, because large changes in volume have little effect on venous pressure (and therefore on the filling of the heart during diastole or capillary blood flow). The arterial system, which overall is less compliant than the venous system, acts as a

pressure reservoir in order to maintain capillary blood flow. Nevertheless, the portions of the arterial system near the heart are elastic in order both to dampen the oscillations in pressure generated by contractions of the heart and to maintain flow in distal arteries during diastole. In summary, a large number of factors affect the relationship between pressure and flow in the circulation. Velocity of flow depends on the total cross-sectional area of the circulation; it is highest in the arteries and veins and lowest in the capillaries because the sum of the crosssectional areas of all the capillaries is higher than that of the arteries or veins (see Figure 12-24).Contractions of the heart generate pressure and flow. The highest pressures in the circulation are found in the ventricles and vessels leading from the heart. Pressures are dissipated as energy is lost overcoming the resistance to flow in the vessels. Changes in kinetic energy are reflected in only very small changes in blood pressure as the blood changes velocity. There are only small pressure drops through the arterial and venous systems, even though blood flow is high, because the vessels are large and resistance to flow is small. The largest pressure drop is seen across the arterioles because at this point in the circulation the flow is high and the vessels are small and have a high resistance (see Figure 12-26). The pattern of the flow of blood through this high-resistance pathway reduces the apparent viscosity of the blood (Fahraeus-Lindqvist effect) and therefore the resistance to flow; even so the largest drop in blood pressure occurs in the arterioles. The capillaries are even smaller than arterioles, but flow is much lower in each capillary; therefore, the pressure drop across the capillaries is much smaller than that across the arterioles.

THE PERIPHERAL CIRCULATION Blood pumped from the left ventricle of the mammalian heart carries oxygenated blood via the arterial system to capillary beds in the tissues, where the oxygen is exchanged for carbon dioxide. The venous system returns the deoxygenated blood to the right atrium (see Figure 12-3). Although all blood vessels share some structural features, the vessels in various parts of the peripheral circulation are adapted for the functions they serve. Figure 12-27 illustrates the structure of various sized arteries and veins. A layer of endothelial cells, called the endothelium, lines the lumen of all blood vessels. In larger vessels, the endothelium is surrounded by a layer of elastic and collagenous fibers, but the walls of capillaries consist of a single layer of endothelial cells. Circular and longitudinal smooth muscle fibers may intermingle with or surround the elastic and collagenousfibers. The walls of larger blood vessels comprise three layers: Tunica adventitia: the limiting fibrous outer coat Tunica media: middle layer consisting of circular and longitudinal muscle

Tunica intima: inner layer, closest to the lumen, composed of endothelial cells and elastic fibers The boundary between the tunica intima and the tunica media is not well defined; the tissues blend into one another. Owing to increased muscularization, arteries have a thickened tunica media, and the larger arteries close to the heart are more elastic, with a wide tunica intima. The thick walls of larger blood vessels require their own capillary circulation, termed the vasa vasorum. In general, arteries have thicker walls and much more smooth muscle than veins of similar outside diameter. In some veins, muscular tissue is absent. Arterial System

The arterial system consists of a series of branching vessels with walls that are thick, elastic, and muscular-well suited to deliver blood from the heart to the fine capillaries that carry blood through the tissues. Arteries serve four main functions, as illustrated in Figure 12-28: 1. Act as a conduit for blood between the heart and capillaries 2. Act as a pressure reservoir for forcing blood into the small-diameter arterioles 3. Dampen oscillations in pressure and flow generated by the heart and produce a more even flow of blood into the capillaries 4. Control distribution of blood to different capillary networks via selective constriction of the terminal branches of the arterial tree

Arterial blood pressure, which is finely controlled, is determined by the volume of blood the arterial system contains and the properties of its walls. If either is altered, the pressure will change. The volume of blood in the arteries is determined by the rate of filling via cardiac contractions and of emptying via arterioles into capillaries. If cardiac output increases, arterial blood pressure will rise; if capillary flow increases, arterial blood pressure will fall. Normally, however, arterial blood pressure varies little, because the rates of filling and emptying are evenly matched (i.e., cardiac output and capillary flow are evenly matched). Blood flow through the capillaries is proportional to the pressure difference between the arterial and venous systems. Because venous pressure is low and changes little, arterial pressure exerts primary control over the rate of capillary blood flow and is responsible for maintaining adequate perfusion of the tissues. Arterial pressure varies among species, generally ranging from 50 to 150 mm Hg. Pressure differences are small along large arteries (less than 1mm Hg), but pressure drops considerably along small arteries and arterioles because of increasing resistance to flow with decreasing vessel diameters. The oscillations in blood pressure and flow generated by contractions of the heart are dampened in the arterial system, because of the elasticity of arterial walls. As blood

493

CIRCULATION

...............................................................................

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membrane Figure 12-27 In the mammalIan per~pheralc~rculatlon,blood flows from the heart v~aprogresslvely smaller arteries, then through the mlcroclrculat~on,and finally back to the heart v ~ aprogresslvely larger velns A layer of endothellal cells, the endothellurn, llnes the lumen of all vessels In

larger vessels, the endothel~umIS surrounded by a muscle layer (tunlca medla) and outer flbrous layer (tun~caadvent~t~a) [Adapted from M a r t ~ n ~ and T~mmons,1995 ]

is ejected into the arterial system, pressure rises and the vessels expand. As the heart relaxes, blood flow to the periphery is maintained by the elastic recoil of the vessel walls, resulting in a reduction in arterial volume (see Figure 12-28). If arteries were simply rigid tubes, pressures and flow in the periphery would exhibit the same stops and starts that occur at the exit of the ventricle during each heartbeat. Although elastic, arteries become progressively stiffer with increasing distension. As a result, they are easily distended at low pressures, but then resist further expansion at high pressures. The response of arterial walls to distension is similar in a wide variety of animals, reflecting similar structural and functional characteristics (Figure 12-29).

According to Laplace's law, the wall tension required to maintain a given transmural pressure within a hollow structure increases with increasing radius (see equation 12-1).Elastic vessels thus are unstable and tend to balloon; that is, since they cannot develop high wall tension as pressure increases, they tend to bulge out. In blood vessels, this instability is prevented by a collagen sheath that limits their expansion. Ballooning of a blood vessel (aneurism) can occur, however, if the collagen sheath breaks down. In general, the elasticity of the arterial wall, as well as the thickness of the muscular layer, decreases with increasing distance from the heart. That is, farther from the heart, the arteries become more rigid and serve primarily as blood conduits. For example, the aorta of a dog becomes


................................................................. Arterial oscillations

Ventricular

/ pressure

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Damping oscillations

Figure 12-28 The systemic arterial system functions as a conduit and pressure reservoir; it also smoothes out pressure oscillations and controls distribution of flow to capillaries. The conduit function (1) is served by the vascular channels along which blood flows toward the peripheryw~thminimal frictional loss of pressure.The distensible walls and high outflow re-

sistance of arteries account for the pressure-reservoirfunction (2) and damping of oscillations in pressure and flow (3). Controlled hydraulic resistance in the peripheral vascular beds controls the distribution of blood t o the various tissues (4). [Adapted from Rushmer, 1965a.l

progressively stiffer and its diameter decreases with increasing distance from the heart (Figure 12-30).In a whale, the aortic arch at the exit from the heart is very elastic and has a large diameter, but the arterial system beyond the aor-

tic arch narrows rapidly and becomes much more rigid than that of a dog. The elastic whale aortic arch expands with each heartbeat, accommodating about 50% -75% of the stroke volume; the remainder flows into the portion of the arterial system downstream of the aortic arch. The change in ventricular volume with each heartbeat can be as much as 35 liters in a large whale, with a heart rate of around 12- 18 per minute. The extent of elastic tissue in arteries varies depending on the particular function of each vessel. In fishes, for example, blood pumped by the heart is forced into an elastic bulbus and a ventral aorta (see Figure 12-15).The blood then flows through the gills and passes into a dorsal aorta, the main conduit for the distribution of blood to the rest of the body. A smooth, continuous flow of blood is required in the gill capillaries for efficient gas transfer. The bulbus, the ventral aorta, and the afferent branchial arteries leading to the gills are very compliant and act to smooth and maintain flow in the gills in the face of large oscillations produced by contractions of the heart. The dorsal aorta, which receives blood from the gills, is much less elastic than the ventral aorta. If the dorsal aorta were more elastic than the ventral aorta, there would be a rapid rush of blood through the gills during each heartbeat. This rush would increase rather than decrease the oscillations in flow through the gills. In this example, then, to ensure a steady blood flow through the gill capillaries, the major compliance must be placed before, not after, the gills to dampen the oscillations in flow through the gills. The ventral aorta must

Relative distension (PIP,) Figure 12-29 The elastic properties of arteries are surprisinglysimilar in a wide variety of animals, with nautilus and octopus being notable exceptions. This similarity is reflected in plots of elastic modulus versus relative distension, expressed as pressure (P) divided by the resting blood pressure (P,) of the species. [Adapted from Shadwick, 1992.1

CIRCULATION

495

...................................... Ventral aortic blood flow (ml min-')

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Dorsal aortic blood flow (ml min-')

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Dog- _,,------Figure 12-31 Blood flow is more pulsatile in the ventral aorta (A) than in the dorsal aorta (B) of fishes. The elasticity of the bulbus and ventral aorta Thoracic aorta Abdominal aorta help to dampen oscillations in pressure and flow. The recordings shown are from cod. [Jones et al., 1974.1

Relative position along aorta Figure 12-30 The arterial system of dogs and whales becomes stiffer and smaller in diameter with distance from the heart. In whales, there is an abrupt decrease in diameter and increase in stiffness between the aortic arch and the thoracic artery. [Adapted from Gosline and Shadwick, 1996.1

be elastic and the large-volume dorsal aorta relatively stiff to achieve a smoothing of flow through the gills (Figure 12-31).

80 mm Hg). Blood is 12.9 times less dense than mercury, so a blood pressure of 120 mm Hg is equal to 120 X 12.9 = 1550 mm (155 cm) of blood. In other words, if the blood vessel were suddenly opened, the blood would squirt out to a maximum height of 155 cm above the cut. To convert pressures in millimeters of mercury to kilopascals (kPa), multiply by 0.1333 kPa (e.g., 120 mm Hg X 0.1333 = 16 kPa). The oscillations in pressure produced by the contractions and relaxations of the ventricle are reduced at the entrance to capillary beds and nonexistent in the venous system. Heart contractions cause small oscillations in pressure within capillaries. The pressure pulse travels at a velocity of 3-5 m-s-l. The velocity of the pressure pulse increases with decrease in artery diameter and increasing stiffness of the arterial wall. In the mammalian aorta, the pressure pulse travels at 3 -5 m sS' and reaches 15-35 m sS' in small arteries. Peak blood pressure and the size of the pressure pulse within the mammalian and avian aorta both increase with distance from the heart (Figure 12-32).This pulse amplification can be large during exercise. There are three possible explanations for this rather odd phenomenon. First, pressure waves are reflected from peripheral branches of the arterial tree; the initial and reflected waves summate; and, where peaks coincide, the pressure pulse and peak pressure are greater than where they are out of phase. If the initial and reflected waves are 180" out of phase, the oscillations in pressure will be reduced. It has been suggested that the heart is situated at a point where initial and reflected waves are out of phase, thus reducing peak arterial pressure in the aorta close to the ventricle. As distance from the heart increases, the initial and reflected pressure waves move into phase, and a peaking of pressures is observed in vessels of the periphery. Second, the decrease in elasticity and diameter of arteries with distance from the heart may cause an increase in the magnitude of the pressure pulse. Third, the pressure pulse is a complex waveform, consisting

.

Blood pressure Blood pressures reported for the arterial system are usually transmural pressures (i.e., the difference in pressure between the inside and outside, across the wall of the blood vessel). The pressure outside vessels is usually close to ambient, but changes in the extracellular pressure of tissues can have a marked effect on transmural pressure and therefore on vessel diameter and consequently blood flow. For example, contractions of the heart raise pressure around coronary vessels and result in a marked reduction in coronary flow during systole. Breathing in is associated with a reduction in thoracic pressure and thus raises transmural pressure in veins leading back to the heart, increasing venous return to the heart. During a heartbeat cycle, the maximum arterial pressure is referred to as systolic pressure and the minimum as diastolic pressure; the difference is the pressure pulse. Transmural pressures are typically given in millimeters of mercury; both the systolic and diastolic pressures generally are indicated with a slash between them (e.g., 1201

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Time (ms) Figure 12-32 In the aorta of mammals and birds, the peak blood pressure and pressure pulse both increase with distance from the heart. Shown are simultaneous recordings of a rabbit's blood pressure in the aortic arch (2 cm from the heart) and at trifurcation of the aorta (24 crn from the heart). Note that the mean pressure is slightly less attrifurcation of the aorta than in the aortic arch near the heart. [Adapted from Langille, 1975.)

above the heart (Figure 12-33A).If the arterial pressure of blood perfusing the brain is to be maintained at around 98 mm Hg, aortic blood pressure must be 195-300 mm Hg near the heart. Aortic blood pressures greater than 195 mm Hg have been recorded in an anesthetized giraffe whose head was raised about 1.5 meters (Figure 12-33B). Arterial pressures in the legs of the giraffe are even higher than aortic pressures; to prevent the pooling of blood, the giraffe has large quantities of connective tissue surrounding the leg vessels. As the giraffe lowers its head to the ground, arterial blood pressure at the level of the heart is reduced considerably, thus maintaining a relatively constant blood flow to the brain. The wide variation in aortic pressure as the giraffe moves its head position could lead to extensive pooling of blood (head raised) or decreased flow (head lowered) in arterioles other than those of the head. Pooling most likely is prevented by vasoconstriction of these peripheral vessels when the head is raised. Conversely, when the head is lowered, extensive vasodilation of arterioles leading to capillary beds other than those

of several harmonics. Higher frequencies travel at higher velocities, and it has been suggested that the change in waveform of the pressure pulse with distance is due to summation of different harmonics. This third explanation is open to question, as the distances are too small to allow summation of harmonics.

Effect of gravity and body position on pressure and flow When a person is lying down, the heart is at the same level as the feet and head, and pressures will be similar in arteries in the head, chest, and limbs. Once a person moves to a sitting or standing position, the relationship between the head, heart, and limbs changes with respect to gravity, and the heart is now a meter above the lower limbs. The result is an increase in arterial pressure in the lower limbs and a decrease in arterial pressure in the head. The height of the column of blood simply results in a higher blood pressure due to gravity. Gravity has little effect on capillary flow, which is determined by the arterial-venous pressure difference rather than the absolute pressure. That is, gravity raises arterial and venous pressure by the same amount and therefore does not greatly affect the pressure gradient across a capillary bed. Because the vascular system is elastic, however, an increase in absolute pressure expands blood vessels, particularly the compliant veins. Thus, pooling of blood tends to occur, particularly in veins, in different regions of the body as an animal changes position with respect to gravity. This effect is related solely to the elasticity of blood vessels and would not occur if the blood flowed in rigid tubes. The problems of pooling and maintaining capillary flow are acute in species with long necks. For instance, when the giraffe is standing with its head raised, its brain is about 6 meters above the ground and over 2 meters

-

Vasoconstriction of vessels in lower body

0

Aortic pressure decreases

Vasodilation of vessels in lower body

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20

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Time (s) Figure 12-33 As animals with long necks raise and lower their heads, the cardiovascular system must adjust to maintain blood flow to the brain and avoid pooling of blood in the lower part of the body. See text for discussion. [Adapted from White, 1972.1

CIRCULATION

497

........................................ in the head probably maintains flow despite the lower aortic pressure. The ability of the giraffe to regulate pressure and flow in peripheral vessels other than those to the head is particularly crucial for kidney function. If the kidney tubule were subjected to the enormous changes in blood pressure associated with the raising and lowering of the giraffe's head, the rate of glomerular filtration would be chaotic. Each time the animal lifted its head, the large increase in arterial blood pressure would lead to a very high rate of ultrafiltrate formation in the kidney; this in turn would require that fluid be reabsorbed at an equally high.rate. In the absence of any appropriate controls, the giraffe could lower its head to drink and then lose any fluid gained as it was filtered through the kidney when the head was raised. Thus, the giraffe must have mechanisms for adjusting peripheral resistance to flow in various capillary beds as it swings its head from ground level to drink to a height of 6 m to eat. Similar problems must have been or are faced by a number of other animals with long necks, like dinosaurs and camels. Pooling of blood, with changes of position with respect to gravity, is not a problem for animals in water, because the density of water is only slightly less than that of blood, whereas air is much less dense than blood. In water, the hydrostatic pressure increases with depth, and effectively matches the increase in blood pressure due to gravity; thus transmural pressure does not change, so the blood does not pool. Clearly, the circulatory problems faced by tall terrestrial dinosaurs were very different from those of aquatic dinosaurs. Velocity of arterial blood flow Blood flow and the oscillations in flow with each heartbeat are greatest at the exit to the ventricle, decreasing with increasing distance from the heart (Figure 12-34).At the base of the aorta, as noted earlier, flow is turbulent and reverses during diastole as closure of the aortic valves creates vor-

Figure 12-34 The maximal velocity of gterial blood and oscillations in flow decrease progressively with distance from the heart. A backflow phase is observed in the large arteries; in the ascending aorta, it is probably related t o a brief reflux of blood through the aortic valves. These tracings were obtained from a dog's arteries. Oscillatory flow is damped out entirely in the capillaries. [Adapted from McDonald, 1960.1

tices in the blood ejected into the aorta during systole. In most other parts of the circulation, flow is laminar, and oscillations in velocity are damped by the compliance of the aorta and proximal arteries. Mean velocity in the aorta-the point of maximal blood velocity-is calculated as about 33 cm -s-' in humans, based on a cross-sectional area of about 2.5 cm2and cardiac output of about 5 La min-'. If we assume that maximal velocity in a vessel is twice the mean velocity (valid only if the velocity profile is a parabola), then the maximal velocity of blood flow in the human aorta would be 66 cm-s-l. If cardiac output is increased by a factor of 6 during heavy exercise, maximal velocity is raised to 3.96 m-s-l. In contrast, the pressure pulse associated with each heartbeat travels through the circulation at 3-35 m s-l; thus, the pressure pulse travels faster than the flow pulse.

-

Venous System

The venous system acts as a conduit for the return of blood from the capillaries to the heart. It is a large-volume, lowpressure system consisting of vessels with a larger inside diameter than the corresponding arteries (see Figure 12-27). In mammals, about 50% of the total blood volume is contained in veins (see Figure 12-3). Venous pressures seldom exceed 11rnrn Hg (1.5 kPa), roughly 10% of arterial pressures. The walls of veins are much thinner, contain less smooth muscle, and are less elastic than arterial walls; venous walls also contain more collagen than elastic fibers. As a result, the walls of veins are easily stretched and exhibit much less recoil than occurs in arteries. The large diameter and low pressure of veins permits the venous system to function as a storage reservoir for blood. If venous pressures were high, then according to Laplace's law (seeequation 12-l),very high wall tensions would develop, requiring the walls to be very strong to prevent them from tearing. In the event of blood loss, venous blood volume, not arterial volume, decreases in order to maintain arterial pressure and capillary blood flow. The decrease in the venous blood reservoir is compensated for by a reduction in venous volume. The walls of many veins are covered by smooth muscle innervated by sympathetic adrenergic fibers. Stimulation of these nerves cause vasoconstriction and a reduction in the size of the venous reservoir. This reflex allows some bleeding to take place without a drop in venous pressure. Blood donors actually lose part of their venous reservoir; the loss is temporary, however, and the venous system gradually expands as blood is replaced due to fluid retention. Venous blood flow Blood flow in veins is affected by a number of factors other than contractions of the heart. Contraction of limb muscles and pressure exerted by the diaphragm on the gut both result in the squeezing of veins in those parts of the body. Because veins contain pocket valves that allow flow only

toward the heart, this squeezing augments the return of blood to the heart. Thus venous return to the heart increases during exercise, as skeletal muscle contractions squeeze veins and drive blood towards the heart. The increase in venous return will increase cardiac output. Activation of this skeletal-muscle venous pump is associated with increased activity in sympathetic fibers innervating the venous smooth muscle, increasing smooth-muscle tone. This increase in venous tone ensures that the skeletalmuscle pump increases venous pressure and therefore return to the heart, rather than simply distending another part of the venous system. In the absence of skeletal muscle contraction, there may be considerable pooling of blood in the venous system of the limbs. Breathing in mammals also contributes to the return of venous blood to the heart. Expansion of the thoracic cage reduces pressure within the chest and draws air into the lungs; this pressure reduction sucks blood from the veins of the head and abdominal cavity into the heart and large veins situated within the thoracic cavity. In sharks contractions of the ventricle reduce pressure in the pericardial cavity, so blood from the venous system is sucked into the atrium (see Figure 12-14). Peristaltic contractions of the smooth muscle of venules, the small vessels joining capillaries to veins, can promote venous flow toward the heart. Such peristaltic activity has been observed in the venules of the bat wing. Blood dzstrzbution in vezns Venous smooth muscle also aids in regulating the dlstribution of blood in the venous system. When a person shlfts from a sitting positlon to a standing posltion, the change In the relatlve posltlons of heart and braln wlth respect to grav~ t yactivates sympathetic adrenergic fibers that Innervate llmb veins, causing contraction of venous smooth muscle and thereby promoting the red~stributlonof pooled blood. Such venoconstrlction 1s inadequate, however; to malntaln good clrculat~on~f the standlng posltion 1s held for long penods in the absence of l ~ m bmovements, as when soldiers stand immobile durlng a review. Under such circumstances, venous return to the heart, cardlac output, arterlal pressure, and flow of blood to the brain are all reduced, whlch can result In faintmg. S~milarproblems affect bedridden patlents who attempt to stand after several days of lnactivlty and astronauts returning to Earth after a long per~odof weightlessness. In these instances other control systems involving baroreceptors (pressurereceptors) and arterioles may be d~srupted as well. In the absence of body changes that shlft the relatlve positions of the heart and brain with respect to gravlty, the system of corrections falls into disrepair, and the result IS the pooling of blood. The reflex control of venous volume IS normally reestablished wlth use. The organization of the venous system is influenced by the degree of support offered by the medlum. There was an extensive reorganization of the 'ystem as vertehates moved Into air and lost the support of water- As ment~onedprev~ously,the effects of gravity on blood d ~ s -

tribution are not important in aquatic animals because the densities of water and blood are not very different. For this reason, pooling of blood due to gravity does not occur in aquatic animals. Because of the large difference between the density of air and blood, pooling became an immediate problem with the evolution of terrestrial forms. The required changes in the venous system are in addition to those required to maintain separation of oxygenated and deoxygenated blood through the heart. Although the effects of gravity are minimal in aquatic animals, venous return to the heart is exacerbated by swimming in fish. As the fish moves forward, blood will collect in the tail due both to inertia and to compression waves that pass down the body associated with the swimming movements of the fish. To diminish these problems most veins returning to the heart pass down the center of the fish's body. Some fish also have an accessory caudal heart in the tail, which aids in propelling blood toward the central heart (Figure 12-35). The flow of water over the pectoral regions of some fish may reduce hydrostatic pressure in that region, so that venous return to the heart is promoted with increased swimming speed. Countercurrent exchangers Countercurrent exchangers are a common feature in the design of animals (seeSpotlight 14-2).In many animals arteries and veins run next to each other with the blood flows

Figure 12-35 Some f~shhave a caudal heart located In the tall, wh~cha~ds In returning deoxygenated blood to the central heart The walls of the heart conta~nskeletal muscle and beat rhythm~cally[Adapted from Kampme~er,19691

CIRCULATION

f

499

............................................................................... moving in opposite directions (i.e., countercurrent blood flow). In many such instances, especially if the vessels are small, there will be exchange of heat between the countercurrent blood flows. Because heat is transferred much more easily than gas, it is possible to have heat exchange with little gas transfer. Countercurent heat exchangers are common in the limbs of birds and mammals and are used to regulate the rate of heat loss via the limbs. A countercurrent arrangement of small arterioles and venules is referred to as a rete mirabile. Before entering a tissue, an artery divides into a large number of small capillaries that parallel a series of venous capillaries leaving the tissue. The "arterial" capillaries are surrounded by "venous" capillaries, and vice versa, forming an extensive exchange surface between inflowing and outflowing blood. These retial capillaries serve to transfer heat or gases between arterial blood entering a tissue and venous blood leaving it. In humans, this type of countercurrent exchanger is found only in the kidney. Tuna have a large number of retia mirabile, which are used to regulate the temperature of the brain, muscles, and eyes (see Figures 16-22 and 16-23). The rete mirabile leading to the physoclist swimbladder of

\

,\

\\

other fish such as the eel function as a carbon dioxide countercurrent exchanger (see Figure 13-59). Capillaries and the Microcirculation

Most tissues have such an extensive network of capillaries that any single cell is not more than three or four cells away from a capillary. This is important for the transfer of gases, nutrients, and waste products, because diffusion is an exceedingly slow process. Capillaries are usually about 1mm long and 3 - 10 pm in diameter, just large enough for red blood cells to squeeze through. Large leukocytes, however, may become lodged in capillaries, stopping blood flow. The leukocytes are either dislodged by a rise in blood pressure or migrate slowly along the vessel wall until they reach a larger vessel and are swept into the bloodstream.

Microcirculatory beds Figure 12-36 illustrates the vessels composing a microcirculatory bed. Small terminal arteries subdivide to form arterioles, which in turn subdivide to form metarterioles and subsequently capillaries, which then rejoin to form venules and veins. The arterioles are invested with smooth

Pericyte covered with

Figure 12-36 A m~croc~rculatory bed conslsts of small arter~es(arterloles), cap~llar~es, and venules Cap~llar~es, wh~chconslst of a s~nglelayer of endothel~alcells surrounded by a basement membrane and have occas~ondcontractile perlcyte cells wrapped around them. D~rectflow

I'

;

from the arter~alto venous system can occur vla the thoroughfare channel, but most blood flows through the network of cap~llar~es The precap~llarysphlncterhelps regulate flow Into the capillary bed Also see Figures 12-27 and 12-37

500


...............................................................................

muscle that becomes discontinuous in the metarterioles and ends in a smooth muscle ring, the precapillary sphincter. Capillary walls, which are completely devoid of connective tissue and smooth muscle, consist of a single layer of endothelial cells surrounded by a basement membrane of collagen and mucopolysaccharides. The capillaries are often categorized as arterial, middle, or venous capillaries, the latter being a little wider than the other two types. A few elongated cells with the ability to contract, called pericyte cells, are found wrapped around capillaries. The venous capillaries empty into pericytic venules, which in turn join the muscular venules and veins. The venules and veins are valved, and the muscle sheath appears after the first postcapillary valve. Even though the walls of capillaries are thin and fragile, they require only a small wall tension to resist stretch in response to pressure because of their small diameter (see equation 12-1). The innervated smooth muscle of the arterioles and, in particular, the smooth muscle sphincter at the junction of arteries and arterioles control blood distribution to each capillary bed. Most arterioles are innervated by the sympathetic nervous system; a few arterioles (e.g., those in the lungs) are innervated by the parasympathetic nervous system. Different tissues have varying numbers of capillaries open to flow and show some variation in the control of blood flow through the capillary bed. In some tissues, opening and closing of the precapillary sphincters, which are not innervated and appear to be under local control, alter blood distribution within the capillary bed. In other tissues, however, most, if not all, of the capillaries tend to be open (e.g., in the brain) or closed (e.g., in the skin) for considerable periods. All capillaries combined have a potential volume of about 14% of an animal's total blood volume. At any one moment, however, only 30%-50% of all capillaries are open, and thus only 5% -7% of the total blood volume is contained in the capillaries.

Material transfer across capillary walls Transfer of substances between blood and tissues occurs across the walls of capillaries, pericytic venules, and, to a lesser extent, metarterioles. The endothelium composing the capillary wall is several orders of magnitude more permeable than epithelial cell layers, allowing substances to move with relative ease in and out of capillaries. However, the capillaries in various tissues differ considerably in permeability. These permeability differences are associated with marked changes in the structure of the endothelium. Based on their wall structure, capillaries are classified into three types (Figure 12-37): Continuous capillaries, which are the least permeable, are located in muscle, nervous tissue, the lungs, connective tissue, and exocrine glands. Fenestrated capillaries, which exhibit intermediate permeability, are found in the renal glomerulus, intestines, and endocrine glands.

A

Continuous capillary

(mucopolysaccharides and collagen fibers)

B

Fenestrated capillary

~ a g e m e nmembrane t

c Sinusoidal capillary

Figure 12-37 D~fferencesin the structure of the capillary endothelium define three types of capillaries, which are found in characteristic tissues. Shown here are portions of the endothelial wall (A) Continuous capillary with 4-nm clefts, a complete basement membrane, and numerous vesicles. (B) Fenestrated capillary with pores through a thin portion of the wall, few vesicles, and a complete basement membrane. (C) Sinusoidal capillary with large paracellular gaps extending through the discontinuous basement membrane. In general, continuous capillaries are the least permeable and sinusoidal capillaries are the most permeable.

Sinusoidal capillaries, which are the most permeable, are present in the liver, bone marrow, spleen, lymph nodes, and adrenal cortex. In the continuous capillaries of skeletal muscle, which have been studied extensively, the endothelium is about 0.2-0.4 pm thick and is underlain by a continuous basement membrane (seeFigure 12-37A).The endothelial cells

CIRCULATION 501 ......................................

are separated by clefts, which are about 4 nm wide at the narrowest point. Most of the cells contain large numbers of pinocytotic vesicles about 70 nm in diameter. Most of these vesicles are associated with the inner and outer membranes of the endothelial cells; the rest are located in the cell matrix. Substances can move across the wall of continuous capillaries either through or between the endothelial cells. Lipid-soluble substances diffuse through the cell membrane, whereas water and ions diffuse through the waterfilled clefts between cells. In addition, at least in brain capillaries, there are transport mechanisms for glucose and some amino acids. Large macromoleculescan move across many capillary walls, but exactly how they are transferred is not always clear. Some evidence indicates that the numerous vesicles in endothelial cells play a role in transferring substances across the capillary wall. For example, electron microscopic studies have shown that when horseradish peroxidase is placed in the lumen of a muscle capillary, it first appears in vesicles near the lumen and then in vesicles close to the outer membrane, but never in the surrounding cytoplasm. This finding suggests that material is packaged in vesicles and shuttled through the endothelial cells. Supporting this concept of vesicle-mediated transport is the observation that endothelial cells of brain capillaries contain fewer vesicles and are less permeable than endothelial cells from other capillary beds. The reduced permeability of brain capillaries, however, is also considered to result from the tight junctions between endothelial cells. Another possibility is suggested by microscopic observations of capillaries in the rat diaphragm in which vesicles have been observed to coalesce, forming pores through the endothelial cells. Conceivably, then, substances diffuse through pores created by coalescence of nonmobile vesicles, rather than being packaged in vesicles that then move across the cell. The continuous capillaries in the lung are less permeable than those in other tissues. In these less-permeablecapillaries, the pressure pulse may play a role in augmenting movement of substances (e.g., oxygen) through the endothelium. As pressure rises, fluid is forced into the capillary wall, but as pressure drops, fluid returns to the blood. This tidal flushing of the capillary wall should enhance mixing in the endothelial barrier and effectively augment transfer. In the capillaries of the renal glomerulus and gut, the inner and outer plasma membranes of endothelial cells are closely apposed and perforated by pores in some regions, forming a fenestrated endothelium (see Figure 12-37B). Not surprisingly,these fenestrated capillaries are permeable to nearly everything except large proteins and red blood cells. The kidney ultrafiltrate is formed across such an endothelial barrier. The basement membrane of fenestrated endothelia normally is complete and may constitute an important barrier to the movement of substances across fenestrated capillaries. These capillaries contain only a few vesicles, which probably play no role in transport.

The endothelium in sinusoidal capillaries is characterized by large paracellular gaps that extend through the basement membrane and an absence of vesicles in the cells (see Figure 12-37C).Liver and bone capillaries always contain large paracellular gaps, and most substance transfer across these capillaries occurs between the cells. As a result, the fluid surrounding the capillaries in liver has much the same composition as plasma. The clefts, pores, and paracellular gaps through which substances can freely diffuse across capillary walls are about 4 nm wide, but only molecules much smaller than 4 nm can get through, indicating the presence of some further sieving mechanism. The diameter of these openings varies within a single capillary network and usually is larger in the pericytic venules than in the arterial capillaries. This is of functional significance because blood pressure, which is the filtration force for moving fluid across the wall, decreases from the arterial to the venous end of the capillary network. Inflammation or treatment with a variety of substances (e.g., histamine, bradykinin, and prostaglandins)increases the size of the openings at the venous end of the capillary network, making it very permeable. Capillary pressure and flow The arrangement of arterioles and venules is such that all capillaries are only a short distance from an arteriole, so that pressure and flow are fairly uniform throughout the capillary bed. Transmural pressures of about 10 mm Hg have been recorded in capillaries (Figure 12-38).High pressures inside a capillary result in the filtration of fluid from the plasma into the interstitial space. This filtration pressure is opposed by the plasma colloid osmotic pressure, which results largely from the higher concentration of proteins in the blood than in the interstitial fluid. Because of their large size, these plasma proteins are retained in the blood and not transported across the capillary wall. To visualize the relationship of these two pressures, consider the schematic situation depicted in Figure 12-39. Generally, blood pressure is higher than the colloid osmotic pressure at the arterial end of a capillary bed, so fluid moves into the interstitial space (area 1).The blood pressure steadily decreases along the length of the capillary, while the colloid osmotic pressure remains constant. Once the blood pressure falls below the colloid osmotic pressure, fluid in the interstitial space is drawn back into the blood by osmosis (area 2). Thus the net movement of fluid at any point along the capillary is determined by two factors: (a)the difference between blood pressure and colloidal 0smotic pressure and (b) the permeability of the capillary wall, which tends to increase toward the venous end. This concept is sometimes referred to as the Starling hypothesis, after its initial proponent, Ernest Starling (1866- 1927),whose prolific research also included studies on the relationship between ventricle work output and venous filling pressure (see Spotlight 12-1). In most capillary beds, the net loss of fluid at the arterial end is somewhat greater than the net uptake at the venous end of the

502

--


A

1s

Small artery

(500 pm)

l5

r

15

[-

5

a,

Metarteriole

(70 ~ m )

L

1 min C--l

0

Arteriole caiary

f'15

(25 w)

; - 0

[ d

L

capillary

g

-

-

1 min

capillary. The fluid, however, does not accumulate ln the tissues, but 1s drained by the lymphatic system and returned to the circulation. Thus, there typically is a circulation of fluid from the arterial end of capillary bed into the mterstitial spaces and back into the blood across the venous end of the capillary bed or via the lymphatic system. Because of this bulk flow of fluid, the exchange of gases, nutrients, and wastes between blood and tissues exceeds that expected by diffusion alone. Net filtration of fluid across capillary walls will result in an increase in tissue volume, termed edema, unless the excess fluid is carried away by the lymphatic system. In the kidney, capillary pressure is high and filtration pressures exceed collold osmotlc pressures; hence, an ultrafiltrate is formed in the kidney tubule, eventually to form urine. The kidney is encapsulated to prevent swelling of the tissue in the face of ultrafiltratlon. In most other tissues, there is only a small net movement of fluid across capillary walls and tissue volume remains constant. A rise in capillary pressure, owing to a rise in either arterial or venous pressure, will result in increased loss of fluid from the blood and tissue edema. In general, though, arterial pressure remains fairly constant t o prevent large oscillations in tissue volume. A drop in colloid osmotic pressure can result from a loss of protein from the plasma by starvation or excretion or by increased capillary wall permeability, leading to movement of plasma proteins into the interstitial space. If filtration pressure remains constant, a decrease in colloid osmotic pressure will also result In an increase in net fluid loss to the tissue spaces.

Figure 12-38 The pressure pulse is reduced and the mean blood pressure decreases as blood flows through a cap~llarybed. (A) Blood pressure tracings recorded throughout capillary bed of the frog mesentery. The pressure is smoothed and falls as blood flows across the capillary bed. (B) Plot of blood pressure versus location in circulation of the subcutaneous layers of the bat wing. Shaded area represents 2 1 SE (standarderror) from the average values indicated by thick line. Also plotted are typical tissue and lymphatic pressures for comparison. [Part A from , Weiderhielm et al., 1964; part B from Weiderhielm and Weston, 1973.1

Capillary

end

Filtration

Uptake

9 3 (0 m

E

a osmotic pressure

1 Arterial end

Venous end Capillary length

Figure 12-39 Net fluid flow across the capillary wall depends on the difference between the blood pressure and the colloid osmotic pressure of the extracellular fluid. At the arterial end of the capillary, blood pressure exceeds colloid osmotic pressure and fluid is filtered from the plasma into the extracellular space (area 1). At the venous end, the reverse is true and fluid is drawn back into the plasma from the extracellular space (area 2). Area 1 is somewhat larger than area 2 in most capillary beds; that is, there is a small net loss of fluid from the circulationto the extracellular space. In general, this tissue fluid is drained and returned to the bloodstream via the lymphatic system.

503

CIRCULATION

.................................... A

Lyrnphatlc trunks

10 s

F

1 min

THE LYMPHATIC SYSTEM Lymph-a transparent, slightly yellow or sometimes milky fluid-is collected from the interstitial fluid in all parts of the body and returned to the blood via the lymphatic system. Because this fluid contains many white blood cells but no red blood cells, it is nearly colorless, making the Iymphatic vessels difficult to see. As a result, even though the lymphatic system was first described about 400 years ago, it has not been nearly as extensively studied as the cardiovascular system. The lymphatic system begins with blind-ending lymphatic capillaries that drain the interstitial spaces. These lymphatic capillaries join to form a treelike structure with branches reaching to all tissues. The larger lymphatic vessels resemble veins and empty via a duct into the blood circulation at a point of low pressure. In mammals and many other vertebrates, the lymph vessels drain via a thoracic duct into a very low pressure region of the venous system, usually close to the heart (see Figure 12-3).The lymphatic system serves to return to the blood the excess fluid and proteins that filter across capillary walls into the interstitial spaces. Large molecules, particularly fat absorbed from the gut and probably high-molecular-weighthormones, reach the blood via the lymphatic system. The walls of lymphatic capillaries consist of a single layer of endothelial cells. The basement membrane is absent or discontinuous, and there are large paracellular gaps between adjoining cells. This feature has been demonstrated by microscopic observation of horseradish peroxidase or china ink particles passing through lymphatic capillary walls. Because lymphatic pressures are often slightly lower than the surrounding tissue pressures, interstitial fluid passes easily into lymphatic vessels. The vessels are valved and permit flow only away from the lymphatic capillaries. The larger lymphatic vessels are surrounded by smooth muscle and, in some instances, contract rhythmically,creating pressures of up to 10 mm Hg and driving fluid away from the tissues (Figure 12-40). The vessels also are squeezed by contractions of the gut and skeletal muscle and by general movements of the body, all of which promote lymph flow. Fats are taken up from the gut by the lymphatic system rather than directly into the blood. Folds in the gut wall, called villi, each contain a lymphatic vessel (central lacteal) into which fats and fat-soluble nutrients (e.g., vitamins A, D, E, and K) pass from the lumen of the gut (see Chapter 15). The lacteal is "milked" of its milky fat-containing lymph by contractions of the gut, pushing the lymph forward and eventually, via the thoracic duct, into the blood. Lymph vessels are innervated, but it is not clear what type of innervation exists nor what function these nerves have.

1 rnin

B

1

rnin

Lymphatic capillary (5 ~ r n )

1 min Figure 12-40 Pressures In the lymphatlc system are slmllar to those In the venous system These recordings are from lymphatlc trunks (A) and lymphat~ccaptllar~es (B) In the wlng of an unanaesthetlzed bat They were obtained by mlcropuncture without prtor surglcal lntervent~onwelderhlelm and Weston, 19731

Lymph flow is variable, 11 ml- h-' being an average value for resting humans. This is 1/3ooo of the cardiac output during the same time period. Nevertheless, although it is small, lymphatic flow is important in draining tissues of excess interstitial fluid. If lymph production exceeds lymph flow, severe edema can result. In the tropical disease filariasis, larval nematodes, transmitted by mosquitoes to humans, invade the lymphatic system causing blockage of lymph channels; in some cases, lymphatic drainage from certain parts of the body is blocked totally. The consequent edema can cause parts of the body to become so severely swollen that the condition has come to be called elephantiasis because of the resemblance of the swollen, hardened tissues to the hide of an elephant. Reptiles and many amphibians have lymph hearts, which aid in the movement of fluid within the lymphatic system. Bird embryos have a pair of lymph hearts located in the region of the pelvis; these hearts persist in the adult bird of a few species. Mammals lack these structures for moving lymph. Frogs have not only multiple lymph hearts but also a very large-volume lymph space, which serves as a reservoir for water and ions and as a fluid buffer between the skin and underlying tissues. The large lymph volume in amphibians is derived from both plasma filtration across capillaries and the diffusion of water across the skin. The ratio of lymph flow to cardiac output is much higher in toads (approximately 1:60) than in mammals (approximately 1 :3000), and toad lymph hearts, although having a much smaller stroke volume, can beat at rates higher than the blood heart. Fish appear to either lack or have only a very rudimentary lymphatic system, although they have a secondary circulation that in the past was described as a lymphatic

system. This secondary circulation, which has a low hematocrit, is connected to the primary circulation via arterio-arterial anastomoses and drains into the primary venous system near the heart (see Figure 12-15). The secondary circulation supplies nutrients but not much oxygen to the skin and gut but is not generally distributed to other parts of the body. The skin exchanges gases directly with the surrounding water. Because of its narrow distribution it is unlikely that the secondary circulation fulfills the lymphatic function of maintaining tissue fluid balance. It is not clear how fish maintain tissue fluid balance, but the absence of lymphatics seems to be related to the fact that fish live in a medium of similar density to their own bodies.

CIRCULATION AND THE IMMUNE RESPONSE Both the circulatory and lymphatic systems are involved in the body's defense against infection. The crucial players in the immune response are lymphocytes, a type of white blood cell (leukocyte).The unique characteristic of lymphocytes is their ability to "recognize" foreign substances

(antigens) including those on the surface of invading pathogens, virus-infected cells, and tumor cells. There are two main types of lymphocytes: B lymphocytes (Bcells) and T lymphocytes (T cells). The latter are subdivided into helper T (TH) cells and cytotoxic T (Tc ) cells. Lymphocytes are aided by other leukocytes, particularly neutrophils and macrophages. Under certain conditions, both neutrophils and macrophages can engulf microorganisms and foreign particulate matter by phagocytosis. These phagocytic cells also produce and release various cytotoxic factors and antibacterial substances. The immune response consists of recognizing the invader, then marking and destroying it. Recognition is carried out exclusively by lymphocytes, whereas destruction can be effected by both lymphocytes and phagocytic cells (phagocytes). The lymphocyte recognition system must be able to discriminate between natural constituents of the body and foreign invaders, that is, to distinguish between self and nonself. Failure to recognize self leads to autoimmune diseases, some of which can be fatal. Lymphocytes respond in three ways to an invasion by a pathogen (Figure 12-41). B cells develop into plasma cells, which secrete antibodies that bind to the pathogen, mark-

Figure 12-41 Three types of lymphocytesB cells, helperT (T,) cells, and cytotoxicT(Tc)

8

Tc cell

I

Develops into antibody secreting plasma cell

receptor

.

Secretes cytokines, which promotes growth and responsiveness of B and Tc cells

I

Antibodies bind to pathogen, which are eliminated by phagocytes

Cytokines

I

Develops into active CTL, which destroys altered self-cell

cells-respond in different ways to antigen. Membrane-boundantibody on B cells and Tcell receptors on T cells recognize and bind antigen specifically. T, and Tc cells can be distinguished by the presence of membrane molecules called CD4 and CD8. See text for discussion. IAdaptedfrom Kuby, 1997.1

ing the cell for degradation by phagocytes. T, cells can recognize tumor cells and pathogen-infected tumor cells; upon recognizing such cells, Tc are stimulated to mature into active cytotoxic T lymphocytes (CTLs),which destroy the altered self-cells. Recognition of antigen by T, cells stimulates them to secrete cytokines, which in turn promote the growth and responsiveness of B cells, T, cells, and macrophages, thereby increasing the strength of the knmune response to a pathogen. Leukocytes circulate in both the blood and lymph. Large numbers of lymphocytes are present in lymph nodes, which are located along the lymphatic vessels (see Figure 12-3). These nodes filter the lymph and help bring antigen into contact with lymphocytes. To get to tissues that have been invaded by pathogens, leukocytes must be able to leave the lymphatic and circulatory systems, a process termed extravasation. Normally, of course, leukocytes are swept along in the bloodstream and do not pass across vessel walls. At sites of infection, however, inflammatory signals are produced that induce the synthesis and activation of adhesive proteins on the blood side of the endothelium. As leukocytes roll past an inflamed vascular endothelium, P-selectin on the blood-facing surface binds to and slows the passing leukocytes (Figure 12-42). This interaction stimulates the leukocytes to produce integrin receptors (e.g., LFA-I), which then bind with intracellular adhesion molecules (ICAMs) on the surface of the endothelium. As

a result of these and other interactions, the cells adhere to the endothelium. Once firmly adhered, the leukocytes can move between the endothelial cells and migrate into the infected tissue.

A A

REGULATION OF ClRCULATlON Regulation of circulation hinges on controlling arterial blood pressure so that three central priorities can be fulfilled: Delivering an adequate supply of blood to the brain and heart Supplying blood to other organs of the body, once the brain and heart supply is assured Controlling capillary pressure so as to maintain tissue volume and the composition of the interstitial fluid within reasonable ranges The body employs a variety of receptors for monitoring the status of the cardiovascular system. In response to sensory inputs from these receptors, both neural and chemical signals induce appropriate adjustments to maintain an adequate arterial pressure. In this section, we first discuss regulatory features affecting the heart and main vessels, and then focus on the microcirculation.

Sectional view of vascular endothelium

/ Rolling

Adhered

Extravasation

Leukocyte migration into infected tissue

/

ICA

+ Selection tethering to inflamed endothelium Figure 12-42 Leukocytes mlgrate from the clrculat~onInto tlssues at sltes of lnflamrnatlon (A) Ovewlew of leukocyte adherence to and extravasatlon across Inflamedvascular endothellurn (B) Some of the Inter-

LFA-1 production

Integrin-ICAM interaction

actlons between cell-surface molecules that cause leukocytes to adhere to an Inflamed endothellurn [Adaptedfrom Kuby, 1997 I

506


...............................................................................

Control of the Central Cardiovascular System

Baroreceptors monitor blood pressure at various sites in the cardiovascular system. Information from baroreceptors, along with that from chemoreceptors monitoring the CO,, '. O,, '. and DHof the blood. is transmitted to the brain. ~~~~l~ or in the composition of the extracellular fluid of muscles activate afferent fibers embedded in muscle tissue, and this in turn causes changes in the cardiovascular system. In addition, inputs from cardiac mechanoreceptors and from a variety of thermoreceptors lead to reflex effects on the cardiovascular system. In mammals, the integration of these sensory inputs occurs in a collection of brain neurons referred to as the medullary cardiovascular center, located at the level of the medulla oblongata and pons. The medullary cardiovascular center also receives inputs from other regions of the brain, including the medullary respiratory center, hypothalamus, amygdala nucleus, and cortex. The output from the medullary cardiovascular center is fed into sympathetic and parasympathetic autonomic motor neurons that innervate the heart and the smooth muscle of arterioles and veins, as well as to other areas of the brain such as the medullary respiratory center. Stimulation of sympathetic nerves increases the rate and force of contraction of the heart and causes vasoconstriction; the result is a marked increase in arterial blood pressure and cardiac output. In general, the reverse effects follow stimulation of parasympathetic nerves, the end result being a drop in arterial blood pressure and cardiac output. The medullary cardiovascular center can be divided

into two functional regions, which have opposing effects on pressure: Stimulation of the pressor center results in sympathetic activation and a rise in blood pressure.

changes

I---------I I

Stimulation of the depressor center results in parasympathetic activation and a drop in blood pressure. In general terms, various sensory inputs affect the balance between pressor and depressor activity: some activate the pressor center and inhibit the depressor center; others have the reverse effect. Thus, the various inputs that converge on the medullary cardiovascular center are modified and integrated. The result is an output that activates the pressor or the depressor center and produces cardiovascular changes in response to changing requirements of the body or disturbances to the cardiovascular system. Figure 12-43presents an overview of this central circulatory control in mammals. Arterial baroreceptors Baroreceptors, which are widely distributed in the arterial system of vertebrates, show increased rates of firing with increases in blood pressure. Unmyelinated baroreceptors have been localized in the central cardiovascular system of amphibia, reptiles, and mammals. These unmyelinated baroreceptors only respond to pressures above normal, initiating reflexes that reduce arterial blood pressure and thus protect the animal from damaging increases in blood pressure. Myelhated baroreceptors, which have been found only in mammals, respond to blood pressures below normal, thus protecting the animal from prolonged periods of

OTHER INPUTS Arterlal blood compos~tlon Lung Inflation Somatic, etc. OTHER CIRCULATORY MECHANORECEPTORS

I I I I I

DISTURBANCE

"set point"

card~ovascular center

EFFECTORS

Cardiac Pulmonary

HEART AND BLOOD VESSELS

..

lntravascular pressures

I

i

I

I-----------

Figure 12-43 The circulatory control system in mammals involves a number of negative-feedback loops. Various receptors monitor changes in the state of the cardiovascular system, sending inputs to medullary cardiovascular center. After integrating these inputs and comparing with the

BARORECEPTOR

arterial set point, this center sends signals via autonomic nerves to maintain an appropriate arterial blood pressure. The arterial set point is altered by inputs from other areas of the brain, which are in turn influenced by a variety of peripheral inputs (dashed lines). [Adapted from Korner, 1971.]

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............................................................................... reduced blood pressure. Baroreceptors in the mammalian carotid sinus have been studied much more extensively than those in the aortic arch or subclavian, common carotid, and pulmonary arteries. In mammals there appear to be only minor quantitative differences between the baroreceptors of the carotid sinus and the aortic arch. Birds have aortic arch baroreceptors. The carotid sinus in mammals is a dilation of the internal carotid at its origin, where the walls are somewhat thinner than in other portions of the artery. Buried in the thin walls of the carotid sinus are finely branched nerve endings that function as baroreceptors. Under normal physiological conditions, there is a resting discharge from these baroreceptors. An increase in blood pressure stretches the wall of the carotid sinus, causing an increase in discharge frequency from the baroreceptors. The relationship between blood pressure and baroreceptor impulse frequency is sigmoidal, the system being most sensitive over the physiological range of blood pressures (Figure 12-44). In addition, the baroreceptor discharge frequency is higher when pressure is pulsatile than when it is constant. The carotid sinus baroreceptors are most sensitive to frequencies of pressure oscillation between 1 and 10 hertz. Since the arterial pressure increases and decreases with each heartbeat, this frequency range is within the normal physiological range of arterial pressure oscillations. Similar observations have been made on the relationship between discharge frequency and pressure for pulmocutaneous arterial baroreceptors in the toad (Figure 12-45).Sympathetic efferent fibers terminate in the arterial wall near the carotid sinus baroreceptors; stimulation of these sympathetic fibers increases discharge of these baroreceptors. Under normal physiological conditions

20 40 60 80 Step increase in pressure (mm Hg)

V

50 100 150 Carotid sinus pressure (mm Hg)

Figure 12-44 The discharge frequency of baroreceptors increases with pressure in a sigmoid fashion. These receptors are most sensitive within the physiological range of pressures and when blood flow is pulsatile. These values were recorded from a multifiber preparation of the carotid sinus nerve and plotted against the mean pressure in the carotid sinus during pulsatile or constant flow. [Adapted from Korner, 1971.I

these efferent neurons may be utilized by the central nervous system (CNS)to control sensitivity of the receptors. Signals from baroreceptors in response to increased blood pressure are relayed through the medullary cardiovascular center to autonomic motor neurons, leading to a reflex reduction in both cardiac output and peripheral vascular resistance (Table 12-1). The reduction in cardiac output results from both a drop in the heart rate and the force of cardiac contraction. The culmination of the various autonomic effects is a decrease in arterial blood pressure. But as the arterial pressure decreases, so does the baroreceptor

Figure 12-45 Baroreceptors are very sensitive to changes in pressure.The effect of step increases in pressure on the discharge frequency of pulmocutaneous baroreceptors in the toad are plotted (A) immediately after the pressure increase and (B) 45 seconds later. Each numbered black line represents one observation corresponding to the pressure increase shown on the horizontalaxis. The rapid initial peak response is greater than the re-

I

I I I I I I I 20 40 60 80 Step increase in pressure (mm Hg)

sponse 45 seconds after the pressure ~ n crease. [Adaptedfrom Van Vilet and West, 1994.1

508


.....................................*...

TABLE 12-1 Reflex effects observed during changes in carotid sinus pressure Carotid sinus pressure* Autonomic effector

Increased

Decreased

Cardiac vagus

++++

-

Cardiac sympathetic

-

+++

Splanchnic b e d Resistance vessels

--

Capacitance vessels

--

Renal b e d

==0

++ ++ +

changes in arterial pressure resulting from peripheral vasoconstriction. Not surprisingly, there are many interactions between the control systems associated with both the respiratory and the cardiovascular system. For example, the discharge pattern from stretch receptors in the lungs has a marked effect on the nature of the cardiovascular changes caused by chemoreceptor stimulation. If the animal is breathing normally, changes in gas levels of the blood will cause one set of reflex changes; if, however, the animal is not breathing, chemoreceptor stimulation results in quite a different series of cardiovascular changes, as we will see in the h e r dscussion on dving.

Muscle b e d Resistance vessels

--

Capacitance vessels

-

Skin Resistance vessels

-

++

Capacitance vessels

?O

0

Adrenal catecholamines

==O

Ant~diuretichormone

?

++ ++

+

*A means increased autonomic effect; a -, decreased autonomic effect; and 0, no autonom~ceffect. Source: Korner, 1971

discharge frequency, causing a reflex increase in both cardiac output and peripheral resistance, which tends to increase arterial pressure. Thus the baroreceptor reflex of the carotid sinus is a negative feedback loop that tends to stabilize arterial blood pressure at a particular set point. The set point may be altered by interaction with other receptor inputs or may be reset centrally within the medullary cardiovascular center by inputs from other regions of the brain (see Figure 12-43). Arterial chemoreceptors Arterial chemoreceptors, which are located in the carotid and aortic bodies, are particularly important in regulating ventilation (see Chapter 13),but they also have some effect on the cardiovascular system. These chemoreceptors respond with an increase in discharge frequency to an increase in CO, or to decreases in 0, and pH of the blood perfusing the carotid and aortic bodies. An increase in discharge frequency results in peripheral vasoconstriction and a slowing of the heart rate if the animal is not breathing (e.g., during submersion). Cardiac output is reduced while birds and mammals dive; peripheral vasoconstriction then ensures the maintenance of arterial blood pressure and therefore brain blood flow in the face of this reduction in cardiac output. Peripheral vasoconstriction can cause a rise in arterial pressure, which then evokes reflex slowing of the heart by stimulation of the systemic baroreceptors. Nevertheless, stimulation of arterial chemoreceptors results in a slowing of the heartbeat even when arterial pressure is regulated at a constant level. Chemoreceptor stimulation thus has a direct effect on heart rate, as well as an indirect effect via

Cardiac receptors Both mechanoreceptiveand chemoreceptiveafferent nerve endings are located in various regions of the heart. Information on the state of the heart collected by these receptors is transmitted via the spinal cord to the medullary cardiovascular center and other regions of the brain. In addition, stimulation of some cardiac receptors causes hormone release either directly from the atria or from other endocrine tissues within the body. Stimulation of cardiac receptors evokes a series of reflex responses including changes in heart rate and cardiac contractility and, under extreme conditions, the pain that can be associated with a heart attack.

Atrial receptors The atrial walls contain many mechanoreceptive afferent fibers, which are classified into three types. Myelinated A-type and B-type afferent fibers are embedded in the atria. A-fiber afferents respond to heart-rate changes and appear to relay information on heart rate to central cardiovascular control centers. B-fiber afferents respond to increases in the rate of filling and volume of the atria. Increases in venous volume result in an increase in venous pressure, which in turn raises the atrial filling and consequently the discharge frequency of the B-fibers. This increased activity is processed by the central cardiovascular centers leading to two major effects, one on the heart and one on the kidney. Stimulation of atrial B-fibers leads to a faster heart rate mediated via increased activity in the sympathetic outflow to the sinus node of the heart. Stimulation of these afferent fibers also causes a marked increase in urine excretion (diuresis),probably mediated by a decrease in antidiuretic hormone (ADH)levels in the blood. Thus, there is a negative feedback loop for regulating blood volume. An increase in blood volume raises venous pressure and atrial filling; this stimulates atrial B-fibers, leading to inhibition of ADH release from the pituitary. The resulting fall in blood ADH levels leads to diuresis and therefore a reduction in blood volume. The third type of atrial mechanoreceptor comprises unmyelinated C-type afferent fibers innervating the junction of the veins and atria. Stimulation of these C-fiber afferents receptors affects both heart rate and blood pressure. If heart rate is low, distension of this region results in an increase in heart rate, whereas if heart rate is high, stimulation results in a fall in heart rate. Stimulation of C-fibers

CIRCULATION

509

...................................... also causes a fall in blood pressure. Both myelinated and unmyelinated sympathetic fibers innervate the atria. Atrial contraction and atrial distension reflexly stimulate these fibers, causing an increase in heart rate. The atrial wall also contains stretch-sensitivesecretory cells that produce atrial natriuretic peptide (ANP). This hormone, which is released into the blood upon stretch of these cells, has several endocrine effects. As its name indicates, ANP causes an increase in urine production and sodium excretion, thereby effectively reducing blood volume and therefore blood pressure. ANP inhibits release of renin by the kidney and production of aldosterone by the adrenal cortex. It thus diminishes the renin-angiotensinaldosterone system, which stimulates sodium resorption and an increase in blood volume (see Chapter 14).In addition to these actions, ANP inhibits release of ADH and acts directly on the kidney to increase water and sodium excretion. ANP has been demonstrated to have a depressor effect, reducing both cardiac output and blood pressure. In addition ANP antagonizes the pressor effect of angiotensin. Atrial natriuretic peptide belongs to a family of natriuretic peptides (A-, B-, C-, and V-type natriuretic peptide) sharing a common 17-amino-acid ring structure linked by a disulfide bridge. Since the initial investigations of ANP in the early 1980s, natriuretic peptides have been found in a wide variety of tissues, including the central nervous system. In many instances they may have an autocrine or paracrine function. For example, receptors for natriuretic hormone have been located in both the atria and ventricles of the hearts of several vertebrates. Binding of locally released natriuretic hormone to these receptors may reduce contractility, indicating a paracrine function within the heart.

Ventricular receptors The endings of both myelinated and unmyelinated sensory afferent fibers are embedded in the ventricle. The myelinated fibers are mechanoreceptive and chemoreceptive, with separate endings for each modality. The mechanoreceptive endings are stimulated by interruption of coronary blood flow. The chemoreceptive endings are stimulated by substances like bradykinin. At low stimulation levels, these fibers cause increased sympathetic outflow and decreased vagal outflow to the heart, raising cardiac contractility as well as blood pressure. At higher stimulation levels, these fibers are necessary for the perception of pain in the heart. Myelinated afferent fibers are much less numerous than unmyelinated C-fiber afferent endings in the left ventricle. Stimulation of the C-fiber af-

ferents at low levels causes peripheral vasodilation and a reduction in heart rate. Increased stimulation of these fibers causes stomach relaxation and, at even higher frequencies, results in vomiting. Skeletal muscle afferent fibers Somewhat surprisingly, most nerves innervating skeletal muscle contain more afferent fibers than efferent fibers. The afferent fibers can be subdivided into four broad groups. Groups I and I1 are sensory fibers from muscle spindles and Golgi tendon organs; these seem to play little or no role in the control of the cardiovascular system. In contrast, stimulation of group 111 fibers, which are myelinated "free nerve endings," or group IV fibers, which are unmyelinated sensory endings, appears to have cardiovascular effects. These fibers are activated by either mechanical or chemical stimulation, with most fibers responding to only one modality. Mechanical stimulation may be due to contraction, squeezing, or stretching of the muscle. The changes in the extracellular fluid associated with muscle contraction also are thought to stimulate chemoreceptive muscle afferent fibers and evoke cardiovascular changes. Large changes in pH and osmotic pressure raise the activity of group IV fibers, but it is not clear if the pH or osmotic changes occurring in vivo are adequate to mediate cardiovascular effects. Electrical stimulation of muscle afferents can result in either an increase or a decrease in arterial blood pressure, depending on the fibers being stimulated or the frequency of stimulation of a particular group of afferent nerves. At low frequencies, stimulation of some afferent fibers results in a fall in arterial blood pressure, whereas stimulation of the same fibers at high frequencies results in a rise in blood pressure. Electrical stimulation of afferent nerves from muscles usually causes a change in heart rate in the same direction as thk change in blood pressure; that is, if blood pressure is elevated, so is heart rate, and vice versa. In those instances where electrical stimulation of muscle afferents causes an increase in heart rate and cardiac output, there is also a change in the distribution of blood in the body. Blood flow to the skin, kidney, gut, and inactive muscle is reduced, thus augmenting flow to the active muscles. The cardiovascular response evoked by muscle contraction has been shown to disappear following dorsal root section, so the response is presumably reflex in origin, resulting from stimulation of afferent fibers in the muscle. The response varies depending on whether muscle contraction is isometric (static exercise) or isotonic (dynamic exercise). Static exercise is associated with an increase in arterial blood pressure with little change in cardiac output, whereas dynamic exercise results in a large increase in cardiac output with little change in arterial blood pressure. The sensory inputs from muscle afferent fibers are processed in the central cardiovascular center, leading to stimulation of the autonomic nerves innervating the heart and vessels, the efferent arm of the reflex arcs.

510 INTEGRATION OF PHYSIOLOGICAL SYSTEMS ......................................... Control of the Microcirculation

Capillary blood flow adjusts to meet the demands of the tissues. If the requirements change suddenly, as in skeletal muscle during exercise, then capillary flow also changes. If requirements for nutrients vary little with time, as in the brain, then capillary flow also varies little. The regulation of capillary flow can be divided into two main types, nervous control and local control. Nervous control of capillary blood flow Nervous control serves to maintain arterial pressure by adjusting resistance to blood flow in the peripheral circulation. The vertebrate brain and heart must be perfused with blood at all times. An interruption in the perfusion of the human brain rapidly results in damage. Nervous control of arterioles ensures that only a limited number of capillaries will be open at any moment, for if all capillaries were open, there would be a rapid drop in arterial pressure and blood flow to the brain would be reduced. The nervous control of capillary flow operates under a priority system. If arterial pressure falls, blood flow to the gut, liver, and muscles is reduced to maintain flow to the brain and heart. Most arterioles are innervated by sympathetic nerves, which release norepinephrine at their endings. Some arterioles, however, are innervated by parasympathetic nerves, which release acetylcholine at their endings, Sympathetic stimulation and circulating catecholamines Binding of the catecholamine norepinephrine to a-adrenoreceptors in the smooth muscle of arterioles usually causes vasoconstriction and therefore a decrease in diameter of the arterioles. This decrease in diameter causes an increase in resistance to flow, thus reducing blood flow through that capillary bed. The generalized effect of sympathetic stimulation is peripheral vasoconstriction and subsequent rise in arterial blood pressure. This overall response is mediated by binding of norepinephrine from nerve endings to a-adrenoreceptors in vascular smooth muscle, resulting in an increase in smooth-muscle tension. Stimulation of p-adrenoreceptors in arterial smooth muscle, however, often results in relaxation of the muscle and an increase in diameter of the arterioles (i.e., vasodilation), thereby decreasing the resistance to flow and increasing the blood flow through that capillary bed. Because p-adrenoreceptors are rarely located near nerve endings, they usually are stimulated by circulating catecholamines. Catecholamines are released into the bloodstream from adrenergic neurons of the autonomic nervous system and from chromaffin cells in the adrenal medulla. Circulating catecholamines are dominated by epinephrine released from the adrenal medulla (see Chapter 8). Epinephrine reacts with both a- and p-adrenoreceptors, causing vasoconstriction and vasodilation, respectively. Although a-adrenoreceptors are less sensitive to epinephrine, when activated they override the vasodilation mediated by p-adrenoreceptors. The result is that high levels of circulating epinephrine cause vasoconstriction and thus an increase in peripheral

resistance via a-adrenoreceptor stimulation. At lower levels of circulating epinephrine, however, P-adrenoreceptor stimulation dominates, producing an overall vasodilation and a decrease in peripheral resistance. Even at levels of epinephrine that produce vasodilation, it causes a rise in arterial blood pressure by stimulating p-adrenoreceptors in the heart, causing a marked increase in cardiac output. The p-adrenoreceptors can be divided into two subgroups: PI-adrenoreceptors, which are stimulated by both circulating catecholamines (epinephrine) and adrenergic nerve stimulation (norepinephrine) and 0,-adrenoreceptors, which respond only to circulating catecholamines. In the peripheral circulation, only p,-adrenoreceptors are present, whereas PI-adrenoreceptors are found in the heart and coronary circulation, where both circulating catecholamines and neurally released norepinephrine can have a marked effect. We can summarize these effects as follows: Stimulation of sympathetic nerves generally causes peripheral vasoconstriction and a rise in arterial blood pressure. An increase in circulating catecholamines causes a decrease in peripheral resistance, with a rise in arterial pressure because of concomitant stimulation of the heart and a rise in cardiac output. The response in any vascular bed depends on several things: the type of catecholamine, the nature of the receptors involved, and the relationship between stimulation of the receptors and the change in muscle tone. Although stimulation of a-adrenoreceptors usually is associated with vasoconstriction and that of p-adrenoreceptors with vasodilation, this is not invariably the case. An additional complicating factor is that not all sympathetic fibers are adrenergic. In some instances, they may be cholinergic, releasing acetylcholine from their nerve endings. Stimulation of sympatheticcholinergic nerves causes vasodilation in the vasculature of skeletal muscle. The action of catecholamines is extensively modulated by a variety of substances, including neuropeptide Y and adenosine. Neuropeptide Y, first isolated from porcine brain in 1982, is structurally related to mammalian pancreatic polypeptide and peptide YY. Neuropeptide Y is widespread throughout the animal kingdom and, so far, has been identified in many vertebrates and in insects. Neuropeptide Y is co-localized with norepinephrine in sympathetic ganglia and adrenergic nerves; it also is found in many nonadrenergic fibers. The atrial and ventricular myocardium and the coronary arteries are surrounded by nerve fibers that contain neuropeptide Y. In addition, it appears that myocardial cells themselves can synthesize and secrete neuropeptide Y. In general, neuropeptide Y decreases coronary blood flow and the contraction of cardiac muscle by reducing the level of inositol triphosphate (InsP,), an intracellular second messenger (see Chapter 9).

CIRCULATION

511

...................................... Neuropeptide Y appears to ameliorate those actions of catecholamines on the heart and coronary circulation mediated via InsP, .The role of neuropeptide Y in the peripheral circulation is less well understood, but it appears to ameliorate the increase in blood pressure resulting from norephinephrine-induced peripheral vasoconstriction mediated by a-adrenoreceptors. ATP, as well as neuropeptide Y, is stored and coreleased with catecholamines. ATP and its breakdown product, adenosine, act to inhibit release of catecholamines. Adenosine is released by many tissues during hypoxia but has only a paracrine or autocrine action because of rapid inactivation. Hypoxia tends to promote catecholamine release into the blood from chromaffin tissue, but this action is modulated by the local release of adenosine. Parasympathetic stimulation Arterioles in the circulation to the brain and the lungs are innervated by parasympathetic nerves. These nerves contain cholinergic fibers, which release acetylcholine from their nerve endings when stimulated. In mammals, parasympathetic nerve stimulation causes vasodilation in arterioles. Some parasympathetic neurons release ATP and other purines from their endings. Some of these purinergic neurons may participate in the control of capillary blood flow. ATP, for instance, causes vasodilation.

&

Local control of capillay blood flow Tissues require a basal capillary blood flow to supply nutrients and 0, and to remove waste products. Active tissues have greater requirements and thus capillary blood flow must increase during activity. In addition to nervous control of the central cardiovascular system, various mechanisms control the microcirculation at the local level. For instance, if a vessel is stretched by an increase in input pressure, the vascular smooth muscle responds by contracting, opposing any increase in vessel diameter. This tendency to maintain vessel diameter within narrow limits prevents large changes in resistance to flow and therefore maintains a relatively constant basal flow through the capillary bed. Local heating of a tissue, which may accompany inflammation, is associated with a marked vasodilation, whereas a reduction in temperature causes a vasoconstriction. Thus an ice pack can reduce the blood flow and, therefore, the swelling associated with damage to a tissue. Numerous compounds also influence capillary blood flow within a tissue. These can be grouped into three types: compounds produced by the vascular endothelium; various vasocontrictors and vasodilators released from other cells; and metabolites associated with increased activity. Endothelium-produced compounds The endothelium is not merely a barrier between blood and the underlying tissues, but an active tissue, producing many compounds. Some of these, such as nitric oxide, endothelin, and prostacyclin, affect vascular smooth muscle and, therefore, capillary blood flow.

Nitric oxide is produced and released continuously by the vascular endothelium, causing relaxation of vascular smooth muscle. Nitric oxide-mediated vasodilator tone regulates blood flow and pressure in mammals and perhaps other vertebrates. Observation of endothelium-dependent vascular relaxation led to the discovery of endotheliumderived relaxing factor (EDRF).It is now known that this phenomenon results largely from the generation and release of nitric oxide, which activates guanyl cyclase, leading to an elevation of the intracellular second messenger cGMP (cyclic 3', 5' guanosine monophosphate). This compound in turn mediates muscle relaxation. A family of enzymes, the nitric oxide synthases, oxidize L-arginine to nitric oxide and L-citrulline in the endothelium. Several nitric oxide synthases are calcium dependent, and calcium entry into endothelial cells has been shown to cause the production and release of nitric oxide and relaxation of surrounding smooth muscle. The finding that some calcium channels in the endothelium are stretch sensitive suggests that nitric oxide production in response to vessel stretch may be due to increased calcium entry into the endothelium. A variety of chemicals (e.g., acetylcholine, ATP, and bradykinin) stimulate release of nitric oxide, as does hy; ,xis, pH change, and increased vessel shear stress. Tht .:is evidence of increased production of nitric oxide wit1 'wreasing pressure associated with each heartbeat. h L r i coxide synthases have been found in a wide variety of animals including horseshoe crabs, the bloodsucking bug Rhodnius, lampreys, and man. Nitric oxide has been shown to have many functions other than maintaining vasodilator tone, so its presence in animals without vascular tone or in nonvascular tissues is not surprising. For example, nitric oxide released in the central nervous system by stimulation of N-methyl-D-aspartate receptors is involved in modulation of synaptic activity. Nitric oxide may also be involved in nonspecific defense reactions, the relaxation of nonvascular smooth muscle in the gastrointestinal and genito-urinary tracts, and the regulation of the release of some hormones. In addition, nitric oxide released by endothelial cells, platelets, and leukocytes modulates both cell adhesion and aggregation and inhibits thrombosis. The vascular endothelium releases endothelins and prostacyclin, as well as nitric oxide. Endothelins are small vasoconstrictive proteins, containing 21 amino acid residues. Prostacyclin causes vasodilation and acts as an anticoagulant. It thus functions as an antagonist of the prostaglandin thromboxane A,, which promotes blood clotting and causes vasoconstriction. Inflammatory and other mediators Thromboxane A, is formed in the plasma from arachidonic acid released by platelets when they bind to damaged tissues. Although thromboxane levels increase in a damaged tissue and cause vasoconstriction, local injury in mammals is accompanied by a marked vasodilation of vessels in the region of the damage, due largely to the local release of histamine. Histamine is released, not from endothelial cells,

512

I N T E G R A T I O N OF P H Y S I O L O G I C A L SYSTEMS

......................................

but from some connective tissue and white blood cells in injured tissues. Antihistamines ameliorate, but do not completely remove, this inflammatory response. Another group of potent vasodilators, plasma kinins, also are activated in damaged tissues. Tissue damage results in the release of proteolytic enzymes that split kininogen, an a2globulin, into kinins. Hypoxia also stimulates formation of kinins. Among the vasocontrictors that act on arterioles are norepinephrine released from sympathetic nerves and angiotensin IT. Angiotensin is formed, primarily in the lungs, from angiotensinogen, which circulates in the blood (see Chapter 14).Finally, serotonin acts as a vasoconstrictor or vasodilator, depending on the vascular bed and on the dose level. It is found in high concentration in the gut and blood platelets. Histamine, bradykinin, and serotonin cause an increase in capillary permeability. As a result, large proteins and other macromolecules tend to distribute themselves more evenly between plasma and interstitial spaces, reducing the colloid osmotic pressure difference across the capillary wall. Filtration thus increases, and tissue edema occurs. On the other hand, norepinephrine, angiotensin 11, and vasopressin tend to promote absorption of fluid from the interstitial fluid into the blood. This absorption can be achieved by reducing filtration pressure and/or the permeability of the capillaries.

Although low 0, levels, indicative of tissue activity, cause vasodilation and increased blood flow in systemic capillaries, the lung capillary bed exhibits the opposite behavior. That is, low 0, in the lung causes local vasoconstriction rather than vasodilation. The functional significance of this difference relates to the direction of gas transfer. In the lung capillaries, 0, is taken up by the blood, and thus blood flow should be greatest in the regions of high 0,. In systemic capillaries, however, 0, leaves the blood for delivery to the tissues, and the highest blood flow should be to the area of greatest need, which is indicated by regions of low 0,. If blood flow to an organ is stopped by clamping the artery or by a powerful vasoconstriction, there will be a much higher blood flow to that organ when the occlusion is removed than there was before the occlusion. This phenomenon is termed reactive hyperemia. Presumably during the ischemic period (a period of no blood flow), O2 levels are reduced, and CO,, H+, and other metabolites build up and cause a local vasodilation. The result is that when the occlusion is removed, blood flow is much higher than normal.

CARDIOVASCULAR RESPONSE TO EXTREME CONDITIONS In the previous sections, we've described the general organization of the circulation and its regulation under usual conditions. The cardiovascular system responds in characteristic ways during exercise, diving, and hemorrhage to meet the physiological challenge of these extreme conditions. Exercise

Metabolic conditions associated with activity When activity in a tissue increases, there must be a concomitant increase in blood flow. Local control of capillary flow ensures that the most active tissue has the most dilated vessels and therefore the most blood flow. The degree of dilation depends on local conditions in the tissue, and those conditions associated with high levels of activity generally cause vasodilation. The term hyperemia means increased blood flow to a tissue; ischemia means the cessation of flow. Active hyperemia refers to the increase in blood flow that follows increased activity in a tissue, particularly skeletal muscle. Active tissues, metabolizing aerobically, are marked by a decrease in 0, and an increase in CO, ,H+,various other metabolites (e.g., adenosine, other ATP breakdown products), and heat. Extracellular K+ also rises in skeletal muscle following exercise. All of these activity-related metabolic changes, as well as nitric oxide and prostacyclin, have been shown to cause vasodilation and a local increase in capillary blood flow. That is, the most active tissue has the most dilated vessels and therefore the highest blood flow.

Regulation of the cardiovascular system during exercise is clearly a complex process involving central neural control mechanisms, peripheral neural reflex mechanisms (especially those involving skeletal muscle afferent fibers), and local control. Many cardiovascular changes seen during exercise can occur in the absence of neural mechanisms, indicating the importance of local control systems In increasing blood flow to active skeletal muscles. The central neural control mechanisms and reflexes from muscle afferent mechanoreceptive and chemoreceptive inputs, however, clearly play a role, the exact form varying with the nature of the exercise. For example, the reflex effect on the cardiovascular system of muscle afferent stimulation depends on the nature of the exercise: Isometric contractions of muscles tend to raise blood pressure with little effect on cardiac output. Isotonic contractions raise cardiac output but cause little change in arterial blood pressure. During exercise, blood flow to skeletal muscle is increased in proportion to the level of activity of the muscle. The increase in flow to a muscle may be as much as twenty

times; at the same time, transfer of oxygen from the blood to muscle may increase threefold, resulting in a sixtyfold increase in oxygen utilization by the muscle. Active hyperemia is primarily responsible for increasing blood flow to muscle; the resulting decrease in peripheral resistance leads to an increase in cardiac output mediated by sympathetic nerves. At the same time, there is a reduction in flow to the gut, kidney, and, at high levels of exercise, the skin (Figure 12-46). Cardiac output can increase up to ten times above the resting level owing to large increases in heart rate and small changes in stroke volume. Much of the increase in cardiac output can be accounted for by a decrease in peripheral resistance to about 50% of the resting value and by an increase in venous return to the heart due to both the pumping action of skeletal muscles on veins and the increase in breathing associated with exercise. The increased sympathetic, but decreased parasympathetic, activity in nerves innervating the heart has the effect of increasing both heart rate and the force of contraction, so as to maintain stroke volume at a relatively constant level. In fact, stroke volume increases by about 1.5 times during exercise in mammals, despite the large increase in heart rate and the associated reduced time available for filling and emptying. Following sympathetic stimulation, however, blood is ejected more rapidly from the ventricles with each beat, maintaining stroke volume at higher heart rates. The relative role of changes in stroke volume and heart rate in generating the increase in cardiac output with exercise varies among animals. In fish, for example, the changes in stroke volume are much greater than the changes in heart rate, whereas in birds there are very large Skin Heart, brain, etc. Viscera

Max

Vo,

0,uptake (L . r n i n - I ) Figure 12-46 During exercise, total cardiac output increases and blood flow shifts to the active muscles. Shown is the approximate distribution of cardiac output at rest and at different levels of exercise up to the maximal oxygen consumption (Max Vo2)in a normal young man. The progressive reduction in the absolute blood flow and percentage of cardiac output distributed to the viscera (splanchnicregion and kidneys) augments muscle blood flow. Even skin is constricted during brief periods of exercise at high oxygen consumption. [Adapted from Rowell, 1974.1

changes in heart rate and little change in stroke volume during exercise. Exercise is associated with only small changes in arterial blood pressure, pH, and gas tensions. The oscillations in PCO2and POI with breathing are somewhat larger, as is the arterial pressure pulse. The increased pressure pulse is dampened to some extent because of increased elasticity of the arterial walls due to a rise in circulating catecholamines. It is probable that arterial chemoreceptors and baroreceptors play only a minor role in the cardiovascular changes associated with exercise. Motor neurons that innervate skeletal muscle are activated by higher brain centers in the cortex at the onset of exercise (see Chapter 10); it is possible that this activating system also initiates changes in lung ventilation and blood flow. Proprioreceptive feedback from muscles may also play a role in increasing lung ventilation and cardiac output (see Chapter 13). A number of other changes augment gas transfer during exercise; for example, red blood cells are released from the spleen in many animals, increasing the oxygen carrying capacity of the blood. Thus, exercise is responsible for a complex series of integrated changes that lead to delivery of adequate oxygen and nutrients to the exercising muscle.

Diving

Many air-breathing vertebrates can remain submerged for prolonged periods. During submersion for any period, all air-breathing vertebrates stop breathing, so the animal must rely on available oxygen stores in the blood (see Chapter 13). The cardiovascular system is adjusted to meter out the limited oxygen store to those organs-brain, heart, and some endocrine structures-that can least withstand anoxia. Much of the information on the responses to submersion has been collected from studies of animals forced to dive, sometimes simply by holding an animal's head under water. Because naturally occurring dives vary considerably in depth, duration, and exercise level, information obtained on forced dives is not always directly applicable to natural dives. Whales and dolphins spend their lives in the water going to the surface to breathe, whereas seals may spend considerable time on land out of water. Other animals may spend most of their time on land and dive only occasionally. Oxygen stores vary in animals, so metabolism may be completely aerobic during some dives but largely anaerobic during others. Figure 12-47 illustrates the typical cardiovascular changes that occur when a seal dives and remains submerged. In mammals, but not in other vertebrates, stimulation of the facial receptors that inhibit breathing cause a marked bradycardia. Although the initial pressurization of

514


......................................

I

Predive

+ Dive 4

Recovery

oxygen content I

I

Time

--+

Figure 12-47 The cardiovascular system undergoes numerous adjustments when a seal dives. Heart rate, cardiac output, and blood O2content decrease during a dive, but blood CO, content increases. During the recovery period after a d~ve,blood lactate increases greatly; the other parameters first overshoot and then gradually return t o predive values.

the lung can lead to a transient increase in blood O2 and CO, levels, the continued utilization of 0, during the dive results in a gradual fall in blood 0, and rise in blood CO, levels. This fall in blood 0, stimulates the arterial chemoreceptors and, in the absence of lung stretch-receptor activity, causes peripheral vasoconstriction and a reduction in heart rate and cardiac output; thus blood flow to many tissues is reduced so as to maintain flow to the brain, heart, and some endocrine organs. The absence of lung stretch-receptor activity is due to the absence of breathing and the compression of the lung as the animal descends in the water column. The increase in peripheral resistance results from a marked rise in sympathetic output and involves constriction of fairly large arteries. Reductions in blood flow to the kidney have been recorded in Weddell seals during a dive. In some instances, blood flow to muscle decreases, but this depends on the level of exercise associated with the dive and the species. Sometimes arterial pressure rises during a dive, causing stimulation of arterial baroreceptors; in such dives bradycardia is maintained by a rise in both chemoreceptor and baroreceptor discharge frequency. The bradycardia is caused by an increase in parasympathetic and, to some extent, a decrease in sympathetic activity in fibers innervating the heart. It has been shown in the seal that the generation of the diving bradycardia can involve some form of associative learning. In some trained seals, bradycardia occurs before the onset of the dive and, therefore, before the stimulation of any peripheral receptors. This psychogenic influence on heart rate can have a marked effect on the change in heart rate during a dive in many animals. In general, if heart rate

is low before a dive, there may be little or no change in heart rate during the dive. If the heart rate is high, then there may be a marked bradycardia due to wetting the face and a decrease in lung stretch-receptor activity. The "water" receptors present in birds are not directly involved in the cardiovascularchanges associated with submersion. A decrease in heart rate is not observed either in submerged ducks breathing air through a tracheal cannula or in submerged ducks following carotid body denervation (Figure 12-48).Thus, activation of the "water" receptors causes suspension of breathing (apnea); the subsequent drop in blood Pol and pH and the rise in PCOlresult in stimulation of chemoreceptors, which then reflexly cause the cardiovascular changes. Stimulation of lung stretch receptors in mammals modifies the reflex response initiated by chemoreceptor stimulation. In the absence of breathing, and hence stimulation of lung stretch receptors, different reflex responses are elicited by chemoreceptor stimulation than when the animal is breathing. In the absence of breathing, lung inflation tends to suppress the reflex cardiac inhibition and peripheral vasoconstriction caused by stimulation of arterial chemoreceptors. As a submerged animal rises in the water column, the lung becomes inflated, possibly activating stretch receptors in the lung and causing cardiac acceleration. When the animal is breathing, stimulation of arterial chemoreceptors results in a marked increase in lung ventilation. In this case, low blood 0, andlor high blood CO, levels cause peripheral vasodilation. This vasodilation leads to an increase in cardiac output to maintain arterial pressure in the face of increased peripheral blood flow. Thus, the hypoxia (low oxygen) caused by cessation of breathing during a dive is associated with bradycardia and a reduc-

A

Control Out

In

B

Rate

After carotid body denervation

In

G 120 I E 80E 40kom0

-

4 -kk

Out

4

h > v.vr

E'

4-

.-

F2

L

-

/-300Rate -

$ I 0

Figure 12-48 The usual decrease in heart rate (bradycardia) that occurs in submerged ducks depends on an intact carotid body innervation. Tracings show heart rate and oxygen tension (PO2)in the brachiocephalic artery during a period of submergence of the head in water indicated by the in and out arrows. (A) Control six-week-old duckwith all nerves intact. (B) The same duck three weeks after denervation of the carotid bodies. [Jones and Purves, 1970a.l

CIRCULATION

515

...................................... tion in cardiac output. In contrast, hypoxia that occurs when the animal is breathing (e.g., at high altitude) is associated with an increase in heart rate and carchac output. Hemorrhage Normally stimulation of arterial and atrial baroreceptors inhibits vasopressin release, as well as sympathetic outflow to the peripheral circulation. Hemorrhage reduces both venous and arterial blood pressure, reducing the discharge frequency of both atrial and arterial baroreceptors. This releases the baroreceptive inhibition of sympathetic outflow causing constriction of both arteries (vasoconstriction)and veins (venoconstriction),and an increase in cardiac output. The peripheral vasoconstriction and increased cardiac output raises arterial blood pressure, while the venoconstriction maintains venous return to the heart. Hemorrhage-induced reduction in baroreceptor inhibition also promotes vasopressin release. In addition, there is an increase in renin/angiotensin/aldosterone activity, resulting from the fall in blood pressure and the associated decreased renal blood flow. Both vasopressin and aldosterone reduce urine formation, thereby conserving plasma volume. There is a marked stimulation of thirst and this helps to restore plasma volume. The reduced renal blood flow promotes kidney production of erythropoietin, which stimulates red blood cell production by the bone marrow. Thus lost red blood cells are replaced by increased production in the days (week)following the hemorrhage. The liver is also stimulated to increase the production of plasma proteins. The increase in production of erythrocytes and plasma proteins, along with the reduction in urine production and increased drinking rate, restores the blood to its original state.

SUMMARY

,

Circulatory systems can be divided into two broad categories-those with open and those with closed circulations. In open circulatory systems, transmural pressures are low, and blood pumped by the heart empties into a space in which blood bathes the cells directly. In closed circulatory systems, blood passes via capillaries from the arterial to the venous circulation. Transmural pressures are high, and fluid that has slowly leaked across capillary walls into the extracellular spaces is subsequently returned to the circulation via a lymphatic system. The heart is a muscular pump that ejects blood into the arterial system. Excitation of the heart is initiated in a pacemaker, and the pattern of excitation of the rest of the muscle mass is determined by the nature of the contact between cells. The junctions between muscle fibers in the heart are of low resistance and allow the transfer of electrical activity from one cell to the next. The initial phase of each heart contraction is isometric; this is followed by an isotonic phase in which blood is ejected into the arterial system. Cardiac output is dependent on venous &ow, and in mammals, changes in cardiac

output are associated with changes in heart rate rather than in stroke volume. Blood flow is generally streamlined (continuous laminar), but because the relationship between pressure and flow is complex, Poiseuille's law applies only to flow in smaller arteries and arterioles. The arterial system acts as a pressure reservoir and a conduit for blood between the heart and capillaries. The elastic arteries dampen oscillations in pressure and flow caused by contractions of the heart, and the muscular arterioles control the distribution of blood to the capillaries. The venous system acts both as a conduit for blood between capillaries and the heart and as a blood reservoir. In mammals, 50% of the total blood volume is contained in veins. Capillaries are the site of transfer of material between the blood and tissues. Only 30% -50% of all capillaries are open to blood flow at any particular time, but no capillary remains closed for long, because they all open and close continuously. Capillary blood flow is controlled by nerves that innervate smooth muscle around arterioles. Changes in the composition of blood and extracellular fluid in the region of a capillary bed cause the vessels either to constrict or to dilate, thereby altering blood flow. The walls of capillaries are generally an order of magnitude more permeable than other cell layers. Material is transferred between blood and tissues by passing either through or between the endothelial cells that form the capillary wall. Endothelial cells contain large numbers of vesicles that may coalesce to form channels for the movement of material through the cell. In addition, some endothelial cells have specific carrier mechanisms for transferring glucose and amino acids. The size of the gaps between cells varies between capillary beds; brain capillaries have tight junctions, whereas liver capillaries have large gaps between cells. Arterial pressure is regulated via central control mechanisms to maintain capillary blood flow, which can be further adjusted locally to meet the requirementsof particular tissues. Arterial baroreceptors monitor blood pressure and reflexly a1 ter cardiac output and peripheral resistance to maintain arterial pressure. Atrial and ventricular mechanoreceptors monitor venous pressure and derivatives of the heart contraction to ensure that activity of the heart is correlated to blood inflow from the venous system and blood outflow into the arterial system. Arterial chemoreceptors respond to changes in the pH and gas levels of the blood. All these sensory receptors feed information into the medullary cardiovascular center, where the inputs are integrated to ensure an appropriate response of the circulatory system to changing requirements of the animal, as during exercise. Natriuretic peptides, vasopressin, and the renin-angiotensin-aldosterone system operate in conjunction with neural reflexes to maintain blood volume following a drink or following hemorrhage. In general, stimulation of sympathetic nerves innervating vascular smooth muscle causes peripheral vasoconstriction and a rise in arterial blood pressure, whereas an increase in circulating catecholamines (especially epinephrine) causes a decrease in peripheral resistance accompanied

516


.........................................

by a rise in arterial pressure due to a concomitant rise in cardiac output. The vascular endothelium releases various compounds (e.g., nitric oxide, endothelin, and prostacyclin) that cause localized vasoconstriction or vasodilation, thereby adjusting blood flow to tissue needs. Inflammatory mediators, including histamine and kinins, act to increase blood flow to sites of tissue injury. Finally, as aerobic metabolism in a tissue increases, there is a local increase in capillary blood flow, termed active hyperemia. This assures that the most active tissues normally have the highest capillary blood flow.

REVIEW QUESTIONS

9.

10. 11. 12.

13.

14. 15.

Describe the properties of myogenic pacemakers. Describe the transmission of excitation over the mammalian heart. Describe the changes in pressure and flow during a single beat of the mammalian heart. Discuss the factors that influence stroke volume of the heart. What is the nature and function of the nervous innervation of the mammalian heart? What is the effect on cardiac function of a rigid versus a compliant pericardium? What is the functional significance of a partially divided ventricle in some reptiles? Discuss the changes in circulation that occur at birth in the mammalian fetus. Discuss the applicability of Poiseuille's equation to the relationship between pressure and flow in the circulation. What are the functions served by the arterial system? Describe the factors that determine capillary blood flow. Describe the location of various baroreceptors and/ or mechanoreceptors in the mammalian circulatory system and their role in cardiovascular regulation. Compare and contrast the cardiovascular responses to breathing air low in oxygen with those associated with diving in mammals. Describe the cardiovascular changes associated with exercise in mammals. What are the consequences of raising or lowering arterial blood pressure for cardiac function and for exchange across capillary walls?

16. Discuss the relationship between capillary structure and organ function, comparing that found in different organs of the body. 17. Describe the ways in which substances are transferred between blood and tissues across capillary walls. 18. What are the functions served by the venous system? 19. Describe the effects of gravity on blood circulation in a terrestrial mammal. How are these effects altered if the animal is in water? 20. Define Laplace's law. Discuss the law in the context of the structure of the cardiovascular system. 21. Discuss the role of the lymphatic system in fluid circulation. Discuss how and why this role may vary in different parts of the body.

SUGGESTED READINGS Bundgaard, M. 1980. Transport pathways in capillaries: in search of pores. Ann. Rev. Physiol. 42:325-326. Crone, C. 1980. Ariadne's thread: an autobiographical essay on capillary permeability. Microvasc. Res. 20:133-149. Heislel; N., ed. 1995. Mechanisms of Systemic Regulation: Respiration and Circulation. Adu. Comp. Environ. Physiol., Vol. 21. Hoar, W. S., D. J. Randall, and A. P. Farrell, eds. 1992. Fish Physiology: Vol XIIIA & B. New York: Academic Press. Johansen, K., and W. Burggren, eds. 1985. Cardiovascular Shunts. (Alfred Benzon Symposium 21.) Copenhagen: Munksgaard. Kooyman, G. L. 1989. Diverse Divers. Zoophysiology. Vol. 23. New York: Springer-Verlag. Kuby, J. 1997. Leukocyte migration and inflammation. In Immunology, 3d ed. New York: W. H. Freeman. Lewis, D. H., ed. 1979. Lymph circulation. Acta Physiol. Scand., Suppl. 463. Radomski, M. W., and E. Salas. 1995. Biological significance of nitric oxide. 4th Int. Congress. Comp. Physiol. Biochem. Physiol. Zool. 68:33-36. Schmidt-Nielsen, K. 1972. How Animals Work. New York: Cambridge University Press. Van Vilet, B. N., and N. H. West. 1994. Phylogenetic trends in the baroreceptor control of arterial blood pressure. Physiol. Zool.67(6):1284-1304.

0

nly 200 years ago Antoine Lavoisier showed that animals utilize oxygen and produce carbon dioxide and heat (Spotlight 13-1).This process was later shown to take place at the level of the mitochondria (see Chapter 3). Animals obtain oxygen from the environment, using it for cellular respiration. The carbon dioxide generated is eventually liberated into the environment. For cellular respiration to proceed, there must be a steady supply of oxygen, and the waste product carbon dioxide must be continually removed. If carbon dioxide accumulates in the body, pH falls and the animal dies. Although the transport of oxygen and carbon dioxide occur in opposite directions, both processes have many elements in common. If gas transport is impaired, animals die due to lack of oxygen rather than accumulation of carbon dioxide, because oxygen is required for metabolism to continue and carbon dioxide is the product of aerobic metabolism. Air contains about 21% oxygen, but almost no carbon dioxide, the remainder being mostly nitrogen. Carbon dioxide added to the environment by animals is removed by photosynthetic bacteria, plants, and algae, which produce oxygen. This cycling of 0, and CO, is part of the vast interdependencythat exists between plants and animals. In this chapter we review the transport of 0, and C 0 2in the blood and the systems that have evolved in animals to facilitate the movement of these two gases both between the environment and the blood and between the blood and the tissues. The main focus is on systems found in vertebrates, particularly mammals, because these have been investigated most thoroughly. A number of systems that transport 0, between the environment and tissues are of particular interest, including the one that moves oxygen into the swimbladder of fish against gradients that can be several atmospheres. This is described at the end of this chapter as an example of one the many intriguing problems of gas transfer in animals.

GENERAL CONSIDERATIONS Oxygen and carbon dioxide are transferred passively from the environment across the body surface (i.e., skin or special respiratory epithelium) by diffusion. Relevant physical

laws regarding the behavior of gases, along with some of the terminology used in respiratory physiology, are reviewed in Spotlight 13-2. To facilitate the rate of gas transfer for a given concentration difference, the surface area of the respiratory epithelium should be as large as possible and diffusion distances as small as possible. The 0, requirements and C 0 2production of an animal increase as a function of mass, but the rate of gas transfer across the body surface is related primarily to surface area. The surface area of a sphere increases as the square of its diameter, whereas the volume increases as the cube of its diameter. In very small animals the distances for diffusion are small, and the ratio of surface area to volume is large. For this reason, diffusion alone is sufficient for the transfer of gases in small animals, such as rotifers and protozoa, which are less than 0.5 mm in diameter. Increases in size result in increases in diffusion distances and reductions in the ratio of surface area to volume. Large surface-area-tovolume ratios are maintained in larger animals by the elaboration of special areas for the exchange of gases. In some animals the whole body surface participates in gas transfer, but in large, active animals there is a specialized respiratory surface. This surface is made up of a thin layer of cells, the respiratory epithelium, which is 0.5 to 15 pm thick. This surface comprises the major portion of the total body surface. In humans, for instance, the respiratory surface area of the lung is between 50 and 100m2, varying with age and lung inflation; the area of the rest of the body surface is less than 2 m2. Gas transfer between the environment and eggs, embryos, many larvae, and even some adult amphibians occurs by simple diffusion. Boundary layers of fluid low in oxygen and high in carbon dioxide are found whenever gas transfer occurs by diffusion alone. The thickness of this hypoxic (low-oxygen) layer increases with animal size, oxygen uptake, and decreasing temperktures. Stagnation of the medium close to the gas-exchange surface is avoided, in most animals, by the movement of air or water by breathing. A circulatory system has evolved in larger animals to transfer oxygen and carbon dioxide by the flow

518


....................................... SPOTLIGHT 13-1

EARLY EXPERIMENTS O N GAS EXCHANGE I N ANIMALS

was burned. According to this theory, coal contained a great deal of phlogiston which was released into the air during combustion leaving behind ash. That is, when substances were burned they lost phlogiston and, therefore, lost weight. Antoine Lavoisier (1743-1 794), however, found that phosphorus gained weight when burned in air and that some other substances, when heated in air, gained weight but did not do so if heated in a vacuum. In other words, something in air was con-

Poul Astrup (1915- ) and John Severinghaus (1922- ), two prominent scientists in the field of gas exchange, described many of the most significant experiments leading to our present

sumed when some substances were heated. This was the end of the phlogiston theory. Lavoisier called the substance that was

understanding of gas transfer in animals in their book The History of Blood Gases, Acids and Bases published in 1986. Studies

consumed during burning and was required to keep animals alive oxygen, from Greek words meaning "to form acid."

of gas exchange in animals began as an extension of Robert Boyle's (1627-1 691) work in the 17th century on the properties of

Lavoisier repeated some experiments of Henry Cavendish (1731-1 810), who found that the inflammable gas evolved when

air. He showed that both animals and flames died in a vacuum,

metals are added to acid can combine with oxygen to form water. Lavoisier named this gas hydrogen, from Greekwords mean-

indicating that something in air was required both to maintain life and to keep a candle burning.

ing "to form water." He also repeated and extended some of

Joseph Priestley (1733-1804), who lived near a brewery, was

Priestley's experiments and found that if mercuric oxide was

fascinated by the large volumes of gas produced during the

heated with coal, fixedair(carbon dioxide) wasformed. Fixed air

brewing process. Continuing Boyle's experiments in modified form, Priestly heated various chemicals, collected the gases pro-

had been described earlier by Joseph Black (1728-1799), who produced it by adding acid to chalk.

duced over water or mercury, and then determined if mice could live in the gases. He noticed that a mouse lived longer and the

Expired air was known to contain some fixed air, and Lavoisier made the next large step. He realized that both burn-

flame burned brighter in the gas produced by heating mercuric

ing coal and animals consumed oxygen and produced heat and carbon dioxide. He then measured oxygen uptake and heat pro-

oxide than in the gas produced from other chemicals. He also observed that mice lived longer if plant material was present in their containers. Priestley's observations caused Benjamin

duction in animals and found that the amount of heat produced

Franklin to remark that the practice of cutting down trees near

burning coal, although the rates of these processes were much slower in animals.

houses should cease as plants were able to restore air, which is spoiled by animals. Thus Priestley demonstrated that plants, as

relative to oxygen uptake was about the same for animals and

well as certain chemicals when heated, could produce some gas

Lavoisier was also a tax collector. Such people are generally not held in high repute and this brilliant scientist was no excep-

that keeps animals and flames alive. He thought this gas could

tion: he was sent to the guillotine in 1794.

absorb phlogiston, something that was released when material

of blood between the tissues and the respiratory epithelium. Blood flows through an extensive capillary network and is spread in a thin film just beneath the respiratory surface, thereby reducing diffusion distances required to distribute the contained gases. The gases are transported between the respiratory surface and the tissues by bulk flow of blood in the circulatory system. Gases diffuse between blood and tissues across the capillary wall. Once again, to facilitate gas transfer, the area for diffusion is large, and the diffusion distance between any cell and the nearest capillary is small. Graham's law states that the rate of diffusion of a substance along a given gradient is inversely proportional to the square root of its molecular weight (or density). Because oxygen and carbon dioxide molecules are of similar size, they diffuse at similar rates in air; they are also utilized (02 ) and produced ( C 0 2) at approximately the same rates by animals. It can therefore be expected that a transfer system that meets the oxygen requirements of an animal will also ensure adequate rates of carbon dioxide removal.

- Figure 13-1 schematically illustrates the components of the gas-transfer system in many animals, which involves four basic steps:

1. Breathing movements, which assure a continual supply of air or water to the respiratory surface (e.g., lungs or gills) 2. Diffusion of 0, and C 0 2 across the respiratory epithelium 3. Bulk transport of gases by the blood 4. Diffusion of 0, and C 0 2 across capillary walls between blood and mitochondria in tissue cells The capacity of each of these steps is matched because natural selection tends to eliminate metabolically costly unutilized capacities. This matching of capacities in a chain of linked events has been referred to as symmorphosis.Presumably the capacities of elements in a chain will be determined by the capacity of the rate-limiting step. Capacities in a chain of events, however; are not always matched, and symmorphosis draws

GAS E X C H A N G E A N D A C I D - B A S E B A L A N C E

519

............................................................................. Diffusion I

I

I I

I I

Diffusion

1

I

:

Figure 13-1 The gas-transport system of a vertebrate consists of two pumps and two diffusion barriers alternatinq - in series between the external environment and the tissues. [Adapted from Rahn, 1967.1

Air or water

Fluid Pump

Blood Pump

attention to these apparently uneconomic design features. One explanationfor over- or under-capacity is that a single element can be a link in several chains; thus its capacity may be appropriate for one chain of events, but be in excess for another, explaining the apparent excess capacity. The rate of flux of gases varies enormously among animals, from 0.08 ml.g-l-h-l in an earth worm to 40 ml .g - l hpl in a hovering hummingbird. The concentrations of aerobic enzymes (e.g., cpochrome oxidase) and cristae area per mitochondrion both increase with metabolic rate. The hummingbird and some insects, however, may have reached the upper limit for rates of oxygen utilization by animals. Clearly mitochondrial volume and density in muscles cannot be increased indefinitely without compromising the capacity of the muscles to contract; that is, there must be some relationship between structures that supply energy (mitochondria) and structures that use energy (myofilaments).The space occupied by mitochondria never exceeds 45% of the total volume in muscle, even in mammals, birds, and insects, the animals with the highest oxygen uptakes. There must also be limits to mitochondrial design in terms of the number of cristae per unit mitochondria1volume, the ultimate miniaturization being determined by the minimum volume required by the enzymes involved in energy production. It would seem that hummingbirds, and perhaps some other small mammals and a few insects, may have approached these limits of design determining the maximum rates of oxygen uptake. Insects are usually much smaller than the smallest birds and mammals. Some large insects seem to have been displaced by small birds, rather like the monoplane displacing the biplane before World War 11. Vertebrate mini'aturization may be limited by the nature of their gas-transfer systems. Insects have a tracheal system that exchanges gases directly between the medium and the tissues, permitting high rates of oxygen uptake in very small animals.

OXYGEN AND CARBON DIOXIDE IN BLOOD In considering the movement of oxygen and carbon dioxide between the environment and the cells, we will first discuss how these gases are transported in the blood, rather than starting with either the environment or the cell. We take this approach because the mechanisms by which oxygen and carbon dioxide are carried in the blood affects their transfer between the environment and the blood and the blood and the tissues. Respiratory Pigments

Once oxygen diffuses across the respiratory epithelium into the blood, it combines with a respiratory pigment that gives the characteristic color to the blood. The best known respiratory pigment, hemoglobin, is red. By binding oxygen, the respiratory pigment increases the 0, content of blood. In the absence of a respiratory pigment, the 0, content of blood would be low. The Bunsen solubility coefficient of oxygen in blood at 37°C is 2.4 m l 0 , per 100 ml of blood per atmosphere of oxygen pressure. Therefore, the concentration of 0, in physical solution (i.e., not bound to a respiratory pigment) in human blood at a normal arterial Po2 will be only 0.3 m l 0 , per 100 ml blood, or 0.3 vol % 0,. In fact, the total 0, content of human arterial blood at a normal arterial Po, is 20 vol %. The 70-fold increase in content is due to the combination of oxygen with hemoglobin. In most animals using hemoglobin as a respiratory pigment, the 0, content in physical solution is only a small fraction of the total 0, content of the blood. The Antarctic icefish is an exception among the vertebrates; the blood of this fish lacks a respiratory pigment and therefore has a low 0, content. It compensates for the absence of hemoglobin with an increased blood volume and cardiac output, but its rate of 0, uptake is reduced compared with that of species from the same habitat that have hemoglobin. Low temperatures probably are a factor in the evolution of fishes lacking hemoglobin. Low temperatures are associated with low metabolic rates in poikilotherms, and oxygen, like all gases, has a higher solubility at low temperatures. Respiratory pigments are complexes of proteins and metallic ions, and each one has a characteristic color. The

520


............................................................................... SPOTLIGHT 13-2

water, but as temperature decreases, water will condense, and thls condensation WIII also reduce the explred gas volume Ifthe

THE GAS LAWS

barometric pressure IS 760 mm ~ gand , the water vapor pressure

Over 300 years ago, Robert Boyle determined that at a glven temperature the product of pressure and volume IS constant for a glven number of molecules of gas Gay-Lussac's law states that elther the pressure or the volume of a gas IS d~rectlyproportlonal to absolute temperature ~fthe other IS held constant

at 37" and 20°C is 47 mm Hg and 17 mm Hg , respectively, then a measured gas volume at 20°C of 500 ml IS converted to BTPS explred volume as follows

500 ml X

(760 - 17) (760 - 47)

(273 (273

+ 37) + 20)

=

551 ml

Combined, these laws are expressed In the equat~onof state Thus, under the conditions stated above, a gas volume of 551 ml

for a gas:

within the lung is reduced to 500 ml following exhalation because of the drop in gas temperature and the condensation of water. where P is pressure, Vis volume, n is number of molecules of a gas, R is the universal gas constant (0.08205 L.atm. K-I. mot-', or 1.987 cal . K-' . mol-I), and

Dalton's law of partial pressure states that the partial pressure of each gas in a mixture is independent of other gases present, so that the total pressure equals the sum of the partial pres-

Kis absolute temperature. For accurate use, the equation should

sures of all gases present. The partial pressure of a gas in a

be modified by using van der Waals constants.

mixture will depend on the number of molecules present in a

or 8.314 x

lo7ergs. O K - ' .mol-',

The equation of state for a gas indicates that equal volumes

given volume at a given temperature. Usually, oxygen accounts

of different gases atthe same temperature and pressure contain

for 20.94% of all gas molecules present in dry air; thus, if the to-

equal numbers of molecules (Avogadro's law). One mole of gas occupies approximately 22.414 liters at O°C and 760 mm Hg. Be-

tal pressure is 760 mm Hg, the partial pressure of oxygen, Po*, will be 760 x 0.2094 = 159 mm Hg. But air usually contains wa-

cause the number of molecules per unit volume is dependent on

ter vapor, which contributes to the total pressure. If the air is 50%

pressure and temperature, the conditions should always be

saturated with water vapor at 22"C, the water vapor pressure is

stated along with the volume of gas. Gas volumes in physiology

18 mm Hg. If the total pressure is 760, the partial pressure of oxy-

are usually reported as being at body temperature, atmospheric

gen will be (760 - 18) x 0.2094 = 155 mm Hg. If the partial

pressure, and saturated with water vapor (BTPS);at ambient temperature and pressure, saturated with water vapor (ATPS); or at

pressure of CO, in a gas mixture is 7.6 mm Hg and the total pressure is 760 mm Hg, then 1% of the molecules in air are CO,.

standard temperature and pressure (O°C, 760 mm Hg) and dry,

Gases are soluble in liquids. The quantity of gas that dissolves at a given temperature is proportional to the partial pres-

or zero water vapor pressure (STPD). Gas volumes measured under one set of conditions (e.g.,

sure of that gas in the gas phase (Henry's law). The quantity of

ATPS) can be converted to another (e.g., BTPS) by using the equation of state for a gas. For example, the volume of air ex-

gas in solution equals a P, where Pis the partial pressure of the gas and a is the Bunsen solubility coefficient, which is inde-

pired from a mammalian lung at a body temperature of 37OC (273 + 37 = 310 K) is often measured at ambient room temper-

pendent of P. The Bunsen solubility coefficient varies with the type of gas, the temperature, and the liquid in question, but is

ature, say 20°C (273 20 = 293 K).The drop in temperature will reduce the expired gas volume. A gas in contact with water will

constant for any one gas in a given liquid at constant temperature. The Bunsen solubility coefficient for oxygen decreases with increases in ionic strength and temperature of water.

+

be saturated with water vapor. The water vapor pressure at 100% saturation varies with temperature. Expired air is saturated with

color of a respiratory pigment changes with its 0, content. Thus, hemoglobin, which is bright red when it is loaded with 02,becomes a dark maroon-red when deoqgenated. Vertebrate hemoglobin, except that of cyclostomes, has a molecular weight of 68,000 and contains four ironcontaining porphyrin prosthetic groups, called heme, associated with globin, a tetrameric protein (Figure 13-2A).The globin molecule consists of two dimers, alp, and alp,, each of which is a tightly cohering unit. The two dimers are more loosely connected to each other by salt bridges, except that the two p chains do not touch. Oxygenation alters these bridges, leading to conformational changes in the hemoglobin molecule. Hemoglobin can be dissociated

into four subunits of approximately equal weight, each containing one polypeptide chain and one heme group. Myoglobin, a respiratory pigment that stores 0, in vertebrate muscles, is equivalent to one hemoglobin subunit and exhibits considerable sequence homology with the hemoglobin a chain. In a hemoglobin molecule, iron in the ferrous state (Fe2+)is bound into the porphyrin ring of the heme, forming coordinate links with the four pyrrole nitrogens (Figure 13-2B). The two remaining coordinate linkages are used to bind the heme group to an 0, molecule and to the imidazole ring of a histidine residue in the globin (Figure 13-2C). If O2is bound, the molecule is referred to

OOC Heme

C

L-Histidine (His)

COO-

I I

+H,N-C-H

Figure 13-2 Hemoglobin, the main respiratory pigment in vertebrates, consists of four globin protein subunits, each containing one heme molecule. (A) Schematic diagram of hemoglobin molecule, showing relationship of the aand pchains. Two of the four heme units (red) are visible in the folds formed by the polypeptide chains. (B) Structure of heme, formed by the combination of ferrous ion (Fez+)and protoporphyrin IX. (C) Schematic diagram of heme in a pocket formed by the globin molecule. The side chain of a histidine (His) residue in globin acts as an additional ligand for the iron atom In heme. When oxygen binds, it displaces the remaining H,O ligand. [Adapted from McGilvery, 1970.1

as oxyhemoglobin; if 0, is absent, it is called deoxyhemoglobin. Binding of 0, to hemoglobin to form oxyhemoglobin does not oxidize ferrous to ferric iron. Oxidation of the ferrous iron in hemoglobin to the ferric state produces methemoglobin, which does not bind 0, and therefore is nonfunctional. Although formation of methemoglobin occurs normally, red blood cells contain the enzyme methemoglobin reductase, which reduces methemoglobin to the functional ferrous form. Certain compounds (e.g., nitrites and chlorates) act either to oxidize hemoglobin or to inactivate methemoglobin reductase, thereby increasing the level of methemoglobin and impairing oxygen transport. The affinity of hemoglobin for carbon monoxide is about 200 times greater than its affinity for oxygen. As a result, carbon monoxide will displace oxygen and saturate hemoglobin, even at very low partial pressures of carbon monoxide, causing a marked reduction in oxygen transport to the tissues. Hemoglobin saturated with carbon monoxide is called carboxyhemoglobin.The effect of such saturation on oxidative metabolism is similar to that of oxygen deprivation, which is why the carbon monoxide produced by cars or improperly stoked coal or wood stoves is so toxic. Even the levels found in city traffic can impair brain function owing to partial anoxia. Hemoglobin is found in many invertebrate groups, but others possess different respiratory pigments, including hemerythrin (Priapulida, Brachiopoda, Annelida), chlorocruorin (Annelida), and hemocyanin (Molluscs, Arthropoda). Many invertebrates do not have a respiratory pigment. Hemocyanin, a large, copper-containingrespiratory pigment, has many properties similar to those of hemoglobin, binding oxygen when the partial pressure is high and releasing it when the partial pressure is low. Hemocyanin binds oxygen in the ratio of 1mol of 0, to approximately 75,000 g of the respiratory pigment. In comparison, 4 mol of 0, bind to 68,000 g of hemoglobin when it is completely saturated. Unlike hemoglobin, hemocyanin is not packaged in cells and is not associated with high levels of carbonic anhydrase in the blood. In its oxygenated form, it is light blue; in its unoxygenated form, it is colorless. Oxygen Transport in Blood Each hemoglobin molecule can combine with four oxygen molecules, each heme combining with one molecule of oxygen. The extent to which 0, is bound to hemoglobin varies with the partial pressure of the gas, Po2.If all sites on the hemoglobin molecule are occupied by 0 2 ,the blood is 100% saturated and the oxygen content of the blood is equal to its oxygen capacity. A millimole of heme can bind a millimole of O,, which represents a volume of 22.4 ml of 0,. Human blood contains about 0.9 mmol of heme per 100 ml of blood. The oxygen capacity is therefore 0.9 x 22.4 = 20.2 vol %. The oxygen content of a unit volume of blood includes the 0, in physical solution as well as that combined with hemoglobin, but in most cases the 0, in physical solution is only a small fraction of the total 0, content.

522

I N T E G R A T I O N O F PHYSIOLOGICAL SYSTEMS

.........................................

Because the oxygen capacity of blood increases in proportion to its hemoglobin concentration, the oxygen content commonly is expressed as a percentage of the oxygen capacity, that is, the percent saturation. This makes it possible to compare the oxygen content of blood of different hemoglobin content. Oxygen dissociation curves describe the relationship between percent saturation and the partial pressure of oxygen. The oxygen dissociation curves of myoglobin and lamprey hemoglobin are hyperbolic, whereas the oxygen dissociation curves of other vertebrate hemoglobins are sigmoid (Figure 13-3). This difference occurs because myoglobin and lamprey hemoglobin have a single heme group, but other hemoglobins have four heme groups. The sigmoid shape of the dissociation curves exhibited by hemoglobins having several heme groups results fr0.m subunit cooperatiuity; that is, oxygenation of the first heme groups facilitates oxygenation of subsequent heme groups. The steep portion of the curve corresponds to oxygen levels at which at least one heme group is already occupied by an oxygen molecule, increasing the affinity of the remaining heme groups for oxygen. As a hemoglobin molecule is oxygenated, it goes through a conformational change from the tense (T) state to the relaxed (R) state. Oxygenation is associated with changes in the tertiary structure near the hemes that weaken or break connections between the a, PI and a, p, dimers, leading to a large change in the quaternary structure from the T to the R state. These conformational changes also produce changes in the dissociation of acidic side chains, so that protons (H+ions) are released as hemoglobin is oxygenated. -I An important property of respiratory pigments is that they combine reversibly with 0, over the range of partial

I,

L

Figure 13-3 Hemoglobinswith multiple heme groups have sigrnoid oxygen dissociation curves, whereas myoglobin with only a single heme group has a hyperbolic dissociation curve. Lamprey hemoglobin, with a single heme group, has a dissociation curve similar to that of myoglobin. P,, the partial pressure at which a respiratory pigment is 50% saturated with oxygen, is a measure of its oxygen affinity.

pressures normally encountered in the animal. At low Po,, only a small amount of 0, binds to the respiratory pigment; at high Po>,however, a large amount of 0, is bound. Because of this property, the respiratory pigment can act as an oxygen carrier, loading at the respiratory surface (a region of high Po>)and unloading at tissues (a region of low Po,). In some animals, the predominant role of a respiratory pigment may be to serve as an oxygen reservoir, releasing 0, to the tissues only when 0, is relatively unavailable. In many animals at rest, the venous blood entering the lung or gills is around 70% saturated with oxygen; that is, most of the oxygen bound to hemoglobin is not removed during transit through the tissues. During exercise, when the oxygen demand by the tissues is increased, this venous reservoir of oxygen is tapped and venous saturation may drop to 30% or less. Hemoglobins that have high oxygen affinities are saturated at low partial pressures of oxygen, whereas hemoglobins with low oxygen affinities are completely saturated only at relatively high partial pressures of oxygen. The affinity is expressed in terms of the P,, ,the partial pressure of oxygen at which the hemoglobin is 50% saturated with oxygen; the lower the P,, ,the higher the oxygen affinity. As the curves in Figure 13-3 demonstrate, myoglobin has a much higher oxygen affinity than hemoglobin. Variations in oxygen affinity among hemoglobins are related to differences in the protein globin, not to differences in the heme group. Each a and P chain of the globin molecule consists of between 141 and 147 amino acids, depending on the chain and the hemoglobin in question. The amino acid sequences of both the a and the P chains from different hemoglobins exhibit many similarities, but there are some differences. Although most amino acid substitutions are neutral, some have a marked impact on function. For example, a genetic defect resulting in substitution of valine for glutamic acid in position 6 of the p chain causes human hemoglobins to form large polymers that distort the erythrocyte into a sickle shape, giving rise to sickle cell anemia. Because these sickle cells cannot pass through small blood vessels, oxygen delivery to tissues is impaired. Individuals with both normal and sickle cell hemoglobins suffer only mild debilitation but have greater resistance to malaria, thus ensuring the continuation of the sickle cell gene in the population. Certain amino acids in globin bind various ligands, and substitution of these residues can cause changes in the oxygen affinity of hemoglobin. The rate of oxygen transfer to and from blood increases in proportion to the difference in Po, across an epithelium. A hemoglobin with a high oxygen affinity facilitates the movement of 0, into the blood from the environment because 0, is bound to hemoglobin at low Po?;i.e., 0, entering the blood is immediately bound to hemoglobin, so 0, is removed from solution and Po2is kept low. Thus, a large difference in PO2is maintained across the respiratory epithelium-and therefore a high rate of oxygen transfer into the blood-until hemoglobin is fully saturated. Only then does blood Po2rise. Hemoglobin with a high oxygen affin-

,

GAS EXCHANGE A N D ACID-BASE BALANCE

523

...................................... ity, however, will not release O2 to the tissues until the Po> is very low. In contrast, a hemoglobin with a low oxygen affinity will facilitate the release of 0, to the tissues, maintaining large differences in Pol between blood and tissues and a high rate of oxygen transfer to the tissues. Thus, a hemoglobin of high oxygen affinity favors the uptake of 0, by the blood, whereas a hemoglobin of low oxygen affinity facilitates the release of 0, to the tissues. From a functional viewpoint, therefore, hemoglobin should have a low O2 affinity in the tissues and a high 0, affinity at the respiratory surface. In light of this, it is highly significant that the oxygen affinity of hemoglobin is affected by changes in chemical and physical factors in the blood that favor oxygen binding at the respiratory epithelium and oxygen release in the tissues. The hemoglobin-oxygenaffinity is labile and dependent on the conditions within the red blood cell. For instance, the hemoglobin-oxygena f f i t y is reduced by the following:

throcytic organophosphate differs among species. For instance, mammalian erythrocytes contain high levels of 2,3diphosphoglycerate (DPG);indeed, hemoglobin and DPG are nearly equimolar in human erythrocytes. DPG binds to specific amino acid residues in the p chains of deoxyhemoglobln, but DPG binding decreases with increasing pH. Increases in DPG levels accompany reductions in blood 0, or hemoglobin concentrations, increases in pH, or both. Low blood O2 levels may result from a climb to a higher

Arterial

Elevated temperature Binding of organic phosphate ligands including 2,3diphosphoglycerate (DPG),ATP, or GTP Decrease in pH (increase in H+ concentration) Increase in CO, The hemoglobin molecule has a much higher affinity for ligands when it is in the T, or deoxygenated, state. Increases in H+ concentration (decreases in pH) cause a reduction in the oxygen affinity of hemoglobin, a phenomenon termed the Bohr effect, or Bohr shift (Figure 13-4). Carbon dioxide reacts with water to form carbonic acid and reacts with -NH2 groups on plasma proteins and hemoglobin to form carbamino compounds. Thus an increase in PCo2causes a reduction in the oxygen affinity of hemoglobin in two ways: by decreasing blood pH (Bohr effect) and by promoting the direct combination of CO, with hemoglobin to form carbamino compounds. Therefore, when C 0 2 enters the blood at the tissues it facilitates the unloading of 0, from hemoglobin, whereas when CO, leaves the blood at the lung or gill, it facilitates the uptake of 0, by the blood. The oxygen dissociation curve for myoglobin, unlike that for hemoglobin, is relatively insensitive to changes in pH. Hemocyanins from the Dungeness crab, Cancer magister, and some other invertebrates exhibit a Bohr shift similar to that of hemoglobin (Figure 13-5). But hemocyanins from several gastropods and from the horseshoe crab, Limulus, show a greater oxygen affinity with a decrease in pH. This phenomenon, referred to as a reverse Bohr effect, may facilitate oxygen uptake during periods of low oxygen availability when prolonged reductions in blood pH,occur in these animals. As noted above, the binding of organic phosphate compounds to hemoglobin reduces the oxygen affinity of most vertebrate hemoglobins, except those from cyclostomes, crocodiles, and ruminants. The dominant ery-

"

30

60

90

'

Po, (mm Hg) Figure 13-4 The oxygen affinity of hemoglobin decreases with decreasing pH. Because of this phenomenon, called the Bohr effect, changes in blood Pco,, wh~chinfluence blood pH, indirectly affect hemoglobinoxygen affinity. Shown are experimental blood oxygen dissociation curves in humans at three pH values. The Po,values of mixed venous and arterial blood are indicated. [Adapted from Bartels, 1971.]

30

60

90

Po, (mm Hg) Figure 13-5 Some hemocyanins, like hemoglobin, exhibit a Bohr shift. Blood oxygen dissociat~oncurves for the crab Cancer magistershown here indicate that the hemocyanin from this crab shows a Bohrshift. [Unpublished data supplied by D. G. McDonald.]

524


.......................................

altitude, as both barometric pressure and the partial pressure of 0, in air decrease with altitude. The resultant DPG rise in humans in response to high altitude is completed in 24 hours with a half-time of about 6 hours. At elevations of 3000 m, the DPG concentration in erythrocytes is 10% greater than it is at sea level. The low 0, levels at altitude result in a decrease in blood O2 levels, which stimulates breathing. The resulting increase in ventilation (i.e., exchange of air between the lungs and ambient air) reduces C0, levels in the blood and raises blood pH, which increases hemoglobin-oxygen affinity. The elevation in DPG at altitude offsets the effects of reduced CO, levels and maintains hemoglobin-oxygen affinity close to that at sea level. In the erythrocytes of some vertebrates, other phosphorylated compounds are present In hlgher concentration than DPG, and consequently they play a more important role in modulating the oxygen affinity of hemoglobin than does DPG. In most fishes ATP and/or GTP has this function, whereas inositol pentaphosphate (InsP, ) is the dominant erythrocytic organophosphate in birds. In the Amazonian fish Arapazrna gzgas, ATP is the dominant erythrocytic organophosphate in the young aquatic form, but InsP, is dominant in the obligate airbreathing adult. Pho~phor~lated compounds in the erythrocyte not only affect the oxygen affinity of hemoglobin but also increase the magnitude of the Bohr effect and may affect subunit interaction. It appears that in mammals the functional significance of increased DPG levels is to maintain hemoglobin-oxygen affinity under hypoxic (low-oxygen) conditions, as at high altitude. In contrast, hypoxia reduces erythrocytic organophosphate levels in fishes. In these animals, however, hypoxia is often associated with a decrease in blood pH (acidosis),rather than the increase in pH (alkalosis) seen in mammals at altitude. The effect of the reduction in ATP (or GTP) in fishes is to offset the effects of this hypoxia-associated acidosis, thereby maintaining blood-oxygen affinity. Thus in a functional sense the effects of changing erythrocytic organophosphate levels are similar in both fishes and mammals; in both instances the result is to maintain hemoglobin-oxygen affinity. Reaction velocities for the binding of oxygen to hemoglobin are rapid and usually do not limit rates of oxygen transfer. The rate at which oxygen can bind to hemoglobin, however, also depends on the hemoglobin concentration. The higher the hemoglobin concentration the more oxygen bound per unit time. The more oxygen bound per unit time the longer the persistence of a large diffusion gradient across the respiratory epithelium for oxygen and, therefore, the higher the rate of oxygen transfer. The presence of a respiratory pigment also increases the transfer of oxygen through the blood, because the oxygenated pigment co-diffuses with oxygen down the concentration gradient. That is, a gradient exists for both oxygen and the oxygenated pigment in the same direction

through the solution; the gradient for the deoxygenated pigment is in the reverse direction from that for oxygen and the oxygenated pigment. Hence, the oxygenated pigment diffuses in the same direction as oxygen, whereas the deoxygenated pigment diffuses in the reverse direction. Thus, a pigment such as hemoglobin may facilitate the mixing of gases in the blood, and myoglobin may play a similar role within tissues.

In some fishes, cephalopods, and crustaceans, an increase in CO, or a decrease in pH causes not only a reduction in the oxygen affinity of hemoglobin but also a reduction in oxygen capacity, which is termed the Root effect, or Root shift (Figure 13-6). In those hemoglobins showing a Root shift, low pH reduces oxygen binding to hemoglobin, so that even at high Po2,only some of the binding sites are oxygenated; that is, 100% saturation is never achieved. An increase in temperature exacerbates problems of oxygen delivery in poikilothermic aquatic animals such as fishes. A rise in temperature not only reduces oxygen solubility in water but also decreases the oxygen affinity of hemoglobin, making oxygen transfer between water and blood more difficult. Unfortunately, this decrease in affinity occurs at a time when tissue oxygen requirements are increasing, also as a result of the rise in temperature. It is generally assumed that a particular hemoglobin has evolved to meet the special gas-transfer and H+ buffering requirements of the animal. Differences in the properties of hemoglobins are due to variation in the amino acid sequence of the peptide chains of the globin portion of the

Plasma only

Figure 13-6 Redua~onsIn pH decrease the blood oxygen capac~ty(Root effect) In hemoglob~ns from some teleost f~shesThese oxygen e q u ~ l ~ b rlum curves of eel blood were obta~nedat 14°Cw ~ t hthe pH from 6 99 to 8 20 The bottom llne descr~besthe 0, content of plasma [Adapted from Steen, 1963.1


525

...................................... molecule, the heme portion being the same in all hemoglobins. Not only do hemoglobins vary among species, but they also may change during development. In humans, for example, several genes encode plike globin chains, and the relative expression of these chains differs during prenatal and postnatal life (Figure 13-7).Human fetal hemoglobin, which contains y chains, rather than adult P chains, has a higher 0, affinity than adult hemoglobin. The higher 0, affinity of fetal hemoglobin enhances oxygen transfer from mother to fetus. As the proportion of fetal hemoglobin decreases and adult hemoglobin increases following birth, the oxygen affinity of the blood decreases (Figure 13-8).Other mammals exhibit similar differences between fetal and adult hemoglobins. It is important to remember that in most animals hemoglobin is contained within red blood cells, but the values of blood parameters usually refer to conditions in the plasma, not in the red blood cell. Differences in these parameters exist between the inside and outside of cells, including red blood cells. For example, the pH of mammalian arterial blood at 37°C is usually 7.4. This is the pH of arterial blood plasma; the pH inside the red blood cell is less, about 7.2 at 37OC. Carbon Dioxide Transport in Blood

Carbon dioxide diffuses into the blood from the tissues, is transported in the blood, and diffuses across the respiratory surface into the environment. Carbon dioxide reacts with water to form carbonic acid, a weak acid, which dissociates into bicarbonate and carbonate ions: CO,

+ H,O

----T H,CO,

H + + C0;-

HC0,-

/

1 I

H + + HC0,-

Fetal hemoglobin (y-chain)

(p-chain)

9k

: Embryonic :hemoglobin : (€-chain)

.,

3

\

A,

(&chain)

Po,

Figure 13-8 In humans, the oxygen affinity of blood decreasesfor about three months after birth as the fetal hemoglobin is replaced by adult hemoglobin (see Figure 13-7).These blood oxygen dissociation curves were determined at a pH of 7.40. [Adapted from Bartels, 1971.1

Carbon dioxide also reacts with hydroxyl ions to form bicarbonate:

=H+ + OHCO, + OH- =HCO, H,O

in solution The proportion of CO,, HC0,-, and C0:depends on pH, temperature, and the ionic strength of the solution. In mammalian blood at pH 7.4, the ratio of CO, to H2C03is approximately 1000: 1, and the ratio of CO, to bicarbonate ions is about 1: 20. Bicarbonate is, therefore, the predominant form of CO, in the blood at normal blood pH. The carbonate content is usually negligible in birds and mammals; in poikilotherms, however, with their low temperature and high blood pH, the carbonate content may approach 5% of the total CO, content of the blood, but bicarbonate is still the predominant form of CO,. Carbon dioxide also reacts with -NH2 groups on proteins and, in particular, hemoglobin to form carbamino compounds. protein -NH,

6

Duration of pregnancy (months)

Birth

3

6

Age (months)

Figure 13-7 Hemoglobins change during development in humans. The relative amounts of the various hemoglobin p-l~kechains synthesized in the fetus changes during the course of pregnancy. Fetal hemoglobin, which contains two a and two y chains, has a h~gheroxygen affinity than adult hemoglobin [Adapted from Young, 1971.]

(mm Hg)

+ CO,

H+ + protein --NHCOO-

The extent of carbamino formation depends on the number of available terminal NH, groups, and it increases with blood pH and increasing CO, levels. The terminal NH, groups of both the a and P chains of mammalian, bird, and reptile hemoglobins are available for carbamino formation. The terminal NH, group of the a chain of fish and amphibian hemoglobins, however, is acetylated and therefore not available for carbamino formation. Because

organophosphates bind to some of the same amino acids that are involved in carbamino formation, organophosphate binding reduces carbamino formation. However, high pH reduces organophosphate binding and so augments carbamino formation by making more NH, groups available. Because fish erythrocytes often have high organophosphate levels as well as acetylated a chains, fish rely less on carbamino formation for CO, transport than mammals. The sum of all forms of CO, in the blood-that is, molecular C 0 2 , H,CO, ,HC0,-, COj2-, and carbamino compounds-is referred to as the total CO, content of the and the relationblood. The CO, content varies with PCO2, ship can be described graphically in the form of a CO, dissociation curve (Figure 13-9).As Pco2increases, the major change is in the bicarbonate content of the blood. The formation of bicarbonate is, of course, pH dependent. The relationships between plasma H C O , concentration and plasma pH at three values of PCo2are shown graphically in Figure 13-10. A decrease in pH at constant PCo2is associated with a fall in bicarbonate. The pH of red blood cells is less than that of plasma, but PCo2is in equilibrium across the cell membrane. Therefore, bicarbonate levels are lower in erythrocytes than in plasma. Erythrocytes usually constitute less than 50% of the blood volume (i.e., plasma volume is greater than erythrocyte volume), and the bicarbonate concentration is higher in plasma than in erythrocytes; it thus follows that most of the bicarbonate in the blood is in plasma.

Deoxygenated blood,

Figure 13-9 The total CO, content of blood increaseswith Pco,, but only the volume of molecular CO, increases linearly. Note that at a glven Pco, oxygenated blood contains less C 0 2 than deoxygenated blood (Haldane effect). A and V refer t o arterial and venous blood levels, respectively.

HC0,- or reacts with-NH, groups of hemoglobin and other proteins to form carbamino compounds. The reverse process occurs when CO, is unloaded from the blood. The largest change is In the HC0,- concentration; changes in the levels of CO, and carbamino compounds usually represent less than 20% of total carbon dioxide excretion. The reaction of CO, with O H to form H C O , is slow and has an uncatalyzed time course of several seconds. But in the presence of the enzyme carbonic anhydrase, this reaction approaches equilibrium In much less than a second. Although plasma has a higher total CO, content than red blood cells, most of the CO, entering and leaving the plasma does so via erythrocytes, because carbonic anhydrase is present in red blood cells but not in the plasma. Therefore, formation of H C O , ions in the tissues and CO, in the lungs occurs predominantly in red blood cells; once

Transfer of Gases to and from the Blood

As CO, is added to the blood in the tissues and removed at the respiratory surface, the levels of CO,, HC0,-, and carbamino compounds all change. Carbon dioxide both enters and leaves the blood as molecular C0, rather than as bicarbonate ion because CO, molecules diffuse through membranes much more rapidly than HC0,- ions. In the tissues, CO, enters the blood and either is hydrated to form

40 -

PCOp

48.0 40 0 35.0

-

-zm

E a 30 C

Figure 13-10 The pH, b~carbonateconcentratlon, and PCo, In human plasma are Interrelated and normallyfall w ~ t h ~qulte n narrow llrn~ts(~nd~cated by red box), However, when blood Pco, IS altered In v~voby hyper- or hypoventilatlon, then plasma pH and blcarbonate are altered beyond the normal range, as ~nd~cated by the whole-body buffer l~ne [Adapted from Davenport,

(mm Hg)

-28

19741

0

%.-

-

0

-23

a

m m a

HC0,-

20-

2

-

Whole-body buffer l ~ n e I

lol

710

I

7.2

I

I

7.4 Plasma pH

I

I

7.6

I

I

7.8


527

............................................................................... formed, H C O , ions and CO, subsequently are transferred from or into the plasma. On entering the blood from the tissues, CO, diffuses into red blood cells, and HC0,- is formed rapidly in the presence of carbonic anhydrase (Figure 13-11A). As the HC0,- level within erythrocytes rises, H C O , ions move from the cells into the plasma. Electrical balance within the cells is maintained by anion exchange; as HC0,- ions leave the red blood cells, there is a net influx of C 1 ions from the plasma into the cells, a process called the chloride shift. Red blood cells, unlike many other cells, are very permeable to both C 1 and H C O , because the membrane has a high concentration of a special anion carrier protein, the band III protein. This transport protein binds C1- and HC0,and transfers them in opposite directions through the erythrocyte membrane. The anion exchange is passive and depends on concentration gradients to drive the process,

COP

TISSUE

which can occur in either direction, bicarbonate flowing out of the erythrocyte in the tissues and into the erythrocyte at the respiratory surface (Figure 13-llB).Band I11 protein is present in all vertebrate erythrocytes except those of lamprey and hagfish. In these animals bicarbonate stays within the red blood cell and there is no anion transfer between the erythrocyte and the plasma. A second reason why most of the CO, entering or leaving the blood passes through the red blood cells is that oxygenation of hemoglobin (Hb) causes H + release, thereby acidifying the cell interior; conversely, deoxygenation results in the binding of H+to Hb. Thus 0, binding to Hb at the respiratory surface facilitates the formation of CO,, whereas release of 0, from Hb in the tissues facilitates the formation of H C O , (Figure 13-12).As a result, changes in pH associated with the transfer of CO, into or out of the blood are minimized because of proton binding to and

T

I

1

Capillary wall

(slow) + H20 = H2C03L=;HCO, + H+

C02

T

I

Figure 13-11 Most of the carbon d~oxideentering the blood In the tlssues and leavlng the blood In the lungs passes through red blood cells (A) Carbon dloxlde produced in the tlssues rapldly forms blcarbonate (HC0,-) In the red blood cell because the hydration reactlon 1s catalyzed by carbonlc anhydrase present In the cell B~carbonateleaves the erythrocyte In exchange for chloride, and excess protons are bound by deoxygenated hemoglobin (Hb) (B) These reactions are reversed In lungs Oxygen enterlng the red blood cell displaces protons from Hb, and carbon d ~ o x ~ d enters e the plasma Carbonlc anhydrase (lndlcated by solld clrcles) In the membrane of the lung endothellal cells converts some of the plasma blcarbonate to carbon dloxlde Movement of carbon dloxlde across the respiratory surface IS augmented by the dlffus~onof blcarbonate and ~ t sconversion back to carbon dloxlde at the outer surface, a process termed facll~tateddiffusion

528


......................................... Capillary endothelium

Respiratory epithelium

OH-

lco2+>

Figure 13-12 The pH changes associatedwith the changes in blood Pco, in the tissues and respiratorysurface are offset by binding and release of H+ ions by deoxygenated and oxygenated blood. For example, transfer of CO, into the blood in the tissues causes a decrease in pH due to formation of bicarbonate; concomitant deoxygenation of hemoglobin frees proton acceptors, which bind the excess H+ ions. The opposite reactions occur at the respiratory epithelium.

proton release from hemoglobin as it is deoxygenated and oxygenated, respectively. For example, as the PC,> increases in the tissues, the subsequent formation of H C O , or carbamino compounds liberates H + ions. At the same time, release of oxygen forms deoxyhemoglobin, which binds protons. As deoxygenation proceeds, however, more proton acceptors become available on the hemoglobin molecule. In fact, complete deoxygenation of saturated hemoglobin, releasing 1 mol O,, results in the binding of 0.7 mol of H+ ions. Thus, when the ratio of CO, production to 0, consumption (calledthe respiratory quotient) is 0.7, the transport of CO, can proceed without any change in blood pH. (As discussed in Chapter 16, the respiratory quotient depends on the type of diet.) Even when the respiratory quotient is 1, the additional 0.3 mol H + is buffered by blood proteins, including hemoglobin, and blood undergoes only a small change in pH. At a given PCo1, deoxyhemoglobin binds more protons, thereby facilitating H C O , formation, and reacts with CO, to form carbamino hemoglobin more easily than does oxyhemoglobin. As a result, the total CO, content of deoxygenated blood at a given Pco, is higher than that of oxygenated blood (seeFigure 13-9).Thus, deoxygenation of hemoglobin in the tissues reduces the change in PCol and pH as CO, enters the blood; this is termed the Haldane effect. In the lungs, two mechanisms are available for transfer of CO, from the blood. As noted already, carbonic anhydrase is absent from plasma, and thus the interconversion of CO, and HC0,- occurs at the slow, uncatalyzed rate in plasma. (Any carbonic anhydrase liberated by the breakdown of red blood cells is excreted via the kidney.) In the endothelial cells of lung capillaries, however, carbonic anhydrase is embedded in the cell surface, accessible to plasma CO, and HC03-. Therefore, the conversion of H C O , to

CO, can occur at the catalyzed rate in plasma as blood perfuses the lung capillaries (see Figure 13-11B).In addition, oxygenation of hemoglobin acidhes erythrocytes in the lung capillaries, facilitating the conversion of HC0,- to CO,, which then diffuses into the plasma and across the lung epithelium. The resulting decrease in erythrocyte bicarbonate levels results in the influx of HC0,- ions from the plasma accompanied by the outward movement of C1- ions. The relative quantities of HC0,- converted to CO, in the erythrocytes and the plasma of the blood perfusing the respiratory epithelium is influenced by the extent of proton production associated with hemoglobin oxygenation and the amount of carbonic anhydrase activity in the walls of the respiratory epithelium. In teleost fish, for instance, the plasma perfusing the gills is not exposed to carbonic anhydrase. In these animals, most excretion of CO, occurs through the red blood cells and is tightly coupled to 0, uptake through proton production by oxygenation of hemoglobin. Carbonic anhydrase activity is also found on the endothelial surfaces of a number of systemic capillary beds, including those in skeletal muscle. In these capillaries, formation of HC0,- catalyzed by carbonic anhydrase can occur in the absence of red blood cells. Thus some of the CO, transferred into the blood in skeletal muscle does not pass through erythrocytes. Carbonic anhydrase also facilitates carbon dioxide transfer, referred to as facilitated diffusion of CO, (see Figure 13-llB), which results from the simultaneous diffusion through the epithelium of bicarbonate and protons, the latter also augmented by release from buffers. Carbonic anhydrase catalyzes the rapid interconversion of CO, and H C O , in this process of facilitated diffusion, with CO, entering and leaving the cell. There are at least seven forms of carbonic anhydrase, designated CA-I through CA-VII. All are similar in structure and catalyze the interconversion of carbon dioxide and bicarbonate. Carbonic anhydrase I (CA-I)and carbonic anhydrase I1 (CA-11),present in human red blood cells, have a molecular weight of about 29,000, containing about 260 amino acid residues. CA-11, an extremely efficient catalyst of the carbon dioxide-bicarbonate hydration-dehydration reactions, is found in a wide variety of tissues, including the brain, eye, kidney, cartilage, liver, lung, pancreas, gastric mucosa, skeletal muscle, and anterior pituitary, as well as red blood cells. This form is involved in a wide variety of functions, augmenting the supply of bicarbonate and/or protons for a number of cellular and metabolic processes. A few humans exhibit an inherited CA-I1 deficiency, the pattern of inheritance being autosomal recessive. Although these individuals have no detectable CA-11, they have normal levels of CA-I in their red blood cells. CA-I1 deficiency not only compromises the gas-exchange process but also produces many other symptoms including metabolic acidosis, renal tubular acidosis, and sometimes mental retardation. In addition, because CA-I1is involved in production of protons needed for bone resorption in osteoclasts, its absence results in osteoporosis, often associated with multiple bone fractures. The wide range of symptoms associated

t.'

\

with inherited CA-I1 deficiency reflects the large number of functions in which CA-I1plays a role in augmenting proton and/or bicarbonate delivery. The rate of movement of CO, and 0, into or out of the red blood cell is determined by the diffusion distance and the diffusion coefficient of these substances through the cell. The diffusion difference and hence the rate of erythrocyte oxygenation might be expected to be related to cell size, which varies considerably among vertebrates. For instance, the amphibian Necturus has erythrocytes that are 600 times the volume of erythrocytes from a goat. Earlier studies demonstrated that small erythrocytes are oxygenated faster than larger cells in vitro (Figure 13-13),but this finding may have little relevance in vivo. Recent experiments using a whole-blood thin-film technique, which is analogous to the in vivo situation, have shown that oxygen uptake rates are independent of cell size. The explanation for this probably lies in the flattened shape of erythrocytes. If the large flat surface of the cells faces the respiratory medium as they pass single file through the respiratory capillaries, then their diffusion distances may be quite similar even though volumes of cells are very different. Thus the in vitro results are probably not applicable to the in vivo situation. Excretion of CO, is considered to be limited by the rate of bicarbonate-chloride exchange across the erythrocyte membrane. The surface-to-volumeratio of erythrocytes, as well as the transport capacity for bicarbonate-chloride exchange mediated by band I11 protein, may be important in determining rates of carbon dioxide excretion. To see the interrelationship of these parameters, let's compare trout and human erythrocytes (Table 13-1).Red blood cells from trout are larger and have a much higher concentration of band I11 protein in their membranes than do red blood cells from humans. The higher concentration of band I11 protein presumably compensates for the increased cellular volume and offsets, at least to some degree, the effects of a lower body temperature in trout, compared with humans, on anion-exchange rates. Even so, anion exchange is slower across trout red blood cells at 15°C than across human red blood cells at 38°C. However, transit times for erythrocytes

TABLE 13-1 Comparison of bicarbonate-chlorideexchange system in trout and human erythrocytes Prolsertv Cell surface (cm2) Band Ill molecules per cell Band Ill molecules per cm2

Trout

2.67 x

Human

1.42 X 10-6

lo6

1 x lo6

30 X 10"

7 x 10"

3.42

17.2

8

X

Half-time for CI- ion exchange (seconds):

O/C

38/C Source: Romano and Passow, 1984.

-

0.05

C

0

.-0

.

d-4

2 (U

X

x

100

~orse

' 1Dog

#Human

Rabbit

o

Bullfroq

Red blood cell volume ( p m 3 )

Figure 13-13 Small erythrocytes are oxygenated faster than large cells in vitro. However, cell size probably is unrelated to oxygenation rates in vivo. [From Holland and Forster, 1966.1

through the gills is longer than that in the lungs, allowing more time for anion exchange across the red blood cell. Despite these considerations, it is still not clear why different species have evolved red blood cells of such different sizes. Those animals with large red blood cells also have large cells generally. Thus cell size may have been selected for reasons other than gas transfer and may be largely unrelated to gas-transfer rates. For instance, triploid salmon, whose red blood cells are 1.5 times the size of those of their diploid cousins but have the same hemoglobin concentration, are able to swim just as fast as their diploid cousins, indicating that the efficiency of gas transfer is comparable. It's important to remember that in vivo gas transfer is a dynamic process that takes place as blood moves rapidly through capillaries. Rates of diffusion, reaction velocities, and steady-state conditions for gases in blood must all be taken into account in analyzing the process. For instance, a Bohr shift (e.g., decrease in hemoglobin-oxygenaffinity with decrease in pH) would have little importance if it occurred after the blood had left the capillaries that supply an active tissue. The Bohr shift, in fact, occurs very rapidly, having a half-time at 37°C of 0.12 seconds in human red blood cells. Although a reduction in temperature always decreases the velocities of reactions involved in gas transfer in a species, these velocities do not vary and are not modulated to regulate gas-transfer rates at constant temperature. Concentration changes, however, are used to adjust gas-transfer rates over hours or days. For example the oxygen content of the . blood depends on the concentration of hemoglobin, which is increased in many vertebrates in response to hypoxia. Rapid changes in gas-transfer rates in vertebrates are achieved either by adjusting the breathing rate and volume andlor by adjusting the flow rate and distribution of blood in both tissues and the respiratory surface.

REGULATION OF BODY pH Animals have a body pH that is on the alkaline side of neutrality; that is, there are fewer hydrogen than hydroxyl ions in the body. The concentrations of hydrogen and hydroxyl ions are very low in aqueous solutions because water is

530


...............................................................................

only weakly dissociated. Human blood plasma at 37°C has a pH of 7.4, or a hydrogen ion activity of 40 nanomoles per liter (1 nM = M). Normal function can be maintained in mammals at 37°C over a blood plasma pH range of 7.0-7.8, that is, between 100 and 16 nM H+. This is, in fact, a rather large percentage deviation from the normal H + concentration of 40 nM compared with the much lower tolerance of variations in Na+ or K+ levels in the body. It is important, however, to bear in mind that the absolute changes in concentration are small, as are the actual concentrations of H+ ions in the body. Blood pH in vertebrates is midway between the pK of the carbon dioxide1bicarbonate and ammonia/ammonium reactions (Figure 13-14A). Most cell membranes are not very permeable to HC0,- and NH,+ ions but are very permeable to CO, and NH,. Some membranes have a relatively low permeability to NH,, but these are the exceptions rather than the rule. A body pH that is midway between these pKs ensures adequate rates of excretion by diffusion of the two major end products of metabolism, namely carbon dioxide and ammonia. Because these pKs vary with temperature, so does the pH of blood, ensuring adequate rates of excretion over a range of temperatures (Figure 13-14B). Changes in body pH alter the dissociation of weak acids and thus the ionization of proteins. The net charge on proteins determines enzyme activity and subunit aggregation, influences membrane characteristics, and contributes to the osmotic pressure of body compartments. Osmotic pressure is affected because the charge on proteins is a major contributor to the total fixed charge within cells. A change in the fixed charge will alter the Donnan equilibrium of ions and therefore could affect the osmotic pressure. Any differences in osmotic pressure between body compartments disappear rapidly because membranes are permeable to water, and water movement will cause changes in the volume of various body compartments. Thus animals regulate their internal pH, in the face of a continual metabolic release of hydrogen ions, to stabilize volume and regulate enzyme activity. Cells also undergo changes in pH either as a result of, or to regulate and control, cellular functions. For example pH plays a central role in such things as sea urchin sperm activation and the stimulation of glycolysis in frog muscle by insulin. Cells also undergo changes in pH as a result of external influences. For example, cells become acidotic during hypoxia because of an imbalance between proton production resulting from hydrolysis of ATP to ADP and proton consumption by NAD in those tissues subjected to anaerobic metabolism. Hydrogen Ion Production and Excretion

Hydrogen ions are produced through metabolism or ingested in foods (e.g., citric acid in oranges) and then excreted on a continuing basis. The largest pool and flux is due, usually, to the metabolic production of CO,, which at the pH of the body reacts with water to form H + and

CO,

6.0

+ OH-=

7.0

pka = 6.08

HCO;

8.0

9.0

10.0

PH

7.6 7.4

6 5

lo

15

20

25

30

35

Temperature ("C) Figure 13-14 In vertebrates, the plasma pH is midway between the pKs of the ammonia/ammonium and carbon d~oxidelbicarbonatereactions. (A) The effect of varying pH on the [CO,]/[HCO,-] and [NH,]/[NH,+] ratios in trout plasma at 15'C. The dashed lines markthe pH values at which the ratios equal 1 (i.e.,the pKvalues).(6)Effect of temperature on plasma pH for several fishes. Red triangles are the calculated pH values at which the CO,/HCO,- and NH,/NH,+ ratios are equal at various temperatures. Thus plasma pH is maintained at levels that ensure both NH, and C0,excretion. [Adapted from Randall and Wright, 1989.1

HC0,- ions (see Figure 13-11A).At the respiratory surface HC0,- is converted to CO,, which is then excreted (see Figure 13-11B).Thus if the production and excretion of CO, are balanced, the overall effect of CO, flux on body pH will be zero. If CO, excretion is less than production, so that CO, accumulates, the body will be acidified; if the reverse occurs, the body pH will rise. Terrestrial vertebrates, however, can vary the rate of CO, excretion to maintain body pH. Ingestion of meat usually results in a net intake of acid, whereas ingestion of plant food often results in a net intake of base. Generally there is a small net production of hydrogen ions as a result of diet and metabolic activity. Thus the overall effect of food ingestion and metabolism is a small continual production of acid. The pH of the body is maintained by excreting this acid via the kidney in terres-

trial vertebrates or across regions of the body surface, such as the gills of fish or the skin of frogs. Changes in blood pH can also occur in response to acid movement between compartments. For example, following a heavy meal, the production of large volumes of acid in the stomach can produce an alkaline tide in the blood owing to transfer of acid from the blood into the stomach. In a similar manner, the production of large volumes of alkaline pancreatic juices can result in an acid tide in the blood.

As discussed in Chapter 3, the relationship between pH and the extent of dissociation of a weak acid, HA, is described by the Henderson-Hasselbalch equation: pH

=

pK'

[A-I + log [HA1

When the pH of a solution of a weak acid is equal to the pK' of the acid, then 50% of the acid is in the undissociated form, HA, and 50% is in the dissociated form, H+ + A-. At 1 pH unit above the pK, the ratio of the undissociated to dissociated form is 10% to 90%, whereas at 2 pH units above the pK, the ratio becomes 1 % to 99%. The Henderson-Hasselbalch equation can be rewritten for the C 0 , / H C 0 3 acid-base pair as [HCO,-]

pH

=

pK'

+ log " Pco2

where PCO2 is the partial pressure of CO, in blood, a is the Bunsen solubility coefficient for CO,, [HCO,-] is the concentration of bicarbonate, and pK' is the apparent dissociation constant. The term "apparent" is used because this pK' is a lumped value for the combined reactions of CO, with water and the subsequent formation of bicarbonate, and is not a true pK. We can see from this equation that changes in pH will affect the ratio of HC0,- to PCO7,and vice versa. The pK' of the CO,/HCO,- reaction is about 6.1, and the pK of the H C 0 , - / C 0 , 2 reaction is around 9.4. At the pH of the body, about 95% of the CO, is in the form of HC0,- ,the remainder being carbon dioxide and carbonic acid; the amount of C032- is negligible. Weak acids have their greatest buffering action when pH = pK. Because the pK of plasma proteins and hemoglobin is close to the pH of blood, these compounds are important physical buffers in the blood. The C02/HC03pair, with an apparent pK' below the pH of the blood, is of less importance than either hemoglobins or proteins in providing a physical buffer system. The importance of the

C0,-bicarbonate system is that an increase in breathing can rapidly increase pH by lowering CO, levels in the blood, and that HC03- can be excreted via the kidney to decrease blood pH. Although bicarbonate is not an important chemical buffer in living systems, it is often referred to as a buffer because the C0,-to-bicarbonate ratio can be adjusted by excretion in order to regulate pH. The most important true buffers in the blood are proteins, especially hemoglobin. Phosphates are also significant buffers in many cells. The importance of buffers in ameliorating pH changes can be seen by considering the effects of acid infusion on mammalian blood. About 28 mmol of hydrogen ions must be added to the blood to reduce pH from 7.4 to 7.0. In fact, only 60 nmol (about 0.2%) are required to change the pH of an aqueous solution to this extent; in blood, however, the bulk of the added 28 mmol of H + is buffered by conversion of HC03- to CO, (18 mmol), hemoglobin (8.0 mmol), plasma proteins (1.7 mmol), and phosphates (0.3 mmol). Thus nearly 500,000 times as many hydrogen ions are buffered as are required to cause the pH to change from 7.4 to 7.0. Clearly, if lung ventilation is reduced so that CO, excretion drops below C 0 2production, body CO, levels will rise and pH will fall. This decrease in body pH is referred to as respiratory acidosis. The reverse effect, that is a rise in pH due to increased lung ventilation, is termed respiratory alkalosis. The word "respiratory" is used to differentiate these pH changes from those caused by changes in metabolism or kidney function. For example, anaerobic metabolism results in net acid production, which reduces body pH; such changes are referred to as metabolic acidosis. Body fluids, like other solutions, are electroneutral;that is, the sum of the anions equals the sum of the cations. The normal electrolyte status of human plasma is illustrated in Figure 13-15. The sum of bicarbonate, phosphates, and protein anions is referred to as the buffer base. The remaining cations and anions are referred to as strong ions (i.e., those completely dissociated in physiological solutions); the difference between the sum of strong cations and the sum of strong anions is referred to as the strong ion difference (SID) and is a reflection of the magnitude of the buffer base. Because a change in blood pH usually results in a change in the buffer base, SID also must change to maintain electrical neutrality. In this situation, the change in SID usually involves either sodium or chloride, since these are the major ions in the blood. For example, a reduction in bicarbonate must be associated with an increase in chloride or a reduction in sodium. Conversely, a change in the ratio of sodium to chloride will be associated with a change in the buffer base and therefore blood pH. Vomiting the stomach contents results in chloride loss and a reduction in blood chloride levels; as a consequence, bicarbonate levels are increased along with blood pH without any change in P o l ; this is referred to as metabolic alkalosis. Vomit originating from the duodenum, rather than the stomach, however, results in the loss of more bicarbonate than chloride, causing a metabolic acidosis.

532


............................................................................... (

meq.1-I

Normal plasma electrolyte status Figure 13-15 All body fluids are electroneutral, containing equal numbers of positive and negative charges. This diagram shows the equivalent concentrations (meq . Lk') of the major electrolytes in human plasma at normal pH. The concentrationof the buffer base (the nonrespiratory acidbase displacement)depends on pH. Thus a pH increase or decrease that changes the buffer base concentration must be accompanied by a corresponding change in the concentration of one or more strong ions, usually sodium or chloride. [Adapted from Siggaard-Andersen, 1963.1

Hydrogen Ion Distribution between Compartments

Cell membranes separating the intracellular and extracellular compartments and layers of cells between two body compartments are much more permeable to carbon dioxide than to either hydrogen or bicarbonate ions. The permeability of most cell membranes to H+ ions, although usually low, is often greater than that for K+, C1-, and HC0,- ions; a notable exception is the erythrocyte membrane, which is very permeable to HC03- and C1- ions, but not very permeable to H + ions. Red blood cells and cells in the collecting duct of the mammalian kidney have

high levels of band 111 protein in their plasma membranes, but other cells do not. As discussed previously, band I11 protein mediates the exchange of HC0,- for CI- ions at high rates. Thus, although all cell membranes are permeable to CO,, only a few membranes can transfer HC03- at high rates via the band I11 anion-exchange mechanism. An increase in extracellular PCo2causes an increase in both bicarbonate and hydrogen ion concentration, thereby creating gradients for CO,, HC0,-, and H+ across the cell membrane. In cells that are very permeable to CO, but not very permeable to H+ or bicarbonate, such a situation leads to rapid movement of CO, into the cell; as the CO, is converted to HC03-, the intracellular pH falls sharply. Acidification associated with increased Pco: often occurs much more rapidly in the intracellular compartment than in the extracellular compartment because carbonic anhydrase, which catalyzes the conversion of CO, to HC0,-, is present inside cells but not always in the extracellular fluid. Even when Pm2remains elevated, intracellular pH slowly returns to the initial level due to the slow extrusion of acid (or uptake of base) across the cell membrane (Figure 13-16A). The rise in intracellular pH is such that if the Pcol level is returned to the original value, cell pH will be higher than the initial value; that is, there is a small overshoot in pH. As noted earlier, most cell membranes are much more permeable to molecular ammonia, NH3, than to ammonium ions, NH4+.If NH4CI levels in the extracellular fluid increase, ammonia penetrates the cell much more rapidly than ammonium ions. The result is, of course, that arnmonia levels in the cell are increased much more rapidly too. Ammonia equilibrates across the membrane and combines with hydrogen ions to form ammonium ions within the cell, thus raising cell pH (Figure 13-16B).After reaching a maximum, pH starts to fall during prolonged NH4Cl exposure because of a slow passive influx of NH4+along with other acid-base regulating mechanisms in the membrane. The return of the external NH4CIlevel to the original value results in a sharp fall in intracellular pH as NH3 diffuses out of the cell. However, because of the accumulation of intracellular NH,+, cell pH falls below the initial level, but slowly returns to the initial level as NH4+diffuses out of the cell. These mechanisms of pH adjustment are activated by either a reduction in intracellular pH or an increase in extracellular pH. In mammalian cells acid extrusion is reduced to low levels if extracellular pH falls below 7.0 or intracellular pH rises above 7.4. If an acid is injected into a cell, it is extruded from the cell at rates that increase in proportion to the decrease in cell pH. Although a portion of the H+ efflux may be related to H+ diffusion out of the cell, some of the efflux is coupled to sodium influx. This coupling of sodium and proton transport could be due to either a cation-exchangemechanism in the membrane or an electrogenic proton pump that increases membrane potential, thereby providing an electrochemicalgradient for diffusion of Na+ ions through sodium-selectivechannels. For exam-


533

......................................

Extracellular fluid OH-

+ CO,

II

I

20 min

I

Increase in extracellular CO,

Overshoot

7.5 CO, influx CO, efflux 7.0 -

Slow H' efflux

Increased NH,CI in extracellular fluid Figure 13-16 Changes in extracellular carbon dioxide and ammonium chloride levels cause changes in intracellularpH of tissue cells. (A) If CO, levels in the extracellular fluid are suddenly increased, CO, diffuses rapidly into the cell, forming bicarbonate and causing a sharp fall in intracellular pH. A subsequent slow efflux of H+ ions (dashed line) leads to

ple, some cells can actively pump protons out via a proton ATPase in the membrane; this proton efflux can result in a sodium influx. Often acid extrusion is accompanied by chloride efflux, presumably in exchange for extracellular HC03-, which has been shown to be required for pH regulation by cells. For instance, the drug SITS (4-acetamido4'-isothiocyanostilbene-2,2'-disulfonicacid), which blocks chloride-bicarbonate exchange in erythrocytes, also inhibits pH regulation in other cells. Thus both proton-exchange and anion-exchange mechanisms in the cell membrane play an important role in adjusting intracellular pH. An acid load in the cell is accompanied by H+ efflux coupled to Na+ influx and by HC0,influx coupled to C 1 efflux. The movement of HC0,into the cell is equivalent to movement of H+ out of the cell because H C O , ions that enter the cell are converted to CO, ,releasing hydroxyl ions and increasing pH. The CO, so formed, leaves the cell and is converted to bicarbonate, releasing protons. This cycling of CO, and HC03-, referred to as the Jacobs-Stewart cycle, functions to transfer H + ions from the cell interior in the face of an intracellular acid load, such as that generated by anaerobic metabolism (Figure 13-17). In most vertebrate red blood cells, unlike most other cells, hydrogen ions are passively distributed across the

a gradual rise in the intracellular pH. (B) If extracellular NH,CI levels rise sharply, NH, diffuses rapidly into the cell and combines with hydrogen ions to form ammonium ions, which diffuse slowly across the cell membrane (dashed line). As a result, the intracellular pH increases.

Figure 13-17 The Jacob-Stewart cycle is the cycling of carbon dioxide and bicarbonate to transfer acid between the extracellular and intracellular compartments. In a red blood cell, depicted here, the cycle generally operates to transfer acid from the plasma to the cell interior. Because carbonic anhydrase is present only inside cells, the slow, uncatalyzed interconversion of CO, and HC0,- in the extracellularfluid determines the rate of acid transfer.

membrane, and the membrane potential maintains a lower pH inside the red blood cell than in the plasma. A sudden addition of acid to the plasma (e.g., following anaerobic production of H + )results in a fall in erythrocyte

534


.........................................

pH. The acid is transferred from the plasma to the interior of the erythrocyte not by diffusion of H+ ions but by bicarbonate-chloride exchange (seeFigure 13-17).The addition of H + to the plasma causes PCo1to increase due to the conversion of HC0,- to CO,, which then diffuses into the red blood cell and is converted to HC03-, thereby reducing intracellular pH. Bicarbonate then diffuses out of the cell via the chloride-bicarbonate exchange mechanism. Thus in erythrocytes, the Jacobs-Stewart cycle operates to transfer acid from the plasma to the cell interior.

fact, catecholaminesreleased into the blood during periods of metabolic acidosis activate the erythrocyte Na+/H+exchanger, which moves H+ out of and Na+ ions into the cell. In fish with a large muscle mass, burst swimming results in a marked acidosis. This drop in plasma pH, if transferred to the red blood cell, would impair oxygen binding to hemoglobin and reduce the ability of the fish to swim aerobically. This does not happen because erythrocytic pH, in these fish is regulated and remains high during the acidosis following burst-swimming activity.

Factors Influencing lntracellular pH

Factors Influencing Body pH

Intracellular pH will be stable if the rate of acid loading, from metabolism or from influx into the cell, is equal to the rate of acid removal. Any sudden increase in cell acidity will be counteracted by the various mechanisms discussed in previously:

A stable body pH requires that acid production be matched to acid excretion. In mammals, this symmetry is achieved by adjusting the excretion of CO, via the lungs and excretion of acid or bicarbonate via the kidneys, so that acid excretion balances production, which is largely determined by the metabolic requirements of the animal. The collecting duct of the mammalian kidney has A-type (acid-excreting) and B-type (base-excreting)cells, the activity of which can be altered to increase acid or base excretion. In aquatic animals, the external surfaces have the capacity to extrude acid in ways similar to that seen in the collecting duct of the mammalian kidney (see Chapter 14).For instance, the skin of frogs and gills of freshwater fish have a proton ATPase, which excretes protons, on the apical surface of the epithelium. Fish gills also have a HC0,-/CI- exchange mechanism. If these mechanisms are inhibited by drugs, body pH is affected. Temperature can have a marked effect on body pH. The dissociation of water varies with temperature, and the pH of neutrality (i.e., [H+] = [OH-]) is 7.00 only at 25°C. The dissociation of water decreases, and the pH of neutrality (pN) therefore increases, with a decrease in temperature. At 37"C, pN is 6.8, whereas at O°C, it is 7.46. Human plasma at 37°C has a pH of 7.4, so it is slightly alkaline. At pN, the ratio of O H to H + concentrations is 1. This ratio increases with increasing alkalinity; at pH 7.4 at 37°C it is about 20. Most animals maintain almost the same alkalinity in many of their tissues relative to pN independent of the temperature of their bodies (Figure 13-18). Fishes at 5°C have a plasma pH of 7.9-8.0; turtles at 20°C, a plasma pH of about 7.6; and mammals at 37"C, a plasma pH of 7.4. Thus, all have a similar relative alkalinity and the same ratio of OH- to H + ions (about 20) in plasma. Tissues are generally less alkaline than plasma; for example, the pHi of erythrocytes is about 0.2 pH units less than plasma, and the pHi of muscle cells is about 7.0. Temperature also has a marked effect on the pK' of plasma proteins and the CO,/HCO,- system, the pK' increasing as temperature decreases. According to the Henderson-Hasselbalch equation, changes in pK' will cause changes in pH or in the dissociation of weak acids. However, the temperature-induced changes in plasma pH (see Figure 13-18) offset the temperature-dependent changes in the pK' of plasma proteins, so that

Buffering by physical buffers (e.g., proteins and phosphates) located within the cell Reaction of HC0,- with H + ions, forming CO,, which then diffuses out of the cell. Passive diffusion or active transport of H+ ions from the cell Cation-exchange (Nat/H+) or anion-exchange (HC0,-/CI-) mechanisms, or both, in the cell membrane In addition, the generation of protons through metabolism may be modulated by pH. Many enzymes are inhibited by low pH, so that the inhibition of glycolysis (and possibly some other metabolic pathways) at low pH may serve to regulate intracellular pH by reducing the net production of protons during periods of increased acidity in cells. In some instances, cell pH may be modulated to control or limit other cellular functions. It is not always clear if these pH changes are a consequence of, or are regulating, the associated cellular activity. In many cells intracellular pH (pHi) and calcium levels are either inversely or directly related. In other cells they are sequentially, rather than directly, related; in these cases changes in pH, may modulate calcium activity and therefore many of its actions on cellular function. For example, when frog eggs are fertilized, intracellular calcium levels increase transiently, followed by a sustained increase in pHl. There is some evidence to indicate that this alkalinization of the cell may prolong the action of elevated calcium. In a few cases the regulation of intracellular pH (pHl) has a clear effect on cellular function. For example, the erythrocytes of many teleosts have a Na+/H+exchanger and a HC0,-/CI- exchanger in the membrane. The hemoglobin in these animals exhibits a Root shift, that is, a decrease in blood oxygen capacity as blood pH falls (see Figure 13-6). Clearly, this effect would impair oxygen transport by erythrocytes during periods of metabolic acidosis in the absence of some countervailing mechanism. In

GAS E X C H A N G E A N D A C I D - B A S E B A L A N C E 535 ......................................

GAS TRANSFER IN AIR: LUNGS AND OTHER SYSTEMS

Temperature ("C) Figure 13-18 The pH at neutrality (pN) and plasma pH decrease with increasing temperature, but the relationship between the two is constant in most animals. In this graph, the effect of temperature on plasma pH in various turtles, frogs, and fishes is compared with the change in pN. [Adapted from Rahn, 1967.1

/

the extent to which the plasma proteins dissociate remains constant. Because the pK' of the CO, hydration-dehydration reaction changes less with temperature than does blood pH, animals must adjust the ratio of CO, to HC0,- in the blood. In general, it appears that as temperature falls, airbreathing, poikilothermic vertebrates keep bicarbonate levels constant but decrease molecular CO, levels. In aquatic animals, on the other hand, CO, levels remain the same and bicarbonate levels increase as temperature drops. This process results in the same adjustment of the C0,-to-bicarbonate ratio and hence pH in both aquatic and air-breathing vertebrates. The important point is that if body pH changes with temperature in the same way as the pK' of proteins, then the Henderson-Hasselbalch equation predicts the charge on proteins should remain unchanged. If there is little or no change in the net charge on proteins, function will be retained over a wide range of temperatures. The ability of the body to redistribute acid between body compartments is of functional significance because some tissues are more adversely affected by changes in pH than others. The brain is particularly sensitive, whereas muscle can and does tolerate much larger oscillations in pH. As a result, the brain has extensive, if poorly understood, mechanisms for regulating the pH of the cerebrospinal fluid (CSF). In the face of a sudden acid load in the blood, hydrogen ions are taken up by the muscles, reducing oscillations in the blood and protecting the brain and other more sensitive tissues. Hydrogen ions are then slowly released into the blood from muscle and excreted either via the lungs as CO, or via the kidney in acid urine. Thus, when there is a sudden acid load in the body, the muscles can act as a temporary H+reservoir, thus reducing the magnitude of the oscillations in pH in other regions of the body.

The previous sections considered the properties of oxygen and carbon dioxide and described how these gases are carried in the blood and the effect they have on body pH. In this section, we examine the ways in which O2and C 0 2 are transferred between air and blood. Focus is placed on the vertebrate lung, but other gas-transfer systems also are considered. In the next section gas transfer between water and blood across gills is discussed. The structure of a gas-transfer system is influenced by the properties of the medium as well as the requirements of the animal. For example, the lungs of mammals have a very different structure from the gills of fish and are ventilated in a different manner. This dissimilarity exists because, although the density and viscosity of water are both approximately 1000 times greater than that of air, water contains only one-thirtieth as much molecular oxygen. Moreover, gas molecules diffuse 10,000 times more rapidly in air than in water. Thus, in general, air breathing consists of the reciprocal movement of air into and out of the lungs, whereas water breathing consists of a unidirectional flow of water over the gills (Figure 13-19A).The design objectives of fish gills are to minimize diffusion distances in water, creating a thin layer of water over the respiratory surface. These variations in the environment, in the structure of the respiratory apparatus, and in the nature of ventilation result in differences in the partial pressures of gases in the blood and tissues of air-breathing and water-breathing animals, particularly in Pco, (Figure 13-19B).

Functional Anatomy of the Lung The vertebrate lung, which develops as a diverticulum of the gut, consists of a complex network of tubes and sacs, the actual structure varying considerably among species. The sizes of terminal air spaces become progressively smaller in the lungs of amphibians, reptiles, and mammals (in that order), but the total number of air spaces per unit volume of lung becomes greater. The structure of the amphibian lung is variable, ranging from a smooth-walled pouch in some urodeles to a lung subdivided by septa and folds into numerous interconnected air sacs in frogs and toads. The degree of subdivision is increased in riGiles, and increases even more in mammals, the total effect being an increase in respiratory surface area per unit volume of lung. In general, the area of the respiratory surface in mammals increases with body weight and the rate of oxygen uptake (Figure 13-20). Teleost fishes typically have a smaller respiratory surface area than mammals of equivalent body weight. The mammalian lung consists of millions of blindending, interconnected sacs, termed alveoli. The trachea subdivides to form bronchi, which branch repeatedly, leading eventually to terminal bronchioles and finally respiratory bronchioles, each of which is connected to a terminal spray of alveolar ducts and sacs (Figure 13-21).The total

Figure 13-19 The different gas-transfer systems in air-breathing and water-breathing animals are associated with characteristic distribution of respiratory gases in the blood and tissues. (A) Schematic diagrams of 0, and CO, flows in air-breathing and water-breathing animals. (B) Relative values of Po2 and Pcq in the inhalant medium, blood, and tissues in air-breathing (top) and water-breathing (bottom) animals.

Air

1

AIR

\ Blood

+==r Capillaries

c02

Tissue Water

loo

Mammals

10

7

1000

Slope = 1.0

Human Monkey

10

100

1,000 10,000100,000 Weight (g)

Figure 13-20Respiratory surface area increases w~thsize (A) Relationship between resp~ratorysurface area and body weight In selected mammals and teleost fishes. (6) Relat~onshipbetween alveolar surface area

(S. A.) and oxygen uptake in mammals. [Part A adapted from Randall, 1970; part B from Tenney and Ternmers, 1963.1

GAS E X C H A N G E A N D ACID-BASE BALANCE

537

...............................................................................

\

\

\

\

Respiratory bronchiole

bronchiole

Figure 13-21 In the mammalian lung, a series of branching, progressively smaller ducts deliver air to the respiratory portion, consisting of terminal and respiratory bronchioles and alveolar duas and sacs. Gas transfer occurs across the respiratory epithelium shown in red.

cross-sectional area of the airways increases rapidly as a result of extensive branching, although the diameter of individual air ducts decreases from the trachea to the terminal bronchioles. The terminal bronchioles, the respiratory bronchioles, the alveolar ducts, and the alveolar sacs constitute the respiratory portion of the lung. Gases are transferred across the thin-walled alveoli found in the regions distal to the terminal bronchioles, termed acini. The airways leading to the terminal bronchioles constitute the nonrespiratory portion of the lung. Alveoli in adjoining acini are interconnected by a series of holes, the pores of Kohn, allowing the collateral movement of air, which may be a significant factor in gas distribution during lung ventilation (Figure 13-22A). Air ducts leading to the respiratory portion of the lung contain cartilage - and a little smooth muscle and are lined with cilia. The epithelium secretes mucus, which is moved toward the mouth by the cilia. This "mucus escalator"

keeps the lungs clean (see Chapter 8). In the respiratory portions of the lung, smooth muscle replaces cartilage. Contraction of this smooth muscle can have a marked effect on the dimensions of the airways in the lungs. Small mammals have a higher resting 0, uptake rate per unit body weight than large mammals because of their greater alveolar surface area per unit body weight. This increase in respiratory surface area is achieved by a reduction in the size but an increase in the number of alveoli per unit volume of lung. In humans the number of alveoli increases rapidly after birth, the adult complement of about 300 million being attained by the age of eight years; subsequent increases in respiratory area are achieved by increases in the volume of each alveolus. The resting 0, uptake rate per unit weight is higher in children than in adults; once again there is a correlation between uptake per unit weight and alveolar surface area per unit body weight. The diffusion barrier in mammals is made up of an aqueous surface film, the epithelial cells of the alveolus, the interstitial layer, endothelial cells of the blood capillaries, plasma, and the wall of the red blood cell (Figure 13-22B). Several cell types compose the lung epithelium. Type I cells, the most abundant, constitute the major part of the lung epithelium. They are squamous epithelial cells, having a thin platelike structure, a single cell extending into the two adjacent alveoli with the nucleus tucked away in a corner. Type I1 cells are characterized by a laminated body within the cell and have surface villi; type I1 cells produce surfactants, discussed later. Type 111cells are mitochondrion-rich cells with a brush border. These rare cells appear to be involved in NaCl uptake from lung fluid. In addition to these cells, a number of alveolar macrophages wander over the surface of the respiratory epithelium. It is generally assumed, but not demonstrated, that the coefficient of diffusion for gases does not vary in the lungs of different animals, the only structural variables being lung area and diffusion distance between air and blood. The following terms are used to describe different types of breathing and lung ventilation: Eupnea-normal, at rest.

quiet breathing typical of an animal

Hyperventilation and hypoventilation-increase and decrease, respectively, in the amount of air moved in or out of the lung by changes in the rate and/or depth of breathing, such that ventilation no longer matches CO, production and blood CO, levels change

.

Hyperpnea-increased lung ventilation due to increase in breathing in response to increased CO, production (e.g., during exercise) Apneahabsence of breathing Dyspnea-labored breathing associated with the unpleasant sensation of breathlessness Polypnea-increase in breathing rate without an increase in the depth of breathing

Figure 13-22 During ventilation of the mammalian lung, the respiratory gases move to and from the alveolar space and blood in the pulmonary capillaries. (A)Three interalveolar septa of dog lung meeting at junction line. Connective tissue fibers lie in the central plane, forming a continuous tensile network with which the capillary network is interwoven. Endothelial cellsand type I epithelial cellsform the lining ofthe thin air-blood barrier. Pores of Kohn connect alveoli. (B) Dimensions and structure of the alveolar capillary membrane. [Part A adapted from Weibel, 1973; part B from Hildebrandt and Young, 1965.1

A

Alveolus

Capillary

Alveolus

B

Alveolus

-

lining (0.01 ~ r n )

Alveolar diameter (50-300 pm)

5 ~m

Air exchanged between the alveoli and the environment must pass through a series of tubes (trachea, bronchi, nonrespiratory bronchioles) not directly involved in gas transfer. At the end of exhalation the air contained in these tubes will have come from the alveoli and will be low in oxygen and high in carbon dioxide. This air will be the first to move back into the alveoli at the next breath. At the end of inhalation the nonrespiratory tubes will be filled with fresh air and this volume will be the first to be exhaled with the next breath. Thus this volume is not involved in gas transfer and, therefore, is referred to as the anatomical dead space volume. Some air may be supplied to nonfunctional alveoli, or certain alveoli may be ventilated at too high a rate, increasing the volume of air not directly involved in gas exchange. This volume of air, termed the physiological dead space, is usually greater than, but includes, the anatomical dead space (Spotlight 13-3). The amount of air moved in or out of the lungs with each breath is referred to as the tidal volume. The amount of fresh air moving in and out of the alveolar air sacs equals the tidal volume minus the anatomical dead-space volume, and is referred to as the alveolar ventilation volume. Only this gas volume is directly involved in gas transfer. The lungs are not completely emptied even at maximal expira-

space (0.02-0.2 pm)

tion, leaving a residual volume of air in the lungs. The maximum volume of air that can be moved in or out of the lungs is referred to as the vital capacity of the lungs. These and other terms used to describe various volumes and capacities associated with lung function are illustrated in Figure 13-23. The 0, content is lower and the CO, content is higher in alveolar gas than in ambient air because only a portion of the lungs' gas volume is changed with each breath. Alveolar ventilation in humans is about 350 ml, whereas the functional residual volume of the lungs exceeds 2000 ml. During inspiration the ducts leading to the alveoli elongate and widen, causing an increase in acinar volume. During breathing, air moves in and out of the acinus and may also move between adjacent alveoli through the pores of Kohn. Mixing of gases in the ducts and alveoli occurs by diffusion and by convection currents caused by breathing (Figure 13-24 ). In the alveolar ducts, 0, diffuses toward the alveoli and CO, away from them. Partial pressures of 0, and CO, are probably fairly uniform across the alveoli, because diffusion is rapid in air and the distances involved are small. The partial pressures of gases within the alveoli oscillate in phase with the breathing movements, the magnitude depending on the extent of tidal ventilation. ~

-

-

-

539 GAS E X C H A N G E A N D A C I D - B A S E B A L A N C E ...............................................................................

-

Maximum inspiratory level reserve

Figure 13-23 Numerous terms are used to describe various volumes and capacities associated with lung function. The t~dalvolume is the volume of air typically moved In and out of the lung, whereas the vital capacity is the maximum volume.

Vital capacity Resting endinspiratory -level

-

Resting endexpiratory level

f

I

~unctional

C

Maximum

Total expiratory volume

I

Primary subdivision of lung volume

lung capacity

level

Special divisions for pulmonary function tests

The 0, and CO, levels in alveolar gas are determined by both the rate of gas transfer across the respiratory epithelium and the rate of alveolar ventilation. Alveolar ventilation depends on breathing rate, tidal volume, and anatomical dead-space volume. Variations in the magnitude of the anatomical dead space will alter gas tensions in the alveolus in the absence of changes in tidal volume.

Thus, artificial increases in anatomical dead space, produced in human subjects breathing through a length of hose, result in a rise in C 0 2and a fall in O2 in the lungs. As discussed in a later section, these changes activate chemoreceptors, leading to an increase in tidal volume. In animals with long necks (e.g., the giraffe and trumpeter swan), the tracheal length and therefore the anatomical dead space is greater than in those with short necks (Figure 13-25).In order to maintain adequate gas tensions in the lungs, longnecked animals have increased tidal volumes. Breathing rate and tidal volume vary considerably in animals. Humans breathe about 12 times per minute and have a tidal volume at rest of about 10% of total lung volume. Such relatively rapid, shallow breathing produces small

Figure 13-24 The direction of airflow (largearrows) in the respiratory portions of the lung changes during inspiration and expiration, but diffusion of oxygen (small arrows) is always towards the alveoli walls.

540


...............................................................................

Figure 13-25 The extremely long trachea of the trumpeter swan results in a large anatomical dead-space volume. For comparison, see Figure 13-29 illustrating the length of the human trachea. 1From Banko, 19Ml.l

oscillations in Pol in the lung and blood. In contrast, the exclusively aquatic but air-breathing amphibian Amphiurna, which lives in swamp water, rises to the surface of the water about once each hour to breathe; its tidal volume, however, is more than 50% of its lung volume. This large tidal volume, coupled with infrequent breathing, produces large, slow oscillations in Po, in the lung and blood, which are more or less in phase with the breathing movements (Figure 13-26). Amphiuma is preyed on by snakes and is most vulnerable when it rises to breathe. Because it lives in water of low oxygen content, aquatic respiration is not a suitable alternative. The hazard of being eaten while surfacing to breathe may have influenced the evolution of its very low breathing rate, its large tidal and lung volume, and its ability to make cardiovascular adjustments that help maintain 0, delivery to the tissues in the face of widely oscillating blood gas levels. Carbon dioxide levels in Amphiuma do not oscillate in the same way as oxygen because carbon dioxide is lost across the skin and is not so dependent on lung ventilation. In summary, 0, and CO, levels in alveolar gas are determined by ventilation and the rate of gas transfer. Ventilation of the respiratory epithelium is determined by breathing rate, tidal volume, and anatomical dead-space volume. The nature and extent of ventilation also influences the magnitude of oscillations in 0, and CO, in the blood during a breathing cycle. Pulmonary Circulation

The lung, like the heart, receives blood from two sources. The major flow is of deoxygenated blood from the pulmonary artery that perfuses the lung, taking up 0, and giving up C0,; this is termed the pulmonary circulation. A second, smaller supply, the bronchial circulation, comes from the systemic (body)circulation and supplies the lung tissues with 0, and other substrates for growth and maintenance. Our discussion here is confined to the pulmonary circulation.

Time (min) Figure 13-26 Breathing frequency tends to vary inverselywith tidal volume and magnitude of oscillat~onsi v o 2 .In Amphiurna, an aquatic, airbreathinq amphibian that breathesSiafrequ'ently, tidal volume and changes in Po, are large. Shown here are pbb of blood pressure, heart rate, P021and Pco2 in a 515-9 Amphiuma during two breathing-diving cycles. Vertical arrows indicate when the animal surfaced and ventilated its lungs. Note that blood pressure, heart rate, and Pco, are nearly constant between breaths, whereas Po, shows large, slow oscillations in the lung and blood. [Adapted from Toews et al., 1971.]

In birds and mammals, pressures in the pulmonary circulation are lower than in the systemic circulation. This pressure difference reduces filtration of fluid into the lung. An extensive lymphatic drainage of lung tissues also helps ensure that no fluid collects in the lung (see Chapter 12). These features are important because any fluid that collects at the lung surface increases the diffusion distance between blood and air and reduces gas transfer. Blood flow through the pulmonary circulation is best described as sheet flow-that is, flow of a liquid between two parallel surfaces. This contrasts with the laminar flow characteristic of flow through a tube and flow in the systemic circulation. The pulmonary capillary endothelium resembles two parallel surfaces, joined by pillar-like structures, with blood flowing between them. As pressure increases, the parallel surfaces move apart, leading to an increase in the thickness of the blood sheet. That is, pressure increases the thickness of the blood sheet rather than spreading out the flow in other directions. The mean arterial pressure in the human lung is about 12 mm Hg, oscillating between 7.5 mm Hg and 22 mm Hg with each contraction of


541

............................................................................... S P O T L I G H T 13-3

and

LUNG VOLUMES

PEC02X VT = (PACOZX VT) - (PAC02X V ),

The alveolar ventllatlon volume, V ,, equals the difference between the tldal ventilation volume, VT, and the dead-space volume, VD: VA = VT -

+ (PICOPx V),

By rearrangement

v,

If fdenotes breathing frequency, the volume of air moved in and out of the lung each minute, VAf, is called the alveolar minute volume, or respiratoryminute volume, symbolized as The dot over the Vindicates a rate function. ,,, is the volume of the nonThe anatomical dead space,,,,V respiratory portion of the lung; the physiologicaldead space, .V, ,ol,,,, is the volume of the lung not involved in gas transfer. If the partial pressure of CO, in expired air is denoted by PECO,, the partial pressure of CO, in alveolar air by PACOP,and the partial pressure of CO, in inspired air by PICOP,then

g. But PICO, approaches zero, and PACO, is the same as the partial pressure of CO, in arterial blood, P,CO,. So the last expression can be written as follows:

PEC02X VT = PACOZ(VT - VD) + (PICO2X V ),

Thus the physiological dead space of the lungs can be calculated from measurements of tidal volume, V,, and the CO, partial pressures in arterial blood, P,CO, and expired air, PEC02.

the heart. In the vertical (upright)human lung, arterial pressure is just sufficient to raise blood to the apex of the lung; hence flow is minimal at the top and increases toward the base of the lung (Figure 13-27).Blood is distributed more evenly to different parts of the horizontal lung.

The pulmonary vessels are very distensible and subject to distortion by breathing movements. Small vessels within the interalveolar septa are particularly sensitive to changes in alveolar pressure. The diameter of these thin-walled collapsible capillaries is determined by the transmural pressure

,, so substituting into this equation, we obtain But VA = V, - V

Apex

I

PA > pa

> P"

Blood flow

Figure 13-27 In the upper portion of the vertical lung, the diameter of alveolar capillaries, and hence blood flow through them, depends on the difference between the arterial pressure, Pa,and the alveolar pressure, PA.In this schematic diagram of blood flow in the vertical human lung, the boxes represent the condition of vessels in the interalveolar septum in different portions of the lung. At the apex of the lung, PAoften exceeds Pa;as a result the capillaries collapse and blood flow ceases. Pv is the venous pressure. [Adapted from West, 1970.1

(arterial blood pressure within capillaries, Pa,minus alveolar pressure, PA). If the transmural pressure is negative (i.e., PA> Pa),these capillaries collapse and blood flow ceases. This collapse may occur at the apex of the vertical human lung, where Pa is low (see Figure 13-27).If pulmonary arterial pressure is greater than alveolar pressure, which in turn is greater than pulmonary venous pressure, then the difference between arterial and alveolar pressure will determine the diameter of capillaries in the interalveolar septa and, in the manner of a sluice gate, control blood flow through the capillaries. Venous pressure will not affect flow into the venous reservoir as long as alveolar pressure exceeds venous pressure. Flow in the upper portion of the vertical lung is probably determined in this way by the difference between arterial blood pressure and alveolar pressure. Arterial blood pressure (and therefore blood flow) increases with distance from the apex of the lung. In the bottom half of the vertical lung, where venous pressure exceeds alveolar pressure, blood flow is determined by the difference between arterial and venous blood pressures. This pressure difference does not vary with position, although both the arterial and venous pressures increase toward the base of the lung. This increase in absolute pressure results in an expansion of vessels and, therefore, a decrease in resistance to flow. Thus, flow increases toward the base of the lung, even though the arterial-venous pressure difference does not change (seeFigure 13-27).The position of the lungs with respect to the heart is therefore an important determinant of pulmonary blood flow. The lungs surround the heart, thus minimizing the effect of gravity on pulmonary blood flow as an animal changes from a horizontal to vertical position. This close proximity of lungs and heart within the thorax also has significance for cardiac function: the reduced pressures within the thorax during inhalation aid venous return to the heart. This is often referred to as the thoraco-abdominal pump. Even though the mammalian pulmonary circulation lacks well-defined arterioles, both sympathetic adrenergic and parasympathetic cholinergic fibers innervate the smooth muscle around the pulmonary blood vessels and bronchioles. The pulmonary circulation, however, has much less innervation than does the systemic circulation and is relatively unresponsive to nerve stimulation or injected drugs. Sympathetic nerve stimulation or the injection of norepinephrine causes a slight increase in resistance to blood tlowv, whereas parasympathetic nerve stimulation or acetylcholine has the opposite effect. Reductions in either oxygen levels or pH cause local vasoconstriction of pulmonary blood vessels. The vasoconstrictor response to low oxygen, which is the opposite to that observed in systemic capillary networks, ensures that blood flows to the well-ventilated regions of the lung. Poorly ventilated regions of the lung will have low alveolar oxygen levels, causing a local vasoconstrict~onand therefore a reduction in flow to that area of the lung' ternativel~,a well-ventllated area of the lung will have high alveolar oxygen levels, so the local blood vessels will be di-

A

lated and blood flow will be high. Although pulmonary hypoxic vasoconstriction is important in directing blood flow to well-ventilated regions of the lung, it leads to problems when animals are exposed to general hypoxia, as may occur at high altitudes (see later section). Cardiac output to the pulmonary circuit is identical to cardiac output in the systemic circuit in mammals and birds. In amphibians and reptiles, with a single or partially divided ventricle that ejects blood into both the pulmonary and the systemic circulation, the ratio of pulmonary to systemic blood flow can be altered. In turtles and frogs, there is a marked increase in blood flow to the lung following a breath due to pulmonary vasodilation. During periods between breaths in the frog Xenopus, pulmonary blood flow decreases, but systemic blood flow is hardly changed possibly because the ventricle is undivided (Figure 13-28). These animals breathe intermittently, and variable blood flow to the gas exchanger, independent of blood flow to the rest of the body, permits some control of the rate of oxygen use from the lung store and rapid renewal of blood oxygen stores during ventilation. In addition, cardiac work is reduced during apnea. Ventilation of the Lung

The mechanism of lung ventilation varies considerably among animals. These variations reflect differences in the functional anatomy of the lungs and associated structures. First we will see how the mammalian lung is ventilated and then consider ventilation in birds, reptiles, frogs, and invertebrates.

m Breathing movements, buccal cavity

Blood flow, right pulmocutaneous

45 E 30E

l5

B I O O ~pressure, left pu~mocutaneous

Time (min)

-

,

L

I

I

0.1 [

0 B I O O ~ flow, left systemic

Blood pressure, r ~ g h systemic t Figure 13-28 Pulmonary blood flow ~ Y P I C ~increases, ~ ~ Y whereas s ~ s temlc flow remalns constant, follow~ngbreathing In turtles and frogs. These traces from the frogxenopus record pressure changes In the buccal caaty produced by lung-ventlatlng movements of the buccal floor (upped, as well as the corresponding flow and pressure In the arter~al arches. [From Shelton, 1970.1


543

............................................................................... Mammals The lungs of mammals are elastic, multi-chambered bags, which are suspended within the pleural cavity and open to the exterior via a single tube, the trachea (Figure 13-29). The walls of the pleural cavity, often referred to as the thoracic cage, are formed by the ribs and the diaphragm. The lungs fill most of the thoracic cage, leaving a lowvolume pleural space between the lungs and thoracic wall; this space is sealed and fluid filled. Because of their elasticity, isolated lungs are somewhat smaller than they are in the thoracic cage. In situ this elasticity creates a pressure below atmospheric in the fluid-filled pleural space. The fluid in the pleural cavity provides a flexible, lubricated connection between the outer lung surface and the thoracic wall. Fluids are essentially incompressible, so when the thoracic cage changes volume, the gas-filled lungs do too. If the thoracic cage is punctured, air is drawn into the pleural cavity and the lungs collapse-a condition known as pneumothorax. When intact lungs are filled to various volumes and the entrance is closed with the muscles relaxed, then alveolar pressure varies directly with lung volume. At low pulmonary volumes, alveolar pressure is less than ambient pressure owing to the resistance of the thorax to collapse, whereas at high pulmonary volumes, alveolar pressure exceeds ambient pressure because of the forces required to expand the thoracic cage. If lung volume is large, then once the mouth and glottis are opened, air will flow out of the lungs because the weight of the ribs will reduce pulmonary volume. At some intermediate volume, Vr, alveolar pressure in the relaxed thorax is equal to ambient pressure (Figure 13-30). During normal breathing, the thoracic cage is expanded and contracted by a series of skeletal muscles, the di-

I

I

Residual volume

Alveolar pressure - ambient pressure (mm Hg)

bronchus Right lobe

"k

Segment bronchi dight lower lobe

\ Diaphragm

"

\

eft lower lobe

Figure 13-29 In mammals, the lungs fill most of the thoracic cavity, formed by the ribs and diaphragm. The right lung has three lobes, and the left lung, two lobes, in humans. The low-volume pleural space between the lungs and thoracic wall is fluid filled and sealed.

aphragm, and the external and internal intercostal muscles. Contractions of these muscles are determined by activity of motor neurons controlled by the respiratory center within the medulla oblongata, which we discuss later. The volume of the thorax increases as the ribs are raised and moved outward by contraction of the external intercostals and by contraction (and therefore the lowering) of the diaphragm

Figure 13-30 During quiet breathing with the thoracic muscles relaxed, the alveolar and ambient pressures are equal between breaths. This plot shows the relationship between lung volume and pressure within the thorax when muscles are relaxed, but the glottis is closed. V, is the lung volume when alveolar pressure is the same as ambient pressure and the lung-chest system is relaxed. The points Iand E represent the pressure and volume of the system following inspiration and expiration during quiet breathing.

544 I N T E G R A T I O N O F PHYSIOLOGICAL SYSTEMS ......................................... (Figure 13-31A). Contractions of the diaphragm account for up to two-thirds of the increase in pulmonary volume. The increase in thoracic volume reduces alveolar pressure, and air is drawn into the lungs. Relaxation of the diaphragm and external intercostal muscles reduces thoracic volume, thereby raising alveolar pressure and forcing air out of the lungs (Figure 13-31B).During quiet breathing, pulmonary volume between breaths is at an intermediate value, V,, at which the alveolar and ambient pressures are equal (see Figure 13-30).Under these conditions exhalation often is passive, simply due to relaxation of the diaphragm and external intercostals. With increased tidal volume, expiration becomes active, owing to contraction of the internal intercostal muscles, which further reduces thoracic volume until it drops below V, at the end of expiration. Birds In birds, gas transfer takes place in small air capillaries (10 pm in diameter) that branch from tubes called parabronchi (Figure 13-32). The functional equivalent of mammalian alveolar sacs, parabronchi are a series of small tubes extending between large dorsobronchi and ventrobronchi, both of which are connected to an even larger A

Inhalation

CJL-, Vertebral column Rib movement

Rib

1

Diaphrag

intercostal

Extdrnal intercostal

Exhalation

P-7

Internal intercostal ~xternal intercostal i J

t~ movement

Figure 13-31 The volume of the thorax increases during inhalation (A) and decreases during exhalation (B) in mammals due to movement of the ribs and diaphragm.

tube, the mesobronchus, which joins the trachea anteriorly (Figure 13-33A). The parabronchi and connecting tubes form the lung, which is contained within a thoracic cavity. A tight horizontal septum closes the caudal end of the thoracic cage. The ribs, which are curved to prevent lateral compression, move forward only slightly during breathing; as a result, the volume of the thoracic cage and lung changes little during breathing. The large flight muscles of birds are attached to the sternum and have little influence on breathing. Although there is no mechanical relation between flight and respiratory movements in birds, "in phase" flight and breathing movements may result from synchronous neural activation of the two groups of muscles involved. How, then, is the avian lung ventilated?The answer lies in the associated air-sac system connected to the lungs (see Figure 13-32).As these air sacs are squeezed, air is forced through the parabronchi. The system of air sacs, which extend as diverticula of the airways, penetrates into adjacent bones and between organs, reducing the density of the bird. Of the many air sacs, only the thoracic (cranial) and abdominal (caudal) sacs show marked changes in volume during breathing. Volume changes in the air sacs are achieved by a rocking motion of the sternum against the vertebral column and by lateral movements of the posterior ribs. Air flow is bidirectional in the mesobronchus, but unidirectional through the parabronchi (Figure 13-33B). During inspiration, air flows into the caudal air sacs through the mesobronchus; air also moves into the cranial air sacs via the dorsobronchus and the parabronchi. During expiration, air leaving the caudal air sacs passes through the parabronchi and, to a lesser extent, through the mesobronchus to the trachea. The cranial air sacs, whose volume changes less than that of the caudal air sacs, are reduced somewhat in volume by air moving from the cranial sac via the ventrobronchi to the trachea during expiration. Oxygen diffuses into the air capillaries from the parabronchi and is taken up by the blood. The air in the parabronchi is changed during both inspiration and expiration, enhancing gas transfer in the bird lung. The unidirectional flow is achieved not by mechanical valves but by aerodynamical valuing. The openings of the ventrobronchi and dorsobronchi into the mesobronchus show a variable, direction-dependent resistance to air flow. The structure of the openings is such that eddy formation, and therefore resistance to flow, varies with the direction of air flow. Reptiles The ribs of reptiles, like those of mammals, form a thoracic cage around the lungs. During inhalation, the ribs are moved cranially and ventrally, enlarging the thoracic cage. As this expansion reduces the pressure within the cage below atmospheric pressure, and the nares and glottis are open, air flows into the lungs. Relaxation of muscles that enlarge the thoracic cage releases energy stored in stretching the elastic component of the lung and body wall, al-


545

......................................

Lungs

Cervical sacs

lnterclavicular

Anterior thoracic sac

Figure 13-32 In bird lungs, gas exchange occurs in air capillaries extending from parabronchi, small tubelikestructuresthat are the functional equivalent of alveoli in mammals. The parabronchi (right) and connect-

ing tubes form the lung. During breathing, volume changes occur in the associated air sacs, not in the thoracic cage and lungs. [Photograph courtesy of H. R. Duncker.]

lowing passive exhalation. Although reptiles do not possess a diaphragm, pressure differences between the thoracic and abdominal cavities have been recorded, indicating at least a functional separation of these cavities. In tortoises and turtles, the ribs are fused to a rigid shell. The lungs are filled by outward movements of the limb flanks andlor the plastron (ventral part of the shell) and by forward movement of the shoulders.The reverse process results in lung deflation. As a result, retraction of limbs and head into the shell leads to a decrease in pulmonary volume.

the buccal cavity with the nares open and glottis closed; then the nares are closed, the glottis is opened, and the buccal floor raised, forcing air from the buccal cavity into the lungs (Figure 13-34).This lung-filling process may be repeated several times in sequence. Expiration also may be a step process, the lungs releasing air in portions to the buccal cavity. Expiration may not be complete, so that some of the air from the lung is mixed with ambient air in the buccal cavity and then pumped back into the lungs. That is, a mixture of pulmonary air, presumably low in 0, and high in CO,, is mixed with fresh air in the buccal cavity and returned to the lungs. The reason for this complex method of lung ventilation is not clear, but it may be directed toward reducing oscillations in CO, levels in the lungs in order to stabilize and regulate blood Pco2 and control blood pH.

Frogs In frogs, the nose opens into a buccal cavity, which is connected via the glottis to paired lungs. The frog can open and close its nares and glottis independently. Air is drawn into

B

Parabronchi

Dorsobronchi

Abdominal sac

Cervlcal sac

r,-

lnterclavicular

sac

.-L

/

/'ble~-

~ r a c h ~ a bronchus

\u' )

~ d s tsac thoracic Trachea

Prethoracic sac Figure 13-33 Squeezing of airs sacs forces air through the parabronchi avian bronchial tree and associated air sacs. The air in bird lungs. ( A ) T ~ & sacs of the cranial group (cervical, interclavicular, and prethoracic sacs) depart from the three cranial ventrobronchi, whereas the air sacs of the caudal group (postthoracic and abdominal sacs) are connected directly

to the mesobronchus. (B) Schematic diagram of airflowthrough the bird lung. Flow in the parabronchi is unidirectional.Solid arrows represent flow during inspiration; open arrows, flow during expiration. [Adapted from Scheid et al., 1972.1

,

In aquatic snails, the lungs serve to reduce the animal's density.

Buccal movement

Buccal plus lung movement

Buccal volume

Open glottis

-

losed glottis

Nares closed

-

pressure

0.5 s

Time

Figure 13-34 Ventilation in the frog is a stepwise process. Shown here are pressure and volume changes in the buccal cavity and lung of a frog during buccal movements alone with the glottis closed and during buccal and lung movements with the glottis open and the nares closed (i.e., lung filling). [Adapted from West and Jones, 1975.1

Invertebrates Invertebrates exhibit a variety of gas-transfer mechanisms. Ventilation does not occur in some invertebrates, which rely only on diffusion of gases between the lung and the environment. In spiders, which have paired ventilated lungs on the abdomen, the respiratory surface consists of a series of thin, blood-filled plates that extend like the leaves of a book into a cavity guarded by an opening (spiracle).The spiracle can be opened or closed to regulate the rate of water loss from these "book lungs." Snails and slugs have ventilated lungs that are well-vascularized invaginations of the body surface, the mantle cavity. The volume change that the snail lung is capable of undergoing enables the animal to emerge from and withdraw into its rigid shell. When the snail retracts into its shell, the lungs empty, a situation similar to that seen in tortoises.

Pulmonary Surfactants The lung wall tension depends on the properties of the alveolar wall and the surface tension at the liquid-air interface. Surface tension is the force that tends to minimize the area of a liquid surface, causing liquid droplets to form a sphere. It also makes a surface film resistant to stretch, so that work must be done to stretch a liquid surface. Because the alveoli are so compliant, the surface tension of their liquid lining contributes about 70% of the lung wall resistance to stretch. If the liquid lining was just water, the alveolar wall tension would be much higher than in fact it is, and large forces would be required to inflate the lung and to separate membranes glued together by surface tension. The explanation for the relatively low surface tension of the liquid lining the lungs is the presence of surfactants, lipoprotein complexes that bestow a very low surface tension on the liquid-air interface. Lung surfactants not only reduce the effort associated with breathing but also help prevent collapse of alveoli. Surfactants are produced by type I1 cells within the alveolar lining and have a half-life of about 12 hours in mammals. The predominant lipid in these lipoprotein complexes is dipalmitoyl lecithin. The lipoprotein film is stable, the lipid forming an outer monolayer firmly associated with the underlying protein layer. Synthesis of surfactants requires cortisol, and their release can be stimulated by sighing. Surfactants are found in the lungs of amphibians, reptiles, birds, and mammals, and they may be present in some fishes that build bubble nests. The small dimensions of the fragile alveolar sacs create mechanical problems that might cause them to collapse. To understand why alveolar collapse is a problem, and how surfactants counteract it, consider a tiny bubble that is alternately inflated and then deflated. As discussed in Chapter 12, Laplace's law states that the pressure differential between the inside and outside of a bubble is proportional to 2y/R, where y is the wall tension per unit length and R the radius of the bubble. If two bubbles have a similar wall tension but a different radius, the pressure in the small bubble will be higher than that in the large bubble. As a result, if the bubbles are joined, the small bubble will empty into the large bubble (Figure 13-35A,B). A somewhat similar situation exists in the lung. We can consider the alveoli as a number of interconnected bubbles. If the wall tension is similar in alveoli of different size, the small alveoli will tend to collapse, emptying into the larger alveoli. This normally does not occur in the lung for two reasons: surrounding tissue helps prevent overexpansion of alveoli, and the properties of the alveolar surfactant lining are such that wall tension increases when the surface film is expanded and decreases when it is compressed. This occurs because the film expands as alveolar volume increases, so the surfactant spreads out and therefore is less effective in

GAS E X C H A N G E A N D A C I D - B A S E B A L A N C E 547 ......................................

lowering surface tension (Figure 13-392).The result of this effect is to minimize pressure differences between large and small alveoli, thus reducing the chance of collapse. Alveoli fold as their volume decreases, and the regions between the folds come to have a thick layer of surfactant. The very low surface tension of this thick surfactant layer permits easy inflation of collapsed and folded alveoli. If only water were present in the folds, large forces would be required to separate the layers and inflate the alveoli. In mammals, surfactants appear in the fetal lung prior to birth, thereby reducing the forces required to inflate the lungs of the newborn. Newborns who produce no lung surfactants cannot inflate their lungs at birth without assistance. This condition, referred to as newborn respiratory distress syndrome, occurs primarily in premature babies. Assistance can be given to the baby by forcing air into the lungs, using positive pressure ventilation, and by surfactant replacement. In addition, pregnant women who are likely to have a premature birth can be given an injection of cortisol during gestation to stimulate surfactant production in the fetus. Heat and Water Loss across the Lung

Increases in lung ventilation not only increase gas transfer but also result in more loss of heat and water. Thus, the evolution of lungs has involved some compromises. Air in contact with the respiratory surface becomes saturated with water vapor and comes into thermal equilibrium with the blood. Cool, dry air entering the lung of mammals is humidified and heated. Exhalation of this hot, humid air results in considerable loss of heat and water, which will be proportional to the rate of ventilation of the lung surface. Many air-breathing animals live in very dry environments, where water conservation is of paramount

A

Two bubbles in air

B

importance. It is therefore not surprising that these animals in particular have evolved means of minimizing the loss of water. The rates of heat and water loss from the lung are intimately related. As air is inhaled, it is warmed and humidified by evaporation of water from the nasal mucosa. Because the evaporation of water cools the nasal mucosa, a temperature gradient exists along the nasal passages. The nose is cool at the tip, increasing in temperature toward the glottis. As the moist air leaving the lung is cooled, water . condenses on the nasal mucosa, since the water vapor pressure for 100% saturation decreases with temperature. Thus the cooling of exhalant air in the nasal passages results in the conservation of both heat and water. The blood circulation to the nasal mucosa is capable of supplying water to saturate the inhalant air, but the temperature gradients established by water evaporation and air movement are not destroyed by the circulation. The structure of the nasal passages in vertebrates is variable, and to some extent it can be correlated with the ability of animals to regulate heat and water loss. Humans have only a limited ability to cool exhaled air, which is saturated with water vapor and is at a temperature only a few degrees below core body temperature. Other animals have longer and narrower nasal passages for more effective wa. ter conservation, as we will discuss in Chapter 14. Poikilotherms such as reptiles and amphibians, whose body temperatures adjust to the ambient temperature, exhale air saturated with water at temperatures about 0.5- l.O°C below body temperature. Pulmonary air temperatures and body surface temperatures are often slightly below ambient because of the continual'evaporation of water. In some reptiles, however, body temperature is maintained above ambient. In the iguana, heat and water loss is

Bubbles joined

C

Properties of surfactant

Laplace's law

I Decreas~ngth~ckness of surfactant film Figure 13-35 The presence of surfactant in the lungs helps prevent alveolar collapse. (A) Laplace's law states that the pressure (P) in a bubble decreases with increased radius (R) if the wall tension (yj remains constant. Thus, if two bubbles have the same wall tension but the radius of one is twice that of the other, the pressure in the small bubble is two times that of the large bubble. The equation is written 4ylR rather than 2ylR because the bubble in air has an inner and an outer surface. (B) If the bubbles are joined, the small bubble with the higher pressure collapses into

'

the large bubble with the lower pressure (C)The tendency of small alve011to collapse Into larger alveoli In the lung IS amellorated by a surfactant l ~ n ~ nAs g the surfactant film expands w ~ t hthe alveolus, the th~cknessof the film decreases and the surface tens~onIncreases Because the surface tens~onis a major component of the wall tenslon, th~seffect tends to mlnlmlze pressure differences between alveol~of different slzes, thereby stab~lrz~ng them

548


.........................................

controlled in a manner similar to that observed in mammals. In addition, this lizard conserves water by humidifying air with water evaporated from the excretory fluid of the nasal salt glands. The rate of water loss is closely correlated with lung ventilation and, therefore, oxygen uptake. Reptiles generally have much lower oxygen requirements than mammals or birds, and so their rate of water loss is much less. Gas Transfer in Bird Eggs

The shells of bird eggs have fixed dimensions but contain an embryo whose gas-transfer requirements increase by a factor of lo3between laying and hatching. Thus, the transfer of 0, and CO, must take place across the shell at ever-increasing rates during development while the dimensions of the transfer surface (eggshell)do not change. Gases diffuse through smabl air-filled pores in the eggshell and then through underlying membranes, including the chorioallantoic membrane (Figure 13-36A).The chorioallantoiccirculation is in close apposition to the eggshell and increases with the development of the embryo. Several factors contribute to the increase in gas-transfer rates during development in the bird's egg: development of an underlying circulation in the chorioallantoic membrane, an increase in blood

Figure 13-36 Duringdevelopment of a bird embryo, gas transfer increases across the eggshell even though the shell structure does not change. (A) Diagram of the diffusion pathway between air and chick embryo blood across the eggshell in the region of the air cell. (6) Plot of Po2versus incubation age in the air cell and allantoic venous blood. (C) Plot of Pco, versus incubationage in the air cell and allantoic venous blood. There is no Pco, difference between air-cell gas and allantoicvenous blood, whereas there is a Po, that increases during development of a chick embryo. [Adapted from Wangensteen, 1972.1

A

co2

0 2

\

Ambient

Allantoic artery

Allantoic vein '

flow and volume, an increase in hematocrit and blood oxygen affinity, and an increase in the Po> difference across the eggshell (Figure 13-36B).The eggshell, once produced, does not change during the development of the embryo. Water is lost from the egg during development, causing a gradual enlargement of an air space within the egg. The volume of this air cell is as much as 12 ml at hatching in the chicken egg. Just before they hatch, birds ventilate their lungs by poking their beaks into the air cell. Blood PCO2 is initially low in the embryo, but it gradually rises to about 45 mm Hg just before hatching (Figure 13-36C). This pressure is maintained after hatching, thus avoiding any marked acid-base changes when the bird switches from the shell to its lungs for gas exchange. The shell and underlying membranes, representing the barrier between ambient air and embryonic blood, can be divided into an outer gas phase (the air cell) and an inner liquid phase. At sea level the outer gas phase represents about 30%-40% of the total diffusive resistance to oxygen transfer, 85% of that to carbon dioxide, and 100% of that to water vapor. Eggs at altitude are exposed to a reduction in both oxygen and total gas pressure. The rate of diffusion of gases increases with a reduction in total pres-

capillaries

B

150

- - - - - - - - - - -Ambient - - - - - -air -- - -----

Allantoic venous blood (oxygenated)

Incubation age (days)

lncubation age (days)


549

........................................ sure. The reduced oxygen pressure at altitude is partially offset by increased rates of oxygen diffusion in the gas phase; despite this, eggs become hypoxic at altitude. If eggs are kept in a hypoxic environment for a period of time, more capillaries develop in the chorioallantoic membrane, increasing oxygen diffusing capacity and offsetting the effects of altitude on oxygen transfer across the eggshell. Because carbon dioxide and water vapor also diffuse more rapidly at the reduced pressures associated with altitude, eggs at altitude also have a reduced blood PCO2and lose water more rapidly than those at sea level. Thus conditions affecting diffusion rates have a marked effect on CO, and water loss b; eggs, and rates of water loss increase markedly in eggs exposed to reduced pressures. The properties of the shell are determined by the adult when the egg is laid. It appears that some birds can reduce the effective pore area of their eggs when acclimated to altitude. Insect Tracheal Systems The system that insects have evolved for transferring gases between the tissues and the environment differs fundamentally from that found in air-breathing vertebrates. The insect tracheal system takes advantage of the fact that oxygen and carbon dioxide diffuse 10,000 times more rapidly in air than in water, blood, or tissues. Tracheal systems consist of a series of air-filled tubes that penetrate from the body surface to the cells, acting as a pathway for the rapid movement of 0, and CO,, thereby avoiding the need for a circulatory system to transport gases between the respiratory surface and the tissues. These tubes, or tracheas, are invaginations of the body surface; thus their wall structure is similar to that of the cuticle. Except in a few primitive forms, the tracheal entrances, called spiracles, can be adjusted to control air flow into the tracheas, regulate water loss, and keep out dust. The bug Rhodnius, for example, dies in three days if its spiracles are kept open in a dry environment. The tracheas branch everywhere in the tissues; the smallest, terminal branches, or tracheoles, are blind-ending and poke between and into individual cells (without disrupting the cell membrane), delivering 0,to regions very close to the mitochondria. Air sacs commonly are located at various intervals throughout the tracheal system; these sacs enlarge tracheal volume and therefore oxygen stores, and sometimes reduce the specific gravity of organs, either for buoyancy or for balance.

Tracheal ventilation Diffusion of gases, even in air, is a slow process. Much more rapid transfer of oxygen and carbon dioxide can be achieved by the mass movement of gases, or convection. Larger insects usually have some mechanism for generating air flow in the bigger tubes of their tracheal system. The air sacs and tracheal tubes are often compressible, allowing changes in tracheal volume. Some larger insects ventilate the larger tubes and air sacs of the tracheal system by alternate compression and expansion of the body

wall, particularly the abdomen. Different spiracles may open and close during different phases of the breathing cycle, thereby controlling the direction of air flow. In the locust, for instance, air enters through the thoracic spiracles but leaves through more posterior openings. Tracheal volume in insects is highly variable; it is 40% of body volume in the beetle Melolontha but only 6% - 10% of body volume in the larva of the diving beetle Dytiscus. Each ventilation results in a maximum of 30% of tracheal volume being exchanged in Melolontha and 60% in Dytiscus. Not all insects ventilate their tracheal system; in fact, many calculations have shown that diffusion of gases in air is rapid enough to supply tissue demands in many species. To augment gas transfer, ventilation of trachea occurs in larger insects and, during high levels of activity, in some smaller insects. In many insects the spiracles open and close, resulting it what is referred to as the insect discontinuous ventilation cycle (DVC).The DVC can be divided into three phases: an open phase, a closed phase, and an intermediate flutter phase when the spiracle oscillates rapidly between the open and closed states. Oxygen utilization and carbon dioxide production by the tissues occurs during all phases, oxygen being supplied from stores in the tracheal system when the spiracles are closed. Pressure in the tracheal system falls during the closed phase because oxygen levels decrease more rapidly than carbon dioxide levels increase. Carbon dioxide levels in the endotracheal space rise slowly during the closed phase because most of the carbon dioxide produced by metabolism is stored in the tissues. Thus during the flutter phase and at the onset of the open phase, gases move into the trachea both by bulk flow down a pressure gradient and by diffusion. Carbon dioxide and water diffuse from the endotracheal space during the open phase and even during the flutter phase, but not during the closed phase (Figure 13-37). In theory, discontinuous ventilation can reduce water loss associated with respiration. The generation of low oxygen levels in the endotracheal space during the closed phase ensures high rates of oxygen diffusion into the tracheal space during the open phase compared with rates of water loss. The functional significance of the flutter phase in determining the rates of gas and water transfer is not clear, but it may enhance gas mixing in the tracheal space. In some instances, however, the role of discontinuous ventilation in water conservation appears to be of little significance. Many xeric species, which require little water, do not show discontinuous ventilation. For example, the lubber grasshopper does not display discontinuous ventilation during desiccation even though it is capable of doing so. In this case only about 5% of total water loss is via the tracheal system, so perhaps it is not surprising that the pattern of ventilation is not changed during desiccation. In water-stressed cockroaches, cuticular water fluxes are more than twice that of loss through the spiracles, and closure of pore structures in the cuticle can conserve water during periods of desiccation. Thus it is not clear why

550


......................................... 0, uptake rate of any tissue, with O2uptake increasing 10to 100-fold above the resting value during flight. In general, more active tissues have more tracheoles, and in larger insects the tracheal system is more adequately ventilated.

Time (min)

"0

20

40

60

80

Time (min) Figure 13-37 Some insects exhibit discontinuous vent~lationas the result of opening and closing of the spiracles. These traces of water and carbon dioxide loss from an alate (would-be queen) harvester ant show that resp~ratorywater loss is concentrated in the spiracle open phase associated with carbon d~oxideexcretion. The spiracles are closed between pulses of CO, excretion. The background cut~cularwater loss rates can be seen between the open phases. [From Lighton, 1994.1

many insects have adopted a pattern of discontinuous ventilation of their tracheal system. Although this mechanism reduces water loss in relation to oxygen uptake, the savings may not always be of much significance to the animal. The functional significance of the flutter phase is an even greater enigma.

Modified tracheal systems There are many modifications of the generalized tracheal system just described. Some larval insects, for example, rely on cutaneous respiration, the tracheal system being closed off and filled with fluid. Some aquatic insects have a closed, air-filled tracheal system in which gases are transferred between water and air across tracheal gills. The gills are evagib nations of the body that are filled with tracheas, the air of which is separated from the water by a 1-pm-thick membrane. Since this tracheal system is not readily compressible, it allows the insect to change depth under water without impairment of gas transfer. Many aquatic insects, such as mosquito larvae, breathe through a hydrofuge (water-repellent) siphon that protrudes above the surface of the water; others take bubbles of air beneath the surface with them. The water bug Notonecta carries air bubbles that cling to velvetlike hydrofuge hairs on its ventral surface when submerged, and the water beetle Dytiscus dives with air bubbles beneath its wings or attached to its rear end. When such insects dive, gases are transferred between the bubble and the tissues via the tracheal system; gases can also diffuse, however, between the bubble and the water.

What are some of the problemsfaced by insects at h~ghaltitude? Are these similar to those faced by bird's eggs?

Gas exchange across tracheolar walls Gases are transferred between air and tissues across the walls of the tracheoles. These walls are very thin, with an approximate thickness of only 40 to 70 nm. The tracheolar area is very large, and only rarely is a particular insect cell more than three cells away from a tracheole. The tips of the tracheoles, except in a few species, are filled with fluid, so that oxygen diffusing from the tracheoles to the tissues moves through the fluid in the tracheoles, the tracheolar wall, the extracellular space (often negligible), and the cell membrane to the mitochondria. This diffusion distance can be altered in active tissues either by an increase in tissue osmolarity, which causes water to move out of the tracheoles and into tissues or by changes in the activity of an ion pump, which results in the net flow of ions and water out of the tracheoles. As fluid is lost from the tracheoles, it is replaced by air, so that oxygen can more rapidly diffuse into the tissues (Figure 13-38).Insect flight muscle has the highest recorded

Figure 13-38 In resting muscle fibers, the terminal parts of tracheoles contain fluid (A), but in active fibers, air may replace this fluid (B), thereby increasing diffusion of oxygen into the muscle. [Adapted from Wigglesworth, 1965.1


551

...................................... Gas exchange in such "bubble-breathing" bugs thus involves diffusion across both tracheolar walls and bubble walls. The rate of 0, transfer between water and the interior of a bubble will depend on the oxygen gradient established and the area of the air-water interface. In a pond the oxygen in the surface water is in equilibrium with the ambient air above the surface. Because surface water mixes with the deeper water, the Po2in the pond water will be in equilibrium with the air and will not vary with depth if the pond is well mixed and no oxygen is removed by aquatic animals or added through photosynthesis by aquatic plants. An air bubble transported to depth by a water bug or beetle will be compressed by hydrostatic pressure; as a result, gas pressure within the bubble will rise, exceeding that in the water. For every 10 m of depth, pressure in a bubble increases by approximately 1 atm. If we consider a bubble just below the surface, the oxygen content of the bubble will decrease owing to uptake by the animal; this will establish an 0, gradient between the bubble and the water (assuming the water is in gaseous equilibrium with air), so oxygen will diffuse into the bubble

A

Air:

from the water. As POLin the bubble is reduced, the nitrogen partial pressure, PN2,will increase; if the bubble is just below the surface, the pressure will be maintained at approximately atmospheric pressure. Nitrogen will therefore diffuse slowly from the bubble into the water (Figure 13-39). (Because of the high solubility of CO, in water, CO, levels in the bubble are always negligible.) If the bubble is taken to depth, however, the pressure will increase by 0.1 atm for every meter of depth, increasing both Po>and PN2and speeding the diffusion of both N, and 0, from the bubble into the water. The bubble will gradually get smaller and eventually disappear as nitrogen leaves it. Thus, the life of the bubble depends on the insect's metabolic rate, the initial size of the bubble, and the depth to which it is taken. The bubble collapses because nitrogen is lost from it as the insect uses the oxygen. It has been calculated that up to seven times the initial bubble 0, content diffuses into the bubble from the water and is therefore available to the insect before the bubble disappears. It is possible that aquatic air-breathing vertebrates like the beaver may take advantage of oxygen diffusing from water into gas bubbles trapped under ice. These

P02 = 150 mm Hg PN2= 592.7 mm Hg

- - - . - .- . -. . - . - - .- . -. - - . - . ... ~

.

.

-

- ~.

-

Water: P o p = 150mmHg PNp= 592.7 mrn Hg

At a depth of 1 m, bubble life is short because of rapid N, loss

Po2 = 150mrnHg

PNz= 592 7 mm Hg

N, loss, 0, uptake from water; bubble size decreases

Figure 13-39 Some aquatic insects carry air bubbles when they dive. Under water, gas exchange occurs between the bubble and the insect5 tracheal system, and between the bubble and water. The direction of gas flow will depend on the partial pressures of 0,, CO,, and N, and the total pressure (P) in bubbles under water. (A) Conditions at start of descent. (B) Condition in bubble immediately after being taken to depth of 1 m. (C)Condition sometime later at same depth. Arrows indicate diffusion of gas molecules. Note that the sum of the gas partial pressures In the water phase (and in the atmosphere) always equals

552


............................................................................... A

"02

=

Water

150 mm ~g

=

2~'

593 mm Hg

f

Water

P

=

743 mm Hg

Air

P

=

Air Cuticle Tissue

Tracheal system Figure 13-40 Hydrofuge hairs on the surface of some insects and insect eggs have an incompressible air space that acts as a gill under water. (A) Schematic diagram of plastron with protruding hydrofuge hairs. Oxygen diffuses from water into the air space contained within the plastron

and then into the animal via the tracheal system. Typically, there are about 106hairs per mm2; only a few are depicted here. (B) Partial pressures of oxygen and nitrogen in the air and water phases.

animals exhale under water and the air bubble produced rests under the ice and gains oxygen from the water; later the animals can inhale the rejuvenated air. If the bubbles were noncollapsible, the insect would not need to surface, because oxygen would continue to diffuse from the water via the bubble into the tracheal system and thence to the tissues. In some insects (e.g.,Aphelocheims)a thin film of air trapped by hydrofuge hairs, called a plastron, in effect provides a noncollapsible bubble (Figure13-40A).The plastron can withstand pressures of several atmospheres before collapsing. In the small air space, N, is presumably in equilibrium with the water, PO2is low, and oxygen therefore diffuses from water into the plastron, wluch is continuous with the tracheal system (Figure 13-40B).

GAS TRANSFER IN WATER: GILLS Gills of fish and crabs are usually ventilated with a unidirectional flow of water (see Figure 13-198). Tidal flow of water, similar to that of air in the lung, would be costly because of the high density and viscosity of water; thus the energetic cost of reversing the direction of flow of water is simply too high. The lamprey and sturgeon are exceptions to the rule that water flow through gills is unidirectional. The mouth of the parasitic lamprey is often blocked by attachment to a host. The gill pouches, although connected internally to the pharyngeal and mouth cavities, are ventilated by tidal movements of water through a single external opening to each pouch (Figure 13-41). This unusual method of gill ventilation is clearly associated with a parasitic mode of life. The ammocoete larvae of lampreys are not parasitic and maintain a unidirectional flow of water over their gills, typical of aquatic animals in general. Water

Figure 13-41 Water flowthrough the gillsof most fishes is unidirectional, but in the adult lamprey, water moves in and out of each gill pouch via the external branchiopore.Shown here is a longitudinal transverse section through the head of an adult lamprey.Arrows markthe direction of water flow. The valves of the external branchiopore move in and out with the oscillatingwater flow.

GAS EXCHANGE AND ACID-BASE BALANCE 553 ......................................

flow through the mouth and gills of sturgeon is normally unidirectional, but if the animal has its mouth in mud while searching for food, it can generate a tidal flow of water through slits in gill coverings. Flow and Gas Exchange across Gills

Blood flow through the fish gills can be described as sheet flow; that is, as pressure increases, the thickness, but not the other dimensions, of the blood sheet increases (Figure 13-42).In this respect, circulation through the gills is similar to the pulmonary circulation. The flow of blood relative to the flow of water in aquatic animals can be either concurrent or countercurrent, or some combination of these two arrangements (Figure 13-43).The advantage of a countercurrent over a concurrent flow of blood and water is that a larger difference in Po2 can be maintained across the exchange surface, thus allowing more transfer of gas. A countercurrent flow is most advantageous if the values for 0, content X flow (capacitylrate) are similar in blood perfusing and water flowing over the gills. If the capacitylrate values for blood and water differ considerably, then countercurrent flow has little advantage over concurrent flow. For example, if water flow were very high in relation to blood flow, there would be little change in Pol in the water as it flowed over the gills, and the mean Po2difference across the gills would be similar in concurrent and countercurrent arrangements of flow. Although the 0, content of fish blood is generally much higher than that of water, the flow rate of water across gills is much higher than the flow rate of blood. Thus the capacitylrate values are similar in the blood perfusing and water flowing over the gills in most fishes, and countercurrent flow is typical. Because water has a much lower oxygen content than air, water-breathing animals require a much higher venti-

Small diffusion resistance

Large diffusion resistance

Countercurrent

Blood

Concurrent

Multicapillary

Figure 13-43 Various arrangements for the flows of water and blood at the respiratory surface are found in aquatic animals. Relative changes in Po, in water and blood are indicated below each diagram. I, inhalant; E, exhalant; a, arterial blood; v, venous blood.

0

15

30

45

60

Transmural pressure (mm Hg) Figure 13-42 In gills, a rise in blood pressure increases the thickness of the blood sheet but not its height or length. In this plot, based on measurements in gill lamellae of the lingcod, Ophiodon elongatus, the black line shows the thickness of the blood sheet, and the red line'tke vascular space to tissue ratio, a measure of the height and length of the blood sheet. [From Farrell et al., 1980.1

lation rate to achieve a given oxygen uptake than do airbreathing animals. This requirement, combined with the much greater density of water compared with air, makes oxygen extraction from the environment a more costly exercise in water. This is offset somewhat by gills having a unidirectional, rather than tidal, flow of water. Water has a much higher heat capacity than air, and heat transfer is more rapid than gas transfer, so blood leaving the gills of a water-breathing animal is usually in thermal equilibrium with the environment. A few fish have some warm tissues; this is only possible because of a countercurrent blood supply to selected tissues. The countercurrent blood supply acts as a heat exchanger, reducing heat loss from the tissue and warming it to above ambient temperatures. Tuna, for instance, have warm muscles, eyes, and brains.

554


......................................... forward motion of the body while swimming. The remora, a fish that attaches itself to the body of a shark, ventilates its own gills only when the shark stops swimming; normally, it relies on the forward motion of its host to ventilate its gills.

Flow of water over the gills of teleost fishes is maintained by the action of skeletal muscle pumps in the buccal and opercular cavities. Water is drawn into the mouth, passes over the gills, and exits through the opercular (gillcovering) clefts (Figure 13-44 ).Valves guard the entrance to the buccal cavity and opercular clefts, maintaining a unidirectional flow of water over the gills. The buccal cavity changes volume by raising and lowering the floor of the mouth. The operculum (gill covering) swings in and out, enlarging and reducing the size of the opercular cavities. Changes in volume in the two cavities are nearly in phase, but a pressure differential is maintained across the gills throughout most of each breathing cycle. The pressure in the opercular cavity is slightly below that in the buccal cavity, resulting in a unidirectional flow of water across the gills throughout most, if not all, of the breathing cycle. Many active fish, such as tuna, "ram-ventilate" their gills, opening their mouths so as to ventilate the gills by the Buccal cavity I

Opercular cavity Gills

I

Operculum closed

Increasing volume

perculum open

Mouth closed

Decreasing volume Figure 13-44 Unidirectionalflow of water through the gills in teleost fish is achieved by sequential opening and closing of the mouth and operculum and by a small pressure differential between the buccal and opercular cavities. In this schematic diagram of gill ventilation, small arrows indicate water flow and large arrows indicate movement of the floor of the mouth.

Functional Anatomy of the Gill The details of gill structure vary among species, but the general plan is similar. The gills of teleost fishes are taken to be representative of an aquatic respiratory surface. The four gill arches on either side of the head separate the opercular and buccal cavities (Figure 13-45A).Each arch has two rows of filaments, and each filament, flattened dors~ventrall~, has an upper and a lower row of lamellae (Figure 13-45B,C).The lamellae of successive filaments in a row are in close contact. The tips of filaments of adjacent arches are juxtaposed, so that the whole gill forms a sievelike structure in the path of water flow. The gills are covered by mucus secreted from mucous cells within the epithelium. This mucous layer protects the gills and creates a boundary layer between the water and the epithelium. Water flows in slitlike channels between neighboring lamellae (see Figure 13-45C,D). These channels are about 0.02-0.05 mm wide and about 0.2-1.6 mm long; the lamellae are about 0.1 -0.5 mm high (Figure 13-46A, on page 556). As a result, the water flows in thin sheets between the lamellae, which represent the respiratory portion of the gill, and diffusion distances in water are reduced to a maximum of 0.01 -0.025 mm (half the distance between adjacent lamellae on the same filament). Gill lamellae are covered by thin sheets of epithelial cells, which are joined by tight junctions (Figure 13-46B, on page 556). The inner lamellar wall is formed by pillar cells, which occupy about 20% of the internal volume of the lamella. The pillar cells are associated with an extensive collagen network, which prevents the lamellae from bulging even though they are subjected to high blood pressures. Blood flows as a sheet in the spaces between the pillar cells, the flow being described by sheet-flow dynamics as in the lung. The diffusion distance between the center of the red blood cell and the water is between 3 and 8 pm, much larger than the diffusion distance across the mammalian lung epithelium (seeFigure 13-22B).The total area of the lamellae is large, varying from 1.5 to 15 cm2 of body weight, depending on the size of the fish and on whether it is generally active or sluggish. Fish gills normally are important in ion regulation and carry out many of the functions of the mammalian kidney. Ion exchange in gills is mediated by at least two types of cells, as discussed in Chapter 14. Because of the metabolic cost of this ion transport, oxygen consumption by gill. tissue may be 10% or more of the total oxygen uptake of the fish. When exposed to air, gills collapse and become nonfunctional, so a fish out of water usually becomes hypoxic, hypercapnic, and acidotic. A few fish and crabs can breathe air, generally using a modified swirnbladder, mouth, gut, og-'


555

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I Blood vessel

Filament c u t across

Septum Lamella

Blood flow

Figure 13-45 The general structure of g~llsis similar in all fishes, although minor variat~onsare found among species. (A) Position of the four gill arches beneath the operculum on the left side of a teleost fish. (B) Enlarged view of part of two gill arches showing the filaments of adjacent rows touching at their tips. Also shown are the blood vessels that carry the blood before and after its passage over the gills. (C) Part of a single fila-

or branchial cavity for this purpose (seeAir-BreathingFishes in Chapter 12).Air-breathing crabs usually show a decrease in oxygen consumption as well as a decrease in body carbon dioxide levels when they move from air to water breathing. The purple shore crab, Leptograspus variegatus, however, shows no change in body oxygen content as it moves between air and water and may regulate body carbon dioxide and therefore pH levels by adjusting the ratio of air to water breathing. Thus this crab truly is amphibious.

REGULATION OF GAS TRANSFER AND RESPIRATION Because the regulation of the rate of 0, and C 0 2 transfer has been studied most extensively in mammals, this section focuses on mammalian regulation of gas transfer. The

canal

rnent with three secondary folds (lamellae) on each side. The flow of blood (red arrows) is in the opposite direction to that of the water (black arrows). (D) Part of the dogfish gill. As in teleost fish, the flow of blood is in the opposite direction to that of the water. [Parts A-C adapted from Hughes, 1964; part D adapted from Grigg, 1970.1

movement of 0, and CO, between the environment and mitochondria in mammals is regulated by altering lung ventilation and the flow and distribution of blood within the body. Here we place emphasis on the control of breathing; Chapter 12 presents details of the control of the cardiovascular system. Ventilation-to-Perfusion Ratios

Energy is expended in ventilating the respiratory surface with air or water and in perfusing the respiratory epithelium with blood. The total cost of these two processes is difficult to assess, but probably amounts to 4%- 10%of the total aerobic energy output of an animal, depending on the species in question and the physiological state of the animal. Thus, gas transfer between the environment and cell accounts for a considerable proportion of the total energy

556


........................................ A

Secondary I , lamellae Water

Mucus

Epithelial cell

Pillar cell

--Blood flow

50 p m

Basement membrane Blood plasma

Water flow

Figure 13-46 Water flows between gill lamellae, which are covered by a thin ep~theliallayer. (A) Scanning electron micrograph of a plastic cast of thevasculatureof a trout gill filament, showing several lamellae. (B)Trans-

verse sertion through a trout gill lamella showing components of the water-blood barrier. [Courtesy of 6. J. Gannon.]

output of the animal and represents a significant selective pressure in favor of the evolution of mechanisms for the close regulation of ventilation and perfusion in order to conserve energy. The rate of blood perfusion of the respiratory surface is related to the requirements of the tissues for gas transfer and to the gas-transport capacities of the blood. To ensure that sufficient oxygen is delivered to the respiratory surface to saturate the blood with oxygen, the rate of ventilation, v*, must be adjusted in accord with the perfusion rate, Q, and the gas content of the two media so that the amount of oxygen delivered to respiratory surface equals that taken away in the blood. The oxygen content of arterial blood in humans normally is similar to that of air. The %lQ ratio, therefore, is about 1 in humans (Figure 13-47A). Water, however, contains only about one-thirtieth as much dissolved oxygen as an equivalent volume of air at the same Pol and temperature. Thus, in fishes, the ratio of water flow, V,, over and blood flow, Q , through the gills is between 10 : 1 and 20 :1 (Figure 13-47B), much higher than the li, /Q ratio in air-breathing mammals. Based on the difference in the oxygen content of water and air, the VG/Qratio in fishes might be expected to be 30: 1. However, it is lower than this because the oxygen capacity of the blood of lower vertebrates is often only half that of mammalian blood. Any changes in the oxygen content of the inhalant medium will affect the VA/Qratio. In order to maintain a given rate of oxygen uptake, a decrease in Po2of inhalant air or water must be compensated for by an increase in ventilation and hence an increase in the ventilation-toperfusion ratio. Conversely, an increase in the inhalant Pol is accompanied by a decrease in ventilation if the rate of oxygen uptake remains the same.

The ventilation-to-perfusion ratio must be maintained over each portion of the respiratory surface as well as over the whole surface. The pattern of capillary blood flow can change in both gills and lungs, changing the distribution of blood over the respiratory surface. The distribution of air or water must reflect the blood distribution. Perfusion of an alveolus without ventilation is as pointless as ventilating an alveolus without blood perfusion of that same alveolus. Although such extreme situations are unlikely to occur, the maintenance of too high or too low a blood flow or ventilation rate will result in energetically inefficient gas transfer per unit of energy expended. For efficient gas transfer, the optimal ventilation-to-perfusion ratio should be maintained over the whole respiratory surface. This optimal maintenance does not preclude differential rates of blood perfusion over the respiratory surface, but requires only that the flows of blood and inhalant medium be matched. The efficiency of gas exchange is diminished if some of the blood entering the lungs or gills either bypasses the respiratory surface or perfuses a portion of the respiratory surface that is inadequately ventilated (Figure 13-48). The magnitude of such venous shunts, expressed as a percentage of total flow to the respiratory epithelium, can be calculated from the arterial and venous 0, content, assuming an ideal arterial 0, content. In the lung, for instance, blood is almost in equilibrium with alveolar gas tensions. If these tensions and the blood oxygen dissociation curves are known, the expected ideal 0, content of arterial blood can be determined. Let us assume that this ideal content is 20 ml of 0, per 100 ml of blood (20 vol %) and the measured values for arterial and venous blood are 1 7 and 5 vol %, respectively. This reduction in measured arterial


557

............................................................................... A

Lung (human)

Figure 13-47 The ventilation-to-perfusion ratio in fish gills is much higher than in the human lung. Approximations of volumes and flows in the human lung and trout gill are shown; actual values may vary considerably.

Lung ventilation = 7500 rnl. min-I

I$ \

Breathing rate = 15 min-I

.

Alveolar ventilat~on(V,) = 5200 ml min-I

Volumes: Alveolar gas = 3000 Pulmonary capillary blood = 70 ml

B

Gill (trout, body weight 200 g, 8°C)

Blood

.

Flow (Q) = 4 ml min-'

.

Flow ( t , ) = 40 rnl min-'

.

Oxygen uptake = 0.1 3 ml min Breathing rate = 75 min" Heart rate = 50 min-I

\;,I&

r

10

L

b

'

0, content from the ideal situation can be explained in terms of a venous shunt, oxygenated arterial blood (20~ 0 1 %being ) mixed with venous blood ( 5~ 0 1 %in) the ratio of 4 : 1to give a final arterial 0, content of 17 ~ 0 1 % ; that is, 20% of the blood perfusing the lung is passing

through one or more venous shunts. This is an extreme example to illustrate a point; in most cases, venous shunts are very small. Flows of blood and inhalant medium (air or water) are regulated to maintain a near optimal ventilation-to-perfu-

Gill

No water flow

i

Lung Figure 13-48 The efflc~encyof gas transfer In the lung and g~llsIS decreased when blood flows to a portlon of the respiratory surface without adequate vent~lat~on (shunt 1) or because blood does not flow close

Gill enough to the resp~ratory eplthel~um(shunt 2) Bloodflow IS regulatedto avo~dthe development of such venous shunts In the lung and gills

558


.........................................

sion ratio over the surface of the respiratory epithelium under a variety of conditions. In general terms, Q is regulated to meet the requirements of the tissues; .ir,and vGare regulated to maintain adequate rates of 0, and CO, transfer. Such mechanisms as hypoxic vasoconstriction of blood vessels help to maintain optimal ventilation-to-perfusion ratios in different parts of the respiratory surface. As discussed earlier, low alveolar oxygen levels cause a vasoconstriction in lung vessels, thereby reducing blood flow to poorly ventilated, and therefore hypoxic, regions and increasing blood flow to well-ventilated regions of the respiratory surface. Blood perfusion of the respiratory surface tends to be less well distributed in resting animals. Blood pressure rises with exercise and blood is distributed more evenly under these conditions, resulting in a more even ventilation-to-perfusion ratio over the respiratory surface.

Neural Regulation of Breathing The integration of breathing movements in all air-breathing vertebrates results from the central processing of many sensory inputs. The central processor consists of a pattern generator, determining the depth and amplitude of each breath, and a rhythm generator, controlling breathing frequency. Several sensory inputs adjust ventilation to maintain adequate rates of gas transfer and blood pH. Other inputs integrate breathing movements with feeding, talking and singing, or other body movements. Certain sensory inputs may cause coughing or swallowing reflexes, which protect the respiratory epithelium from environmental hazards. Other inputs function to optimize breathing patterns to minimize energy expenditure. Medullary respiratory centers As noted earlier, the mammalian lung is ventilated by the action of the diaphragm and muscles between the ribs (see Figures 13-29 and 13-31). These muscles are activated by spinal motor neurons and the phrenic nerve, which receive inputs from groups of neurons that constitute the medullary respiratory centers. The control of respiratory muscles can be very precise, allowing extremely fine control of air flow, as is required for such complex actions in humans as singing, whistling, and talking, as well as simply breathing. Microsections of the neonatal rat brain stem indicate that the ere-Botzinger complex in the ventral medulla is capable of generating the respiratory rhythm and may represent the central rhythm generator that maintains breathing rhythm in the adult. Rhythmic activity is enhanced by neurons in the pons and medulla, and some neurons just anterior to the medulla cause prolonged inspiration in the absence of rhythmic drive from the pons. In 1868 Ewald Hering and Josef Breuer observed that inflation of the lungs decreases the frequency of breathing. (Breuer later became an early proponent of psychoanalysis and collaborated with Sigmund Freud in producing a book on hysteria.) The Hering-Breuer reflex is abolished by cutting the vagus nerve. Inflation of the lung stimulates pulmonary stretch receptors in the bronchi and/or bronchioles,

which have a reflex inhibitory effect, via the vagus nerve, on the medullary inspiratory center (nucleus tractus solitarius) and therefore on inspiration. Thus the medulla contains a central rhythm generator that drives the pattern generator within the medullary respiratory center to cause breathing movements. This system is modified by inputs from other areas of the brain and from various peripheral receptors. The medullary respiratory center contains inspiratory neurons, whose activity coincides with inspiration, and expiratory neurons, whose activity coincides with expiration. The respiratory rhythm was once considered to arise from reciprocal inhibition between inspiratory and expiratory neurons, with reexcitation and accommodation occurring within each set of neurons. But several lines of evidence indicate this model of the central rhythm generator is not tenable, and more recent studies suggest that respiratory rhythm depends primarily on the activity of inspiratory neurons. Inspiratory neuronal activity, recorded from either the phrenic nerve or some individual neurons in the medulla, shows a rapid onset, a gradual increase, and then a sharp cutoff with each burst of activity associated with inhalation. This neuronal activity results in a contraction of the inspiratory muscles and a decrease in intrapulmonary pressure (Figure 13-49A).Increased blood CO, levels cause the progressive growth of inspiratory activity to increase more rapidly (Figure 13-49B). Thus, the rate of rise of inspiratory activity is increased by inputs from chemoreceptors, resulting in a more powerful inspiratory phase. The "off switch" of inspiratory neurons occurs once activity in the neuron has reached a threshold level. Expansion of the lung stimulates pulmonary stretch receptors, whose activity reduces the threshold for the inspiratory off switch (Figure 13-49C). Thus the pulmonary stretch receptors, through their action on inspiratory neurons, prevent overexpansion of the lung. The interval between breaths is determined by the interval between bursts of inspiratory neuronal activity, which is related to the level of activity in the previous burst and in afferent nerves from pulmonary stretch receptors. In general, the greater the level of inspiratory activity (i.e., the deeper the breath), the longer the pause between inspirations. The result is that the ratio of inspiratory to expiratory duration remains constant in spite of changes in the length of each breathing cycle. This ratio is affected by the level of activity of the pulmonary stretch receptors. If, for example, the lung empties only slowly during expiration, the pulmonary stretch receptors will remain active while the lung remains inflated; the continued activity of the stretch receptors will prolong the duration of expiration and increase the time available for exhalation. The neuronal mechanisms causing phasic activation of inspiratory neurons are poorly understood, as is the nature of the central rhythm generator, possibly located in the pre-Botzinger complex in the ventral medullary region of the brain. Exhalation often is a largely passive process, which does not depend on activity in expiratory neurons. This is


559

...............................................................................

lntrapulmonary pressure Decreasing pressure

3rnrnHg

L

lnsplration

200 rns

lncreasing activity

t

Activity f r o m pulmonary stretch receptor Increasing activity /

activity

'

u

activity

Figure 13-49 Phrenic nerve activity, stimulated by increasing alveolar Pco,, induces insp~rat~on. (A) Relationship between phrenic nerve activity and intrapulmonary pressure during inspiration. Note the sudden onset, gradual rise, and then "off switch," or termination, of inspiratory activity (B)Effect of increasing alveolar Pco, levels (P,CO,) on discharge in the phrenic nerve. Recordings were made at P,C02 ranging from 28.5 mm H g (bottom trace) to 60 m m Hg (top trace). The higher the

P,C02, the more rapid the rise of phrenic nerve adivity during inspiration. (C) Effect of increasing activity from pulmonary stretch receptors o n activity in the phrenic nerve. In the absence of stretch receptor activity, the switch off in phrenic nerve activity is delayed (red traces). An increase in receptor activity results in an earlier termination of activity in the phrenic nerve but does not affect the rate of increase in phrenic activity before the switching off (black traces).

especially true during quiet, normal breathing. Expiratory neurons are active only when inspiratory neurons are quiescent, and then they show a burst pattern somewhat similar to, but out of phase with, that of inspiratory neurons. Inspiratory neuronal activity inhibits expiratory activity, showing the dominance of inspiratory neurons in the generation of rhythmic breathing. In the absence of inspiratory activity, expiratory neurons are continually active. Inspiratory neuronal activity, however, imposes a rhythm, via inhibition, on expiratory neurons. Fish, birds, and awake mammals usually breath rhythmically and continuously, whereas amphibians and reptiles often show episodic breathing, with pauses between episodes of rhythmic breathing. Recent studies of the bullfrog brain stem have shown that these episodic patterns of breathing are an intrinsic property of the brain stem and do not depend on sensory feedback. The nucleus isthmi in the bullfrog brain stem is involved not only in the integration of chemoreceptor input but also appears to be essential for the maintenance of episodic breathing. In sleeping mammals episodic breathing appears to be the result of the interaction between peripheral and central components of the control system. During sleep, central respiratory drive is reduced in mammals, and breathing is maintained by input from peripheral chemoreceptors. A breathing period increases oxygen and decreases carbon dioxide levels in the

blood, reducing peripheral chemoreceptor input to the respiratory center. Breathing stops until oxygen levels fall sufficiently to increase chemoreceptor drive enough to initiate breathing again. This results in the periodic breathing typical of many sleeping mammals. In the awake mammal central respiratory drive is sufficient to maintain continuous rhythmic breathing.

Factors affecting the rate and depth of breathing Several types of receptors respond to stimuli that influence ventilation, causing reflex changes in the rate and/or depth of breathing. Among the stimuli affecting ventilation are changes in O,, CO,, and pH; emotions; sleep; lung inflation and deflation; lung irritation; variations in light and temperature; and the requirements for speech. These influences are integrated by the medullary respiratory centers. Breathing can also, of course, be controlled by conscious volition. In most, if not all, animals, changes in 0, and CO, lead to reflex changes in ventilation. The chemoreceptors involved have been localized in only a few groups of animals. Chemoreceptors monitor changes in 0, and CO, in arterial blood in the carotid bodies and aortic bodies of mammals, in the carotid body of birds, and in the carotid labyrinth of amphibians. In teleost fish chemoreceptors located in the gills respond to reductions in O2levels in the

560


.......................................

water and the blood. In all cases, the chemoreceptors are innervated by branches,of the ninth (glossopharyngeal)or tenth (vagus)cranial nerve. Mammals and probably other air-breathingvertebrates also have central chemoreceptors, located in the medulla, that drive ventilation in response to decreases in the pH of the cerebrospinal fluid (CSF),usually caused by elevations in Pco,. Stimulation of this system is required to maintain normal breathing: if body PCol falls, or is held at a low level experimentally, breathing will cease. These central chemoreceptors have little ability to respond to falling O2 levels; the peripheral chemoreceptors have this role and are important in increasing ventilation during periods of hypoxia. The carotid and aortic bodies of mammals receive a generous blood supply and have a high oxygen uptake per unit weight (Figure 13-50A). These arterial chemoreceptors consist of a number of lobules, or "glomoids," that surround very convoluted capillaries. The blood vessels can be divided into small and large capillaries and arteriovenous shunts. The arterioles are innervated by both sympathetic and parasympathetic postganglionic efferents. Each lobule consists of several glomus (type I) cells covered

by sustentacular (type 11) cells. The glomus cells, thought to be the actual receptors, are small ovoid cells with a large nucleus and many dense-core vesicles, or granules (Figure 13-SOB).These cells are interconnected by synapses and often possess cytoplasmic processes of different lengths. They are innervated by afferent fibers of the glossopharyngeal nerve and possibly preganglionic sympathetic efferent~.A single nerve fiber may innervate 10 or 20 glomus cells. A glomus cell may be either presynaptic or postsynaptic, or both (reciprocal),with respect to a nerve fiber. A single nerve fiber may be postsynaptic (afferent)to one glomus cell with a presynaptic connection (efferent) to a neighboring glomus cell or even another region of the same glomus cell. Many glomus cells lack innervation but are synaptically connected to other glomus cells in the lobule. A few glomus cells may be innervated by sympathetic efferent fibers. The chemoreceptors in the carotid and aortic bodies are stimulated by decreases in blood 0, and pH and increases in blood CO, .It is possible that the observed response to increasing CO, is due to changes in pH within these receptors rather than to changes in CO, per se. The result of chemoreceptor stimulation is to recruit new fibers and increase the

Carotid body chemoreceptors /

'Aortic bodies

Figure 13-50 In mammals, chemoreceptors in the carotid and aortic bodies monitor blood gas levels and pti. (A) Diagram showing the location of carotid and aortic body chernoreceptors and carotid sinus and aortic arch baroreceptors (small red dots) in the dog. The baroreceptors help regulate arterial blood pressure (see Chapter 12). (B) Small portion of the rat carotid body, which consists of several lobules containing glo-

mus cells. These are connected by synapses and innervated by glossopharyngeal afferentfibers. Some regions of afferent nerve endings are presynaptic to the glomus cell, some postsynaptic, and some form reciprocal synapses. A , presynaptic regions. [Part A adapted from Comroe, 1962; part B adapted from M.cDonald and Mitchell, 1975.1

firing rate in the afferent nerves innervating glomus cells. The chemoreceptors .adapt to changing arterial CO, levels. The carotid body chemoreceptors show a much larger response to pH and/or C 0 2 changes than the aortic body chernoreceptors. Stimulation of these chemoreceptors leads to an increase in lung ventilation, mediated via the medullary respiratory center. The actual increase, in response to a given decrease in arterial Pol, depends on the blood CO, level, and vice versa (Figure 13-51). Efferent activity to the carotid body modulates the response. Increased sympathetic efferent activity constricts arterioles in the carotid body via an a-adrenergic mechanism, thereby reducing blood flow, which in turn increases the chemoreceptor discharge and lung ventilation. Nonsympathetic efferent activity in the carotid nerve reduces the response of the carotid body to changes in arterial blood Pol and Pco, and/or pH. Increases in temperature and osmolarity also stimulate the arterial chemoreceptors, and stimulation of the carotid nerve causes increased ADH release. Thus the carotid body chemoreceptors may play a role in osmoregulation as well as in the control of breathing and circulation. As mentioned previously, mammals and possibly other air-breathing vertebrates have central chemoreceptors that are necessary for normal breathing. These H+-sensitive receptors are located in the region of the medullary respiratory center and are stimulated by a decrease in the pH of the CSF. The CSF of mammals, and possibly of other vertebrates, is very low in protein and is essentially a solution of NaCl and NaHCO,, with low but closely regulated levels of K+, Mg2+,and Ca2+.The CSF is also poorly buffered; therefore small changes in PCo2have a marked effect on CSF pH. Because the blood-brain barrier is relatively impermeable to H+,the central H+-sensitive chemoreceptors are insensitive to changes in blood pH. However, changes cause corresponding changes in the PCo2of in blood PCOL the CSF, and these in turn result in changes in the pH of the CSF. An increase in PC,> leads to a decrease in the pH of the CSF; subsequent stimulation of the H+-sensitive receptors causes reflex increases in breathing (Figure 13-52). Prolonged changes in PC,! result in the adjustment of the pH of the CSF by changes in H C 0 3 levels.

"

120

240

360

Arterial oxygen Po2(mm Hg) Figure 13-51 Lung ventilation rates increase with a decrease in arterial PO2and an increase in arterial Pco2.The relationships shown here are from measurements in the duck. [From Jones and Puwes, 1970.1

Figure 13-52 Central H+-sensitivereceptors are influenced by the pH of cerebrospinalfluid (CSF) and by arterial Pco, Carbon dioxide molecules diffuse readily across the walls of the brain capillaries and alter CSF pH, but there is a barrier to other molecules.An increase in Pco2causes a decrease in the CSF pH; the resulting stimulation of H+ receptors reflexively increases breathing.Across some capillarywalls, exchange of HCO, and C I helps to maintain a constant pH of the CSF in the face of a prolonged change in PC02.

In mammals and other air-breathing vertebrates, carbon dioxide rather than oxygen levels dominate in the control of breathing. In aquatic vertebrates, however, oxygen is the major factor in the control of breathing. In fact, fishes exposed to high oxygen levels will reduce breathing to the extent that there is a marked increase in Pco2in the blood. Two factors account for this difference. First, oxygen concentration is much more variable in the aquatic environment than in air. Second, oxygen is much less soluble than carbon dioxide in water; as a result, if ventilation is adequate to deliver oxygen to the gills, it will also be adequate to remove carbon dioxide from the blood. Under most conditions ventilation does not limit carbon dioxide excretion in aquatic animals. Only in the rare condition of very high oxygen levels in the water is ventilation reduced sufficiently to curtail carbon dioxide excretion in fish. The lungs contain several types of receptors that help regulate inflation and prevent irritation of the respiratory surface. We saw earlier that stimulation of pulmonary stretch receptors prevents overinflation of the lung (HeringBreuer reflex).In mammals increased CO, levels reduce the r~ receptors on inhibitory effects of these ~ u l m o n a stretch the medullary respiratory center, thereby increasing the depth of breathing and lung ventilation. It is not clear whether the C02-sensitive receptors found in the lungs of birds are pure C 0 2 chemoreceptors or C0,-sensitive mechanoreceptors, as observed in mammals. Increased CO, in the lungs of birds, however, has a greater effect on sensory discharge from the lung than that observed in mammals. In addition to pulmonary stretch receptors, a variety of irritant receptors are present in the lung. Stimulation of these receptors by mucus and dust or other irritant particles causes reflex bronchioconstriction and coughing. A third

group of receptors in the lung is positioned close to the pulmonary capillaries in interstitial spaces; these are called juxtapulmonary capillary receptors, or type J receptors. These receptors were previously termed "deflation receptors," but their natural stimulus appears not to be lung deflation but an increase in interstitial volume, as seen, for example, during pulmonary edema. Stimulation of type J receptors elicits a sensation of breathlessness. Violent exercise probably results in a rise in pulmonary capillary pressure and an increase in interstitial voluine, which could cause stimulation of type J receptors and therefore breathlessness.

RESPIRATORY RESPONSES TO EXTREME CONDITIONS Variations in the levels of respiratory gases, diving by airbreathing animals, and exercise all induce respiratory responses. Let's see how animals adjust to these extreme conditions. Reduced Oxygen Levels (Hypoxia)

Aquatic animals are subjected t o more frequent and rapid changes in oxygen levels than are air-breathing animals. Both mixing and diffusion are more rapid in air compared with water, so regions of local hypoxia develop more often in aquatic environments. Although photosynthesis can cause very high oxygen levels during the day in some aquatic environments, oxygen consumption by both biological and chemical processes can produce localized hypoxic regions. The changes in oxygen levels in water may or may not be accompanied by changes in carbon dioxide. Many aquatic animals can withstand very long periods of hypoxia. Some fishes (e.g., carp) overwinter in the bottom mud of lakes where the Pol is very low. Many invertebrates also bury themselves in mud with a low Pol but high nutritive content. Some parasites live in hypoxic regions, such as the gut, during one or more phases of their life cycle. Limpets and bivalve mollusks close their shells during exposure at low tide to avoid desiccation, but as a consequence are subject to a period of hypoxia. Many of these animals utilize a variety of anaerobic metabolic pathways to survive the period of reduced oxygen availability. Others also adjust the respiratory and cardiovascular systems to maintain oxygen delivery in the face of reduced oxygen availability. For instance, aquatic hypoxia causes an increase in gill ventilation in many fish, as a result of stimulation of chemoreceptors on the gills. The increase in water flow offsets the reduction in oxygen content and maintains delivery of oxygen to the fish. In fishes, such as tuna, that ram ventilate their gills by swimming forward with their mouth open, the size of the gap increases with hypoxia to increase water flow over the gills. Compared with aquatic environments, oxygen and carbon dioxide levels are relatively stable in air, and local regions of low oxygen or high carbon dioxide are rare

and easily avoided. There is, of course, a gradual reduction in Pol with altitude, and animals vary in their capacity to climb to high altitudes and withstand the accompanying reduction in ambient oxygen levels. The highest permanent human habitation is at about 5800 m, where the Po, is 80 mm Hg compared to about 155 mm Hg at sea level. Many birds migrate over long distances at altitudes above 6000 m, where atmospheric pressures would cause severe respiratory distress in many mammals. High altitudes are associated with low temperatures as well as low pressures, and this also has a marked effect on animal distribution. A reduction in the Po, of ambient air results in a decrease in blood P,.,-' which in turn stimulates the carotid and aortic body chemoreceptors, causing an increase in lung ventilation in mammals. The rise in lung ventilation then leads to an increase in CO, elimination and a decrease in blood PCo2.The decrease in blood PCoLcauses a reduction in Pco, and therefore an increase in p H of the CSF. Decreases in blood PC,> and increases in CSF p H tend to reduce ventilation, thereby attenuating the hypoxia-induced increase in lung ventilation. If, however, hypoxic conditions are maintained, as occurs when animals move to high altitude, both blood and CSF pH are returned to normal levels by the excretion of bicarbonate. This process takes about one week in humans. Thus, as CSF p H returns to normal, the reflex effects of hypoxia on ventilation predominate; the result is a gradual increase in ventilation as the animal acclimatizes to altitude. The response to prolonged hypoxia may also involve modulation of the effects of CO, on the carotid and aortic bodies to reset these chemoreceptors to the new lower CO, level at high altitude.

As mentioned earlier, low oxygen levels cause a local vasoconstriction in the pulmonary capillaries in mammals, producing a rise in pulmonary arterial blood pressure. This response normally has some importance in redistributing blood away from poorly ventilated, and therefore hypoxic, portions of the lung. When animals are subjected to a general hypoxic environment, however, the increase in the resistance to flow through the whole lung can have detrimental effects. Some mammals that live at high altitudes exhibit a reduced local pulmonary vasoconstriction in response to hypoxia; this is probably a genetically determined acclimation. Humans residing at high altitudes are usually small and barrel chested, and have large lung volumes. Lung development is oxygen insensitive, but the growth of limbs is reduced under hypoxic conditions. The h ~ g hlung to body ratio enables these people to maintain oxygen up-


563

....................................... take under hypoxic conditions. Pulmonary blood pressures are high, and there is often hypertrophy of the right ventricle. High pulmonary pressures produce more even distribution of blood in the lung, and so augment the diffusing capacity for oxygen. Long-term adaptations also occur during prolonged exposure to hypoxia. Most vertebrates respond by increasing the number of red blood cells and the blood hemoglobin content-and therefore the oxygen capacity of the blood. A reduction in blood oxygen levels stimulates production of the hormone erythropoietin in the kidney and liver. Erythropoietin acts on the bone marrow to increase production of red blood cells (erythropoiesis). Under hypoxic conditions, the levels of hemoglobin-binding organophosphates (e.g., DPG) changes, thus altering the oxygen affinity of hemoglobin. In humans, a climb to high altitude is accompanied by an increase in DPG levels and a reduction in the hemoglobin-oxygen affinity. The increasing DPG levels offset the effects of high blood pH on hemoglobin-oxygen affinity. The high blood pH results from hyperventilation in response to the low 0, availability. Hypoxia due to travel to high altitude also results in systemic vasodilation and an increase in cardiac output. The higher cardiac output lasts only a few days and returns to normal or drops below normal as O2 supplies to tissues are restored by the compensatory increases in ventilation and blood hemoglobin levels. Exposure to hypoxia stimulates a proliferation of capillaries in tissues, ensuring a more adequate oxygen delivery to the tissues. The gills of fishes and amphibians are larger in species exposed to prolonged periods of hypoxia. Similar enlargement of the respiratory surface apparently does not occur in mammals. These processes augment the transfer of oxygen, its transport in the blood, and its delivery to the tissues, but they take from several hours to days or weeks to reach completion. Increased Carbon Dioxide Levels (Hypercapnia)

In many animals, an increase in blood PCo2results in an increase in ventilation. In mammals the increase is proportional to the rise in the CO, level in the blood. The effect is mediated by modulation of the activity of several receptors that send messages to the medullary respiratory center. These receptors include the chemoreceptors of the aortic and carotid bodies and the mechanoreceptors in the lungs, but the response is dominated by the central H + receptors (see Figure 13-52).Correction of CSF pH, in the face of altered PCO2levels, is very important in the return of ventilation to normal. A marked increase in ventilation occurs almost immediately in response to a rise in CO, .The increase is maintained for long periods in the presence of increased CO,, but ventilation eventually returns to a level slightly above the volume that prevailed before hypercapnia. This return to a value only slightly greater than the initial ventilation level is related to increases in levels of plasma bicarbonate and CSF bicarbonate, with the result that pH returns to normal even though the raised CO,?levels are maintained.

Diving by Air-Breathing Animals

Many air-breathing vertebrates live in water and dive for varying periods of time. Dolphins and whales rise to the surface to breathe but spend most of their life submerged. The time between breaths varies with the diver, but is around 10-20 minutes for many diving vertebrates (Table 13-2).The elephant seal dives regularly to depths of 400 m, subjecting itself to a pressure of over 40 atm at the bottom of the dive. These pressures would crush the thoracic cage of man. There are reports of sperm whales diving to nearly 2000 m, and staying submerged for over an hour. These are of course maximum estimates; most dives are much shorter and to less depth. Diving mammals and birds are, of course, subjected to periods of hypoxia during submergence. The mammalian entral nervous system (CNS)cannot withstand anoxia and must be supplied with oxygen throughout the dive. Diving animals solve the problem by utilizing oxygen stores in the lungs, blood, and tissues (Figure 13-53). Many diving animals have high hemoglobin and myoglobin levels, and their total oxygen stores generally are larger than those in nondiving animals. To minimize depletion of available stores, oxygen is preferentially delivered to the brain and the heart during a dive; blood flow to other organs may be reduced, and these tissues may adopt anaerobic metabolic pathways. There is a marked slowing of heart rate (bradycardia) and a reduction of cardiac output during a prolonged dive or if the animal is forcibly submerged in an experimental setting (see Figure 12-47).Air-breathing animals that spend prolonged periods submerged at sea must have sufficient oxygen stores to sustain aerobic metabolism, because they cannot tolerate the high accumulation of lactic acid that results from anaerobic metabolism. During prolonged dives, metabolic rates and thus oxygen needs often are reduced in such animals (e.g., elephant seals). Some diving animals, such as the Weddell seal, exhale before diving, thus reducing the oxygen store in their

TABLE 13-2 Total oxygen stores, mean dive time, and mean dive depth in diving vertebrates

0, stores Spec~es

(ml . kg-')

Mean dr~ve tlme (m~nutes)

Mean depth of d ~ v e (meters)

Leatherback turtle

20

11

-

Pengu~ns*

58

6

100

Weddell seal

60

15

100

Norhern elephant seal

-

20

400

Humant

20

2

shallow

*The 0, stores are for; King penguin; the dive time and depth are for an Emperor penguin. +Leatherbackturtles have similar oxygen stores as humans but can dive for much longer times because of their lower rate of oxygen use. Source: Adapted from Kooyman, 1989.

564


............................................................................... Muscles

lungs

~ i ~ u13-53 r e Air-breath~ng an~malsdraw on cxygen stores In the lungs, blood, and t~ssues(espec~allymuscles)when submerged The generalzed total oxygen stores (expressed In ml 0,. kg body we~ght)of the major taxa of marlne d~versare compared w~thhumans The d~str~butlon of the total 0, stores In the lungs (gray), blood (red), and muscle (whlte) vanes somewhat among specles [Adapted from Kooyman, 1989 1

'

lungs. During a deep dive, the increase in hydrostatic pressure results in lung compression. In those animals that reduce lung volume before a dive, air is forced out of the alveoli as the lungs collapse and is contained within the trachea and bronchi, which are more rigid but less permeable to gases. If gases remained in the alveoli, they would diffuse into the blood as pressure increased. At the end of the dive, the partial pressure of nitrogen in the blood would be high, and a rapid ascent would result in the formation of bubbles in the blood the equivalent of decompression sickness, or the "bends," in humans. Thus exhalation before diving reduces the chances of the bends occurring. Since only about 7% of a Weddell seal's total oxygen stores are in the lungs, pre-dive exhalation appears to be a reasonable trade-off. Receptors that detect the presence of water and that inhibit inspiration during a dive are situated near the glottis and near the mouth and nose (depending on the species). The decrease in blood 0, levels and increase in CO, levels that occur during a dive do not stimulate ventilation because inputs from the chemoreceptors of the carotid and aortic bodies are ignored by the respiratory neurons while the animal is submerged. During birth, a mammal emerges from an aqueous environment into air and survives a short period of anoxia between the time the placental circulation stops and the time air is first The respirator). and responses of the fetus during this period are similar in several respects to those of a diving mammal.

Exercise Exercise increases 0, utilization, C 0 2 production, and metabolic acid production. Cardiac output increases to meet the higher demands of the tissues. Even though the transit time for blood through the lung capillaries is reduced, nearly complete gas transfer still occurs (Figure 13-54). Ventilation volume increases in order to maintain gas tensions in arterial blood in the face of increased blood flow. The increase in ventilation in mammals is rapid, coinciding with the onset of exercise. This initial sudden increase in ventilation volume is followed by a more gradual rise until a steady state is obtained both for ventilation volume and oxygen uptake (Figure 13-55).When exercise is terminated, there is a sudden decrease in breathing, followed by a gradual decline in ventilation volume. During exercise, 0, levels are reduced and CO, and H+ levels in arteraised in venous blood, but the mean P02and PCO7 rial blood do not vary markedly, except during severe exercise. The oscillations in arterial blood Pg and PcOlassociated with each breath increase in magnitude, although the mean level is unaltered. Exercise covers a range from slow movements up to maximum exercise capacity. The phrase moderate exercise refers to exercise above resting levels that is aerobic, with only minor energy supplies derived from anaerobic glycolysis. Severe exercise refers to exercise in which oxygen uptake is maximal and further energy supplies are derived from anaerobic metabolism. Heavy exercise is a term sometimes used to denote the exercise level between moderate and severe exercise. The onset of exercise involves many changes in lung ventilation and the cardiovascular system, as well as muscle contraction. In the initial stages, during the transition from rest to exercise, the animal is not in a steady state and part of the energy supply is derived from anaerobic

- -Alveolar - - - -gas -----------------

m

t

Time in capillar~es

61ood enters lung Figure 13-54 Blood Po, rapidly reaches near equilibration with alveolar Po2even during exercise. Although blood flow increases, and therefore blood spends less time in the lung capillaries, during exercise, increased ventilation allows equilibration to occur. [Adapted from West, 1970.1


565

......................................

lpzlp-

Exercise

1

Recovery

Plateau phase Secondary increase

Rapid l n ~ t i aphase l Slow decline

Time (min) Figure 13-55 An increase in iung ventilation is one of several adjustments to meet the increased oxygen demand during exercise. Typical changes in lung ventilation during exercise and recovery in humans are depicted.

processes. If the exercise level is moderate and sustained, the animal moves into a new steady state typical of that exercise level, with increased lung ventilation, cardiac output, blood flow to the exercising muscles, and oxygen uptake. The relationship between lung ventilation and oxygen uptake is linear during moderate exercise, the slope of the relationship varying with the type of exercise. A number of receptor systems, some not yet identified, appear to be involved in the respiratory responses to exercise. Contractions of muscles stimulate stretch, acceleration, and position mechanoreceptors in muscles, joints, and tendons. Activity in these receptors reflexively stimulates ventilation, and this system probably causes the sudden changes in ventilation that occur at the beginning and end of a period of exercise. The increase in ventilation varies with the group of muscles being stimulated. Leg exercise, for example, results in a larger increase in ventilation than arm exercise; the same is true for bicycle exercise versus exercise on a treadmill. It has also been suggested that changes in neural activity in the brain and spinal cord leading to muscle contraction may also affect the medullary respiratory center, causing an increase in ventilation. Muscle contraction generates heat and raises body temperature, thereby increasing ventilation via action on temperature receptors in the hypothalamus. The exact response elicited by stimulation of the hypothalamus depends on the ambient temperature. The increase in ventilation is more pronounced in a hot environment. Since the rise and fall of temperature that follow exercise and subsequent rest are gradual, they would appear to account for only slow changes in ventilation during exercise. In the absence of exercise, large changes in carbon dioxide and oxygen are required to produce equivalent changes in ventilation. It would seem that the chemoreceptors in both the aortic and the carotid bodies and in the medulla are probably not directly involved in the ventilatory responses to exercise, because mean Po>and PCol levels in arterial blood do not change very much during exercise. Howevel;

the sensitivity of these receptors may increase during exercise, so that relatively small changes in gas partial pressures can cause an increase in ventilation. In this regard, it is significant that catecholamines, which are released in increased quantities during exercise, increase the sensitivity of medullary receptors to changes in carbon dioxide. Threshold levels of carbon dioxide are required to drive ventilation during exercise, as in resting conditions. Exercising sheep connected to an external artificial lung to maintain low PCoiand high Po: levels in their blood do not breathe. Ventilat~onin the intact mammal increases in proportion to the CO, delivery to the lung, but the location of any receptors involved is not known. There are chemical changes in exercising muscle, and these may play a role in reflexively stimulating ventilation via muscle afferent fibers. Ventilation increases more during severe exercise than during moderate exercise, and the relationship between ventilation and oxygen uptake during severe exercise is no longer linear but becomes exponential. This large increase in ventilation is probably driven by the same mechanisms as in moderate exercise, with the added stimulation of a marked metabolic acidosis and h g h circulating catecholamine levels.

SWIMBLADDERS: OXYGEN ACCUMULATION AGAINST LARGE GRADIENTS Fish are denser than the surrounding water and must generate upward hydrodynamic forces if they are to maintain position in the water column and not sink to the bottom. They can generate lift by swimming and using their fins and body as hydrofoils. The minimum speed below which sufficient lift cannot be generated is about 0.6 m . spl for Skipjack tuna; thus these fish must, and in fact do, swim continually to maintain position in the water column. Other fish hover like a helicopter, using their pectoral fins to maintain position. In both cases there is an energetic cost to maintaining position that can be reduced by incorporation of a buoyancy device. To avoid expending energy to maintain lift, many aquatic animals maintain a neutral buoyancy, compensating for a dense skeletal structure, by the incorporation of lighter materials in specialized organs. These "buoyancy tanks" may be NH,Cl solutions (squids),lipid layers (many animals, including sharks), or air-filled swimbladders (many fishes). Ammonium chloride and lipid floats have the advantage of being essentially incompressible, not changing volume with the changes in hydrostatic pressure that accompany vertical movement in water. These float structures, however, are not much lighter than the other body tissues and so must be large if the animal is t o achieve neutral buoyancy. Swimbladders are less dense and can be much smaller than NH,Cl and lipid floats, but they are compressible and change in volume, thus changing the buoyancy of the animal with changes in depth.

566


........................................

Hydrostatic pressure increases by approximately 1 atrn for every 10 m of depth. If a fish is swimming just below the surface and suddenly dives to a depth of 10 m, the total pressure in its swimbladder doubles from 1 to 2 atrn and the bladder volume is reduced by one-half, thus increasing the density of the fish. The fish will now continue to sink because it is more dense than water. Similarly, if the fish rises to a shallower depth, its swimbladder will expand, decreasing the fish's density, so that it continues to rise. AIthough the low density of swimbladders is an advantage, they are essentially unstable because of the volume changes they undergo with changes in depth. One means of preventing volume changes is for gas to be removed or added as the fish ascends or descends, respectively. Many fishes do have mechanisms for increasing or decreasing the amount of gas in the swimbladder in order to maintain a constant volume over a wide range of pressures. Fishes with swimbladders spend most of their time in the upper 200 m of lakes, seas, and oceans. The pressure in the bladder will range from 1 atrn at the surface to about 21 atrn at 200 m. Gases dissolved in water are generally in equilibrium with air, and neither the partial pressure nor the gas content in water will vary with depth, because water is virtually incompressible (Figure 13-56). The swimbladder gas in most fishes consists of 0 2 , but in some species the swimbladder is filled with CO, or N2 . If the fish dives to a depth of 100 m, 0, is added to the swimbladder to maintain buoyancy. The aquatic environment is the source of this O,, which is moved from the surrounding water to the swimbladder against a pressure differencein this example, a difference of nearly 10 atrn (water PO' = 0.228 atm; bladder Po, = 10 atrn). To understand how this occurs, let us review the structure of the swimbladder.

/

Bladder PO,(atm)

Water Po,

-

20

40 60 Depth (m)

80

100

Figure 13-56 The volume of the swimbladder decreases and bladder Po, increases as a fish descends. Hydrostatic pressure increases by approximately 1 atm every 10 m. In this example, oxygen is assumed to be the only gas present and is neitheradded to nor removedfrom the bladder. Fishes can maintain constant density only by maintaining constant bladder volume, which is achieved by add~tionof oxygen to the bladder with increasing depth. Note the increasing Po, difference between water and bladder with depth. Oxygen must be moved from water into the swimbladder agalnst this increasing Po, gradlent.

The Rete Mirabile The teleost swimbladder is a pouch of the foregut (Figure 13-57).In some fishes, there is a duct between the gut and bladder; in others, the duct is absent in the adult. The bladder wall is tough and impermeable to gases, with very little leakage even at very high pressures, but the wall expands easily if pressures inside the bladder exceed those surrounding the fish. Those animals capable of moving oxygen into the bladder against a high pressure gradient have a rete mirabile. The rete consists of several bundles of capillaries (both arterial and venous) in close apposition, so that there is countercurrent blood flow between arterial and venous blood. It has been calculated that eel retia have 88,000 venous capillaries and 116,000 arterial capillaries containing about 0.4 ml of blood. The surface area of contact between the venous and arterial capillaries is about 100 cm2. Blood passes first through the arterial capillaries of the rete, then through a secretory epithelium (gas gland) in the bladder wall, and finally back through the venous capillaries in the rete. The arterial blood and the venous blood in the rete are separated by a distance of about 1.5 pm. The rete structure allows blood to flow into the bladder wall without a concomitant large loss of gas from the swimbladder. Blood leaving the secretory epithelium at high Po>passes into the venous capillaries. The partial pressure of oxygen decreases in both arterial and venous capillaries with distance from the secretory epithelium. The Poldifference between arterial and venous blood at the end of the rete distal to the swimbladder is small compared with the Pol difference between the environment and the swimbladder, reducing the loss of oxygen from the swimbladder. It was thought that the reason oxygen levels dropped in the rete was because of the diffusion of oxygen from venous to arterial capillaries, the rete acting as a countercurrent exchanger (see Spotlight 14-2). H. Kobayashi, B. Pelster, and P. Scheid (1993),howevel; were unable to detect any significant transfer of oxygen across the rete. The Pol does fall in the blood flowing away from the gas gland because oxygen binds to hemoglobin, not because of any loss of oxygen to arterial blood entering the rete. Exactly how and why this occurs will be discussed later. Oxygen Secretion The rete structure reduces gas loss from the swimbladder, but how is oxygen secreted into the swimbladder? First, consider the relationship between Po,, oxygen solubility, and oxygen content. Oxygen is carried in blood bound to hemoglobin and in physical solution. If oxygen is released from hemoglobin into physical solution, Pol will increase. The release of oxygen from hemoglobin can be caused by a reduc-

GAS E X C H A N G E A N D A C I D - B A S E B A L A N C E 567 ...............................................................................

To liver Rete

Esophagus Duct

Swimbladder

-

____------------_ ----_

To heart

To l ~ v e r

Oval (gas resorbtion)

---.1

(gas secretion)

Figure 13-57 Two main types of swimbladder are found in fish. (A) A physostome swimbladder (e.g., from the eel, Anguilla vulgaris) is connected to the outside via a duct to the esophagus. (B) A physoclistswim-

bladder (e.g.,from the perch, Perca fluviatilis) lacks a dud. Gas enters and leaves the bladder vla the blood. [Adapted from Denton, 1961.I

tion in pH via the Root-off shift (Figure 13-58).An increase in ionic concentration reduces oxygen solubility and also results in an increase in Po>,as long as the oxygen content in physical solution remains unchanged. Thus, an increase in blood can be achieved by releasing oxygen from hemoglobin or increasing the ionic concentration of the blood. The cells of the gas gland have few mitochondria and negligible Krebs cycle activity. For this reason, even in an oxygen atmosphere, glycolysis in the secretory epithelium (gas gland) of the swimbladder yields two lactate molecules

and two protons for each glucose molecule. The pentose phosphate shunt, however, is active in the gas gland, producing carbon dioxide via decarboxylation of glucose without oxygen consumption. The production of carbon dioxide, lactate, and protons by gas-gland cells results in (1)a decrease in pH, which causes the release of oxygen from hemoglobin (Root-off shift) and (2)an increase in ionic concentration and therefore a reduction in oxygen solubility (sometimes termed the "salting-out effect"). Both changes cause the Po2in the secretory epithelium to increase more

Root-on shift

Root-off shift

Figure 13-58 Anaerob~cmetabolism of glucose to lactate and CO, In the gas gland, located In the wall ofthe flsh sw~mbladder,leads to a decrease in erythrocyte pH and release of oxygen from hemoglobin As a result, the Po2 In the blood flowlng through the gas gland becomes greater than the PO2In the lumen of the swlmbladder, so oxygen dlffuses Into the lumen Root-off sh~ft,leadlng to Increase In P02, occurs on arter~al slde of the rete, whereas Root-on shlft, leadlng t o decrease In Po,l occurs on the venous s~de

than that in the swimbladder, so that oxygen diffuses from blood into the gas space of the swim bladder (see Figure 13-58).The salting-out effect will also reduce the solubility of other gases, such as nitrogen and carbon dioxide, and may explain the high levels of these gases sometimes observed in swim bladders. Let's return now to the situation in the rete. As discussed earlier, erythrocytes are not very permeable to H+ ions, so the drop in pH in the gas gland is transferred into the red blood cells by CO,, which crosses cell membranes with ease (Figure 13-59).Acid produced in the gas gland reacts with HC0,-, probably taken up from the plasma, producing CO, .Thus blood leaving the gas gland and entering the venous capillaries of the rete has a high CO, content. As the high-CO, venous blood flows through the rete, CO, diffuses into the arterial blood flowing towards the gas gland. This raises the pH of the venous blood, which in turn increases oxygen binding by hemoglobin (Root-on shift); as more oxygen is bound, the Po> in the venous blood falls as it flows away from the gas gland (see Figure 13-58). On the arterial side of the rete, the entering C 0 2lowers blood pH, which drives oxygen from the hemoglobin (Root-off shift), thereby raising the blood Po2. Thus the Po? changes in the rete result from loading and unloading of hemoglobin with oxygen, with the rete serving as a countercurrent exchanger for carbon dioxide and not oxygen. In fact the rete has a relatively low oxygen permeability. The gas gland and associated rete enable fish to transfer oxygen into the swimbladder even though the bladder may contain oxygen at several atmospheres pressure. The bladder wall is slightly permeable to gases so there is a continual loss of gas that increases with depth (bladder pressure). Gases, therefore, must be secreted continually to maintain volume in the face of this loss. Eels migrating at depth across the oceans enlarge their rete and gas gland and decrease the permeability of the bladder wall, enabling them to maintain bladder volume at higher pressures. The permeability of the bladder wall is decreased by an increase in its thickness due to

increased deposition of guanine. Eels turn from yellow to silver as a result of these guanine deposits. This occurs as they leave the rivers and begin their migration across the oceans.

SUMMARY At the level of the mitochondria, the number of oxygen molecules that an animal extracts from the environment and utilizes is approximately the same as the number of carbon dioxide molecules it produces and releases into the environment. In very small animals, gases are transferred between the surface and the mitochondria by diffusion alone, but in larger animals a circulatory system has evolved for the bulk transfer of gases between the respiratory surface and the tissues. Respiratory surfaces are characterized by large surface areas and small distances for diffusion between the inhalant medium and the blood to facilitate gas transfer. Breathing movements assure a continual supply of oxygen and prevent stagnation of the medium close to the respiratory epithelium. The design of the respiratory surface and the mechanism of breathing are related to the nature of the medium (i.e., gills in water, lungs in air). Bulk transport of 0, and CO, in the blood is augmented by the presence of a respiratory pigment (e.g., hemoglobin). The pigment not only increases the oxygencarrying capacity of the blood, but also aids the uptake and release of 0, and CO, at the lungs and tissues. The rate of gas transfer across a respiratory surface depends on the ratio of ventilation rate of the respiratory surface to blood flow, ~ I Qas, well as on the absolute ventilation volume and cardiac output. These factors are closely regulated to maintain adequate rates of gas transfer to meet the requirements of the tissues. The control system, which has been studied extensively only in mammals, consists of a number of mechanoreceptors and chemoreceptors that feed information into a central integrating region, the medullary respiratory center. This center, through a variety

Venous blood

Swim bladder

lumen RETE

Figure 13-59 The rete associated with the gas gland acts as a countercurrent exchanger for carbon dioxide. Venous blood coming from the gas gland is high in CO,, which diffuses into the arterial side ofthe rete, lowering pH and causing a Root-offshift (see Figure 1358) and increased Po, in the arterial blood entering the gland. The CO, is recycled through the rete, further increas~ngPo2 in the arterial blood and decreasing it in the venous blood.

of effectors, causes appropriate changes in breathing and blood flow to maintain rates of 0, and C 0 2 transfer at a level sufficient to meet the requirements of metabolism. Animals regulate body pH in the face of continual production and excretion of H+ ions. Production of H + varies with the metabolic requirements of the animal; H+ excretion via the lungs and kidney is adjusted to match production. Buffers, particularly proteins and phosphates, ameliorate any oscillations in body pH due to an imbalance between acid production and excretion. The muscle tissues are utilized as a temporary H+ ion reservoir, thereby further protecting more sensitive tissues such as the brain from wide swings in pH until the excess H + can be excreted from the body. Intracellular pH is adjusted by modulation of Na+/H+and HC0,-/CI exchange mechanisms located in the cell membrane. Insects have evolved a tracheal system that takes advantage of the rapid diffusion of gases in air and avoids the necessity of transporting gases in the blood. The tracheal system consists of a series of air-filled, thin-walled tubes that extend throughout the body and serve as diffusion pathways for 0, and CO, between the environment and the cells. In some large active insects, the tracheal system is ventilated. Bird eggs and fish swimbladders present interesting problems in gas transfer. A bird's egg contains an embryo whose oxygen must be transferred across a shell of fixed dimensions, the transfer requirements increasing a thousandfold between laying and hatching. Gas tensions in the fish swimbladder often exceed that in the blood by several orders of magnitude, but the design of the blood supply and gas gland is such that gases move from the blood into the swimbladder.

REVIEW QUESTIONS Calculate the percent change in volume when dry air at 20°C is inhaled into the human lung (temperature = 37°C). Define the following terms: (a) oxygen capacity, (b) oxygen content, (c)percent saturation, (d)methemoglobin, (e) Bohr effect, and (f)Haldane effect. Describe the role of hemoglobin in the transfer of oxygen and carbon dioxide. Describe the effects of gravity on the distribution of blood in the human lung. What effect does alveolar pressure have on lung blood flow? Compare and contrast ventilation of the mammalian lung and the bird lung. What is the functional significance of the presence of surfactants in the lung? How have insects avoided the necessity of transporting gases in the blood? The number and dimensions of air pores in eggshells are constant for a given species. What effect would doubling the number of pores have on the transfer of oxygen, carbon dioxide, and water across the eggshell?

9.

10. 11. 12.

13. 14. 15.

16.

17.

18.

19.

20.

Discuss the role of the rete mirabile in the maintenance of high gas pressures in the fish swimbladder. How is oxygen moved into the swimbladder of teleost fishes? Describe the structural and functional differences between gills and lungs. Why is the ventilation-to-perfusion ratio much higher in water-breathing than in air-breathing animals? Describe the role of central chemoreceptors in the control of carbon dioxide excretion. What is the importance of the Hering-Breuer reflex in the control of breathing? Describe the processes involved in the acclimation of mammals to high altitude. What is the effect on intracellular pH of elevating extracellular NH,C1 levels at either low or high extracellular pH? Describe the role of the C0,-bicarbonate systems in pH regulation in mammals. Explain the consequences of the localization of the enzyme carbonic anhydrase within the red blood cell and not in the plasma. Describe the possible mode of operation of the medullary respiratory center. Discuss the interaction between gas transfer and heat and water loss in air-breathing vertebrates.

SUGGESTED READINGS Asrtrup, P., and J. W. Severinghaus. 1986. The History of Blood Gases, Acids and Bases. Copenhagen: Munksgaard. Brauner, C. J., and D. J. Randall. 1996. The interaction between oxygen and carbon dioxide movements in fishes. Comp. Biochem. Physiol. 113A: 83-90. Dejours, P. 1988. Respiration in Water and Air. Amsterdam: Elsevier. Diamond, J. 1982. How eggs breathe while avoiding desiccation and drowning. Nature 295:10- 11. Evans, D. H. 1993. The Physiology of Fishes. Boca Raton, Fla.: CRC Press. Euler, C. von. 1980. Central pattern generation during breathing. Trends Neurosci. 3:275-277. Heisler, N., ed. 1995. Mechanisms of Systemic Regulation: Respiration and Circulation. Advances in Comparative and Environmental Physiology, Vol. 21. Berlin: Springer-Verlag. Hochachka, P. W., and G. N. Somero. 1983. Strategies of Biochemical Adaptation. 2d ed. Princeton, N.J.: Princeton University Press. Jensen, F. B. 1991. Multiple strategies in oxygen and carbon dioxide transport by haemoglobin. In A. J. Woakes, M. K. Grieshaber, and C. Bridges, eds., Physiological Strategies for Gas Exchange and Metabolism. Society of Experimental Biology Seminar Series, Vol. 41. Cambridge: Cambridge University Press.

570


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Kobayashi, H., B. Pelster, and P. Scheid. 1993. Gas exchange in fish swimbladder. In P. Scheid, ed., Respiration in Health and Disease: Lessons from Comparative Physiology. Stuttgart: Gustav Fischer. Krogh, A. 1968. The Comparative Physiology of Respiratory Mechanisms. New York: Dover. Milvaganam, S. E. 1996. Structural basis for the Root effect in haemoglobin. Nature Struct. Biol. 3:275-283. Nikinmaa, M. 1990. Vertebrate red blood cells: adaptations of function to respiratory requirements. In S. D. Bradshaw, W. Burggren, H. C. Heller, S. Ishii, H. Langer, G. Neuweiler, and D. J. Randall, Zoophysiology, Vol. 28. New York: Springer-Verlag. Perutz, M. F. 1996. Cause of the Root effect in fish heamoglobins. Nature Struct. Biol. 3:211-212. Rahn, H. 1966. Aquatic gas exchange theory. Resp. Physiol. 1:l-12.

Richter, D. W., K. Ballanyi, and S. Schwarzacher 1992. Mechanism of respiratory rhythm generation. Curr. Opin. Neurobiol. 2:788-793. Roos, A., and W. F. Boron. 1981. Intracellular pH. Physiol. Rev. 61:296-434. Schmidt-Nielsen, K. 1972. How Animals Work. Cambridge: Cambridge University Press. Weber, R.E. 1992. Molecular strategies in the adaptation of vertebrate hemoglobin function. In S. C. Wood, R. E. Weber, A. R. Hargens, and R. W. Millard, Physiological Adaptations in Vertebrates:Respiration, Circulation and Metabolism. New York: Marcel Dekker. West, J. B. 1974. Respiratory Physiology: The Essentials. Baltimore: Williams and Wilkins. Zhu, X. L., and W. S. Sly. 1990. Carbonic anhydrase IV from human lung. J. Biol. Chem. 15:8795-8801.

T

he unique physical and chemical properties of water undoubtedly played a major role in the origin of life, and all life processes take place in a watery milieu (see Chapter 3). Water is, in fact, indispensable for all biochemical and physiological processes. Indeed, the physicochemical nature of Earth life is largely a reflection of the special properties of water. The presence of water here on Earth made it possible for life as we know it to arise several billion years ago in a shallow, salty sea. Extracellular fluids surrounding living cells to this day reflect, to some extent, the composition of the primeval sea in which life evolved (Table 14-1). The ability to survive in a variety of osmotic environments was achieved in the more advanced animal groups by the evolution of a stable internal environment, which acts to buffer the internal tissues against the vagaries and extremes of the external environment. Thus, the ability to maintain a suitable internal environment in the face of osmotic stress (something that tends to disturb the ionic and osmotic homeostasis of the animal) has played a most important role in animal evolution. There are two main reasons for this. First, animals are restricted in their geographic distribution by environmental factors, one of the most important being the osmotic nature of the environment. Second, geographic dispersal followed by genetic isolation is an important mechanism for the divergence of species in the process of evolution. If, for example, the arthropods and the vertebrates had not evolved mechanisms for regulating their extracellular compartments, they would have been unsuccessful in their invasion of the osmotically hostile freshwater and terrestrial environments. In the absence of competition from terrestrial arthropods and vertebrates, other groups would have evolved with greater diversity to fill the vacant terrestrial niches, and the living world would be quite different from the one we know. In this chapter, we consider the osmotic environment, osmotic exchange between the animal and its environment, and mechanisms used by various animals to cope with environmental osmotic extremes. The movement of water and solutes across cell membranes and multicellular ep-

ithelial layers has been covered, along with other cellular mechanisms, in Chapter 4. That discussion forms an essential background for understanding the osmoregulatory processes in organs such as the kidney, gill, and salt gland covered in this chapter. Toward the chapter's end we discuss the closely related problem of elimination of toxic nitrogenous wastes produced during the metabolism of amino acids and proteins.

PROBLEMS OF OSMOREGULATION One of the requirements in the regulation of the internal environment is that appropriate quantities of water be retained. Another major requirement for cell survival is the presence, in appropriate concentrations, of various solutes (e.g., salts and nutrient molecules) in the extracellular and intracellular compartments (Table 14-2). Some tissues require an extracellular ionic environment that is an approximation of seawater-namely, fluid high in sodium and chloride and relatively low in the other major ions, such as potassium and the divalent cations. For many marine invertebrates, the surrounding seawater itself can act as the extracellular medium; for most of the more complex forms, the internal fluids are in near ionic equilibrium with seawater. In contrast, the extracellular fluids of vertebrates, with the exception of the hagfishes, have an ionic concentration that is about one third that of seawater with much of the magnesium sulfate removed and some of the chloride replaced by bicarbonate anion (see Table 14-1). This presumably reflects the freshwater origin of most vertebrates, including marine teleost fishes. The extracellular fluids of marine teleosts are much more dilute than seawater, and these fishes maintain both an ionic and osmotic difference between their body fluids and seawater. Elasmobranchs, on the other hand, maintain an ionic difference but only minor osmotic differences; the high levels of urea in the body of elasmobranchs brings the osmolarity to slightly above that of seawater. The intracellular environment of most animals is low in sodium but high in potassium, phosphate, and proteins

TABLE 14-1 Composition of extracellular fluids of representative animals* Osmolar~ty (mosM)

Habitat*

l o n ~ cconcentrations (mM) Na+

K+

Ca2+

Mg2+

C I

SO-:

10

53

540

27

460

10

SW

454

10 2

97

510

554

14.6

SW

428

95

11 7

492

487

26.7

SW Ter

459 76

10 1 40

100 29

524

537 43

24 4

SW SW

492 419 156

97 20 6 049

133 113 84

49 516 019

543 522 11 7

28 2 69 073

SW

146 47 2

39 10 0

81 156

43 67

139 470

Ter Ter

60 161

12 79

17 40

25 56

144

1000

Seawatert

HP0;

Urea

Coelenterata Aurelia (jellyf~sh)

Echinodermata Astenas (starf~sh)

Annelida

-

Arenrcola (lugworm) Lumbrrcus (earthworm)

Mollusca Aplysra (sea slug) Lolrgo (squid) Anodonta (clam)

FW

Crustacea Cambarus (crayf~sh) Homarus (lobster)

FW

lnsecta Locusta Perrplanta (cockroach)

Cyclostomata Eptatretus (hagf~sh) Lampetra (lamprey)

SW FW

1002 248

554 120

6.8 32

88 19

234 21

532 96

17 27

SW

1075

FW

269 200

43 8

32 3

11 2

258 180

1 0.5

SW

181

51 3

69

287

199

3 6

1 3

160 107

23

16

2.1

3 0.4

Chondrichthyes Dogflsh shark Carcharhrnus

1.1

4.0

376 132

Coelacantha Latrmerra

355

Teleostei Paralichthys (flounder) Carassius (goldfish)

SW RIV

337 293

180 142

4 2

FW FW 80% SW

210 290 830

92 125 252

3 9 14

FW

278

140

36

51

FW

294

138

31

24

142 145

40 62

50 31

02

Amphibia Rana esculenta (frog) Rana cancrrvora

70 98

2 40 350

227

Reptilia All~gator

30

111

Aves Anas (duck)

103

1.6

Mammalia Homo saprens Lab rat

Ter Te r

20 16

104 116

1

2

*The osmolar~tyand compos~tlonof seawater vary, and the values glven here are not intended to be absolute The composltlon of body flu~dsof osmoconformers will also vary, depending on the composltlon of the seawater In wh~chthey are tested

+SW = seawater, FW = freshwater, Ter

=

terrestr~al.

Sources Schm~dt-Nlelsenand Mackay, 1972, Prosser, 1973

(Table 14-3).There are only minor and transient osmotic differences between the intracellular and extracellular fluids of animals. Thus the cell membrane maintains ionic, but not osmotic, differences between the intracellular and extracellular fluids, whereas the epithelium surrounding the body often maintains both ionic and osmotic differences between animals and their environments. In most multicellular animals, the entire body surface is not usually involved in ionic and osmotic regulation; rather, this regulation is effected by a specialized portion of the body surface such as the gills of fish or some internal structure like the

salt gland of elasmobranchs or the kidney of mammals. The rest of the body surface, with the exception of the lining of the gut, is relatively impermeable to ions and water. Animals require nutrients and oxygen to maintain metabolism, and as a result of metabolism, they produce waste products. Cell membranes that are permeable to oxygen are also permeable to water, and energy must be expended to maintain the ionic and osmotic balance of the animal. An animal cannot reduce osmotic and ionic problems by sealing itself off from the environment because nutrients must be acquired and waste products excreted. Some animals en-

573

I O N I C A N D OSMOTIC BALANCE

............................................................................... TABLE 14-2 Major inorganic ions of tissues Ion

D~str~butlon

Major funct~ons

Main extracellular cation

Is major source of extracellular osmotic pressure Provides potential energy for transport of substances across cell membranes Provides inward current for membrane excitation

K+

Main cytosolic cation

Is source of cytosolic osmotic pressure Establishes the restlng potential Provides outward current for membrane repolarization

Ca2+

Low concentration in cells

Regulates exocytosis and muscle contraction Is involved in "cementing" cells together Regulates many enzymes and other cell proteins; acts as second messenger.

Mg2+

Intra- and extracellular

Acts as cofactor for many enzymes (e.g., ATPases)

HPO?; HCO,

Intra- and extracellular

Buffers H+ concentration

CI-

Main extracellular anion of tissues

Is counterion for inorqanic cations

cyst themselves, but this is feasible only if their metabolic rate is very much reduced. Brine shrimp larvae, for example, can survive in a state of suspended animation for many years with little or no growth; when in this state, they can be placed in water and revived. This is only possible because energy turnover is very reduced during encystment, limiting nutrient utilization and waste-product accumulation. Most animals, however, do not exist in a state of suspended animation and must ingest nutrients at a high rate and cope with the associated osmotic and ionic problems. The waste products generated during metabolism are often toxic and cannot accumulate to high levels in the body without serious consequences. Thus the cellular environment must be freed of these toxic by-products of metabolism. In the smallest aquatic organisms, this purification happens simply by diffusion of the wastes into the surrounding water. In animals that have circulatory sys-

tems, the blood typically passes through excretory organs, generally termed kidneys. In terrestrial animals, the kidneys not only play an important role in the removal of organic wastes but are also the primary organs of osmoregulation.

A number of mechanisms are employed to handle osmotic problems and regulate the differences (1)between intracellular and extracellular compartments and (2)between the extracellular compartment and the external environment. These are collectively termed osmoregulatory mechanisms,a term coined in 1902 by Rudolf Hober to refer to

TABLE 14-3 Electrolyte composition of the human body fluids

Electrolytes

Serum (meq kg-' H,O)

.

Interstitial fluid (meq kg-' H20)

.

lntracellular fluid (muscle) (meq kg-' H,O)

Cations Na+

K+ Ca2+ Mg2+ Totals

Anions CI-

104

114

HC03-

27

31

HPO,~-

sotOrganlc aclds Proteln Totals

2 1 6 13

153

145

2 8 95 20 55 180

Note: Some of the ions contained within cells are not completely dissolved within the cytosol, but may be partially sequestered within cytoplasmic organelles. Thus, the true free Ca2+concentration in the cytosol is typically below the overall value given in the table for intracellular Ca2+.Failure of anion and cation totals to agree reflects incomplete tabulation.

574


........................................

the regulation of osmotic pressure and ionic concentrations in the extracellular compartment of the animal body. The evolution of efficient osmoregulatory mechanisms had extraordinarily far-reaching effects on other aspects of animal speciation and diversification. The various adaptations and physiological mechanisms evolved by animals to cope with the rigors of the osmotic environment form especially fascinating examples of the resourcefulness of evolutionary adaptation. This is the theme of an excellent book by the late Homer Smith entitled From Fish to Philosopher. Although there may be hourly and daily variations in osmotic balance, an animal is generally in an osmotic steady state over the long term. That is, on the average, the input and output of water and of salts over an extended period are equal. Water enters terrestrial animals with their food and drink. For animals living in a freshwater environment, water enters the body primarily through the respiratory epithelium-the gill surfaces of fish and invertebrates, and the integument of amphibians and many invertebrates. Water leaves the body in the urine, in the feces, and by evaporation through the lungs and integument, the outer covering. The problem of osmotic regulation does not end with the intake and output of water. If that were so, osmoregulation would be a relatively simple matter: A frog sitting in freshwater that is far more dilute than its body fluids would merely have to eliminate the same amount of water as leaked in through its skin, and a camel would just stop urine production between oases. Osmoregulation also involves the maintenance of favorable solute concentrations in the extracellular compartment. Thus, the frog immersed in hypotonic pond water is faced not only with the need to eliminate excess water, but also with the problem of retaining salts, which tend to leak out through the skin, because the skin in amphibians is generally more permeable than that in the other vertebrate classes. The osmotic exchanges that take place between an animal and its environment can be divided into two classes (Figure 14-1): Obligatory osmotic exchanges, which occur mainly in response to physical factors over which the animal has little or no physiological control

Regulated osmotic exchanges, which are physiologically controlled and serve to aid in maintaining internal homeostasis. Regulated exchanges generally serve to compensate for obligatory exchanges. The flux of a substance across a membrane is determined by its concentration gradient, the surface area of the membrane involved, the thickness of the membrane (i.e. the diffusion distance), and the permeability of the membrane. The same factors influence both obligatory and regulated exchanges. In the next section, we consider obligatory exchange and then, in the following sections, various mechanisms of regulated exchange.

OBLIGATORY EXCHANGE OF IONS AND WATER The integument, respiratory surfaces, and other epithelia in contact with the surrounding milieu act as the barriers to obligatory exchange between an organism and its environment. The various factors that contribute to the obligatory exchange are outlined next. Gradients Between the Animal and the Environment

The greater the difference between the concentration of a substance in the external medium and that in the body fluids, the greater the tendency for net diffusion in the direction of the lower concentration. Thus, although a frog immersed in a pond tends to take up water from its hypotonic environment, a bony fish in seawater is faced with the problem of losing water to the surrounding hypertonic seawater. Similarly, a marine fish with a lower NaCl content than that of seawater faces a continual diffusion of salt into the body, whereas a freshwater fish faces a continual loss of salt. The rate of transfer depends on the size of the gradient and the permeability and area of the animal's surface. Surface-to-VolumeRatio

The volume of an animal varies with the cube of its linear dimensions, but its surface area varies with the square of its linear dimensions. Therefore, the surface-to-volume ratio is greater for small animals than for large animals. It fol-

Figure 14-1 Two major classes of osmotic exbechange-obligatory and controlled-occur tween an animal and its environment.Obligato* exchanges occur in response to physical factors over which the animal has little short-term physiological control. Controlled exchanges are those that the animal can vary physiologically to maintain internal homeostasis. Substances entering the animal by either path can leave by the other path.

-------------Transepithelial diffusion, in defecation, metabolic water p

-------

Active epithelial transport

CONTROLLE

.

.

I O N I C AND OSMOTIC BALANCE Flea

Figure 14-2 Small an~malsdehydrate more rap~dlythan large an~rnalsbecause of the~rh ~ g hsurface-to-mass (and thus surface-to-volume) rat~osThls log-log plot shows the amount of water, as percentage of body we~ght,that IS lost per hour under hot desert cond~t~ons versus

3-

-c I

=. 8 2

n o n

575

2-

body we~ght [Edney and Nagy, 1976.)

C

0 .C

F

0

a

m>

20

1-

(I)

C

F rn 0

J

70,000g (1.47%)

0 -4

-3

-2

-1

0

1

2

3

4

5

Log body weight (g)

lows that the surface area of the integument, through which water or a solute can exchange with the environment, is greater relative to the water content of a small animal than it is for a large animal. This means that for a given net rate of exchange across the integument (in moles per second per square centimeter), a small animal will dehydrate or hydrate more rapidly than a larger animal of the same shape (Figure 14-2). Permeability of the Integument

The integument acts as a barrier between the extracellular compartment and the environment. Movement of water across the integument occurs through cells (transcellular) and between cells (paracellular). Pure phospholipid bilayers, however, are not very permeable to water, and transcellular movement of water across biological membranes depends on the presence of water channels. For example, erythrocytes swell or shrink rapidly in response to changes in the osmotic strength of the extracellular fluid because of the presence of a 28-kDa protein, appropriately called aquaporin. It appears that water channels in membranes are formed of a tetramer of identical aquaporin molecules. The role of aquaporin as a water channel was demonstrated in experiments with frogs eggs and oocytes, which are not very permeable to water and thus do not swell much when placed in pond water. When mRNA encoding the aquaporin protein was injected into frog oocytes, they became very water permeable and swelled when placed in water. Membrane permeability to water is presumably related to the concentration of aquaporin water channels within the phospholipid bilayer. Tight junctions between cells reduce the permeability of the paracellular pathway to water. The absence of aquaporin water channels in the membrane reduces the transcellular water permeability The permeability of the integument to water and solutes varies among animal groups. Amphibians generally

have moist, highly permeable skins, through which they exchange oxygen and carbon dioxide and through which water and ions move by passive diffusion. Amphibian skin compensates for loss of electrolytes by active transport of salts from the aquatic environment into the animal. Fish gills are necessarily permeable, as they engage in the exchange of oxygen and carbon dioxide between the blood and the aqueous environment. The gills, like the frog skin, also engage in active transport of salts. The volume of blood perfusing the gills of fish has been shown to decrease as respiratory demand drops and to rise in response to increased oxygen need. This reduction in blood perfusion of the gills effectively limits osmotic transfer through the gill epithelium during periods of low oxygen uptake. Thus, as oxygen transfer increases so does osmotic and ionic exchange across the gills. In contrast, reptiles, some desert amphibians, birds, and many mammals have relatively impermeable skins and thus generally lose relatively little water through this route. In fact, the skin of some mammals (e.g., cow hide) is so impermeable that it can be used to carry water or even wine. The low permeability of the integument of terrestrial animals is maintained in those species that have secondarily become marine or aquatic, such as pond insects and marine mammals. Not all vertebrates, however, have a relatively impermeable integument. Many amphibians, as well as mammals that perspire, can become dehydrated at low humidity because of water loss through the integument. Animals with highly permeable skin are simply not able to tolerate very hot, dry environments. Most frogs stay close to water. Toads and salamanders can venture a bit farther, but they also are limited to moist woods or meadows not far from puddles, streams, or bodies of water in which they can replenish their supply of -body water. These animals also minimize water loss by behavioral strategies, avoiding

Epidermis

desiccation by staying in cool, damp microenvironments during hot, dry times of day. The desert toads, Chiromantis xerampelina and Phyllomedusa sauuagii, have extremely low evaporative loss of water from their skin because it is covered by a secreted wax coating. These toads also excrete uric acid rather than ammonia or urea (seethe later section Excretion of Nitrogenous Wastes). Frogs and toads are endowed with a large-volume lymphatic system and an oversized urinary bladder in which they store water until needed. When these animals wander from a body of water or during periods of low rainfall, water will move osmotically from the lumen of the bladder into the partially dehydrated interstitial fluid and blood. The epithelium of the bladder, like the amphibian skin, is capable of actively transporting sodium and chloride from the bladder lumen into the body to compensate for the loss of salts that accompanies excessive hydration during times of plentiful water. Thus, the anuran bladder serves a dual function as a water reservoir in times of dehydration and as a source of salts during times of excessive hydration. The high water permeability of amphibian skin is used to advantage to take up water from hyposmotic sources such as puddles. Many anurans have specialized regions of skin on the abdomen and thighs, termed the pelvic patch, that when immersed can take up water at a rate of three times the body weight per day. The permeability of amphibian skin is controlled by the hormone arginine vasotocin (Am) or more simply uasotocin; like the mammalian hormone vasopressin, or antidiuretic hormone (ADH),vasotocin enhances water permeability. The outer layers of toad skin

Figure 14-3 The waxy lipid layer on the outside of the insect integument serves as the major water barrier, reducing evaporative water loss in insects. The waxy layer is deposited through minute canals in the integument. [Adapted from Edney, 1974.1

contain minute channels that draw water by capillary action to moisten the skin, conserving internal water during cutaneous evaporation. Because insects have a waxy cuticle that is highly impermeable to water, their evaporative water loss is much lower than in many other animal groups (Table 14-4).The wax is deposited on the surface of the exoskeleton through

24

28

32

Cuticular temperature

36

("C)

Figure 14-4 The rate of water loss from insects is much higher at temperatures above the melting point of the waxylayercovering the cuticle. The sharp break in this plot of water loss versus cuticular temperature In a cockroach corresponds to the melt~ngpolnt of the waxy cuticle. [From Beament, 1958.1

I O N I C A N D OSMOTIC BALANCE 577 ......................................

TABLE 14-4 Evaporative water loss of representative animals under desert conditions Water loss (mg .cm-' h-')

Species -

--

Remarks*

-

Antropods Eleodes armata (beetle) Hadrurus arizonensis

(scorpion) Locusta mrgratoria (locust)

Amphibian Cyclorana alboguttatus (frog)

Reptiles Gehrydra variegata (gecko) Uta stansburiana (lizard)

30°C; dry air 30°C

Birds Arnphispiza bell; (sparrow) Phalaenpitus nutalllii (poo'will)

Mammalst Peromyscus eremicus (Cactus mouse) Oryx beisa (African oryx) Homo sapiens

70 kg; nude, sitting in sun;

35°C *r.h. stands for relative humidity.Where not indicated, relative humidity is not available. +The cactus mouse and African oryx are desert animals and employ various water-conservation measures. Thus their evaporative water loss is much less than that of humans. Source: Hadley, 1972

,

fine canals that penetrate the cuticle (Figure 14-3).The importance of the waxy layer for water retention by insects has been demonstrated by measuring the rate of water loss at different temperatures. In Figure 14-4, we see that there is a sudden jump in the rate of water loss coincident with the melting point of the wax coating. The major route of water loss in terrestrial insects is via the tracheal system, which consists of air-filled tracheoles that penetrate the tissues. As long as the tracheoles are open to the air, water vapor can diffuse out while oxygen and carbon dioxide diffuse down their respective gradients. The entrances to the tracheoles are guarded by valvelike spiracles that are closed periodically by the spiracular muscles, conserving water. The importance of this mechanism in water conservation in insects, however, has been questioned (see Chapter 13 for further discussion). Feeding, Metabolic Factors, and Excretion

Water and solutes are taken in during feeding. Those end products of digestion and metabolism that cannot be used by the organism must be eliminated. Carbon dioxide diffuses into the environment from the respiratory surfaces. Al-

though water is another end product of cellular metabolism, it is produced in small enough quantities that its elimination is no problem (Table 14-5).In fact, this so-called metabolic water is the major source of water for many desert dwellers. Osmotic problems are posed by the inevitable production of nitrogenous end products of metabolism (e.g., ammonia and urea) and by the ingestion of salts, because water is required for their elimination from the body. The diet may include excess water or excess salts. A seal feeding on marine invertebrates with an osmolarity similar to seawater ingests a relatively high quantity of salt relative to water but requires water to excrete the salt load. If the seal feeds on marine teleost fish, which are more dilute than seawater, the ingested salt load is much less. The seal burns fat to produce both energy and water when eating marine invertebrates but stores fat when eating fish. The burning of fat produces the water required to excrete the salt load associated with eating marine invertebrates (see Table 14-5). Thus the seal becomes fat when eating fish but gets thin eating marine invertebrates. In terrestrial animals, the regulation of plasma ion concentrations and the excretion of nitrogenous wastes are accompanied by unavoidable losses of body water. A number of physiological adaptations tend to minimize the loss of water associated with these important functions of excretory systems. Among terrestrial invertebrates, insects are highly effective in conserving water in the course of eliminating nitrogenous and inorganic wastes. The extent to which ions are reabsorbed in the insect rectum or eliminated with the feces is regulated according to the osmotic condition of the insect. This is illustrated by an experiment in which locusts were allowed to drink either pure water or a concentrated saline solution containing NaCl and KC1 (450 mosm- L-l). The salt concentration of the rectal fluid after the insect drank saline was several hundred times higher than after it drank pure water, whereas the salt concentration of the hemolymph increased by only about 50% after drinking saline (Table 14-6). The kidney is the chief organ of osmoregulation and nitrogen excretion in most terrestrial vertebrates, especially mammals, which have no other provision for the excretion of salts or nitrogen. The kidneys of birds and mammals utilize countercurrent multiplication to produce a TABLE 14-5 Production of metabolic water during oxidation of foodstuffs Foodstuff Carbohydrates

Fats

Proteins

Grams of metabolic water per gram of food

0.56

1.07

0.40

Kilojoules expended per gram of food

17.58

39.94

17.54

Grams of metabolic water per k~lojoule expended

0.032

0 027

0.023

Source. Edney and Nagy, 1976

578


.........................................

TABLE 14-6 lonic regulation in locusts*

Concentration (mean values In meq. L-') Fluld

Na

K

CI

Sallne for drlnk~ng

300

150

450

108 158

11 19

5 569

1

22 24 1

5 569

Hernolyrnph Wlth water t o drlnk Wlth sallne t o d r ~ n k

Rectal fluid Wlth water t o drlnk Wlth sallne t o drlnk

405

*Desert locusts were given strong saline or pure water to drink. When they drank saline, the ionic concentrations in the hemolymph rose, but not to the level of the saline. lonic concentrations in their rectal fluid became higher than those in saline. Source: Edney and Nagy, 1976

hyperosmotic urine, which is more concentrated than the plasma. This specialization, centered on a hairpin-like bend in the kidney tubules, called the loop of Henle, has undoubtedly been of major importance in allowing birds and mammals to exploit dry terrestrial environments. The loop of Henle reaches its highest degree of specialization in desert animals such as the kangaroo rat and Australian hopping mice, which can produce a urine of up to 9000 mosm L-I. In birds, the countercurrent organization of the loop of Henle is less efficient, perhaps because the avian kidney contains a mixture of "reptilian-typem tubules, which lack the loop of Henle, and "mammalian-type'' tubules, which contain this specialized structure. The highest osmolarities determined in avian urine (in the salt-marsh Savannah sparrow) have been around 2000 mosm L-l. Reptiles and amphibians, whose kidneys are not organized for countercurrent multiplication, are unable to produce a hyperosmotic urine. As an adaptive consequence, some amphibians, when faced with dehydration, are able to cease urine production entirely during the period of osmotic stress.

.

.

Temperature, Exercise, and Respiration

Because of its high heat of vaporization, water is ideally suited for the e h n a t i o n of body heat by evaporation from

epithelial surfaces. During evaporation, those water molecules w ~ t hthe highest energy content enter the gaseous phase and thus take with them their thermal energy. As a result, the water left behind becomes cooler. The importance of water in temperature regulation leads to conflicts and compromises between physiological adaptation to environmental temperatures and osmotic stresses in terrestrial animals. Desert animals, faced with both high temperatures and a meager water supply, are especially hard pressed as they must avoid becoming overheated and yet avoid losing large quantities of body water. In some instances desert mammals and birds will let their body temperature rise above 40°C rather than expend water for evaporative cooling. Strenuous exercise generates heat owing to muscle metabolism and must be compensated by a high rate of heat dissipation. This compensation can be accomplished best by evaporative cooling over respiratory surfaces (e.g., the lungs, air passages, and tongue) or by evaporative water loss through the skin. In some very active mammals, body temperature rises during exercise, but brain temperature remains normal due to a countercurrent heat exchanger in the nasal region that cools the brain blood supply. Even during basal conditions (no exercise beyond breathing), the nature of the respiratory mechanism of many terrestrial animals leads to the loss of water through the respiratory surfaces. The nose of mammals plays an important role in reducing water loss through this pathway, As we have noted, respiratory surfaces are, by their very nature, a major avenue for water loss in air-breathing animals. The internalization of the respiratory surfaces in a body cavity (i.e., the lung) reduces evaporative loss in terrestrial vertebrates. Even within the lung, however, ventilation of the respiratory epithelium by unsaturated air will cause evaporation of the moisture wetting the epithelial surface. Such evaporative loss of water is enhanced in birds and mammals because their body temperature generally is higher than the ambient temperature. The same holds for those reptiles and amphibians that raise their body temperature by behavioral strategies. In such animals, the warmer expired air contains more water than the cooler inspired air as the water-holding capacity of air increases with temperature (Figure 14-5).

70 Figure 14-5 Water loss associated with respiration de-

60 Lung air saturated

- 4050 3 30 .k

m

F

20 10 0

10 20 30 Ambient air temperature

40

pends on the relationship between the body temperature and the inhaled-air temperature, as well as on the relative humidity of the inspired air. As unsaturated air is warmed in the lungs, ~ t spicks up moisture until it is saturated (gray bars). During exhalation, the air is cooled in the nasal passages, so that much of the water is recovered (red bars). The data here are for the kangaroo rat when the inhaled air is at 25% relative humidity (r.h.1and 15°C (left bars) or 30°C (right bars). Clearly, the amount of water recovered is greaterand hence the amount lost is less when the inhaled air is at the lower temperature. Indeed, under these climatic conditions, the kangaroo rat exhales air at 13'C (lower than ambient!). [Adapted from Schmidt-Nielsen et al., 1970.1

I O N I C A N D OSMOTIC BALANCE 579 ...............................................................................

The respiratory loss of water is minimized through a mechanism first discovered in the nose of the desertdwelling kangaroo rat, Dipodomys merriami, by Knut Schmidt-Nielsen. This mechanism, termed a temporal countercurrent system, retains most of the respiratory water vapor by condensing it on cooled nasal passages during expiration. Air entering the nasal passages is warmed to about 37-38°C and humidified by heat and moisture absorbed from the tissues of the nasal passages, trachea, and bronchi (Figure 14-6A). The nasal passages are cooled by this evaporative water loss and the flow of cool air through the nasal passages. The tissue temperature is lowest at the tip of the nose and increases along the nasal passages towards the lung. The nose has a large blood supply to maintain the delivery of water to humidify the incoming air. The blood supply does not warm the nose because it is arranged in a countercurrent fashion, so that warm blood entering the nasal region is cooled by cold blood leaving the nose. During exhalation, the process of heat exchange between air and nasal tissues is reversed. The warm expired air is cooled to somewhat above ambient as it passes back out through the nasal passages, which had been cooled by the same air during inhalation. As the expired air gives up some of its heat to the tissues of the nasal passages, most of the acquired moisture condenses on the cool nasal epithe-

A Inspiration

I

lium (Figure 14-6B). Mammals, Including humans, employing this mechanism to humidify inhalant air have "cold" noses, which can be wet or even occasionally drip. With the next inhalation, this condensed moisture again contributes to the humidification of the inspired air, and the cycle is repeated, most of the vapor being recycled within the respiratory tract. The nose, therefore, plays an important role in reducing the loss of water and heat from the body. The importance of the nose in cooling expired air can be detected easily by placing your hand in front of your nose and mouth and breathing out via your mouth and via your nose; the temperature difference is usually obvious. Because there is little cooling of air expired through the mouth, ,the loss of water and heat is greater when breathing out via the mouth (e.g., when the nose is clogged due to a cold) compared with expiration through the nose. If air flow through the nasal passages is bypassed by placing a tube in the trachea, as during human or animal surgical operations, heat and. water loss may increase; and surgical patients must be given additional food and water to compensate for the increased water loss. The increased water loss from the trachea can contribute to post-surgical sore throat, a common problem. A similar mechanism for trapping exhaled moisture occurs in numerous birds and lizards. Where salt glands drain

B Expiration

Outgo~ngair is cooled and loses water, wetting

Air

Figure 14-6 Temporal countercurrent exchange in the respiratory systerns of many vertebrates acts to conserve body heat and body water. (A) During inspiration, cool air (e.g., at 28°C) is warmed and humidified as it flows toward the lungs, removing heat and water from the nasal pas-

sages. (B) During expiration, the same air loses most of the heat and water it gained earlier, as it warms and deposits water on the cooled nasal passages on its way out. Small red arrows indicate direction of heat and water movement; long arrows indicate direction of air flow.

580


.......................................

into the nasal passages, as they do in iguanas, the water in the excreted salt solution enters the incoming air during inspiration and is largely conserved by being condensed during exhalation. Occassionally all the water from the salt solution is evaporated, leaving salt deposits around the nose of these seawater-drinking lizards. This will occur, however, only if the animal's temperature is above that of the environment. Water loss via the lungs is small for mammals living in hot humid climates and large for those living in cold dry climates. The rate of ventilation and the ventilation pattern (e.g., breathing through either the mouth or nose) also affect the rate of water loss via the lungs. The problem of respiratory water loss is much less in animals with body temperatures similar to the ambient temperature; in this case the air has only to be humidified at ambient temperature. A reptile with a low metabolic rate and, therefore, a low vefitilation rate and with a body temperature equal to the ambient temperature will have only minimal rates of water loss via the lung. This gives reptiles a n advantage over mammals in regions where water is scarce.

What is the effect of exerclse on water flux In

from marine invertebrates continue to function for many hours when placed in seawater. Ionic concentrations in the body fluids of freshwater and terrestrial invertebrates differ from seawater; in these animals the body fluids are invariably more dilute than seawater but considerably more concentrated than freshwater. Some aquatic invertebrates are strict osmoregulators like the vertebrates, some are limited osmoregulators, and some are strict osmoconformers. These classes are illustrated in Figure 14-7, in which the osmolarity of the extracellular compartment is plotted against the osmolarity of the aqueous environment. As the osmolarity of the environment changes, the osmolarity of a strict osmoconformer changes by an equal amount, paralleling the line that describes internal-external osmolar equality. In contrast, a strict osmoregulator maintains a constant internal osmolarity over a large range of external osmolarities, so as to produce a horizontal plot parallel with the abscissa. Limited osmoregulators regulate over a limited range of osmolarities and conform at other environmental osmolarities.

Do str~ctosmoconformers and strrct osmoregulators really ex1st7

freshwater and marine teleosts?

OSMOREGULATORS AND OSMOCONFORMERS Animals that maintain an internal osmolarity different from the medium in which they are immersed have been termed osmoregulators. An animal that does not actively control the osmotic condition of its body fluids and instead conforms to the osmolarity of the ambient medium is termed an osmoconformer. Table 14-1 reveals these two extremes of adaptation. Most vertebrates, with the notable exception of elasmobranchs and hagfish, are strict osmoregulators, maintaining the composition of the body fluids within a small osmotic range. Although some osmotic differences do exist among vertebrate species, the blood of vertebrates is hyposmotic (or slightly hyperosmotic, as in sharks) to seawater and significantly hyperosmotic to freshwater. This is true, as well, of fishes that migrate between freshwater and saltwater environments; these employ endocrine mechanisms to meet the changing osmotic stresses accompanying environmental change. Many terrestrial invertebrates also osmoregulate to a large degree. Aquatic, brackish-water, and marine invertebrates are, of course, exposed to various environmental osmolarities. Marine invertebrates, as a rule, are in osmotic balance with seawater, and the ionic concentrations in their body fluids generally parallel the seawater in which the species live. This similarity has allowed the use of seawater as a physiological saline in studies of the tissues of marine species. For example, some large neurons removed

"

1 b . "

r..

S Q I l U .

L1

Osmoconformers display a high degree of cellular osmotic tolerance, whereas osmoregulators maintain strict extracellular osmotic homeostasis in the face of the large environmental differences in electrolyte concentration. In osmoregulating animals, the internal tissues are generally not able to cope with more than minor changes in extracellular osmolarity and must depend entirely on osmotic regulation of the extracellular fluid to maintain cell volume. The cells of osmoconformers, on the other hand, can cope with high plasma osmolarities by increasing their intracellular osrnolarities, thereby maintaining cell volume. This is

Internal-external

/ osmolar equality

Strict osmoregulator Limited osrnoregulator

Osrnolarity of environment Figure 14-7 Aquat~canlmals can be classlf~edInto three groups based between the osmolar~tyof thew body flu~dsand that on the relat~onsh~p of the environment In these plots of Internal versus external osmolar~ty, the behavlor of a strlct osmoconformer parallels the l~nerepresenting equal~tyof ~nternal-external oqmolar~ty(blgck I~ne).

...................................... achieved by increasing the concentration of intracellular organic osmolytes, which are substances that by their presence in high concentrations act to increase intracellular osmolarity. The use of such substances reduces the need to maintain osmotic pressure with inorganic ions, which could give rise to other problems (e.g., lower enzyme efficiency). In some marine vertebrates and invertebrates, organic osmolytes also are present in the blood and interstitial fluids, as well as inside cells, so that both extracellular and intracellular osmolarity are brought close to that of seawater. The best-known examples of such organic osmolytes are urea and trimethylamine oxide, both utilized by various marine elasmobranchs, the primitive coelacanth fish Latimeria, and the crab-eating, brackish-water frog Rana cancrivora of Southeast Asia (see Table 14-1, page 572).

OSMOREGULATION IN AQUEOUS AND TERRESTRIAL ENVIRONMENTS Animals face quite distinct osmotic problems in aqueous and terrestrial environments. In this section, we first discuss osmoregulation by water-breathing animals and then consider air-breathing animals. Figure 14-8 presents an overview of water and salt exchange in various osmoregulating animals. Water-Breathing Animals

Many aquatic animals find themselves and all their respiratory surfaces immersed in water. The osmolarities of aqueous environments range from a few milliosmoles per liter in freshwater lakes to about 1000 mosm-L-' in ordinary seawater, or even more in landlocked salt seas. Intermediate environments, such as brackish bogs, marshes, and estuaries, have salinities ranging between these extremes. As a rule, the body fluids (i.e., interstitial fluids and blood) tend away from the environmental osmotic extremes. Euryhaline aquatic animals can tolerate a wide range of salinities, whereas stenohaline animals can tolerate only a narrow osmotic range. In this section, we consider the nature of the osmotic problems faced by freshwater and marine animals and their mechanisms for dealing with them. Freshwater animals The body fluids of freshwater animals, including invertebrates, fishes, amphibians, reptiles, and mammals, are generally hyperosmotic to their aqueous surroundings (see Table 14-1). Freshwater vertebrates have blood osmolarities in the range of 200 to 300 mosm. L-', while the osmolarity of freshwater is generally much less than 50 mosm-L-l. Because they are hyperosmotic to their aqueous surroundings, freshwater animals face two kinds of osmoregulatory problems: They are subject to swelling by movement of water into their bodies owing to the osmotic gradient They are subject to the continual loss of body salts to the surrounding medium, which has a low salt sontent,

Thus freshwater animals must prevent the net gain of water and net loss of salts, which they accomplish by several means. One way to avoid a net gain of water is production of a dilute urine. Among closely related fishes, for example, those that live in freshwater produce a more copious (i.e., plentiful and hence dilute) urine than their saltwater relatives (see Figure 14-8). The useful salts are largely retained by reabsorption into the blood from the ultrafiltrate in the tubules of the kidney, and thus a dilute urine is excreted. Nonetheless, some salts pass out in the urine, so there is a potential problem of gradually washing out biologically important salts such as KCI, NaCl, and CaCI,. Lost salts are replaced, in part, from ingested food. An important specialization for salt replacement in freshwater animals is active transport of salt from the external dilute medium across the epithelium into the interstitial fluid and blood. This activity is accomplished across transporting epithelia such as those in the skin of amphibians and in the gills of fishes. In fishes and many aquatic invertebrates, the gills act as the major osmoregulatory organs, having many of the functions located in the kidneys of mammals. Freshwater animals have remarkable abilities to take up salts from their dilute environment. Freshwater fishes are able, for example, to extract Na+ and C1- ions with their gills from water containing less than 1 mM NaC1, even though the plasma concentration of the NaCl exceeds 100 mM (Figure 14-9A). Thus, the active transport of NaCl in the gills takes place against a concentration gradient in excess of 100-fold. The mechanism of sodium reabsorption appears to be similar in the gills of freshwater fishes, frog skin, turtle bladder, and the mammalian kidney. In all cases the cells of these epithelia are joined together by tight junctions. Transport of Na+ into these cells is dependent on an electrogenic proton ATPase, which actively transports protons out of the cells into the environment. The mechanism of sodium reabsorption is discussed in detail later. In some freshwater animals, including fishes, reptiles, birds, and mammals, water uptake and salt loss are minimized by an integument having low permeabilities to both salts and water. As a general rule, animals living in freshwater refrain from drinking fresh water, reducing the need to expel excess water. Marine animals In general, the intracellular and extracellular body fluids of marine invertebrates and the ascidians (primitive chordates) are close to seawater both in osmolarity (isosmotic) and in the plasma concentrations of the individual major inorganic salts (see Table 14-1). Such animals therefore need not expend much energy in regulating the osmolarity of their body fluids. A rare example of a vertebrate whose plasma is also isosmotic to the environment is the hagfish. It differs from most marine invertebrates, however, in that it does regulate the concentrations of individual ions. In are maintained at particular, blood Ca2+,Mg2+,and SO-:

582


...............................................................................

Type of animal

Mar~ne elasmobranch

Blood concentration relative t o environment

Isotonic

Urine concentration relative t o b l o o d

Osmoregulatory mechanisms

Does not drink seawater

lsoton~c Hypertonic NaCl from rectal gland

Marine teleost

Hypoton~c

Isotonic

-

--_

seawater Drinks Secretes salt from gills

Freshwater teleost

Hypertonic

Strongly hypotonic

Drinks no water Absorbs salt with gills

Amph~blan

Hyperton~c

Marine rept~le

Hypotonic

Strongly hypotonic

@

Absorbs salts through s k ~ r

-

Isotonic

Drinks seawater

a

secretion

Strongly hypertonic

Desert mammal

Drinks no water

Depends on metabolic water

Marine mammal

Hypotonic

Strongly hypertonic

Does not drink seawater

c Drinks seawater

Marine bird

Weakly hypertonic

0

Weakly hypertonic urine

Hypertonic salt-gland secretion

c Dnnks fresh water b

Terrestrial bird

Weakly hypertonic

Figure 14-8 Animals living in different environments exhibit various osmoregulatory mechanisms. The active exchange of water and salt in

some vertebrates is illustrated here. Passive loss of water through the skin, lungs, and alimentary tract is not indicated.

significantly lower concentrations than they are in seawater, whereas Naf and C1- are higher. Since various functions of excitable tissues such as nerve and muscle of vertebrates are especially sensitive to the concentrations of Ca2+ and Mg2+,the regulation of these divalent cations

may have evolved to accommodate the requirements of neuromuscular function. Like the hagfish, the cartilaginous fishes such as sharks, rays, and skates, as well as the primitive coelacanth Lab timeria, have plasma that is approximately isosmotic to


583

...............................................................................

Food

+

only

Freshwater teleost

Marine teleost

Figure 14-9 Salt and water exchange is in the opposite direction in freshwater and marine teleosts. (A) Freshwater teleosts prevent the net gain of water and loss of salt by copious excretion of a dilute urine from which most of the salt has been reabsorbed. (B) Marine teleosts face the opposite osmotic problems, namely, avoiding a water deficit and excess of

salts. They achieve this by drinking seawater and then eliminating salts by several routes. Solid arrows indicate active processes; broken arrows, passive processes. Note the active role of the gills In salt transport in both groups. [Adapted from Prosser, 1973.1

seawater. They differ, however, in that they maintain far lower concentrations of electrolytes (i.e., inorganic ions), making up the difference with organic osmolytes such as urea and trimethylamine oxide (TMAO).High urea concentrations tend to cause the breakup of proteins into constituent subunits, whereas TMAO has the opposite effect, canceling the effect, of urea and stabilizing protein structure in the face of high urea levels. In the elasmobranchs and coelacanths, excess inorganic electrolytes such as NaCl are excreted via the kidneys and also by means of a special excretory organ, the rectal gland, located at the end of the alimentary canal. The body fluids of marine teleosts (modern bony fishes), like those of most higher vertebrates, are hypotonic to seawater, so there is a tendency for these fishes to lose water to the environment, especially across the gill epithelium. To replace the lost volume of water, they drink saltwater. Most of the net salt uptake is due to drinking seawater rather than salt uptake across the body surface or gills. By absorption across the intestinal epithelium, 70% to 80% of the ingested water enters the bloodstream, along with most of the NaCl and KC1 in the seawater. Initially the ingested seawater is diluted by about 50% by diffusional uptake of salts across the esophagus. Active salt uptake occurs in the small intestine, via a Na/2Cl/K cotransporter across the apical membrane and then by active transport via a Na+/K+ ATPase across the basolateral membrane. Left behind in the gut and expelled through the anus are most of the divalent cations such as Ca2+, Mg2-, and SO:-. The excess salt absorbed along with the water is subsequently eliniinated from the blood by active transport of Na+, C1-, and some K+ across the gill epithelium into the seawater, and by secretion of divalent salts by the kidney (see Figure 14-9B).The urine is isotonic to the blood, but rich in those salts (especiallyCa2+,Mg2+,and SO:-) that are not secreted by the gills. The net result of the combined osmotic work of gills and kidneys in the marine teleost is a net retention of water. The gills of marine teleosts, as might be exoected, are organized differently from those of freshwater fish. ma-

rine teleosts, the gill epithelium contains specialized cells, called chloride cells, that mediate transport of NaCl from the blood into the surrounding water. The mechanism of this transport, which makes it possible for these fishes to live in saltwater; is described in a later section.

Some teleost species-for example, the salmon of the Pacific Northwest and eels in eastern North America and Europe-are able to maintain a more-or-less constant plasma osmolarity even though they migrate between marine and freshwater environments. Such fish undergo a physiological adaptatlon that enable them to maintain a more-or-less constant ionic composition In both environments. Some of the physiological changes that occur when teleosts migrate from freshwater to seawater begin before the animal enters seawater. Eels, for instance, reduce the permeability of the integument, changing from yellow to silver in the process. Likewise, the gill reorganization that characterizes adaptation of salmon to seawater begins as the fish migrate down river to the ocean. We'll discuss the adaptation of migrating teleosts in detail later in this chapter. To summarize, freshwater animals tend to take in water passively and to remove it actively through the osmotic work of kidneys (vertebrates)or kidney-like organs (invertebrates). They lose salts to the dilute environment and replace them by actively absorbing ions from the surrounding fluids into their bodies through skin, gills, or other actively transporting epithelia. Marine fish, on the other hand, lose water osmotically through the gills or through the integument, if it is permeable. To replace lost water, marine fish drink seawater and actively secrete the excess

salt ingested with the seawater back into the environment. This process takes place through active transport in extrarenal osmoregulatory organs such as gills and the rectal gland. Air-Breathing Animals

Animals in a terrestrial environment can be thought of as submerged in an ocean of air rather than water. Unless the humidity of the air is high, animals having a waterpermeable epithelium will be subject to dehydration very much as if they were submerged in a hypertonic medium such as seawater. Dehydration would be avoided if all epithelial surfaces exposed to air were totally impermeable to water. The evolutionary process has not found this to be a feasible solution to the problem of desiccation, since an epithelium that is impermeable to water (and thus dry) will have limited permeability to oxygen and carbon dioxide, and will thus be unsuited for the respiratory needs of a terrestrial animal. As a consequence, air-breathing animals are subject to dehydration through their respiratory epithelia. Air-breathing animals utilize various means to minimize water loss into the air by this route and others (see Figure 14-8). Marine reptiles (e.g., iguanas, estuarine sea turtles, crocodiles, sea snakes) and marine birds drink seawater to obtain a supply of water but, like marine teleosts, are unable to produce a concentrated urine that is significantlyhyperosmotic to their body fluids. Instead, they are endowed with glands specialized for the secretion of salts in a strong hyperosmotic fluid. These salt glands are generally located above the orbit of the eye in birds and near the nose or eyes in lizards. Brackish-water crocodiles were long suspected of using extrarenal means of excreting salts, and eventually salt glands were discovered in the tongue of this reptile. Although neither reptilian nor avian kidneys are capable of producing a very hypertonic urine, the salt glands of marine reptiles and birds secrete a sufficiently concentrated salt solution to enable them to drink saltwater even though their kidneys are unable to produce urine more concentrated than seawater (Figure 14-10A). Salt glands compensate in these groups for the inability of their kidney to produce a urine that is strongly hypertonic relative to body fluids. Marine mammals, which lack salt glands or similar specializations, avoid drinking seawater, get their water entirely from their food intake and its subsequent metabolism, and depend primarily on their kidneys for maintaining osmotic balance. Human beings, like other mammals, are not equipped to drink seawater. The human kidney can remove up to about 6 g of Nat from the bloodstream per liter of urine produced. Because seawater contains about 12 g.L-' of Na+, imbibing seawater will cause a human being to accumulate salt without adding a physiologically equivalent amount of water (Figure 14-10B).Stated differently, to excrete the salt ingested with a given volume of seawater, the human kidney must pass more water than is contained in that volume. This, of course, will lead rapidly to dehydra-

Seawater (3%salt)

Seawater (3%salt)

Nasal

fluid

(5% salt)

Urine (3%salt)

Ur~ne salt)

(2'%

Figure 14-10 Marine reptiles and birds drink seawater to obtain water, whereas most mammals become dehydrated if they drink seawater. (A) When marine birds drinkseawater,they secrete NaCl via the salt glands, thereby eliminating 80% of the ingested salt along with only 50% of the ingested water. As a result, these birds can produce a hypotonic urine without dehydrating. (B) When humans and other mammals, who lack salt glands, drink seawater, they cannot concentrate the urine sufficiently t o conserve water while eliminating the ingested salt. Like terrestrial mammals, marine mammals cannot drink seawater; these animals use various water-conservation mechanisms to survive. [From "Salt Glands" by K. Schmldt-Nielsen,Copyright 0 1959 by Scientific American, Inc. All r~ghtsreserved.] I

tion. Thus humans stranded at sea will die unless they have access to freshwater. They cannot replace lost water by drinking seawater. If they do so, it will only make the problem worse. Humans require a constant source of fresh drinking water to excrete accumulated salts and metabolic waste products. Mammals cannot drink seawater, and yet marine mammals such as pinnipeds (e.g., sea lions, seals) and cetaceans (e.g., porpoises, whales) live in the ocean, even though they do not have extrarenal salt-secreting organs like the salt glands of birds and reptiles. Camels and many other mam-

IONIC AND OSMOTIC BALANCE

585

............................................................................... mals can survive in the desert. Thus, unlike humans, many mammals can survive in habitats where drinking water is not available. Joseph Priestley (1733- 1804),who was the first to isolate many gases, including oxygen, observed that he could keep mice alive without water for three or four months, in a cage on a shelf above the kitchen fireplace in his home in Yorkshire, England. That is, he observed that mice could survive for many months without drinking water. Another small mammal, the kangaroo rat, Dipodomys merriami, a native of the American Southwest, has become a classic example of how small mammals survive without drinking water in the arid conditions of the desert. Let's see how these mammals, as well as certain terrestrial arthropods, survive in the absence of freshwater.

Desert-living mammals The survival strategies practiced by the kangaroo-rat exemplify a variety of osmoregulatory adaptations characteristic of many small desert mammals (Figure 14-11).The kangaroo rat and other desert mammals are faced with a physiological double jeopardy-excessive heat and near absence of free freshwater. Water regulation and temperature regulation are, of course, closely related, since one important means of channeling excess heat out of the body into the environment is by evaporative cooling. Since evaporative cooling is at odds with water conservation, most desert animals cannot afford this method and have devised means of circumventing it. The kangaroo rat, like many desert mammals, avoids much of the daytime heat by remaining in a burrow during daylight hours and coming out only at night. This nocturnal lifestyle is an important and widespread behavioral adaptation to desert life. Not only does the cool burrow reduce the animal's temperature load, but it reduces respiratory water loss. The nasal countercurrent mechanism for conserving respiratory moisture depends, of course, on the ambient temperature in the burrow being significantly lower than the 37°C to 40°C characteristic of the core temperatures of birds and mammals. If the rodent ventures out of its cool burrow into air close to its own temperature, its respiratory water loss will rise

abruptly, since the cooling properties of the nasal epithelium will be reduced. Desert mammals also generally avoid heat-generating exercise during the day, when removal of excess heat from the body is slowed by the higher ambient temperature. Because of its efficient kidneys, the kangaroo rat excretes a highly concentrated urine, and rectal absorption of water from the feces results in essentially dry fecal pellets. By using all these adaptations for desert survival, the kangaroo rat greatly reduces its potential water loss. In spite of this extreme osmotic economy, the small amount of lost water must, of course, be replaced, or the animal will eventually dry up. Since the kangaroo rat eats dry seeds that contain only a trace of free water, is not known to drink, and in fact survives quite well in the near absence of free water, it must have a cryptic source of water. This, it turns out, is the metabolic water noted earlier (see Table 14-5). The exquisite conservation of water by the kangaroo rat allows it to survive primarily on the water produced during the oxidation of foods, so that over the long term water gains equal water losses (Table 14-7). Unlike the kangaroo rat, camels are too large to hide from the hot desert sun in burrows. When deprived of drinking water, camels do not sweat but allow their body temperature to rise rather than losing water by evaporative cooling during the heat of the day. During the cooler night, the camel's body temperature drops and increases only slowly the next day because of the animal's large body mass TABLE 14-7 Sources of water gain and loss by the kangaroo rat Gains

Losses 90%

Evaporat~on+ persp~rat~on

70%

Free water In "dry" food

10%

Ur~ne

25%

Dr~nk~ng

0% -

Feces

5% -

Metabol~c water

100%

100%

Source Schm~dt-N~elsen, 1972

Figure 14-11 The water-conserving strategies of the kangaroo rat are characteristic of many small desert dwellers.

Animal remains in cool burrow during daytime

ndensed in nasa

es dehydrated prior

586


............................................................................... Dehydrated

-e -$s

42

40

a,

n

Watered

-

38

>.

-0

CS; 36

37 36

r 34 1 Time (days)

V

I

6 A.M.

camel

I

I

12

6 PM.

Time

Figure 14-12 When water is scarce, large desert animals such as the camel exhibit a large, but slow, increase in body temperature during the day, whereas smaller animals heat up rapidly when exposed to the sun. (A)The daily temperature fluctuation in a well-watered camel and in a dehydrated one. When the camel is depr~vedof drinking water, the daily fluctuation may increase to as much as 7°C. This has a great influence on

the use of water for temperature regulation. (B) Diagrammatic representation of the daily patterns of body temperature in a large and a small mammal subjected to heat stress under desert conditions. Small animals must enter burrows per~odicallyto avoid overheating. [Part A from Schmidt-Nielsen, 1963; part B from Bartholomew, 1964.1

and thick fur, which acts as a heat shield. Nevertheless, the body temperature of a dehydrated camel may vary from 35°C at night to 41°C during the day (Figure 14-12A). This strategy of heating during the day and cooling at night is impossible in small rodents, whose body temperatures oscillate much more rapidly than in the larger camel (Figure 14-12B).Because of their small size, desert rodents heat up- rapidly . in the sun and must return to their burrow to cool down. The camel also reduces heating by orienting to give minimal surface exposure to direct sunlight. The camel, like other desert animals, produces dry feces and concentrated urine. When water is not available the camel does not produce urine but stores urea in the tissues. The camel can tolerate not only dehydration but also high - urea levels in the body. When water becomes available, these ships of the desert can rehydrate by drinking 80 liters in 10 minutes.

flow through the blow hole is high because both inspiration and expiration are rapid, and large volumes of gas are moved with each breath. It is possible that the expansion of air passing through the blow hole of a whale also cools the air, resulting in water condensation in the region of the blow hole that can be used to wet inspired air. This would reduce water loss via ventilation.

Marine mammals Marine mammals face problems similar to those of desert animals because they live in an environment without available drinking water. E e r . water everywhere and not a &to drink! The physiological responses of marine mam--- mals, although different in detail, are generally similar to those of desert mammals. The emphasis is on water conservation. They are endowed, as are other mammals, with highly efficient kidneys capable of producing a very hypertonic urine. Seals have a characteristic labyrinth-like proliferation of epithelial surfaces in the nasal passages which reduces water loss via breathing. Whales and dolphins have a blow hole rather than the typical mammalian nose. These animals have large lung tidal volumes. The velocity of air

-

A remarkable example of water retention in a marine mammal faced with desert-like problems of water conservation occurs in a recently weaned baby elephant seal. After being abandoned by its mother, the baby seal goes for 8-10 weeks without food or water. During this time the baby seal's only source of water is that derived from the oxidation of its body fat. It weighs about 140 kg at the time of weaning and loses only about 800 g of water per day, of which less than 500 g is lost through respiration. This economy is ascribed both to its nasal countercurrent heat exchanger and to a slowing of metabolic rate, which allows it to stop breathing for 40 minutes and then alternate with 5 minutes of deep breathing. The ability to suspend breathing is, of course, not uncommon for marine mammals such as the elephant seal, which can dive for prolonged periods. The ability to conserve water is also seen in the adult elephant seal. The large males spend up to three months on


587

................................... the beach and when on land do not drink or eat. The females suckle their young for about four weeks on the beach and then the pup is abandoned when the female goes to sea for four months. She returns for about a month to molt and then goes to sea for another six months. While at sea, the female elephant seal does not drink but relies on the water in fish in her diet and metabolic water to maintain her water requirements.

Terrestrial arthropods Certain terrestrial arthropods have the ability to extract water vapor directly from the air, even, in some species, when the relative humidity is as low as 50% (Table 14-8). To date, this poorly understood ability has been demonstrated only in certain arachnids (ticks, mites) and in a number of wingless forms of insects, primarily larvae. Those species that exhibit this ability live in habitats devoid or nearly devoid of free water. The ability to remove water from the air is all the more remarkable in these arthropods because it normally occurs even when the vapor pressure of the hemolymph exceeds that of the air, which it does at all values of relative humidity below 99%. TABLE 14-8 Critical equilibrium humidities for some arthropods that can extract water from the vapor phase Relatlve hum~dlty

Arachnida lxodes ricinus Rhiprcephalus sangurneus

92 0 84 0-90 0

lnsecta Thermobia domestrca Tenebrio molrtar larvae

45 0 88 0

Note At relatlve hum~dltlesbelow crltlcal, the an~malIS unable to extract molsture from the alr Source Edney and Nagy, 1976

The water vapor pressure associated with a solution decreases with increasing ionic content, so highly concentrated salt solutions will absorb water from air. Insects take advantage of this by creating very concentrated solutions that can absorb water from air. In insects that extract water from air, the site of entry is often the rectum, which reduces the water content of fecal matter to remarkably low levels. As water is removed from the feces, the latter can take on new water from the air, if the water vapor pressure is high enough and if the air has access to the rectal lumen. In ticks, tissues in the mouth have been implicated in the uptake of water vapor. Here it appears that the salivary glands secrete a highly concentrated solution of KC1 that in turn absorbs water from the air.

OSMOREGULATORY ORGANS The osmoregulatory capabilities of metazoans depend to a great extent on the properties of transport epithelia located in gills, skin, kidneys, and gut. The highly specialized

epithelial cells composing these epithelia differ from all other cell types in being anatomically and functionally polarized. The apical surface (sometimes referred to as the mucosal or luminal surface) of an epithelial cell faces a space that is continuous with the external world (the sea, the pond, the lumen of the gut, the lumen of a kidney tubule, etc.). The other side of the epithelial cell, the basal surface (sometimes referred to as the serosal surface) generally bears deep basal clefts and faces the internal compartment containing extracellular fluid. This internal compartment is the one that contains all the other cells of the remaining body tissues. These exist, so to speak, in their own private "pond," composed of the extracellular fluid in which they are bathed. The proper composition of this internal pond depends on the osmoregulatory work and barrier functions performed by epithelial cells. The excretion of nitrogenous wastes varies among species depending on water availability. The nature of the nitrogenous end product varies and many different organs are involved in the excretion of ammonia, urea, andlor uric acid. In freshwater fish, for example, ammonia is usually the major nitrogenous end product and the gills are the major site of excretion. In mammals, by contrast, the major nitrogenous end product is urea and the kidneys the site of excretion. Because the excretion of nitrogenous end products is variable and not organ specific, it is discussed at the end of this chapter. The mechanisms for transporting substances across epithelia were discussed in Chapter 4 and the same basic cellular machinery is used in all excretory or osmoregulatory organs. For example, similar salt-excreting cells are found in the nasal gland of birds and reptiles, the mammalian kidney, the rectal gland of elasmobranchs, and the gills of marine teleost fishes. Not only are the cells similar but their activities are regulated by similar arrays of hormones. The detailed functioning of organs with similar cellular structure may be different because of the gross organization of the organ. The capabilities of transport epithelia are greatly enhanced in osmoregulatory organs by their anatomic organization, as is exquisitely evident in the kidneys of mammals. Here, in addition to a high degree of cellular differentiation for transepithelial transport, the epithelium is organized into tubules arranged so as to enhance the transport efficiency of the tubular epithelium. This combination of cell function and tissue organization has produced a marvelously efficient osmoregulatory and excretory organ. The next several sections describe and compare the operation of various types of osmoregulatory organs found in different animals.

MAMMALIAN KIDNEY The mammalian kidney is the osmoregulatory organ of which we have the most complete understanding, thanks to intensive research over the past four to five decades. The mammalian kidney performs certain functions that in lower vertebrates are shared by other organs such as the

......................................

skin and bladder of amphibians, the gills of fishes, and the salt glands of reptiles and birds. Thus, the mammalian kidney is not representative of all vertebrate kidneys, which are organized somewhat differently in different groups of vertebrates.

Renal

Anatomy of the Mammalian Kidney

The gross anatomy of the mammalian kidney is shown in Figure 14-13. Each individual normally has two kidneys, one located on each side against the dorsal inner surface of the lower back, outside the peritoneum. In view of their small size (about 1% of total body weight in humans), the kidneys receive a remarkably large blood flow, equivalent to about 20%-25% of the total cardiac output. The kidney filters the equivalent of the blood volume every 4-5 minutes. The outer functional layer, the cortex, is covered by a tough capsule of connective tissue. The inner functional layer, the medulla, sends papillae projecting into the pelvis. The pelvis gives rise to the ureters, which empty into the urinary bladder. The urine leaves the bladder during micturition (urination) via the urethra, which leads to the end of the penis in males and into the vulva in females. Human adults produce about a liter of slightly acidic (pH approximately 6.0) urine each day. Urine production rates vary diurnally, being high during the day and low at night, reflecting the time course of water intake and production of metabolic water. Urine contains water and other by-products of metabolism, such as urea, as well as NaC1, KCl, phosphates, and other substances that are present at concentrations in excess of the body's requirements. The objective is to maintain a more-or-less constant body composition; hence the volume and composition of urine reflects the volume of fluid taken in and the amount and composition of ingested food. The actual volume of urine produced is determined by the volume of water ingested plus the water produced during metabolism minus evaporative water loss via the lungs and sweating and, to a lesser extent, that lost with the feces. When voided, urine is normally clear and transparent, but after a rich meal the urine may become alkaline and slightly turbid. The smell and color of urine is determined by the diet. For example, consumption of methylene blue will give urine, which typically is yellow, a distinctive blue color, and consumption of asparagus will completely change the more usual, slightly aromatic odor of urine. The release of urine is accomplished by the simultaneous contraction of the smooth muscle of the bladder wall and the relaxation of the skeletal muscle sphincter around the opening of the bladder. As the bladder wall is stretched by gradual filling of the bladder, stretch receptors in the wall of the bladder generate nerve impulses that are carried by sensory neurons to the spinal cord and brain, producing the "associated" sensation of fullness. The sphincter can then be relaxed by inhibition of motor impulses, allowing the smooth muscle of the bladder wall to contract under autonomic control and empty the contents. The presence of a bladder allows the controlled release of stored urine

Figure 14-13 The functional units of the mammalian kidney, called nephrons, are arranged in a radiating fashion within the renal pyramids. The distal end of each nephron within a pyramid empties into a collecting duct, which passes through a papilla into a calyx. The renal calyces drain In a central cavity termed the renal pelvis. The urine passesfrom the pelvis into the ureter, which takes it t o the urinary bladder. In this crosssectional drawing, only one nephron is depicted, although each pyramid contains many nephrons.

rather than a continual dribble paralleling the flow of urine from the kidney into the bladder. Such controlled release is used by some animals to mark out their territory. The functional unit of the mammalian kidney is the nephron (Figure 14-14), an intricate epithelial tube that is closed at its beginning but open at its distal end. Each kidney contains numerous nephrons, which empty into collecting ducts. These ducts combine to form papillary ducts, which eventually empty into the renal pelvis. At the closed end, the nephron is expanded-somewhat like a balloon that has been pushed in from one end toward its neck-to form the cup-shaped Bowman's capsule. The lumen of the capsule is continuous with the narrow lumen that extends through the renal tubule. A tuft of capillaries forms the renal glomerulus inside Bowman's capsule. This remarkable structure is responsible for the first step in urine formation. An ultrafiltrate of the blood passes through the single-cell layer of the capillary walls, through a basement membrane, and finally through another single-cell layer of epithelium that forms the wall of Bowman's capsule. The ultrafiltrate accumulates in the lumen of the capsule to begin its trip through the various segments of the renal tubule, finally descending the collecting duct and eventually into the renal pelvis. The wall of the renal tubule is one cell layer thick; this epithelium separates the lumen, which contains the ultrafiltrate, from the interstitial fluid. In some portions of the nephron, these epithelial cells are morphologically specialized for transport, bearing a dense pile of microvilli on their luminal, or apical, surfaces and deep infoldings of their

A

Juxtamedullary nephron

B

Cortical nephron

basal membranes (Figure 14-15). The epithelial cells are tied together by leaky tight junctions, which permit limited paracellular diffusion between the lumen and interstitial space surrounding the renal tubule. The nephron can be divided into three main regions: the proximal nephron, the loop of Henle, and the distal nephron. The proximal nephron consists of Bowman's capsule and the proximal tubule. The hairpin loop of Henle comprises a descending limb and an ascending limb. The latter merges into the distal tubule, which joins a collecting duct serving several nephrons. The nun-ber of nephrons per kidney varies from several hundred in lower vertebrates to many thousands in small mammals, and a million or more in humans and other large species. The loop of Henle, found only in the kidneys of birds and mammals, is considered to be of central importance in concentrating the urine. Vertebrates that lack the loop of Henle are incapable of producing a urine that is hyperosmotic to the blood. In mammals, the nephron is so oriented that the loop of Henle and the collecting duct lie parallel to each other (see Figure 14-14).The glomeruli are found in the renal cortex, and the loops of Henle reach down into the papillae of the medulla; thus the nephrons are arranged in a radiating fashion within the kidney (seeFigure 14-13). Nephrons can be divided into two groups:

Juxtamedullary nephrons, which have their glomeruli in the inner part of the cortex and long loops of Henle that plunge deeply into the medulla (seeFigure 14-14A)

Figure 14-14 The mammalian nephron is a long tubular structure, which is closed at its beginning in Bowman'scapsule but open at its terminus, where it empties into a collecting duct. The renal tubule and collecting duct are shown in yellow; the vascular elements in red or blue. Juxtamedullary nephrons (A) have a long loop of Henle, which passes deep into the renal medulla and is associated with a vasa recta. The blood first passes through the capillaries of the glomerulus and then flows through the hairpin loops of thevasa recta, which plunges into the medulla of the kidney along with the loop of Henle. The more common cortical nephrons (B) have a short loop of Henle, only a small portion of which enters the medulla, and lack a vasa recta. In these nephrons, the blood passes from the afferent arteriole to the glomerular capillaries and then leaves the ne~hron via the efferent arteriole.

Brush border

\

Mucosal side or apical side or luminal side

Serosal side or basolateral side or blood side

Figure 14-15 The cells of the proximal tubule are specialized for the transport of salts and other substances from the luminal (apical) side to the serosal (blood) side. The apical membranefacing the lumen is thrown into fingerlike projections (microvilli),greatly increasing its surface area. This surface is referred to as a brush border. Mitochondria are concentrated near the basolateral (serosal) surface, which is thrown into deep basal clefts. These features allow the concentration of salts in the renal interstitium by active transport of salts across the basal membrane.

590


.

............................................................................... Figure 14-16 Blood pressure in the renal glomerulus is high because of the low-resistance input pathway (afferentarteriole) and the high-resistanceoutput pathway. Regulation of glomerular blood pressure, which influences the filtration rate, is largely through modulation of the diameter of the afferent arteriole.

Bowman's capsule

Cortical nephrons, which have their glomeruli in the outer cortex and relatively short loops of Henle that extend only a short distance into the medulla (see Figure 14-14B) The anatomy of the renal circulatory system is important in the function of the nephron. The renal artery subdivides to form a series of short afferent arterioles, one of which supplies each nephron (see Figure 14-14). The glomerular capillaries within Bowman's capsule are subjected to somewhat higher pressures than other capillaries because of the low-resistance input pathway and highresistance output pathway (Figure 14-16).The capillaries of the glomerulus recombine to form an efferent arteriole. Unlike most other vessels, which would join to form veins, the efferent arteriole in juxtamedullary nephrons then subdivides again to form a second series of capillaries surrounding the loop of Henle. Thus the blood, on leaving the glomerulus located in the cortex, enters the efferent arteriole and is carried into the medulla in a descending and subsequently ascending loop of anastomosing (interconnecting) capillaries before leaving the kidney via a vein. The hairpin loops, which parallel the loops of Henle of juxtamedullary nephrons, are referred to as the vasa recta (see Figure 14-14A).Flow in the efferent arteriole is less than that in the afferent arteriole because around 10% of the blood is filtered across the Bowman's capsule. For humans this amounts to about a liter of filtrate formed every 10 minutes. Clearly urine flow rate is much less, so much of the initial filtrate formed in the Bowman's capsule is reabsorbed into the blood across the kidney tubule. Urine Production

Three main processes contribute to the ultimate composition of the urine (Figure 14-17): Glomerular filtration of plasma to form an ultrafiltrate in the lumen of the Bowman's capsule

Tubular reabsorption of approximately 99% of the water and most of the salts from the ultrafiltrate leaving behind and concentrating waste products such as urea Tubular secretion of a number of substances via active transport in nearly all instances Formation of the ultrafiltrate is the initial step in urine production; reabsorption and secretion occur along the length of the renal tubule. In addition to these processes, the excretion of nitrogenous wastes, discussed at the end of the

Urine Figure 14-17 Urineformation in the mammalian nephron involvesthree main processes. Filtration, the initial step, takes place in Bowman's capsule, followed by reabsorption and secretion, which occur along the renal tubule. The final product of these processes is a hypertonic urine, whose composition differs from that of blood.

chapter, involves the synthesis of certain excreted products in the tubular cells and lumen.

Glomerular filtration The glomerular ultrafiltrate contains essentially all the constituents of the blood except for blood cells and nearly all blood proteins. Filtration in the glomerulus is so extensive that 15%-25% of the water and solutes are removed from the plasma that flows through it. The glomerular ultrafiltrate is produced atthe rate of about 125 rnl-min-l, or about 180 L day-', in human kidneys. When this number is compared to the normal intake of water, it is evident that unless most of the glomerular ultrafiltrate is subsequently reabsorbed into the bloodstream, the body would be quickly dehydrated, thus much of the ultrafiltrate must be reabsorbed.

.

The process of ultrafiltration in the glomerulus (Figure 14-18)depends on three factors: (1)the net hydrostatic pressure ddference between the lumen of the capillary

and the lumen of Bowman's capsule, which favors filtration; (2)the colloid osmotic pressure, which opposes filtration; and (3) the hydraulic permeability (sievelikeproperties) of the three-layered tissue separating these two compartments. The net pressure gradient results from the sum of the hydrostatic pressure difference between the two compartments and the colloid osmotic pressure difference. The latter arises because of the separation of proteins during the filtration process. In humans, the proteins remaining in the capillary plasma produce an osmotic pressure difference of about -30 mm Hg, and the hydrostatic pressure difference (capillary blood pressure minus the back pressure in the lumen of the Bowman's capsule) is about +40 mm Hg (Table 14-9).The result is a net filtration pressure of only about + 10 mm Hg. This small pressure differential acting on the high permeability of the glomerular sieve produces a phenomenal rate of ultrafiltrate formation by the millions of glomeruli in each human kidney. It is important to note that the filtration process in the kidney is entirely passive, depending on hydrostatic pressure that derives its energy from the contractions of the heart. In lower vertebrates such as the salamander, the blood pressure in the glomerular capillaries is much lower than in humans, but the net filtration pressure is not that much less than in the human kidney, because of a lower intracapsular and osmotic pressure in the salamander (see Table 14-9). TABLE 14-9 Balance sheet o f pressures (in m m Hg) involved in glomerular ultrafiltration as illustrated i n Figure 14-20

Afferent arteriole

Efferent arteriole

Salamandar

I

Glomerular cap~llarypressure

Capillary pressure, +55 rnrn Hg

lntracapsular pressure

17 7

Man

55

-1 5

-

-

Net hydrostatic pressure

162

40

Colloid osmotlc pressure

-104 -

Net f~ltrat~on pressure

58

-15

- 30 -

10

Source Pltts, 1968, Brenneret al , 1971 Collo~dosmot~c

Prox~maltubule

Ultrafiltrate Figure 14-18 The net hydrostatic pressure affecting the glomerular filtration is determined by the sum of the various forces indicated at the left. Samples of the glomerular filtrate can be obtained by insertion of a micropipette, as shown on the right. The mercury in the plpette is pushed to the t ~ by p pressure before penetration of the capsule. Asample is then sucked into the calibrated tip for subsequent microanalysis. [Adapted from Hoar, 1975.1

Fluid filtered from the blood into the Bowman's capsule must cross the capillary wall, then the basement membrane, and finally the inner layer of the capsule. The glomerulus consists of fenestrated capillaries, which contain many large pores and are about 100 times more permeable than the continuous capillaries found in other parts of the body (see Figure 12-37).The basement membrane contains collagen for structural purposes and negatively charged glycoproteins that repel albumin and other negatively charged proteins. The hydraulic properties of the glomerular apparatus depend primarily on the sievelike properties of filtration slits, formed by a rather remarkable assemblage of fine cellular processes termed pedicels. These extend from larger processes of podocytes ("foot cells"), the cells composing the visceral layer of Bowman's capsule (Figure 14-19A). The pedicels are aligned in an array covering the endothelium (vascular epithelium) of glomerular capillaries. These fingerlike processes interdigitate so as to leave very small

592


............................................................................... A

B

Afferent artenole

Podocyte of visceral layer of Bowman's capsule

Pedicels

,Filtration slits

7

Endothelial pores

-Endothelium of glomerulus

Basement membrane

of glomerular capillary Figure 14-19 The inner (visceral)surface of Bowman's capsule is specialized for filtering the blood in the glomerular capillaries (A) Overview of glomerulus. The podocytes composing the visceral layer have long processes, termed pedicels, which cover the vascular epithelium. (B) En-

largement of the portion of partA that is enclosed in a box. Substances pass from the blood through the endothelial pores, across the basement membrane, and then through the filtration slits between pedicels.

spaces, the filtration slits, between them (Figure 14-19B). The filtrate, driven by the net pressure drop across the endothelium, passes through the pores formed in the walls of the glomerular capillaries and then through the filtration slits. The three-layered membrane separating the capillary lumen and lumen of Bowman's capsule acts as a molecular sieve, excluding almost all proteins from the ultrafiltrate

based mainly on molecular size, but also on shape and charge (Table 14-10).There is a bulk flow of water through the sieve carrying with it ions, glucose, urea, and many other small molecules. The kidneys are perfused by 500-600 ml of plasma per minute, or 20%-25% of the cardiac output, yet constitute less than 1% of the body weight. This preferential perfu-

TABLE 14-10 Relation between the molecular size of a substance and the ratio of its concentration in the filtrate appearing in Bowman's capsule t o its concentration in the plasma, [filtrate]/[filtrand] D~mens~ons (In nanometers) Substance

Mol wt

Rad~usfrom d~ffus~on coeff~c~ent

D~mens~ons from x-ray d~ffract~on

[flitrate] [f~ltrand]

Water

18

0.1 1

10

Urea

6

0.16

1.O

180

0.36

1.O

Glucose Sucrose Insulin Myoglobin Egg albumin Hemoglobin Serum albumin Source P~tts,1968


593

. . . . . . . . . . . . .< . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . sion takes place in a relatively low-resistance vascular bed within the kidney. A high renal blood pressure is the result of the relatively direct arterial supply; because arteries and arterioles are large in diameter and short in length, the loss of pressure due to friction is minimized. The efferent arterioles (those taking the blood away from the glomeruli) are of smaller diameter and along with the capillaries of the vasa recta, constitute the major resistance of the renal vascular bed, ensuring high pressures within the glomeruli. As noted already, the glomerular filtration rate depends largely on the net filtration pressure and the permeability of Bowman's capsule. The net filtration pressure depends on the blood pressure (glomerular capillary pressure), the intracapsular pressure, and the colloid osmotic pressure of the blood plasma (see Table 14-9). Under normal conditions, the colloid osmotic pressure and intracapsular pressure do not vary. The colloid osmotic pressure of plasma can be elevated during dehydration, and the intracapsular pressure can be increased by the presence of kidney stones obstructing the renal tubules; in both cases glomerular filtration rate will be reduced. In contrast, the seepage of plasma through burned skin can lower the colloid osmotic pressure of plasma, which in turn could increase the glomerular filtration rate. These examples, howevel; are exceptions rather than the rule. Although blood pressure and cardiac output normally increase during exercise, these changes have little effect on the glomerular filtration rate in mammals because of regulatory processes that control blood flow to the kidney. This regulation is achieved by modulating the resistance to flow in the afferent arteriole leading to each nephron and de-

pends on a number of interrelated mechanisms involving both paracrine and endocrine secretions as well as neural control. Several intrinsic mechanisms provide autoregulation of the glomerular filtration rate. First, an increase in blood pressure will tend to stretch the afferent arteriole, which would be expected to increase the flow to the glomerulus. The wall of the afferent arteriole, however, responds to stretch by contraction, thus reducing the diameter of the arteriole and therefore increasing the resistance to flow. This myogenic mechanism thus reduces variations in flow to the glomerulus in the face of oscillations in blood pressure. Second, cells in the juxtaglomerular apparatus (JGA),which is located where the distal tubule passes close to the Bowman's capsule between the afferent and efferent arterioles, secrete substances that modulate renal blood flow. The juxtaglomerular apparatus is composed of three types of cells (Figure 14-20): Modified distal-tubule cells, which form the macula densa and may monitor the osmolarity and flow of fluid in the distal tubule Specialized vascular cells, called granular cells, located between the afferent and efferent arterioles Secretory juxtaglomerular cells, modified smooth-muscle cells that are located primarily in the wall of the afferent arteriole Under certain conditions, the juxtaglomerular cells release the hormone renin, which indirectly affects blood pressure Bowman's caosule

Endothelial cell

Distal tubule

~4c;culadensa Figure 14-20 The juxtamedullary apparatus plays a key role in controlling blood flow through the glomerulus. This structure is composed of several cell types including modified distal-tubule cells, which constitute

~ f f e r e n arteriole t the macula densa; secretory juxtaglomerular cells in the wall of the afferent arteriole; and granular cells. [Adapted from Sherwood, 1993.1

594


.........................................

and, therefore, renal blood flow as described below. The juxtaglomerular apparatus also releases various substances that act in a paracrine fashion to cause vasoconstriction or vasodilation of the afferent arteriole in response to increased or decreased flow, respectively, through the distal tubule. Thus myogenic and i ~ x t a ~ l o m e r u l afeedbackr control mechanisms work together to autoregulate the glomerular filtration rate over a wide range of blood pressures. In addition to these autoregulatory mechanisms, the glomerular filtration rate is subject to extrinsic neural control. The afferent arterioles are innervated by the sympathetic nervous system. Sympathetic activation causes vasocontriction of afferent arterioles and a reduction in glomerular filtration. This response, which overrides any autoregulation, occurs when there is a sharp drop in blood pressure, for example, as a result of extensive blood loss. The reduction in filtration rate helps to restore blood volume and pressure to normal. Conversely, an elevation in blood pressure reduces sympathetic vasoconstriction and enhances glomerular filtration, decreasing blood pressure and volume. Sympathetic activation can also cause contractions of elements within the glomerulus, closing off portions of the filtering capillaries and effectively reducing the area available for filtration. The podocytes also are contractile, and when they contract, the number of filtration slits decreases. Thus contraction of either or both of these elements can effectively reduce the hydraulic permeability of Bowman's capsule. In the past, the hydraulic permeability of the glomerular membrane was thought to change only in disease states that caused the membrane to become leaky. It is now apparent that normal regulation of the glomerular filtration rate can involve changes in the hydraulic permeability of the glomerular membrane. A reduction in renal blood pressure, a fall in solute delivery to the distal tubule, andlor activation of the sympathetic innervation induces release of the hormone renin from the secretory juxtaglomerular cells located in the wall of the afferent arteriole that carries blood into the glomerular capillaries in Bowman's capsule. Renin is a proteolytic enzyme whose release leads to increased levels of angiotensin I1 in the blood. This hormone has several actions, one of which is t o cause general vasoconstriction (constriction of arterioles), which raises the blood pressure, thereby increasing both renal blood flow and the rate of glomerular filtration. Angiotensin I1 may also cause constriction of the efferent arterioles, raising glomerular blood pressure and increasing filtration. Angiotensin I1 also stimulates release of the steroid aldosterone from the adrenal cortex and vasopressin from the posterior pituitary. The role of these hormones in promoting the tubular reabsorption of salts and water is discussed later. Tubular reabsorption As the glomerular filtrate makes its way through the nephron, its original composition is quickly modified by re-

absorption of various metabolites, ions, and water. The human kidneys produce about 180 liters of filtrate per day, but the final volume of urine is only about 1 liter. Thus, over 99% of the water is reabsorbed. Of the 1800 g of NaCl typically occurring in the original filtrate, only 10 g (or less than 1%) appear in the urine of persons consuming 10 g of NaCl per day. Varying amounts of many other filtered solutes are also reabsorbed from the tubular lumen. In addition, some substances are secreted into the tubular fluid. The renal clearance of a substance is a measure of the extent to which it is reabsorbed or secreted in the kidneys, as explained in Spotlight 14-1. To understand the relationship between clearance and reabsorption, let's consider glucose. A healthy mammal exhibits a plasma glucose clearance of 0 ml smin-'. That is, even though the glucose molecule is small and is freely filtered by the glomerulus, normally it is completely reabsorbed by the epithelium of the renal tubule (Figure 14-21). Glucose is fully reabsorbed because its loss in the urine would mean a loss of chemical energy to the organism. Usually glucose appears in the urine only when the glucose concentration in the blood plasma, and hence in the glomerular filtrate, is very high. Figure 14-21 reveals that there is a maximum rate (milligrams per minute) at which glucose can be removed from the tubular urine by reabsorption. This transfer maximum, or Tm, is about 320 mg min-' in humans. Below plasma glucose levels of about 1.8 mg-ml-', all the glucose appearing in the glomerular filtrate is reabsorbed. At about 3.0 mg-mlkl, the carrier mechanism is fully saturated, so that any additional amount of glucose appearing in the filtrate will be passed out in the urine. The arterial plasma glucose concentration in humans is normally held at about 1mg mlkl by an endocrine feedback loop involving insulin. Since this level is well below the Tm for glucose, normal urine contains essentially no glucose. Because the high plasma glu-

.

.

/

/

/

Filtered /

Reabsorbed Excreted

Plasma glucose (mg %) Figure 14-21 The concentrat~onof glucose in the glomerular filtrate (broken line) is proportional to the plasma glucose concentration. The renal tubules are capable of reabsorbing the glucose by active transport (colored line) at rates up to 320 mg.min-' (Trn,). Glucose entering the filtrate in excess of this rate is necessarily excreted via the urine (black line).

IONIC A N D OSMOTIC BALANCE 595 ...............................................................................

SPOTLIGHT 14-1

In thls speclal case, the substance used, ~nulln,1s freely flltered and unchanged by tubular absorption or secretion Therefore,

RENAL CLEARANCE

the GFR and the clearance, C, of the substance are equal. substltutlng Cfor GFR glves, for lnulln,

The renal clearance of a plasma-borne substance 1s the volume of blood plasma from whlch that substance 1s "cleared" (I e., completely removed) per unlt tlme by the kldneys. A substance that IS freely flltered Into the nephron along wlth water, but that IS nerther reabsorbed nor secreted in the kidney, permits the cal-

vu

-- -

I

CP SO

the renal clearance IS glven by

culatlon of the glomerular filtration rate (GFR) merely by dlvldIng the amount of the nontransported substance appearing In the urine by the concentration of that substance ~nthe plasma

- = C = renal clearance (ml - m ~ n - I ) VU

P

One such molecule is inulin (not insulin), a small starchlike carbohydrate (molecularweight 5000). Since the inulin molecule is neither reabsorbed nor secreted by the renal tubule, the inulin

isidentical with the rate atwhich the glomerularfiltrate is produced-that is, the GFR, generally given in per minute.

1fwe

know the GFR andthe concentration o f a freelyfiltered

substance in the plasma (thus also its concentration in the ultra-

If the amount of a substance, x, appearing in the urine per minute deviates the amount present in the of plasma that is filtered Per minute, this will be reflected in a value of C,that dlffersfrom the inulin renal clearance, C. For example, if the inulin clearance of a subject, and hence the GFR, is 125 ml min-' and substance x exhibits a clearance of 62.5 ml'min-l~ then

.

filtrate), we can easily calculate whether the substance undergoes a net reabsorption or net secretionAduring the passage of the ultrafiltrate along the renal tubule. Thus, if less of the substance appears in the urine than was filtered in the glomerulus, it must have undergone some reabsorption in the tubule. This is true for water, NaCI, glucose, and many other essential constituents of the blood. If, however, the quantity of a substance appearing in the urine over a period of time is greater than the amount that passed into the nephron because of glomerularfiltration, it can be concluded that this substance is actively secreted into the lumen of the tubule. Unfortunately,the clearance technique is of limited usefulness in studies of renal function, since it indicates only the net output of the kidney relative to input and fails to provide insight into the physiological details. In renal clearance studies, a test substance such as inulin is first injected into the subject's circulation and allowed to mix to uniform concentration in the bloodstream. Asample of blood is removed, and the plasma concentration of inulin, P, is determined from the sample. The rate of appearance of inulin in the urine is determined by multiplying the concentration of inulin in the urine, U, by the volume of urine produced per minute, V. The amount of inulin appearing in the urine per minute (VU) must equal the rate of plasma filtration (GFR) multiplied by the plasma concentration of inulin:

vu

--

(GFR)P

-

amt. inulin appearing in urine. min-I amt. inulin removed from blood min-'

Vux -

-

.

Cx = 62.5 ml min-I

=

0.5 (GFR)

px In this case, a volume of plasma equivalentto half that filtered each minute is cleared of substance x. Stated differently, only half the amount of substance xpresent in a volume of blood plasma equal to the volume filtered each minute actually appears in the urine per minute. There are two possible reasons why the renal clearance for a, substance would be less than the GFR. First, it may not be freely filterable. For example, filtration of a substance may be hindered by its binding to serum proteins, by its large molecular size, or by some other factor. Second, a substance may be freely filtered, but it may be reabsorbed in the kidney tubules, thus reducing the amount that appears in the urine. As a matter of fact, most molecules below a molecular weight of about 5000 are freely filtered, but many of these are either partially reabsorbed or partially secreted (see Table 14-10).The extent of reabsorption or secretion can be gauged by the renal clearance of a substance. Reabsorption reduces the renal clearance to below the GFR. Tubular secretion, however, will cause more of a substance to appear in the urine than is carried into the tubule by glomerular filtration.

=1

3

cose levels typical of diabetes mellitus exceed the reabsorption ability of the renal tubule, diabetics commonly have glucose in their urine. The details of tubular function vary from species to species. Our knowledge of the changes in urinary composition along different portions of the nephron is based to a large extent on the technique of micropuncture, first devel-

oped by Alfred Richards and his coworkers in the 1920s. A glass capillary micropipette is used to remove a minute sample of the tubular fluid from the lumen of the nephron. The osmolarity of the sample (expressed as milliosmoles per liter1 is then determined by measuring its freezing point. The lower the freezing point, the higher its osmolarity. The stopped-flow perfusion technique, a modification of

......................................

L

;"'

Richards' original technique, can be used to isolate a portion of the lumen and analyze its action on injected samples of a defined solution in vitro (Figure 14-22). Microchemicalmethods are now used to determine the concentrations of individual ion species in the sample. In a technique developed more recently, a given segment of renal tubule is dissected from the kidney and perfused in vitro with a defined test solution; analysis of the perfusate provides insight to the movement of substances across the isolated tubule segment (Figure 14-23).The results of numerous studies using these techniques have detailed the roles of various portions of the nephron in the reabsorption of salts and water, which are summarized in Figure 14-24. The proximal tubule, which initiates the process of concentrating the glomerular filtrate, is most important in active reabsorption of salts. In this segment, about 70% of the Na+ is removed from the lumen by active transport, and a nearly proportional amount of water and certain other solutes, such as C1-, follow passively. So about 75% of the filtrate is reabsorbed before it reaches the loop of Henle.

n:

Pipette Proximal tubule

Bowman's capsule 1

Bowman's capsule Figure 14-22 The stopped-flow perfusiontechnique is used to study the . function of various portions of the renal tubule in vitro. A micropipette is

inserted into Bowman's capsule (I), and oil (color)then is injected until it enters the proximaltubule. Perfusionfluid (gray) is injected through a second pipette into the middle of the column of oil, forcing a droplet ahead of it (2). The tubule is full when the droplet reaches the far end of the tubule (3).After about 20 minutes, the perfusion fluid is collected by the injection of a second liquid behind the oil remaining nearthe glomerulus (4). The ability of the tubule segment to reabsorb or secrete substances can be determ~nedby comparing the composition of the perfusate before and after it is injected. [From "Pumps in the Living Cell" by Arthur K. Solomon. Copyright 0 1962 by Scientific American, Inc. All rights reserved.]

The result is a tubular fluid that is isosmotic with respect to the plasma and interstitial fluids. Stopped-flow perfusion experiments revealed that when the NaCl concentration inside the tubule is decreased, the movement of water also decreases. This result is just the opposite of what would be expected if the outward movement of reabsorbed water occurred by simple osmotic diffusion, and it indicates that water transport is coupled to active sodium transport (see Chapter 4). The actual pumping of Naf takes place at the basolateral (serosal) surface of the epithelial cells of the proximal tubule, just as it does in frog skin and gallbladder epithelia. In amphibians, this active transport leaves the tubular lumen about 20 mV negative relative to the fluid surrounding the nephron. This potential difference probably accounts for the passive net diffusion of chloride out of the proximal tubule as the counterion for sodium. In the proximal tubule, NaHCO, is the major solute reabsorbed proximally and NaCl the major solute reabsorbed distally. At the most distal portion of the proximal tubule (where it joins the thin descending limb of the loop of Henle), the glomerular filtrate is already reduced to one fourth of its original volume. As a result of the reduction in the volume of tubular fluid, substances that are not actively transported across the tubule or that do not passively diffuse across it are four times as concentrated toward the end of the proximal tubule than in the original filtrate. In spite of this great reduction in the volume of tubular fluid, the fluid at this point is isosmotic relative to the fluid outside the nephron, having an osmolarity of about 300 mosm. L-l. It is interesting to note that the active transport of NaCl alone can account for the major changes in volume of the fluid along the proximal tubule and for the increased concentration of urea and many other filtered substances. The proximal tubule is ideally structured for the massive reabsorption of salt and water. Numerous microvilli at the luminal border of the tubular epithelial cells form the so-called brush border (see Figure 14-15). These projections greatly increase the absorptive surface area of the membrane, thereby promoting diffusion of salt and water from the tubular lumen into the epithelial cell. Glucose and amino acids are also reabsorbed in the proximal tubule by a sodium-dependent mechanism and are not normally present in the ultrafiltrate beyond the proximal tubule. Carriers on the apical membrane cotransport sodium and glucose or amino acids from the ultrafiltrate into the cell. The uptake process, which is uphill for glucose and amino acids, depends on the sodium electrochemical gradient created by the Na+/K+ATPase in the basolateral membrane of the tubular cell. Once in the tubular cell, glucose and amino acids diffuse into the blood. Phosphates, calcium ions, and other electrolytes normally found in the blood are reabsorbed up to that required by the body, and any excess is excreted. Parathyroid hormone modulates the reabsorption of phosphates and calcium by the kidney. Parathyroid hormone stimulates kidney la,25-hydroxylase activity, which in turn stimulates


597

............................. Perfusing

Figure 14-23 Perfusionof a dissected segment of re-

Fluxes

nal tubule and chemical analysis of the perfusate permits determination of the fluxes of ions across the tubular wall in vitro.

Isolated segment of renal tubule

Test solution

the production of calcitriol, the active form of vitamin D. Calcitriol released into the blood stimulates calcium reabsorption and phosphate excretion from the kidney, as well as calcium absorption from the gut and release from bone (see Figure 9-29). The descending limb and thin segment of the ascending limb of the loop of Henle are made up of very thin cells containing few mitochondria and no brush border. In vitro perfusion studies have demonstrated that there is no active salt transport in the descending limb. Moreover, this segment exhibits very low permeability to NaCl and low permeability to urea but is permeable to water. As discussed later, this differential permeability plays an important role

Bowman's capsule

in the urine-concentrating system of the nephron. The thin segment of the ascending limb has also been shown by perfusion experiments to be inactive in salt transport, although it is highly permeable to NaCl. Its permeability to urea is low, and to water, very low. Again, this differing perme' ability plays a key role in the urine-concentrating mechanism of the nephron. The medullary thick ascending limb differs from the rest of the loop of Henle in that it exhibits active transport of NaCl outward from the lumen to the interstitial space (see Figure 14-24).This portion, along with the rest of the ascending limb, has a very low permeability to water. As a result of NaCl reabsorption, the fluid reaching the distal

Figure 14-24 The movement of ions, water, and other substances in and out of the filtrate along the renal tubule determines the composition of the urine. In this schematic diagram, the fluxes of NaCI, water, and urea are shown in different portions of the rnammalian renal tubule. The numbers indicate the flltrate tonicity in milliosrnolesper liter. The relative rates of active transport of NaCl are indicated by the size of the arrow. The permeability of the stippled portion of the collecting duct IS regulated by antidiuretic hormone (ADH).[Adapted from Pitts, 1959.1

Distal tubule

+ /+ -

'

Proximal

1

Cortex

400 Outer medulla

duct

Inner medulla

Loop o f Henle

-,

1200

Active transport of NaCl

7 Passive diffusion of urea I I )Passive

diffusion of H,O

Pass~ved ~ f f u s ~ o n of NaCl

598


.........................................

tubule is somewhat llyposmotic relative to the interstitial fluid. The importance of salt reabsorption by the thick ascending tubule is discussed later in the section on the urineconcentrating mechanism. The movement of salt and water across the distal tubule is complex. The distal tubule is important in the transport of K+, H+,and NH3 into the lumen and of Na-, C1-, and HC0,- out of the lumen and back into the interstitial fluid. As salts are pumped out of the tubule, water follows passively. The transport of salts in the distal tubule is under endocrine control, and is adjusted in response to osmotic conditions. Because the collecting duct is permeable to water, water flows from the dilute urine in the duct into the more concentrated interstitial fluid of the renal medulla (see Figure 14-24).This is the final step in the production of a hyperosmotic urine. The water permeability of the duct is variable and controlled by antidiuretic hormone (ADH). Thus the rate at which water is absorbed is under delicate feedback control. The collecting duct reabsorbs NaCl by active transport of sodium. The inner medullary segment of the collecting duct, toward its distal end, is highly permeable to urea. The significance of this will become clear in our later discussion of the countercurrent mechanism that concentrates the urine in the collecting duct. Now that we have summarized the movement of watel; ions, and glucose out of the tubular filtrate, let's examine sodium reabsorption in the nephron more closely. In the proximal tubule and ascending limb of the loop of Henle, sodium is transferred across the apical membrane of the tubular epithelium via cotransporters and then is actively transported into the blood via a Na+/K+ ATPase (Figure 14-25A). The electrochemical gradient for Nai between the ultrafiltrate and the blood favors the diffusion of Na+ through channels in the apical membrane from the ultrafiltrate into the tubular cells. Sodium is also exchanged for a proton via an electrically neutral Na+/H+exchanger; in this case, the downhill movement of Na+ energizes the uphill movement of H+into the lumen (Figure 14-25B). Farther along in the distal tubule and collecting duct, sodium reabsorption is coupled t o secretion of protons into the urine by acid-secreting cells, which are involved primarily in p H regulation. These cells are described in detail later. Sodium chloride represents more than 90% of the 0smotic activity of the extracellular fluid. Because reabsorption of salt results in the reabsorption of water, the amount of salt in the body is an important determinant of the volume of the extracellular fluid (ECF).If ECF volume is large, then blood pressure tends to rise. The converse is true for a reduction in ECF, for example, as a result of blood loss. Thus blood pressure is an indication of blood volume, which in turn is a reflection of the salt content of the body. When cells of the macula densa portion of the juxtaglomerular apparatus sense a decrease in blood pressure andlor solute delivery to the distal tubule, they stimulate release of renin from the juxtaglomerular cells in the walls of the afferent arteriole (see Figure 14-20). As outlined in

Tubular lumen

A

Na+ Glucose

Figure 14-25 Several transport systems are involved in reabsorption of Na+ in the proximaltubule and ascending limb ofthe loop of Henle in the mammalian kidney. (A) Sodium passively crosses the apical membrane via Na/ZCI/K and glucose/Na+ cotransporters. A Na7/K+ATPase in the basolateral membrane actively removes Na+from the cell into the blood; K+ and CI- exit via ion channels down their concentration gradient. (B) The movement of Na+ down its electrochem~calgradient into the cell also energizes the outward movement of protons via an electrically neutral Na+/H+exchanger. CO, in the blood diffuses into the cell, where carbonic anhydrase (c.a.) ensures a high rate of proton delivery to the exchanger. A basolateral sodium pump transports Na* from the cell into the blood. K+ and HCO, exit vla ion channels down their electrochemical gradient.

Figure 14-26A, renin acts to cause a rise in blood levels of angiotensin I1 and subsequently aldosterone; the latter promotes sodium reabsorption from the filtrate. Renin, a proteolytic enzyme, cleaves angiotensinogen, a glycoprotein molecule that is manufactured in the liver and is present in the a,-globulin fraction of plasma proteins. Cleavage of angiotensinogin releases a 10-residue peptide, angiotensin I. Angiotensinogen-converting enzyme (ACE) then removes two additional amino acids to form the 8-residue peptide, angiotensin I1 (Figure 14-26B). Much of the formation of angiotensin I1 occurs during the passage of blood through the lungs. Angiotensin I1 stimulates the secretion of aldosterone from the adrenal cortex and also causes a general vasoconstriction, which raises blood pressure. Removal of the amino-terminal aspartic acid residue from angiotensin II yields angiotensin 111, which also causes secretion of aldosterone from the adrenal cortex but to a lesser extent than angiotensin 11. Like other steroid hormones, aldosterone diffuses across the cell membrane and binds to cytoplasmic recep-


599

............................. A

Aldosterone

&

\

Increased N a f reabsorptlon

tubule

Adrenal

....

I

General systemic vasoconstriction

Angiotensin II

Figure 14-26 The renin-angiotensin system plays an important role in controlling sodium reabsorption in the mammalian kidney. (A) Renin is liberated by secretory cells in the juxtaglomerular apparatus (JGA) in responseto decreased pressure in the afferent arteriole and to low Na+ concentration in the distal tubule. Circulating renin leads to an increase in the titer of angiotensin II and aldosterone. Aldosterone stimulates Na+ reabsorption from the filtrate in the renal tubule. (B) Renin is a proteolytic enzyme that cleaves angiotensinogen, an a2-globulin, yielding angiotensin I. Another proteolytic enzyme' then removes the two carboxyl-terminal residues to give angiotensin II.

/ \ ,

Converting enzyme

\

Angiotens~nogen (Renin Substrate)

Asp-Arg-Val-Tyr-lle-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser-Protein

1 1

Renin

Angiotensin l

Asp-Arg-Val-Tyr-lle-His-Pro-Phe-His-Leu Converting enzyme

Angiotensin II

Asp-Arg-Val-Tyr-lle-His-Pro-Phe

tors in target cells, leading to an increase in transcription of specific genes and ultimately synthesis of the encoded proteins (see Figure 9-9). Aldosterone acts on the cells of the tubular epithelium to increase sodium reabsorption but does so without affecting water permeability. Three mechanisms have been proposed to account for the aldosteroneinduced increase in sodium reabsorption across tubular epithelial cells (Figure 14-27):

1. Sodium-pump hypothesis: Increased activity of Na+/K+ATPase in the basolateral membrane, perhaps due to changes in membrane structure that enhance ATPase activity as well as to increased synthesis of the pump protein.

2. Metabolic hypothesis: Increase in the production of ATP to power the sodium pump, perhaps due to aldosterone-stimulated increase in fatty acid metabolism. 3. Permease hypothesis: Increased permeability of the apical membrane to Na+ ions, presumably due to an increase in the number of sodium channels in the membrane. Quite possibly, all three mechanisms operate in tubular cells stimulated by aldosterone. Increased circulating levels of angiotensin I1 also increase the synthesis of vasopressin, also called antidiuretic hormone (ADH),in the hypothalamus and its release from the posterior pituitary (see Figures 9-5 and 9-7). Vaso-

Figure 14-27 Aldosterone, a steroid hormone that stimulates gene expression, increases sodium reabsorption in the kidney. Depicted here are three mechanisms proposed t o explain the effect of aldosterone: an increase in the activity of the Na+/K+ ATPase directly (sodium-pump hypothesis) or indirectly by increasing ATP levels (metabolic hypothesis), and an increase in the activity of sodium channels (permease hypothesis). [Adapted from M. E. Hadley, 1992.1

pressin acts through cyclic AMP to increase the water permeability of the principal cells in the distal tubule and collecting duct, increasing the number of water channels in the apical membrane and, therefore, promoting- water reabsorption. Aldosterone, unlike vasopressin, does not act through cyclic AMP, but does act with vasopressin to enhance both sodium and water reabsorption by the kidney. Atrial natriuretic peptide (ANP), released from the atrium of the heart into the blood in response to an increase in venous pressure, causes an increase in urine production and sodium excretion. It thus has the opposite effect of the renin-angiotensin system on the kidney. ANP inhibits the release of vasopressin and renin and the production of aldosterone from the adrenal gland. ANP acts directly on the kidney to reduce sodium and, therefore, water reabsorption (see Chapter 12).

Tubular secretion The nephron has several distinct systems that secrete substances by transporting them from the plasma into the tubular lumen. Most thoroughly investigated are the systems for secretion of K+, H+,NH3,organic acids, and organic bases. Although the number of secretory mechanisms and transport molecules must be limited, nevertheless, the nephron is capable of secreting innumerable "new" substances, including drugs and toxins, as well as endogenous, naturally occurring molecules. How is the nephron able to recognize and transport all these diverse substances? The answer seems to reside in the role of the vertebrate liver in modifying many such molecules so that they can react with the transport systems located in the wall of the nephron. These secretory mechanisms are important because they remove potentially dangerous substances from the blood. In the liver, many of these substances, along with normal metabolites, are conjugated with glucuronic acid or its sulfate. Both these classes of conjugated molecules are actively transported by the system that recognizes and secretes organic acids. Since they are highly polar, these conjugated molecules, once deposited by the transport machinery in the lumen of the nephron, cannot readily diffuse back across the wall of the nephron into the peritubular space and from there into the blood, and so these substances are excreted in the urine.

Normally, most of the potassium ions, which are freely filtered at the glomerulus, are reabsorbed from the filtrate in the proximal tubule and the loop of Henle due to the presence of a Na/2CI/K cotransport system in the apical membrane and Na+/K+ATPase in the basal membrane (see Figure 14-25A).Potassium channels in the basal membrane allow potassium to be recycled across the basal membrane. The rate of active reabsorption in the proximal tubule and loop of Henle continues unabated even when the level of K+ in the blood and filtrate rises to high levels in response to excessive intake of this ion. However, the distal tubule and collecting duct are able to secrete K+ into the tubular filtrate to achieve homeostasis in the face of a high body load of potassium. The secretion of K+ involves the active transport of K+ from the interstitial fluid into the tubular cell by the usual Na+/K+ATPase in the basolateral membrane and subsequent leakage of cytosolic Kt through potassium channels in the apical membrane into the tubular fluid (Figure 14-28). The latter is electronegative with respect to the cytosol, so K+ can simply diffuse down its electrochemical gradent from inside the renal tubular cell into the lumen.

'

Tubular lume

Figure 14-28 In the distal tubule and collecting duct, K+ can besecreted into the tubular filtrate. A Na+/K+ ATPase in the basolateral membrane actively transports K+ into the tubular epithelium; it then passively moves down its electrochemical gradient via potassium channels in the apical membrane into the lumen.

The rate of potassium secretion (and sodium reabsorption) by these mechanisms is stimulated by aldosterone, which is released in response to elevated plasma potassium levels as well as reduced sodium levels. Reduced potassium levels directly stimulate the adrenal glands, whereas .reduced blood sodium levels stimulate the adrenals via activation of the renin-angiotensin system. Stimulation of sodium reabsorption is therefore coupled to potassium secretion through the action of aldosterone; one cannot be corrected without affecting the other. Release of aldosterone in responge to low blood sodium levels will enhance sodium reabsorption but could also lead to abnormally low blood potassium levels due to enhanced potassium secretion and excretion. Because high extracellular potassium levels may cause cardiac arrest and convulsions, excess K + ions must be quickly removed from the plasma. Insulin is released in response to high potassium levels and stimulates potassium uptake by cells, especiallyfat cells. Potassium is then slowly released from these cells and removed by the somewhat slower renal mechanisms. Thus, like aldosterone, insulin release can also lead to low plasma potassium levels. Regulation of pH by the Kidney

As discussed in detail in Chapter 13, the carbon dioxidehicarbonate buffering system is primarily responsible for determining the pH of the extracellular space in mammals. This system involves three reactions:

+ OH- + H+---T HCO, + H+ (3) HOH ---T OH- + H+

(2) CO,

Reaction (1)occurs very slowly at body temperatures, but reaction (2)is catalyzed by the enzyme carbonic anhydrase

and is therefore rapid. Two factors have the most effect on the CO,/HCO,- system in mammals: excretion of CO, via the lung and excretion of acid via the kidney. The ratio of lung ventilation to CO, production largely determines the CO, concentration of the body. For example, when lung ventilation is reduced, CO, levels increase and the blood pH drops as hydrogen ions and bicarbonate ions accumulate (see Figure 13-10). Changes in breathing can adjust carbon dioxide excretion and, therefore, modulate body pH in the short term (see Chapter 13).The excretion of acid (H+ ions) in the urine is ultimately responsible for maintaining the plasma HC0,- concentration in mammals. Acid excretion across the skin of amphibia or the gills of fish supplements or takes over the role of acid excretion by the kidney in these animals. The concentration of HC0,- in mammalian plasma is around 25 X lo-, mole L-l, whereas the H+ concentration mol -L-'. The concentrations of biis around 40 x carbonate and protons in the glomerular ultrafiltrate are similar to those in the plasma; that is, the filtrate contains large quantities of bicarbonate but a very low concentration of protons. Yet urine has a pH of around 6.0 and contains little or no bicarbonate. Thus acid is added to the filtrate and most, if not all, of the bicarbonate is removed in the process of urine formation. At pH 6 the urine still has a very low concentration of protons, and the change in H + concentration alone, as the filtrate flows down the tubule, would not be sufficient to maintain body pH in the face of continual metabolic production of acid. In fact most of the acid added to the urine is buffered by either phosphate or ammonia. Because protons are added to the tubular filtrate along the entire length of the tubule, the filtrate becomes progressively more acidic. In the proximal tubule and loop of Henle, protons are secreted via a H+1Naf exchanger discussed earlier (see Figure 14-25B).The distal tubule and the collecting duct contain cells, referred to as A-type cells, that have a proton ATPase in the apical membrane and a chloridelbicarbonate exchange system in the basolateral membrane. (This anion exchanger is similar to the band 3 protein in the red blood cell membrane.) These cells also contain high levels of carbonic anhydrase, so that intracellular carbon dioxide is rapidly hydrated forming bicarbonate ions and protons; the protons are transported across the apical membrane and the bicarbonate ions move across the basal membrane. The secreted protons can react with bicarbonate in the ultrafiltrate to form carbon dioxide and water, which can diffuse back into the cell. Thus the secretion of protons from the A-type cell can result in the net uptake of bicarbonate into the blood through the cycling of carbon dioxide (Figure 14-29A). Clearly the A-type cell is an acid-secreting cell. Removal of protons from an A-type cell makes the intracellular potential more negative, thereby enhancing sodium reabsorption from the filtrate. The intracellular sodium level is kept low by the activity of a Na+lK+ATPase in the basolateral membrane, which transports Na+ from the cell into

A

A-type cell of kidney

Tubular lumen

B-type cell of kidney Figure 14-29 Body pH in mammals can be modulated by regulating the relative act~vityof acid-secreting (A-type)cells and base-secreting(B-type) cells in the distal tubule and collecting duct of the kidney. (A) A-type cells pump protons into the lumen vla an apical H+ ATPase, acidifying the filtrate; the resulting increase in the potential across the apical membrane favors reabsorption of Na'. (B) B-type cells use the H+ ATPase in the basolateral membrane to pump protons into the blood, accompanied by the reabsorption of CIk. Both cell types contain carbonic anhydrase (c.a.), which rapidly forms Ht and HCO, ions from C02diffusing into the cell from the blood.

the extracellular flood. The basolateral membrane of the A-type cell also contains K+ channels, and K + is cycled through this membrane by the Na+/K+ATPase. Thus acidification of the filtrate by A-type cells is coupled to sodium reabsorption. The distal tubule and collecting duct also contain basesecreting cells, called B-type cells. These cells have a chloridelbicarbonate exchanger in the apical membrane; this exchanger differs from the band 3-type protein found in the basal membrane of the A-type cell. As illustrated in Figure 14-29B, B-type cells contain carbonic anhydrase and secrete bicarbonate into the lumen of the tubule in exchange for chloride. Protons and chloride ions move across the basolateral membrane via a proton ATPase and chloride channels. A mammal can regulate its body pH by altering the activity of these A-type and B-type cells. The activity of A-type cells and, therefore, acid secretion increase during acidosis, whereas increased B-type cell activity and bicarbonate secretion are associated with alkalosis. Changes in the activity of the A-type cells involve alteration in both the proton ATPase activity in the apical membrane and the number of bicarbonatelchloride exchangers present in the basal membrane.

Proton secretion by renal tubular cells reduces the pH of the ultrafiltrate, thereby increasing the gradient against which protons are transported. Thus the ability to secrete protons decreases with filtrate pH; when the pH of the filtrate drops below 4.5, acid secretion stops. If the ultrafiltrate is buffered, however, more protons can be secreted across the tubular epithelium without a drop in pH sufficient to inhibit the proton pump. The ultrafiltrate is buffered by bicarbonate, phosphates, and ammonia. Acid secreted into the ultrafiltrate reacts with bicarbonate to form carbon dioxide, with HP0,2- to form H2P04-, or with NH, (ammonia) to form NH,+ (ammonium) ions (Figure 14-30). The tubular membrane is essentially impermeable to both phosphates and ammonium ions. Phosphates are filtered from the blood in the glomerulus, whereas ammonia diffuses from the blood across the tubular cells into the lumen, where it is converted to ammonium ions. Both phosphates and ammonium ions are trapped in the filtrate and then excreted from the body. The bicarbonate, phosphate, and ammonia buffer systems compete for protons secreted into the filtrate. Phosphate levels depend on diet, with excess phosphate being filtered into the ultrafiltrate. Thus the capacity of the phosphate buffer system (i.e., the number of protons that it can bind) depends o n what the animal eats and is independent of the acid-base requirements of the animal. Body pH is not generally regulated by selection of appropriate foods. Under acidotic conditions, plasma bicarbonate levels often fall; as a result, bicarbonate levels in the filtrate are reduced and less is available to act as a buffer. Under such conditions, ammonia is a major vehicle for elimination of excess acid. Ammonia is produced within the renal tubular cells by enzymatic deamination of amino acids, especially glutamine (see Figure 14-30). In its nonpolar, un-ionized Tubular lumen

Blood

Glutamine

Basolateral membrane Figure 14-30 Buffering of the renal filtrate by HPO, and NH,' permits greater secretion of protons. The phosphate ions in the lumen arise by filtration, whereas the ammonium ions arise by passive diffusion of NH, from the blood across the tubular cells or by intracellular breakdown of glutamine. Glutamine (and other amino acids) enter the tubular cells via basolateral transporters and is deaminated, yield~ngNH,, which diffuses across the apical membrane into the lumen. Because the membrane is largely impermeable to both H,P04- and NH4+,both ions are trapped in the urine and excreted.

at

form, ammonia freely diffuses across the cell membrane into the lumen where it reacts with protons forming NH,+ ions. Because the highly polar NH4+is impermeant, it traps both nitrogen atoms and protons in the urine, thus serving as a vehicle for their excretion. If acidotic conditions continue in the body for a few days, ammonia production by the tubular epithelium increases, NH4+concentration in the filtrate rises, and acid excretion by the kidney is increased. The secretion of ammonia is highly adaptive. Mammals that have entered a state of metabolic acidosis (excess acid production) show dramatic increases in ammonia production and secretion, as this is the body's major adaptive long-term mechanism for correcting an acid load.

I

1

I

Medulla

I lJ---i 'Ortex

Outer

zone

Urine-Concentrating Mechanism The urine of birds and mammals becomes concentrated by osmotic removal of water from the filtrate in the collecting ducts as they course through the renal medulla. There is a clear-cut correlation between the architecture of the vertebrate kidney and its ability to manufacture a urine that is hypertonic relative to the body fluids. Kidneys capable of producing a hypertonic urine (i.e., those of mammals and birds) all have nephrons featuring the loop of Henle. Moreover, the ability of a mammal to concentrate the urine is directly related to the length of the loops of Henle in its kidneys. The loops of Henle are longest in desert dwellers, such as the kangaroo rat; these longer loops produce larger overall gradients in osmolarity from renal cortex to medulla, thus permitting more efficient osmotic extraction of water from the collecting duct. In general, the longer the loop and the deeper it extends into the renal medulla, the greater the concentrating power of the nephron. Thus, desert mammals have both the longest loops of Henle and the most hypertonic urine. In addition to this correlation between the anatomy and concentrating ability of the nephron, the tonicity of the interstitial fluid progressively increases toward the deeper regions of the renal medulla (Figure 14-31)for reasons discussed later. These findings led B. Hargitay and Werner Kuhn to propose in 1951 that the loop of Henle acts as a countercurrent multiplier (Spotlight 14-2).Though a very attractive and plausible hypothesis, it was initially hard to test because of the difficulty of sampling the intratubular fluid in the thin loop of Henle. Determinations of the melting point of the fluid in slices of frozen kidney and subsequently in situ perfusion experiments of segments of the loop provided experimental support for the countercurrent hypothesis. These studies showed that the fluid entering the descending limb of the loop of Henle from the proximal

Inner medulla

/ Outer medulla

Figure 14-31 Solute concentrations in the interstitiurnof the mammalian kidney progressively increase from the cortex into the depths of the medulla. Shown here are the interstitial concentrations(in millimoles per liter) of urea, sodium, and chloride at different depths Note that most of the increase in urea concentration occurs across the inner medulla, whereas most of the increase in NaCl concentration occurs across the outer medulla. Since the osmotic contributions of Na+ and CI- sum, the total osmotic contributions of NaCl and urea are about equal deep within the medulla. [Adapted from Ulrich et al., 1961.1

tubule is isosmotic with respect to the extracellular fluid at that point (i.e., the outer portion of the renal medulla), having a concentration of about 300 mosm-L-' (see Figure 14-24).The concentration of the fluid gradually increases as it makes its way down the descending limb toward the hairpin turn in the loop, where its concentration reaches 1000-3000 mosm. L-l in most mammals. At this point, too, it is nearly isosmotic relative to the surrounding extracellular fluid in the deep portion of the renal medulla. This increase in the osmolarity of the tubular fluid flowing down the descending limb occurs because the wall of the descending limb is relatively permeable to water, but far less

.

604


....................................... gressively higher. As the fluid rounds the bend and begins flowing out the other limb, its salt concentration progressivelyfalls as a result of the cumulative effect of outward NaCl transport along

SPOTLIGHT 14-2

COUNTERCURRENT

the length of the outflow limb. By the time it reaches the end of that limb, its osmolarity is slightly lower than that of the fresh fluid beginning its inward flow in the other limb. This establishes a salt

SYSTEMS

,

In 1944 Lyman C. Craig published a method of concentrating chemical compounds based on the countercurrent principle. This method has proved useful in many industrial and laboratory applications. As in many other instances, human ingenuity turns

gradient along the tube. This example resemblesthe loop of Henle in principle but not in detail. The loop of Henle has no common wall dividing the two

out to be a reflection of Nature's inventiveness; countercurrent

limbs; nevertheless, the limbs are coupledfunctionally through the interstitial fluid, so that the NaCl pumped out of the ascending

mechanisms have been found to operate in a variety of biologi-

limb can diffuse the short distance toward the descending limb

cal systems, including the vertebrate kidney, the gas-secreting organ of swim bladders and gills of fishes, and the limbs of vari-

and cause osmotic reabsorption of water from that limb. Several

ous birds and mammals that live in cold climates.

important points should be noted about countercurrent systems

The principle can be illustrated with a hypothetical counter-

such as the loop of Henle and the simple model illustrated here. First, the standing concentration gradient set up in both

current multiplier that employs an active-transport mechanism much like the one that operates in the mammalian kidney. The

system and the cumulative effect of transfer from the outflow limb

model shown in part A of the accompanyingfigure consists of a

to the inflow limb. The gradient would disappear if either fluid

tube bent into a loop with a common dividing wall between the

movement or transport across the membrane were to cease.

limbs is due to both the continual movement of fluid through the

two limbs. A NaCl solution flows into one limb of the tube and

Second, the difference in concentrationfrom one end to the

then out the other. Let us assume that, within the common wall

other of each two limbs of the countercurrent multiplier is far

separating the two limbs of the tube, there is a mechanism that actively transports NaCl from the outflow limb to the inflow limb

greater than the difference across the part~tionseparating the

of the tube, without any accompanying movement of water. As bulk flow carries the fluid along the inflow limb, the effect of NaCl

the countercurrent multiplier can produce greater concentration

transport is cumulative, and the salt concentration becomes pro-

lium without the configuration of a countercurrent system. The

limbs at any one point (part B of the figure). As a consequence, changes than would be attained by a simple transport epithelonger the multiplier, the greater the concentration differences

A

that can be attained. Third, the multiplier system can work only if it contains an

Increasing salt gradient Bulk

-

b

asymmetry. In the model in part A, there is an active energy-

flow

requiring net transport of NaCl in one direction across the partition. A passive countercurrent system, such as one used to conserve heat, does not requlre the expenditure of energy (part C of the figure). In the extremities of birds and mammals that inhabit cold climates, fgr example, a temperature differential exists between the arterial and venous flow of blood, because the blood is cooled as it descends into the leg. As a result of this asymmetry and the countercurrent arrangement of the vessels, the arterial blood gives up some of its heat to the venous blood leaving the leg, thereby reducing the amount lost to the environment.

Distance along loop

L;

Warm water

Decreasing temperature gradient

+

I

I

Active countercurrent systems require the expenditure of energy, whereas passive ones do not (A) Model of an active system in which a salt solution flows through a U-shapedtube with a common dividingwall. The active transport of NaCl from the outflow to the inflow limb constitutes an asymmetry necessary for the multiplier system to work. (6)A plot of salt concentration along the two limbs. Note that the concentration difference across the wall at any point is small relative to the total concentration difference along the length of the loop. The length of the loop as well as the efficiency of transport across the wall WIII determine the overall concentration gradient along the entire length ofthe loop (C)Model of a passive system in which warm water flows down the input limb and gives up part of its heat to cooler water flowing in the opposite direction in the outflow limb. Some heat is lost to the heat sink representedby the ice, but much more of the heat is conserved by passive transfer from the inflow to the outflow limb.

permeable to NaCl or urea. Thus the osmotic loss of water allows the tubular fluid to approach osmotic equilibrium with the interstitial fluid around the hairpin turn of the loop. As the tubular fluid flows up the ascending limb, it undergoes a progressive loss of NaCl (but not water). Most of the NaCl is actively transported across the wall of the thick segment of the ascending limb, although there is some passive loss of NaCl across the thin segment. Both the thin and the thick segments of the ascending limb are relatively impermeable to water. The functional asymmetry between the descending and the ascending limbs of the loop of Henle, together with the countercurrent principle, accounts for the observed interstitial corticomedullary osmotic gradient of NaCl and urea represented by the gray wedge in Figure 14-32. The interstitial osmotic gradient is believed to be established by a combination of features that include the active transport of NaCl from the ascending thick segment and selective passive permeabilities to water, salt, and urea along specific segments of the nephron. Recall that the descending limb of the loop of Henle has high-wateq low-urea, and low-salt permeability, whereas

the ascending limb has low-water, low-urea, and high-salt permeability. As shown in Figure 14-32 (step I), NaCl is actively transported out of the tubular fluid in the thick segment of the ascending limb of the loop of Henle and in the distal tubule. The mechanism of this active salt transport is similar to salt secretion in the rectal gland of sharks and in the gills of saltwater teleosts (discussed in later section) except that in the kidney salt is transferred from the tubular lumen into the blood. The loss of NaCl from these segments and its addition to the surrounding interstitiurn leads to the osmotic loss of water (step 2) from the distal tubule and from the salt-impermeant descending limb in the cortex and outer medulla. Because of the net loss of water and salt from the filtrate in the loop of Henle and the distal tubule, the filtrate entering the collecting duct has a high urea concentration. The renal tubule up to this point is largely impermeable to urea, but as the collecting duct passes into the depths of the medulla, it becomes highly permeable to urea. As a result, urea leaks out down its concentration gradient (step 3), raising the interstitial osmolarity of the inner medulla. The resulting high interstitial osmolarity draws water from the Figure 14-32 The steady-state corticomedullary osmotic gradlent in the renal interstltium depends on differing permeabilities and active salt transport in different segments of juxtaglomerular nephrons, as well as on the anatomic layout of the nephrons and their circulatory supply (the vasa recta, not shown). The gray wedge depicts the osmotic gradient in the extracellular fluid with the small numbers indicating the total osmolarity.Active transport of NaCl from the ascendingthick limb of the loop of Henle and distal tubule (step I)is largely responsibleforthe interstitial osmolarity in the cortex and outer medulla. The high osmolarity of the inner medulla depends largely on the passive diffusion of urea from the lower collecting duct (step 3), the only portion of the nephron highly permeable to urea. Some urea reenters the filtrate in the thin limb of the loop of Henle, where the urea level is relatively low, leading to recycling of urea (thin red arrow).See text for further discussion of the various transport steps depicted. [Adapted from Jamison and Maffly, 1976.1

Collecting duct gggx;b Act~vetransport of NaCl

Passive diffusion of urea

@ Passive diffusion

of H,O Passive diffusion of NaCl

606


.......................................

i

descending limb of the loop of Henle (step 4), producing a very high intratubular solute concentration at the bottom of the loop. As the highly concentrated tubular fluid then flows up the highly salt-permeable thin segment of the ascending limb, NaCl leaks out (step 5) down its concentration gradient. The lower collecting duct is the only section of the nephron with a high urea permeability. The high 0smolarity of the inner medullary interstitium thus depends largely on the passive accumulation there of urea by the countercurrent mechanism of the nephron. If the ascending limb were as permeable to urea as is the collecting duct, this accumulation would not occur. If NaCl were not actively removed (with water following passively), urea would not become concentrated in the collecting duct, and the high medullary accumulation of urea would not take place either. It is interesting that the interstitial medullary urea gradient is established largely by passive means, although the active transport of NaCl is an essential component of the system and accounts for most of the metabolic energy expenditure necessary to set up the NaCl and urea gradients. The result of this combination of cellular specialization and tissue organization is a standing corticomedullary gradient of urea and NaCl in which the osmolarity becomes progressively higher with distance into the depths of the renal medulla, both inside the tubule and in the peritubular interstitium. This gradient is responsible for the final osmotic loss of water from the collecting ducts into the interstitium and the consequent production of a hyperosmotic urine. A countercurrent feature in the organization of vasa recta, the blood vessels around the nephron, is essential in maintaining the standing concentration gradient in the interstitium. Blood descends from the cortex into the deeper portions of the medulla in capillaries that form looplike networks around each juxtamedullary nephron and then ascends toward the cortex (see Figure 14-14A).In this circuit, the blood takes up salt and gives up water osmotically as the surrounding interstitial fluid becomes increasingly hyperosmotic. Thus the osmolarity of the blood increases as it descends via the vasa recta into the medullary depths (Figure 14-33).The reverse occurs as the blood returns to the cortex and encounters an interstitium of progressively lower osmolarity. As a result, there is little net change in blood osmolarity during the circuit through the vasa recta, although the water and solutes removed from the glomerular filtrate in its passage through the nephron are carried away by the blood. However, this represents only a small percentage of the large volume of blood that perfuses the kidney. An important consequence of the countercurrent organization of the vasa recta is that it allows a high rate of renal blood flow (essential for effective glornerular filtration) without disrupting the corticomedullary standing gradient of NaCl and urea concentration. As the blood leaves the glomerulus and moves down the vasa recta into the medulla, it passively accepts interstitial NaCl and urea as it encounters ever-increasing interstitial osmolarities. NaCl

Figure 14-33 The countercurrent arrangement of the vasa recta helps maintain the interstit~al corticomedullary osmotic gradient. This schematic diagram of the vasa recta indicates the passive fluxes of NaCl and water and the osmolarity of the blood atvarious points. Note thatthe blood osmolarity is the same at the beginning and end of the vasa recta.

and urea in the blood reach their peak concentrations as the blood traverses the loop of the vasa recta in the depths of the medulla. On ascending back toward the cortex, the excess NaCl and urea diffuse back into the interstitiurn, staying behind as the blood leaves the kidney. But before leaving the kidney, the blood, in fact, regains some of the water it lost during glomerular filtration. This happens because the colloid osmotic pressure of the blood plasma is elevated during ultrafiltration.

Control of Water Reabsorption The tubular fluid is concentrated by the osmotic removal of water as it passes down the collecting duct into the hyperosmotic depths of the renal medulla (see Figure 14-32). This concentration provides a means of regulating the amount of water passed in the urine. The rate at which water is osmotically drawn out across the wall of the collecting duct from the urine into the interstitial fluid depends on the water permeability of the wall of the collecting duct. Vasopressin, also known as antidiuretic hormone (ADH),regulates the water permeability of the collecting duct and thereby controls the amount of water leaving the animal via the urine. The higher the level of ADH in the blood, the more permeable the epithelial wall of the collecting duct, and hence the more water is drawn out of the urine as it passes down the duct toward the renal pelvis. The effect of ADH on water reabsorption .from the duct is shown in Figure 14-34.


607

............................................................................... -

ADH I

I

I

I

L

Time (min) Figure 14-34 Antidiuretic hormone (ADH) increases the water permeability of portions of the collecting duct (see Figure 14-35). The data shown are from a perfusion experiment in which the fluid perfused through the duct and in the external bath were held constant at 125 mosM and 290 mosM, respectively. In the absence of ADH little water was reabsorbed from the perfusate, but application of ADH caused a dramatic increase in reabsorption. [Adapted from Grantham, 1971.I

The blood ADH level is a function of the osmotic pressure of the plasma and blood pressure. The neurosecretory cells that produce ADH have their cell bodies in the hypothalamus and their axon terminals in the neurohypophysis (posterior pituitary gland). These osmotically sensitive cells respond to increased plasma osmolarity by increasing the rate at which ADH is released into the bloodstream from their axon terminals, thereby increasing the blood level of ADH and reabsorption of water from the collecting duct

Impulse:

-

Hypothalamic

Figure 14-35 The osmolarity of the blood is under feedback regulation by the action of antidiuretic hormone on the collecting duct. Antidiuretic hormone (ADH) increasesthe water permeability of the stippled region, enhancing the rate of osmotic removal of water from the urine. The increased recovery of water counteracts worsening of the conditions that stimulate ADH secretion.

+

neurosecretory cells

Neurosecretory termrnals rn pltultary

Hlgh plasma

L

absorptlon H,O perm

(Figure 14-35).If, for example, the osmolarity of the blood is increased as a result of dehydration, the activity of the neurosecretory neurons is increased, more ADH is released, the collecting ducts become more permeable, and water is osmotically drawn from the urine at a higher rate. This process results in the excretion of a more concentrated urine and the conservation of body water. The hypothalamic cells that produce and release ADH receive inhibitory input from arterial and atrial baroreceptors that respond to increases in blood pressure. Hemorrhage, for example, results in a fall in blood pressure, reducing the activity of these baroreceptors (see Figure 12-44); the resulting decreased inhibitory input to the ADH-producing cells in the hypothalamus leads to increased release of ADH and reduced loss of water in the urine, thus helping to restore blood volume. Conversely, any factor that raises the venous blood pressure (e.g., an increase in blood volume due to dilution by ingested water) will inhibit the ADH-producing hypothalamic cells, causing an increased loss of body water via the urine. The ingestion of drinks containing ethyl alcohol inhibits the release of ADH and therefore leads to copious urination and an increase of plasma osmolarity beyond the normal set-point level. Some degree of dehydration results, and this contributes to the uncomfortable feeling of a hangover. The action of mammalian ADH and the related peptide, arginine vasotocin, of nonmammalian vertebrate species, is not limited to the kidney. If these antidiuretic hor-

,

608


.........................................

mones are applied to frog skin and toad bladder, they increase the water permeability of those epithelia. To summarize the mechanisms we've discussed, the formation of urine in the mammalian kidney begins with the concentration of the glomerular filtrate into a hyperosmotic fluid in the proximal tubule. About 75% of the salt and water are removed from the filtrate in osmotically equivalent amounts as it passes through the proximal tubule, leaving urea and certain other substances behind. As the filtrate moves through the loop of Henle and the distal tubule, there is little net change in its osmolarity, but the countercurrent mechanism sets up a standing concentration gradient in the medullary interstitium along the length of the loop. This gradient provides the basis for the osmotic removal of water from the urine as it makes its way down the collecting duct within the medulla. Interestingly, this process takes place without active transport of water at any place along the nephron. An animal can experience osmotic stress owing to changes in temperature or salinity and owing to the ingestion of food and drink. Perturbations in the osmotic state of the body fluids are minimized through feedback mechanisms by which the osmoregulatory organs adjust their activity so as to maintain the internal status quo. These feedback control mechanisms may be neural, endocrine, or a combination of the two. In mammals, adjustments in the volume and concentration of the urine are the primary means for maintaining osmotic homeostasis. In response to osmotic stress and other signals, mammals can regulate several aspects of urine formation including (1) the glomerular filtration rate, (2) the rate at which salts and water are absorbed from the lumen of the renal tubule, (3) secretion of unwanted substances, and (4)the rate at which water is osmotically drawn out of the pre-urine in the collecting duct.

NONMAMMALIAN VERTEBRATE KIDNEYS In the kidneys of the marine hagfishes (class Cyclostomata), the nephrons possess glomeruli but no tubules, so the Bowman's capsules empty directly into collecting ducts. The kidneys are used largely to excrete divalent ions (e.g., Ca2+, Mg2+,and S 0 4 2 - ) and carry out little or no osmoregulation. Thus, the extracellular fluids of the most primitive living vertebrate, the hagfish, are relatively similar to seawater in concentration of major salts, and their plasma is essentially isotonic relative to seawater (see Table 14-1). As a general rule, the kidneys in freshwater teleosts have larger glomeruli and more of them than do those in their marine relatives. Because their bodies are hypertonic to the environment and water diffuses into their bodies, freshwater teleosts maintain water balance by producing large volumes of dilute urine. The kidney nephrons in certain marine teleosts have neither glomeruli nor Bowman's capsules. In such aglomerular kidneys, the urine is formed entirely by secretion because there is no specialized mecha-

nism for the production of a filtrate. These fish are hypotonic to their environment and so lose water continually across the skin and gills. Their problem is water conservation and they produce only small volumes of urine. Little urea is formed and ammonia is excreted across the gills. Amphibians and reptiles appear incapable of producing a hypertonic urine (i.e., of higher osmolarity than the plasma), because they lack the countercurrent system of the loop of Henle that is necessary to produce urine of significantly greater osmolarity than the plasma. Only mammals and birds are known to have a renal countercurrent organization, and thus only these animals, apparently, have their plumbing so organized as to allow osmotic countercurrent multiplication. The avian kidney contains a mixture of reptilian-type and mammalian-type nephrons. That is, some avian nephrons lack a loop of Henle, and in some birds the loop is oriented perpendicular to the collecting duct, producing a less efficient concentrating mechanism. The elasmobranch Raja erinacea (a skate) has been shown to have a complex renal tubule organization that has the anatomic requisites for countercurrent multiplication. However, the skate nephron is functionally quite different from the mammalian nephron. As we have seen, the mammalian kidney excretes urea and retains water to produce a hypertonic urine. The elasmobranch kidney, in contrast, retains urea (which is used as an osmolyte) and does not produce a concentrated urine. Tubular bundles constitute the countercurrent system in elasmobranch kidneys. These tubular bundles have been described in the kidneys of marine elasmobranchs, which have high levels of urea in their tissues and reabsorb urea from the kidney ultrafiltrate. Freshwater stingrays, on the other hand, do not reabsorb filtered urea and their kidneys lack tubular bundles, indicating that the bundles are the site of urea reabsorption. Thus, the function of the countercurrent organization of the elasmobranch nephron may be to conserve urea.

EXTRARENAL OSMOREGULATORY ORGANS IN VERTEBRATES As indicated in the previous section, many vertebrates rely on extrarenal osmoregulatory organs to maintain osmotic homeostasis. First, we consider specialized glands for excreting salt found in some animals, and then see how fish gills are used for osmoregulation.

Salt Glands Elasmobranchs, marine birds, and some reptiles possess glands that secrete salt by cellular mechanisms similar to sodium reabsorption in the mammalian kidney.

Elasmobranch rectal gland Marine elasmobranchs, although slightly hypertonic to seawater, have a much lower NaCl content than seawater. As a result there is a continual influx of NaCl into the body of the animal. The excess salt is removed largely by the rectal gland, which produces a concentrated salt solution and is


609

...................................... the major (perhaps the only) extrarenal site for secretion of excess NaCl by marine elasmobranchs. The gland functions to regulate extracellular volume by controlling the amount of NaCl in the body. The rectal gland consists of a large number of blindending tubules that drain into a duct, which opens into the intestine near the rectum. The fluid produced by the gland can have a slightly higher salt concentration than seawater but is isosmotic to the plasma of the fish. The blood of elasmobranchs is also slightly hyperosmotic to seawater but has a much lower salt concentration, the osmolarity of the blood being made up by high concentrations of urea and trimethylamine oxide (TMAO).Elasmobranchs are able to tolerate high levels of urea, which normally causes the dissociation of multi-subunit enzymes, thus inhibiting their activity. In contrast, TMAO promotes the association of subunits, thereby counteracting the effect of urea. Urea and TMAO do not appear in the rectal gland fluid, only NaCl. Formation of the secreted fluid in the rectal gland does not involve filtration of the blood; rather NaCl is secreted into the lumen of the tubule and water follows. The cells of the tubule wall of the rectal gland consist of a single type of cell, a salt-secreting cell similar to the chloride cell found in the gills of marine teleosts. This cell has a very extensive, folded, basolateral membrane, whose surface area is much larger than that of the apical (mucosal) membrane. The basolateral (serosal) membrane contains high concentrations of a Na+/K+ATPase, which pumps Na+ out and K+ into the cell; the K+, however, cycles back out through the many potassium channels also present in the basolateral membrane (Figure 14-36).The activity of the Na+/K+ATPase generates a large sodium gradient across the basal membrane, which drives NaCl uptake via a Na12CVK cotrans-

Figure 14-36 Salt-secreting cells present in the rectal gland of sharks, the bird and reptile nasal gland, and the gills of marine teleosts all use the same basic mechanism for transporting salt from the blood. Operation of the Na+/K+ ATPase and Na/2CI/K cotransporter in the basolateral membrane results In net movement of C I from the blood Into the gland tubules or seawater in the case of fish. The transmembrane potential created by this movement increases the sodium electrochem~calgradient sufficientlyso that Na' can diffuse via paracellular channels even against a high concentration gradient.

port system also in the basolateral membrane. Thus as Naf and K+ cycle across the basal membrane, the C1- level inside the cell rises above that in the tubular lumen; eventually C 1 exits via chloride channels in the apical (mucosal) membrane moving down its concentration gradient. The overall effect is the movement of C1- from the serosal (blood)side of the tubular wall into the lumen. This creates an electrical potential, with the serosal side positive and the lumen negative; the resulting electrochemical gradient for sodium permits diffusion of Nat from the serosal side through paracellular pathways into the lumen. Water follows the transport of NaCl and is distributed passively across the tubular wall, but the wall is impermeable to urea and TMAO. Thus the rectal gland produces a solution that has a much higher NaCl concentration than the blood but is isosmotic with the blood. The hearts of dogfish sharks contain a natriuretic peptide hormone that stimulates chloride secretion in perfused rectal glands. Although circulating natriuretic peptide levels have not yet been measured in elasmobranchs, it is possible that natriuretic peptides released from the heart into the circulation stimulate secretion by the rectal gland, reducing extracellular volume. The appropriate stimulus for the release of the natriuretic peptide would seem to be a rise in venous pressure, that is, filling pressure of the heart. In fact, the heart of a teleost fish, the rainbow trout, has been hormone, which is reshown to contain natriuretic -peptide leased into the circulation by increased venous pressure.

EiI

Do you think dinosaurs had salt glands?

Salt gland in birds and reptiles In 1957, Knut Schmidt-Nielsen and his coworkers, investigating the means by which marine birds maintain their osmotic balance without access to freshwater, discovered that the nasal salt glands secrete a hypertonic solution of NaC1. It was found in those early studies that if cormorants or gulls are administered seawater by intravenous injection or by stomach tube, the increase in the plasma salt concentration leads to a prolonged nasal secretion of fluid with an osmolarity two to three times that of the plasma. Salt glands have subsequently been described in many species of birds and reptiles, especially those subjected to the osmotic stress of a marine or desert environment. These species include nearly all marine birds, ostriches, the marine iguana, sea snakes, and marine turtles, as well as many terrestrial reptiles. Crocodilians have a similar salt-secreting gland in the tongue. The salt glands of birds and some reptiles occupy shallow depressions in the skull above the eyes. In birds, the salt gland consists of many lobes about 1mm in diameter, each of which drains via branching secretory tubules and a central canal into a duct that, in turn, runs through the beak and empties into the nostrils (Figure 14-37A,B). Active

610


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Lumen of Lobe

Gland

Blood flow

Figure 14-37 Marine birds malntain osmotic balance by excretion of a concentrated salt solution from glands located above the orblt. (A) The avian salt gland consists of a longitudinal arrangement of many lobes, which drain via a central canal into a duct that carries secretions to the nasal passages.(B) Each lobe consists of tubules and capillaries arranged radially around a central canal. Single tubules are surrounded by capillaries In which blood flows counter to the flow of secretory fluid in the tubule. This countercurrent flow facilitates the transfer of salt from the

blood to the tubule, since the uphill gradient of salt concentration between cap~llaryand tubule lumen is thereby minimized at each point along the length of the tubule. (C)The secretory cells constituting the tubularwall transport NaCl from the blood into the lumen via the mechanism depicted in Figure 14-%.These cells have a brush border and contain many mitochondria. [Part A adapted from Schmidt-Nielsen, 1960; part B from "Salt Glands," by K. Schmidt-N~elsen.Copyright O 1959 by Scientific American, Inc. All rights reserved.]

secretion takes place across the epithelium of the secretory tubules, which consists of characteristic salt-secretingcells. These have a profusion of deep infoldings in the basolateral membrane and are heavily laden with mitochondria. As in many other transport epithelia, adjacent cells are tied together by junctions, which preclude the massive leakage of water past the cells, from one side of the epithelium to the other. These cell junctions, however, are not as tight as those binding cells of the frog skin together but are leaky allowing the paracellular movement of ions, as in the rectal gland. The formation of fluid in the nasal gland, as in the rectal gland, does not include filtration of the blood. The absence of filtration can be deduced from the failure of small filterable molecules (e.g., inulin or sucrose) that are injected into the bloodstream to appear in the gland fluid. High concentrations of a Na+/K+ATPase have been demonstrated in the basolateral membrane of the tubular cells. Application of ouabain to the basal surface of the epithelium blocks

salt transport. Since this inhibitor does not pass across epithelia and can block the pump only by direct contact with the ATPase, the sodium-transport mechanism appears to operate in the basal membrane of the epithelial cells, as it does in the rectal gland. Increased salt secretion is associated with increased Na+/K+ ATPase activity in the salt gland. The Na+/K+ ATPase also occurs to some extent in the apical membrane of the bird nasal gland. The basal membrane of the salt-gland epithelium also contains a Na12CVK cotransporter and potassium channels, and the apical membrane contain chloride channels. The net result is movement of NaCl from the blood across the epithelium into the lumen of the salt gland (Figure 14-37C). As we saw earlier, the salt solution produced by the elasmobranch rectal gland is isosmotic to plasma; in contrast, the fluid produced by the nasal gland is hyperosmotic to plasma. In both cases, the gland fluid has a high salt concentration, but the osmolarity of the blood of elasmobranchs is much higher than that of birds and reptiles. It is


611

............................................................................... not clear how the solution produced by the nasal glands of birds and reptiles is concentrated. It is possible that the initial solution in the apex of the tubule is isosmotic to plasma and becomes more concentrated as it passes down the tubule. The cells of the secretory epithelium of a single tubule become larger, with deeper paracellular channels, towards the base of the tubule, indicating that the fluid may become more concentrated towards the base of the tubule. Those birds that can produce the most concentrated salt solutions have the largest secretory cells, with long paracellular channels between cells. In addition, the avian salt gland and its blood flow are organized as a countercurrent system and this might aid in concentration of the salt solution. The capillaries are so arranged that the flow of blood is parallel to the secretory tubules and occurs in the direction opposite to the flow of secretory fluid (see Figure 14-37B). This flow maintains a minimal concentration gradient from blood to tubular lumen along the entire length of a tubule; it thereby minimizes the concentration gradient for uphill transport from the plasma to the secretory fluid. The salt gland is not always active but responds to a salt load and/or expansion of the extracellular space. When birds drink seawater, water will diffuse from the body into the gut because seawater has a higher osmolarity than the body fluids. At the same time NaCl will diffuse from the seawater in the gut into the body. Thus the initial effect of

A

drinking saltwater is to reduce extracellular volume while increasing NaCl levels in extracellular fluid and the blood (Figure 14-38A).The salt level in the gut thus will drop because of salt loss to the body and diffusion of water from the body into the gut. After a while the osmolarity of the gut fluids will fall below that of the body, so that water movement between the body into the gut will reverse, that is, water moves into the body, following the movement of salt and expanding extracellular volume. The initial reduction in extracellular volume inhibits nasal fluid production immediately after drinking seawater. The subsequent elevation of both extracellular volume and salt content, acts as a strong stimulus for salt secretion, thus there is often a short delay between drinking saltwater and secretion from the nasal gland. Since the solution secreted from the salt gland is more concentrated than the seawater taken in, the bird ends up gaining osmotically free water, as illustrated in Figure 14-38B. Regulation of the secretory activity of the avian salt gland involves both parasympathetic neural control and neuroendocrine control through the pituitary (Figure 14-39). Osmoreceptors in the hypothalamus respond to an increase in plasma tonicity by a sensory discharge. This response, together with input from extracranial osmoreceptors and/or volume receptors, activates parasympathetic cholinergic neurons that innervate the salt

Figure 14-38 Because the salt-gland secretion is more concentrated than seawater, birds that drink seawater gain osmotically free water. Before drinking seawater, the gull in this example has an extracellular fluid (ECF) volume of 0.2 liters and an ECF [Na+] of 0.15 M; thus the ECF contains 0.03 moles of Na+. The bird then drinks 0.025 liters of seawater with a [Na+]of 0.45 M, so it ingests 0.011 moles of Na-, Initiallythe ECF volume decreases and ECF [Na+] increases (A) because Na+ moves from the seawater in the gut into the ECF (down its concentrationgradient), while water moves into the gut until osmotic equilibrium is established between the ECF and gut. The initial decrease in ECF volume inhibits salt-gland secretion. As the ECF [Na+] rises, water from the gut moves back into the ECF. When both the ECF volume and [Na'] are above their base levels, the salt gland is stimulated (B). If the secretion has a [Na+] of 0.9 M (twice as concentrated as the ingested seawater), then the gull can secrete all the ingested salt in half the volume. In this example, the gull has a net profit of 12.5 ml of osmotically free water; this water can be used to excrete other ions (and molecules) via the kidneys, which continue to filter actively. [Adaptedfrom unpublished material courtesv of Dr. Maryanne Hughes.]

Bird drinks 25 ml seawater

Time (min)B

L

volume x 0.025 L 0.011 moles Intake

Volume x 0.200 L 0.030 moles

ECF

0.225 L

= 0.183 M

Intake + ECF

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Neurohypophys~s Dehydrat~on, hypersalln~ty

Figure 14-39 The increase in avian salt-gland secretion in response t o a rise in blood osmolarity and decrease in blood pressure is mediated by direct and indirect mechanisms.Stimulation of osmoticallysensitive neurons in the hypothalamus and sensory input from peripheral osmoreceptors activate direct parasympathetic pathwaysto the salt gland and to the vessels supplying blood to the gland. Atrial natriuretic peptide (ANP), re-

gland. Acetylcholine liberated from the terminals of those neurons not only stimulates the secretion of salt but also enhances secretion by causing vasodilation and thus increased blood flow to the secretory tissue. Acetylcholine acts on muscarinic receptors in the secretory cells of the gland, triggering the inositol phospholipid intracellular signaling system that leads to a rise in cytosolic calcium levels (see Figure 9-14).The increased intracellular calcium levels activate chloride and potassium channels in the plasma membrane of the secretory cells. A variety of other agents can stimulate secretion by increasing CAMPlevels, which, in turn, activate chloride channels. The end result of an intracellular increase in the levels of inositol phosphate andlor CAMPis salt secretion. Secretion is also stimulated by adrenocorticosteroids and by prolactin. Although direct neural control is most important in making short-term adjustments to osmotic stress, corticosterone is required for maintaining the responsiveness of the salt gland. For instance, when an animal's adrenal cortex, the source of corticosteroids, is removed, the infusion of a high-tonicity salt solution is no longer effective in stimulating salt-gland secretion (Figure 14-40).But if corticosterone is then injected into the experimental animal, salt-gland function is retained. Atrial natriuretic peptide ( A m ) ,secreted from the heart in response to increased venous pressure, also stimulates secretion by acting directly on the secretory cells of the bird salt gland. This hormone

leased from the heart in response t o low venous pressure, also directly stimulates secretion. Pituitary secretion of ACTH in response to increasing blood osmolarity indirectly promotes salt secretion by stimulating release of corticosterone (CS) from the adrenal cortex. This hormone acts directly on the gland making it responsive to blood tonicity.

E

./(,

Sham operation

Duration of infusion (min) Figure 14-40 The responsivenessof the avian salt gland to high blood osmolarity is dependent on corticosterone. Two days after adrenalecthe blood of animals with tomy (adrenal-X), 10% NaCl was infused ~ n t o (black squares) and without (blacktriangles) corticosterone replacement therapy. [Adapted from Thomas and Phillips, 1975.1

causes a transient increase in salt secretion, presumably reducing blood volume and, therefore, venous pressure. In addition, increased blood osmolarity stimulates secretion of AVT, an ADH-like neurohypophyseal peptide. Although AVT has no effect on salt-gland secretion, it reduces glomerular filtration and water and salt excretion in the bird's kidneys, which are capable of producing urine that is only slightly hypertonic to plasma. The action of AVT on the kidney, combined with salt excretion via the salt gland, results in water retention by the body and a decrease in blood osmolarity. As in mammals, low blood pressure andlor solute delivery to the distal tubule of bird kidneys stimulates release of renin and subsequent formation of angiotensin I1 (see Figure 14-26). Angiotensin I1 inhibits salt secretion by the nasal gland via action on the central nervous system, having no direct effect on the gland. The reason that birds and reptiles can drink seawater and survive is because, unlike mammals, they have a nasal gland that can excrete hypertonic salt solutions. Mammals have salt-secretingcells located in the thick ascending limb of the loop of Henle that are similar to those found in the nasal $and of birds and the rectal gland of elasmobranchs. In addition, these mammalian cells seem to be controlled by the same array of hormones, namely natriuretic peptides and the renin-angiotensin system. In mammals, however, these salt-secreting cells are not arranged in a way that permits production of a hypertonic salt solution that can be excreted from the body. Thus organization at the organ, as well as the cellular and molecular levels, is important in determining the ability of an animal to survive in a range of environments.

Fish Gills The epithelial surface area of a gill must be large if it is to function efficiently as an organ for respiratory gas exchange. Although this feature makes gills an osmotic lia-

bility for animals such as fishes, which are out of osmotic equilibrium with their aqueous environment, it does make gills suitable as organs for osmoregulation. Thus, the gills of numerous aquatic species, vertebrate and invertebrate, are active not only in gas exchange but also in such diverse functions as ion transport, excretion of nitrogenous wastes, and maintenance of the acid-base balance. In teleost fishes, for example, the gills play the central role in coping with 0s-. motic stress. The structure of a teleost gill is illustrated in Figure 14-41. The epithelium separating the blood from the external water consists of several cell types including mucous cells, chloride cells, and pavement cells (Figure 14-42). The epithelium of the lamellae consists mostly of flat pavement cells no more than 3-5 pm thick, but containing some mitochondria. These are clearly best suited for respiratory exchange, acting as minimal barriers for diffusion of gases. The epithelium covering the gill filaments also contains chloride cells, which are more columnar in shape and several times thicker from base to apex than the pavement cells. Chloride cells are deeply invaginated by infoldings of the basolateral membrane and are heavily laden with mitochondria and with enzymes related to active salt transport. Pavement cells and chloride cells are joined by tight junctions limiting the paracellular movement of water and ions.

Secretion of salt in seawater Chloride cells were first described in 1932 by Ancel Keys and Edward Willmer, who ascribed to them the transport of chloride because they exhibit histochemical similarities to cells that secrete hydrochloric acid in the amphibian stomach, and because it had already been shown that the gill of marine teleosts is the site of extrarenal excretion of chloride (and sodium). Subsequent histochemical studies confirmed the presence of high levels of.chloride in these

GILL STRUCTURE

Gill

Operculum

b

vessel

Figure 14-41 Fish gills function as both respiratory and osmoregulatory organs. These drawings show a portion of the teleost gill in increasing magnification. In addition to gas exchange between the blood and wa-

cut across

ter, Na+can move in and out of the blood in the larnellae. Black arrows indicatewater flow; red arrow and dotted lines indicate blood flow.

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I N T E G R A T I O N OF PHYSIOLOGICAL SYSTEMS

...................................... Figure 14-42 The eplthellum of the teleost gill 1s composed largely of pavement cells Interspersed w ~ t ha few mucous and chlor~decells (A) Drawlng of lamellar eplthellum showlng typlcal dlstrlbutlon of pavement, mucous, and chlorlde cells The chlorlde cells tend t o be at the base of the lamellae (B) Electron mlcrograph of a freshwaterteleost chloride cell wlth adjacent pavement cells (C) Electron mlcrograph of dogfish g ~ lmucous l cell contalnlng many large mucous granules [Electron mlcrographs courtesy of Jonathan W~lson]

cells, especially near the pit that develops on the apical (mucosal or external) border of these cells in fishes that have become adapted to high salinities. The mechanism of salt transport by chloride cells is similar to that of the salt-secreting cells illustrated in Figure 14-36. Thus, chloride cells have high levels of a Na+/K+ATPase associated with Na/2Cl/K cotransporters in the basolateral membrane and a chloride channel in the apical membrane. Each chloride cell is associated with an accessory cell (distinct from a pavement cell), and Na+ diffuses from blood to seawater through the less-tight paracellular channel between the chloride and accessory cell. In

the case of marine teleosts, the secretion of salt occurs against an osmotic gradient and there is no movement of water following the movement of salt. Thus the shark rectal gland, avian nasal gland, seawater teleost gill, and the thick ascending limb of the loop of Henle in the mammalian kidney tubule all appear to contain salt-secreting cells that transport NaCl by the same basic mechanism (see Figures 14-25A and 14-36). In the mammalian kidney, however, the direction of salt transport is into the blood rather than into the environment, as in the other cases. Since chloride cells were first characterized and associated with the transport of C1- across the gills of marine

d

I O N I C A N D O S M O T I C BALANCE

615

............................................................................... teleosts, they have been found to also mediate the exchange of other ions including ca2+. F~~ instance, ca2+ in the water is taken up via calcium channels in the apical membrane of the chloride cells and then actively transported into the blood via a Ca2+ATPase, present at high levels in the basolateral membrane.

Uptake of salt in freshwater The pavement cells in the gills of freshwater fish appear to have a proton ATPase and sodium channels in the apical membrane. The proton ATPase is presumably electrogenic and pumps protons out of the gills, generating a potential that draws Na+ into the cell, a mechanism similar to that demonstrated in frog skin and the mammalian kidney (see Figure 14-29A).A clear relationship between the activity of the proton pump and the apical membrane potential has yet to be demonstrated in fish gills. A Na+/Kf ATPase in the basolateral membrane pumps Naf out of the cell into the blood, and K+ cycles through potassium channels in the membrane. Thus a proton ATPase appears to energize Na+ uptake across the apical membrane, whereas a Na+/K+ATPase moves sodium across the basolateral membrane of the gills of freshwater fish. The gill epithelium of freshwater fish also possess chloride cells, which mediate uptake of Ca2+from the water. These cells, which differ from the chloride cells in marine teleost fish, have an anion transporter on the apical membrane and high levels of proton ATPase within the cell. They may be involved in the uptake of C1-, as well as Ca2+, by freshwater fish. Physiological adaptation in migrating fish In species (e.g., salmon and eels) that regularly migrate between seawater and freshwater, the gill epithelium changes so as to adapt to changes in environmental salinity. These fishes actively take up NaCl in freshwater and actively excrete it in saltwater by the mechanisms described above. The physiological adaptation of the gills involves the synthesis andlor destruction of molecular components of the epithelial transport systems and changes in the morphology and number of the chloride cells. When fishes that are able to tolerate a wide range of salinities are transferred between freshwater and saltwater, it can take up to a few days for physiological adaptation to the new environment to occur and for the animal to regain osmotic homeostasis. It is now known that osmoregulatory adaptation is mediated by endocrine hormones that influence epithelial differentiation and metabolism. The steroid hormone cortisol and growth hormone stimulate the changes in gill structure associated with the transition from freshwater to seawater, whereas prolactin stimulates the changes in gill structure that accompany the reverse transition. First, let's consider what happens when fish migrate from freshwater to saltwater (Table 14-11, part A). When the fish are in freshwater, the proton ATPase in pavement cells is active. But when they move from freshwater to seawater, the proton ATPase is down-regulated because Na+

TABLE 14-11 Physiological adaptation accompanying movement of fish to water of differing salinity (A) Freshwater + saltwater

1. Proton ATPase that powers active uptake of NaCl is downregulated.

2. Increased Na+ influx into body raises plasma Na+, stimulating an increase in plasma cortisol and growth hormone levels.

3. Hormones induce an increase in the number of chloride cells and an elaboration of their basolateral membrane, resulting in increased infoldings.

4. As a result, Na+/K+ ATPase activity and NaCl secretion increases. 5. Plasma Naf levels return t o normal. (6) Saltwater + freshwater 1. Low external sodium closes paracellular gaps between chloride and accessory cells, so NaCl efflux falls rapidly.

2. Plasma prolactin levels increase. 3. Hormone causes the number of chloride cells t o decrease and the apical pits t o disappear.

4. As a result Na+/K+ ATPase activity falls.

5. Up-regulation of proton ATPase returns fish t o the freshwater condition.

uptake is no longer required. The influx of sodium from seawater causes plasma Na+ levels to rise, which in turn stimulates secretion of cortisol (Figure 14-43A). Cortisol, along with growth hormone, induces an increase in the number of typical seawater chloride cells. As a result of these changes, the gill Na+/K+ ATPase activity and salt secretion increase (Figure 14-43B). In salmon, cortisol release begins while the fish is moving down river, thus preadapting the fish for a seawater existence. This process is termed smelting, and the end product is a smolt, a fish ready for seawater transfer. The increase in plasma Na+ that occurs when the fish enters seawater causes additional release of cortisol and starts the changes that will allow the fish to live in seawater. After reaching the ocean, it normally takes about a week for the elevated plasma Na+ to return to normal levels, similar to those of freshwater fish (see Figure 14-43A). When a marine teleost moves from seawater to freshwater, more-or-less opposite changes occur, adapting the fish to low-salt water (see Table 14-11, part B). Initially, the paracellular gaps in the gill epithelium close, reducing salt loss. A rise in the plasma prolactin level stimulates changes in the chloride cells so that the Na+/K+ ATPase activity falls. Finally, up-regulation of the proton ATPase allows the uptake of salt necessary for survival in freshwater.

What are t h e design conflicts that exist in the gills, a structure used for b o t h gas exchange and ~ o n i creaulation? I

.

*&-~~,L;jl*rIplriWd.a

*

r

-

I I I 0x1

I I 2 4 Transfer to seawater Time (days)

I 8

Figure 14-43 Cortisol plays a major role in inducing the physiological adaptation that occurs when coho salmon are transferred from freshwater to seawater. (A) Initially after a f~shmoves into seawater, the plasma sodium level begins to rise, stimulating secretion of cortisol. (B)The spike in plasma cortisol levels mediates a number of changes in the gills in-

INVERTEBRATE OSMOREGULATORY ORGANS In general, invertebrate osmoregulatory organs employ mechanisms of filtration, reabsorption, and secretion similar in principle to those of the vertebrate kidney to produce a urine that is significantly different in osmolarity and composition from the body fluids. Insects and possibly some spiders are the only invertebrates known to produce a concentrated urine. These mechanisms are used to differing extents in various organs in different groups of animals. That there has been convergent evolution of physiological mechanisms in nonhomologous organs underscores the utility of these mechanisms.

Filtration-Reabsorption Systems Several lines of evidence indicate that filtration of plasma, similar in principle to that which occurs in the Bowman's capsule of vertebrates, underlies the formation of the primary urine in both mollusks and crustaceans. For example, when the nondigestible polysaccharide inulin is injected into the bloodstream or coelomic fluid, it appears in high concentrations in the urine. (This also occurs in mammals.) Since it is unlikely that such substances are actively secreted, they must enter the urine during a filtration process in which all those molecules below a certain size pass through a sievelike membrane of tissue. During the reabsorption of water and essential solutes, these polymers remain behind in the urine. As in vertebrates, the normal urine of some invertebrates contains little or no glucose, even though substantial levels occur in the blood. However, studies with several mollusks have shown that when the blood glucose is elevated by artificial means (e.g., by injection), glucose appears in the urine. In each species, glucose appears in the urine at a characteristic threshold concentration of blood glucose; the urine glucose concentration rises linearly with

I I I 0 x 1

I 2

I 4

I 8

Transfer to seawater Time (days) cluding an increase in the gill Na+/K+ATPase activity.As this activity increases, sodium secretion from the gill rises; thus, after several days in seawater, plasma sodium returns to values close to those observed in freshwater fish. [Unpublished data supplied by N. M. Whiteley and J. M. Wilson.]

blood glucose concentration beyond the threshold level. This behavior parallels that in the mammalian kidney (see Figure 14-21)and probably results from saturation of the transport system by which glucose filtered into the tubular fluid is reabsorbed from the filtrate into the blood. Once the transport system is saturated, the "spillover" of glucose in the urine is proportional to its concentration in the blood. More conclusive evidence is obtained with the drug phlorizin, which is known to block active glucose transport. When phlorizin is administered to mollusks and crustaceans, glucose appears in the urine even at normal blood glucose levels. The most reasonable explanation for this effect is that glucose enters the urine as part of a filtrate and remalns in the urine when the reabsorption mechanism is blocked by phlorizin. Further support for the filtration-reabsorption mechanism comes from analyses of tubular fluids near suspected sites of filtration indicating that their composition is similar to that of the plasma. Finally, the rate of urine formation in some invertebrates has been found to depend on the blood pressure. This relationship is consistent with a filtration mechanism, but the change in blood pressure may also produce a change in the circulation to the osmoregulatory organ. The site of primary urine formation by filtration is known in only a few invertebrates. In a number of marine and freshwater mollusks, filtration takes place across the wall of the heart into the pericardial cavity, and the filtrate is conducted to the "kidney" through a special canal. Glucose, amino acids, and essential electrolytes are reabsorbed in the kidney. In the crayfish, the major organ of osmoregulation is the so-called antennal gland (Figure 14-44). Part of that organ, the coelomosac, resembles the vertebrate glomerulus in ultrastructure. Micropuncture measurements have shown that the excretory fluid that collects in the coelomosac is produced by ultrafiltration of the blood. The antennal gland of Crustacea is clearly involved

, Coelomosac Labyrinth Nephr~dial

The filtering of large quantities of plasma requires the active uptake of large quantities of salts, either in the excretory organ itself or in other organs, such as gills or skin. In frog skin, for example, it has been shown that 1mol of oxygen must be reduced in the synthesis of ATP for every 16-18 mol of sodium ions transported. In freshwater clams, the cost of maintaining sodium balance amounts to about 20% of the total energy metabolism. In marine invertebrates, however, the filtration-reabsorption system proves metabolically less expensive, since salt conservation is much less of a problem. Secretory-Reabsorption Systems

Crayfish antennal "gland" Figure 14-44 Osmoregulation in some invertebrates depends on filtration-reabsorption organs that differ structurally from the mammalian kidney but are functionally analogous. In this schematic diagram of the crayfish antennal gland, the osmotically active tissues are shown in color. Filtration of the blood produces the initial excretory fluid, which then is modified by selective reabsorption of various substances. [Adapted from Phillips, 1975.1

in regulating the concentration of ions (e.g., Mg2+)in the hernolymph. Since the final urine in mollusks and crustaceans differs in composition from the initial filtrate, there must be either secretion of substances into the filtrate or reabsorption of substances from the filtrate. The reabsorption of electrolytes is well established in freshwater species, for the final urine has a lower salt concentration than either the plasma or the filtrate. Glucose must also be reabsorbed, since it is present in the plasma and in the filtrate but is either absent or present at very low concentrations in the final urine. It is interesting that the filtration-reabsorption type of osmoregulatory system has appeared in at least three phyla (Mollusca,Arthropods, Chordata) and perhaps more. This kind of system has the important advantage that all the low-molecular-weight constituents of the plasma are filtered into the ultrafiltrate in proportion to their concentration in the plasma. Such physiologically important molecules as glucose and, in freshwater animals, such ions as Na+, K+, C1-, and Ca2+are subsequently removed from the filtrate by tubular reabsorption, leaving toxic substances or unimportant molecules behind to be excreted in the urine. This process avoids the need for active transport into the urine of toxic metabolites or, for that matter, unnatural, man-made substances of a neutral or toxic nature encountered in the environment. Thus, an advantage of the filtration-reabsorption system is that it permits the excretion of unknown and unwanted chemicals taken in from the environment without the necessity for a large number of distinct transport systems. A disadvantage of the filtration-reabsorption osmoregulatory system is its high energetic cost for the organism.

Insects can survive in both freshwater and arid terrestrial environments; given their often large surface-to-volume ratios, the osmotic demands placed on these insects can be extreme. The locust, for example, has a large capacity to regulate the ionic strength of the hemolymph (blood). During dehydration the hemolymph volume may decrease by up to 90%, but its ionic composition is maintained. In addition, when this insect is given solutions to drink that range in osmotic strength from that of seawater to that of tapwater, hemolymph osmotic pressure changes by only 30%. This capacity to regulate hemolymph composition depends on a secretory-type osmoregulatory system. In broad outline, the osmoregulatory system of locusts and other insects consists of Malpighian tubules and the hindgut (ileum, colon, and rectum). The closed ends of the long, thin Malpighian tubules lie in the hemocoel (the blood-containing body cavity); the tubules empty into the alimentary canal at the junction of the midgut and hindgut (Figure 14-45).The secretion formed in the tubules passes into the hindgut, where it is dehydrated and passes into the rectum and voided as a concentrated urine through the anus. The presence of a tracheal system for respiration in insects (describedin Chapter 13)diminishes the importance of an efficient circulatory system. As a consequence, the Malpighian tubules do not receive a direct, pressurized arterial blood supply, as the mammalian nephron does. Instead, they are surrounded with blood, which is at a pressure essentially no greater than the pressure within the tubules. Since there is no significant pressure differential across the walls of the Malpighian tubules, filtration cannot play a role in urine formation in insects. Instead, the urine must be formed entirely by secretion, with the subsequent reabsorption of some constituents of the secreted fluid. This process is analogous to the formation of urine by secretion in the aglomerular kidneys of marine teleosts. The serosal surface of the Malpighian tubule exhibits a profusion of microvilli and mitochondria, a specialization often associated with a highly active secretory epithelium.

Midgut

2

colon

7

Rectum

~ a l p i ~ h i atubule n

crbp

cecae

tubules

Figure 14-45 Osmoregulat~onIn Insects ~nvolvesa secretory-reabsorpt~onmechan~sm(A) External s ~ d evlew and cross-sect~onalvlew of locust (B) S ~ m p l ~ f ~ d~agram ed showlng relat~onof Malp~gh~an tubule to gut of the locust The pre-urlne 1s produced by secretion Into the tubules, wh~chIle In the blood-conta~n~ng lumen of the Malp~gh~an hemocoel The pre-urlne flows ~ n t othe rectum, where ~tIS concen-

trated by the reabsorpt~onof water, although Ions also are reabsorbed, the excreted urine 1s hyperton~cto the hemolymph The arrows 1nd1cate the clrcular pathway of water and Ion movement The Insect body contalns numerous Malpcgh~antubules, even though only two are dep~cted

The details of urine formation by tubular secretion differ among different insects, but some major features seem to be common throughout. KC1 and, to a lesser extent, NaCl are transported from the hemocoel into the tubular lumen, along with such waste products of nitrogen metabolism as uric acid and allantoin. It appears that the transport of K+ is the major driving force for the formation of the pre-urine in the Malpighian tubules, with most of the other substances following passively. This has been concluded from the following observations:

mands. The fluid formed in the Malpighian tubules passes into the hindgut, where several important changes in its composition occur. In the hindgut, water and ions are removed in amounts that maintain the proper composition of the hernolymph. Thus, it is in the hindgut that the composition of the final urine is determined. The water and ions removed from the urine by the hindgut are transferred through intimate connections to the lumen of the Malpighian tubules. These substances are thereby retained and recycled in the Malpighian tubule-hindgut circuit (see Figure 14-45B). The most complete study of the osmoregulatory behavior of the hindgut has been done with the desert locust Schistocerca. The serosal surface of the ileum and rectum is a highly specialized secretory epithelium (Figure 14-46). When a solution similar to hernolymph is injected into the hindgut of this insect, water, Kf, Na+, and C1- are absorbed into the surrounding hemolymph. Evidence from electrical measurements suggests that the ions are transported actively with water following. An electrogenic chloride pump and potassium channels in the apical membrane appear to mediate KC1 uptake from the hindgut lumen into the cells of the gut lining. Sodium uptake from the lumen is coupled to amino acid uptake andlor ammonium ion excretion. The KC1 then moves from the cell into the hemolymph via appropriate channels in the basolatera1 membrane, while sodium is removed from the cell into the hemolymph by a Na+/K+ATPase (Figure 14-47). Acid is excreted into the lumen of the hindgut by a proton ATPase. The locust hindgut is capable of removing a large amount of ions plus water, leaving behind an excess of

The pre-urine is isotonic or slightly hypertonic relative to the hemolymph. The pre-urine has a high K+ concentration in all insects. The rate of pre-urine formation is a function of K+ concentration in the fluid surrounding the tubule, higher K+ concentrations producing more rapid preurine accumulation. The formation of pre-urine is largely independent of the Na+ concentration of the surrounding fluid. Although K+ is osmotically the most important substance actively transported, there is evidence that active transport plays an important role in the secretion of uric acid and other nitrogenous wastes. The pre-urine formed in the Malpighian tubules is relatively uniform in composition from one species to another, and in each species it remains isotonic relative to the hemolymph under different osmoregulatory de-

I O N I C A N D OSMOTIC BALANCE 619 ............................................................................... Lumen of

Ap~calmembrane

mbrane Infolds

Figure 14-46 The h~ndgutof Insects 1s spec~al~zed for transport of water and lons from the lumen to the surround~nghemocoel Shown here are the ultrastructural organlzatlon and of the ep~thel~um of the gross d~mens~ons Ileum and rectum In the locust, both of wh~ch are lnvolved In reabsorpt~onNote the extensive ~nfold~ng of the ap~calmembrane and extensive lateral ~ntercellularspaces [Adapted from lrvlne et al , 1988 ]

Lateral scalarlform

Dllated lntercellular layer

~ntercellular spaces (hemocoel)

Intercellular slnus Subepltheltal space (hemocoel) Basal cell

ions and waste products so that the excreted urine is hypertonic with an osmolarity up to four times that of the blood. In the mealworm Tenebrio, the urine-to-blood osmolarity ratio can be as high as 10, which is comparable to the concentrating ability of the most efficient mammalian kidneys. It has been suggested that uphill transport of water in Tenebrio and some other species results from a countercurrent arrangement of the Malpighian tubules, the perirectal space, and the rectum (Figure 14-48).Water is drawn

Rectal lumen

Hemocoel

osmotically from the rectum into the Malpighian tubules because of the KC1 gradient produced by active transport. The direction of bulk flow in these compartments is such that the osmotic gradient along the length of the complex is maximized, with the absolute osmolarities highest toward the anal end of the rectum. This gradient may allow the concentrations near the anal end to exceed those of the hemolymph by several times. There is evidence for the feedback regulation of osmolarity among the invertebrates, especially in insects. The bug Rhodnius becomes bloated after sucking blood from a mammalian host. Within 2-3 minutes, the Malpighian tubules increase their secretion of fluid by more than a thousand times, producing a copious urine. Artificially bloating the insect with a saline solution does not produce such diuresis in an unfed Rhodnius. It has also been found that isolated Malpighian tubules immersed in the hemolymph of unfed individuals remain quiescent, but if immersed in the hemolymph of a recently fed Rhodnius they produce a copious secretion. A factor that stimulates the secretion of these tubules can be extracted from the neural tissue containing the cell bodies or axons of neurosecretory cells, primarily those of the metathoracic ganglion. Thus, it appears that these cells release a diuretic hormone in response to a factor present in the ingested blood. The only identified neurohumor that stimulates the diuretic action of the neurosecretory cells is serotonin. Similar findings in other insect species suggest that diuretic and antidiuretic hormones produced in the nervous system regulate the secretory activity of the Malpighian tubules or the reabsorptive activity of the rectum. In earthworms, removal of the anterior ganglion results in the retention of water and a concomitant decrease in plasma osmolarity. Injection of homogenized brain tissue reverses these effects, suggesting a humoral mechanism. - -

KC1

w

reabsorption Figure 14-47 lons are transported In and out of locust rectal cells by numerous rnechan~smsThe prlmary effect 1s the reabsorptlon of KC1 and water and excretion of arnrnonla and acld

Hemocoel Perinephric membrane

Perirectal

space

Rectal

lumen

Figure 14-48 The countercurrentarrangement of the water-extractionapparatus of the rectum of the mealworm beetle, Tenebrio, probably accountsforthe high urine-concentrating ability of this organism. Most of the water and KC1 entering the rectal lumen is recycled into the Malpighian tubules. See text for further discussion. [Adapted from Phillips, 1970.1

T L

Anus

*

-

Direction of bulk water flow

Net water transfer

@ Net KC1 transfer EXCRETION OF NITROGENOUS WASTES When amino acids are catabolized, the amino group is released or transferred to another molecule for removal or for reuse. Unlike the atoms of the carbon skeleton of amino acids, which can be oxidized to CO, and water, the amino group must either be salvaged for the resynthesis of amino acids or be excreted dissolved in water to avoid a toxic rise in the plasma concentration of nitrogenous wastes. Elevated body ammonia levels have several deleterious effects on metabolism and amino acid transport; also, NH,+ can substitute for K+ in ion-exchange mechanisms, leading to convulsions, coma and eventually death. Thus, in most animals, there is a close link between osmoregulatory functions and processes involved in the elimination of excess nitrogen. In those animals faced with a limited water supply, this relation gives rise to a serious problem-namely, the inevitable conflict between conserving water, on the one hand, and preventing toxic accumulation of nitrogenous wastes, on the other. As we will see, animals have evolved excretory strategies appropriate to their water economies. Animals generally excrete most excess nitrogen as ammonia, urea, or uric acid (Figure 14-49).Lesser quantities of nitrogen are excreted in the form of such compounds as creatinine, creatine, or trimethylamine oxide; very small quantities of amino acids, purines, and pyrimidines also may be excreted. The three primary nitrogenous excretory compounds differ in their properties, so that in the course of evolution some animal groups have found it more opportune to produce one or the other of these forms for excretory purposes during all or part of their life cycles (Figure 14-50). Ammonia is more toxic than urea or uric acid and must be kept at low levels in the body. Because excretion of ammonia occurs by diffusion, a large volume of water is re-

-

-

-

quired to maintain the concentration of ammonia in the excretory fluid below that in the body, which is necessary for diffusion to occur. This means that about 0.5 liters of water is needed to excrete 1g of nitrogen in the form of ammonia. Urea is less toxic than ammonia and requires only 0.05 liters of water to excrete 1g of nitrogen as urea, that is, only 10% of the water required to excrete the equivalent amount of nitrogen as ammonia. Urea synthesis, however, consumes ATP; therefore, if plenty of water is available and ammonia levels in the body can be kept low enough, excreting nitrogenous waste as ammonia saves energy. Even less water is required to excrete uric acid, with only 0.001 liters required for uric acid excretion per gram of nitrogen, or only 1%of that needed for ammonia excretion. Uric acid is only slightly soluble in water and is excreted as a white pasty precipitate, characteristic of bird feces. The low solubility of uric acid has adaptive significance in that, in its precipitated form, uric acid contributes nothing to the tonicity of the "urine" or feces. Thus, in general, water availability normally determines the nature and pattern of nitrogenous excretion. Aquatic animals normally excrete ammonia across their gills, whereas terrestrial animals normally excrete urea or

Ammonia

Urea

Uric acid

Figure 14-49 Most excess nitrogen is eliminated in the form of ammonia, urea, and/or uric acid. Of these common nitrogenousexcretory products, the amount of water utilized in excretion of 1 g of nitrogen is greatest for ammonia, which is highly soluble, and least for uric acid, which is relatively insoluble. Note the differences in number of nitrogen atoms per molecule.

Hydrolysis Cellular proteins

/

-

=

-

-

Amino acids

Retention as osmolyte

Ingested proteins

,

\

+ ma~ntenance

some excretlo

Excreted

Ureotelic

Ur~cotelic

& Ammonotelic

Figure 14-50 Although exceptions occur, water availability correlates with the predominant nitrogen excretory product (pink boxes) found in different animals. This general overview of nitrogen metabolism and excretion in animals shows the polnts at which they differ. Based on the

main nitrogen excretory product used by an animal, it can be classified as ammonotelic, ureotelic, oruricotelic. In some animals certain nitrogenous compounds act as osmolytes, which are substances used to adjust body osmolarity. [Adapted from Wright, 1995.1

uric acid via their kidney. That is, the type of excretion is generally related to habitat: Terrestrial birds excrete about 90% of their nitrogenous wastes as uric acid and only 3%-4% as ammonia, but semiaquatic birds, such as ducks, excrete only 50% of their nitrogenous wastes as uric acid and 30% as ammonia. Mammals excrete most of their waste as urea. Frog tadpoles are aquatic and excrete ammonia; after they metamorphose into the terrestrial adult form, they excrete urea. Avian embryos produce ammonia for the first day or so, and then switch to uric acid, which is deposited within the egg as an insoluble solid and thus has no effect on the osmolarity of the precious little fluid contained in the egg. Lizards and snakes have various developmental schedules for switching from the production of ammonia and urea to the production of mainly uric acid. In species that lay their eggs in moist sand, the switch to uric acid production occurs late in development, but before hatching. The switch to uric acid production is a kind of biochemical metamorphosis that prepares the organism for a dry, terrestrial habitat. It is evident, however, that there are overlaps of different excretory products in animals in similar environments.

Ammonia-Excreting (Ammonotelic) Animals Most teleosts and aquatic invertebrates are ammonotelic; that is, they excrete their nitrogenous wastes primarily as ammonia, producing little or no urea. As just noted, most land animals convert nitrogenous wastes to either uric acid or urea to save water. Interesting exceptions are the terrestrial isopod (sowbug),as well as some terrestrial snails and crabs; these animals eliminate a significant portion of their nitrogen waste by ammonia volatilization. Cell membranes are generally permeable to un-ionized ammonia (NH,) but not very permeable to ammonium (NH,+) ions. Ammonia excretion is due largely to the passive diffusion of un-ionized ammonia. In most teleosts, nearly all ammonia is excreted as NH,.The associated excretion of H + and carbon dioxide acidifies the water next to the gill surface, trapping NH, as the largely impermeant NH,+ and enhancing ammonia excretion. Some membranes, however, have a low permeability to NH3, as well as NH,+. Membranes of Xenopus eggs and those of the cells of the thick ascending limb of the loop of Henle in the mammalian kidney are examples of structures having a low NH, permeability.

-

toxic, should act as the amino-group carrier through blood and tissues until its deamination in the ammonotelic kidney. A blood concentration of only 0.05 mmo1.L-' ammonia is toxic to most mammals, causing convulsions and death. Similar acute toxic effects have been observed in many other animals, including birds, reptiles, and fish. Mexican guano bats are unusual among mammals in that they can withstand very high levels of ammonia (1800 ppm) in the atmosphere of the caves in which they live. This level is sufficient to kill humans, so enter guano bat caves with care! The toxicity of NH, is due in part to the elevation of p H it produces, which causes changes in the tertiary structure of proteins. Ammonia also interferes with some ion-transport mechanisms, because NH,+ substitutes for K+ in some cases. Ammonia can also effect brain blood flow and some aspects of synaptic transmission, particularly glutamate metabolism.

Amino groups of various amino acids are transferred, with the aid of a transaminase enzyme, to glutamate, which is then deaminated to form ammonium ions and a-ketoglutarate in the liver. In the liver, glutamate is also converted to glutamine, which is much less toxic than ammonia and crosses membranes easily but is not normally excreted in any quantity. Although mammals excrete most nitrogenous waste as urea, they excrete small amounts of ammonia in the urine. The less toxic glutamine, rather than ammonia, is released from the mammalian liver into the blood and taken up by the kidney. The glutamine is then deaminated in the cells of the kidney tubules, and ammonia is liberated into the tubular fluid. The excreted ammonia can take up a proton to form the NH,+, which cannot diffuse back into the tubular cells and thus leaves the body via the urine (see Figure 14-30). Since ammonia in both its free and ionized forms is highly toxic, it makes good sense that glutamine, which is non-

W#-C

T l3

t

nvr

I ,C=O I

Carbamoyl synthetase

Ornithine p h O s p h ~ carbamoyl wansferase

HC-NH,

I

\

COOH L-Ornlthine

H

-

1

HN 1

;

iH2

HOOC-CH~-C-COOH L-Aspartlc acid

Argininosucc~nate synthetase

I COOH C=N-C-H

AHz Arginase

I

Argininosucc~nate I HC-NH, lyase

I

I FH2 I

HC-NH,

COOH L-Argininosuccinate

I

COOH

cycle In all vertebrates Figure 14-51 Urea IS formed bythe orn~th~ne-urea except teleosts Because ATP IS required for the f~rststep, ureotel~canl-

mals consume more energy In the excretion of nltrogen than do other an~mals.

are the effects of variations in t

Some squid, shrimps, and tunicates sequester NH,+ in high concentration in specialized acidkied chambers that act as a float making the animal more buoyant. In the floats of these marine animals, NH,+ is substituted for heavier Ca2+, Mg2+,and S042- ions (see Figure 14-50).Ammonium levels in the floats are very high and the tissues that make up the float must be resistant to the toxic actions of ammonia. Ammonia levels in other regions of the body are relatively low. Urea-Excreting (Ureotelic) Animals

Ureotelic animals excrete most nitrogen wastes as urea, which is quite soluble in water, is far less toxic than ammonia, and requires much less water for excretion. Moreover, urea contains two nitrogen atoms per molecule. Ureotelic animals utilize one of two pathways for urea formation. With the exception of most teleost fishes, vertebrates synthesize urea primarily in the liver via the ornithine-urea cycle (Figure 14-51). In this cycle, two -NH, groups and a

'

molecule of CO, are added to ornithine to form arginine. The enzyme arginase, which is present in relatively large quantities in these animals, then catalyzes removal of the urea molecule from arginine, regenerating ornithine. The Lake Magadi tilapia, Oreochromis alcalicus grahami, is a completely aquatic freshwater teleost fish; unlike most teleosts, it excretes all nitrogenous waste as urea. The high p H of Lake Magadi (about 10) impairs ammonia excretion to an extent that leads to ammonia accumulation and death in other fish. Oreochromis alcalicus grahami can live in Lake Magadi because it converts ammonia to urea via the ornithine-urea cycle, thereby avoiding ammonia toxicity. Elasmobranch fish use urea produced from ammonia via the ornithine-urea cycle to increase their body osmolarity; they also excrete most of their nitrogenous waste as urea across their gills. So not all aquatic animals excrete ammonia. Most teleosts and many invertebrates utilize the socalled uricolytic pathway in which urea is produced from uric acid that either arises from a transamination via aspartate or is produced during nucleic acid metabolism. In this pathway, uric acid is converted first to allantoin and allantoic acid with the help of the enzymes uricase and allantoinase, respectively, and then to urea using allantoicase (Figure 14-52). During evolution most mammals have lost Amino acids

Figure 14-52 U r ~ ca c ~ dand urea are produced vta the ur~colyttcpathway. U r ~ ca c ~ d arlses from a purlne rlng that IS synthesized by a complex unlon of aspartlc ac~d,form~c aad, gycne, and CO, Humans lack the enzymes needed t o break down urlc a c ~ d and thus excrete urlc a c ~ das the end product of nucle~ca c ~ dmetabol~sm

I

Transaminase

Glycine, Aspartate, Glutamate

(Purine ring)

Uric acid

Uricase +

0 2

N"2 O=C
reported iq a paper can accurately he converted into other units such asMo, or Mco, only if the author has reported temperature and pressure, which is not always done. L

.&

successive determinationsof the decreasing amount of oxygen dissolved in the water or present in the air contained in the chamber. These measurements can be obtained very conveniently with the aid of an oxygen electrode and the appropriate electronic circuitry. The partial pressure of oxygen of the water or air directly determines the signal produced in the electrode. In gaseous phase (that is, an airfilled respirometer), 0, can be measured by a mass spectrometer or an electrochemical cell in addition to an oxygen electrode. In water or air, C0, can be determined with a CO, electrode, but the complex chemistry of CO, dissolved in water (see Chapter 13) makes the interpretation of these values more complex. In gases, CO, can be accurately measured with a C 0 2electrode, an infrared device, a gas chromatograph, or a mass spectrometer. Generally, 0, is easier to measure than CO,, so MO2is more commonly reported thanM,,, as a measure of metabolic rate. All of these methods of gas analysis allow mass-flow analytical techniques in which the flow of gas or water into and out of a chamber is monitored, and the difference in gas concentrations or partial pressures is used to calculate respiratory exchange. Such systems employ flow-through or open respirometry. Importantly,.the chambers in which the animals reside must be well stirred so that the gas exiting the chamber is in equilibrium with that throughout the chamber. Open system respirometry can also be carried out on animals fitted with breathing masks, a method especially useful in wind-tunnel tests of flying animals or on animals running on treadmills. Closed and open respirometry can be combined in a single experiment, as illustrated in Figure 16-3. Such combined systems are frequently used in partitioning total gas exchange into pulmonary, branchial, and cutaneous locations in such animals as amphibian larvae and air-breathing fishes that simultaneously employ water and air breathing. The determination of metabolic rate from O2 consumption rests on important assumptions:

1. The relevant chemical reactions are assumed to be aerobic. This assumption usually holds for most animals at rest, because energy available from anaerobic reactions is relatively minor except during vigorous activity. However, anaerobiosis is important in animals that live in oxygen-poor environments, as do gut parasites and invertebrates that dwell in deep lake-bottom muds. Oxygen consumption would be an unreliable index of metabolic rate in such animals and would underestimate the true metabolic rate. 2. The amount of heat produced (i.e., energy released) when a given volume of oxygen is consumed is assumed to be constant irrespective of the metabolic substrate. This assumption is not precisely true: more heat is ~roducedwhen 1 liter of 0, is used in the breakdown of carbohydrates than when fat or protein is the substrate. However, the error resulting - from this assumption is no greater than about 10%. Unfortunately, it is generally difficult to precisely identify the

670


............................................................................... Figure 16-3 Open and closed respirometery can be combined in a single experiment to measure an animal's gas exchange partitioning between various sites. In this experiment on the reedfish Calamoicthys calabaricus, two independent open systems are used to determine branchial and cutaneous aquatic oxygen uptake. A third, closed respirometery system includes the air in the funnel above the head, from which the animal breathes air. Gas samples taken after an air breath are used to calculate aerial oxygen consumption. [Adapted from Sacca and Burggren, 1982.1

i

i

Airequilibrate water

3.

substrate(s)being oxidized in correcting for differences in caloric yield. The 0, stores in the body are small, so the minute-tominute oxygen consumption from air or water flowing over the gas-exchange organs fairly accurately represents the metabolic rate. (Note that the ability to store CO, in body tissues is much greater than the ability to store 0,, so the minute-to-minute elimination of CO, is a much less reliable indicator of metabolic rate.)

A final important method for measuring metabolic rate employs isotopic techniques. These techniques first came to prominence in the measurement of water fluxes in animals: deuterium- or tritium-labeled water is injected into an animal, and the specific activity in serial blood or other body fluid samples is determined. The decline in specific activity with time indicates the loss of labeled water and therefore the outward water flux. The use of isotopes was then extended to the measurement of CO, production as a measure of metabolic rate. In essence, isotopes of oxygen and hydrogen are injected into an animal. The subsequent decline in the abount of isotopic oxygen ("0)in body water is related to the rates of CO, loss through exhalation and through water loss, the latter measured by the disappearance of deuterium- or tritium-labeled water. Although the numerous assumptions required must be validated for each experimental setting, the great advantage of the technique

is that it can be employed on intact, unrestrained, normally behaving animals. The many studies by Ken Nagy and his colleagues have shown the usefulness of this technique in measuring field metabolic rate. Respiratory Quotient

To translate the amount of oxygen consumed into equivalent heat production, we must know the relative amounts of carbon and hydrogen oxidized. The oxidation of hydrogen atoms is hard to determine, however, because metabolic water (i.e., that produced by oxidation of hydrogen atoms available in foodstuffs),together with other water, is lost in the urine and from a variety of body surfaces at a rate that is irregular and determined by unrelated factors (e.g., osmotic stress and ambient relative humidity). It is more practical to measure, along with the 0, consumed, the amount of carbon converted into CO,, as explained earlier. As noted in Chapter 13, the ratio of the volume of CO, produced to the volume of 0, removed from within a given time is called the respiratory quotient (RQ): R

[rate of C 0 2 production] - [rate of O2 consumption] -

Q

(16-2)

Under resting, steady-state conditions, the RQ in Table 16-1 is characteristic of the type of molecule catabolized

TABLE 16-1 Heat production and respiratory quotient for the three major foodstuff types Heat production (kJ) Per gram of foodstuff

Per h e r of 0, consumed

RQ Per h e r of CO, produced

17.1 38.9

21 .I 19 8

21.1

1.00

Fats

27.9

0.71

Protelns (to urea)

17 6

18 6

23.3

0.80

Carbohydrates

USING ENERGY: MEETING E N V I R O N M E N T A L CHALLENGES

671

............................................................................... (carbohydrate, fat, or protein). Thus, the RQ reflects the proportions of carbon and hydrogen in the food molecules. The following examples illustrate how the RQ of the major food types may be calculated from a formulation of their oxidation reactions: Carbohydrates. The general formula of carbohydrates is (CH,O),. In the complete oxidation of a carbohydrate, oxygen is used in effect only to oxidize the carbon to form CO,. Upon complete oxidation, each mole of a carbohydrate produces n moles of both H,O and CO, and consumes n moles of 0,. The RQfor carbohydrate oxidation is thus 1. The overall catabolism of glucose, for example, may be formulated as C6H,,0,

+ 6 0,

6 CO,

+ 6 H,O

R - 6 volumes of C 0 2 Q 6 volumes of 0 2

Fats. The Rq characteristic of the oxidation of a fat such as tripalmitin may be calculated as follows: 2 C,,H,,06

+ 145 0, '=;102 CO, + 98 H,O

RQ =

102 volumes of C 0 2 145 volumes of 0,

Because different fats contain different ratios of carbon, hydrogen, and oxygen, they differ slightly in their RQs. Proteins. The RQ characteristic of protein catabolism presents a special problem because proteins are not completely broken down in oxidative metabolism. Some of the oxygen and carbon of the constituent amino acid residues remains combined with nitrogen and is excreted as nitrogenous wastes in urine and feces. In mammals, the excreted end product is urea, (NH,),CO; in birds, it is primarily uric acid, C,H,N,O, . To obtain the RQ, it is therefore necessary to know the amount of ingested protein as well as the amount and kind of nitrogenous wastes excreted. The oxidation of carbon and hydrogen in the catabolism of protein typically produces R Q

96.7 volumes of O2

- 77.5 volumes of C 0 2

It is routinely assumed in making deductions from RQ that (1)the only substances metabolized are carbohydrates, fats, and proteins; (2) no synthesis takes place alongside breakdown; and (3)the amount of CO, exhaled in a given time equals the CO, produced by the tissues in that interval. These assumptions are not strictly true, so caution must be exercised in using RQvalues at rest and in postabsorp-

tive (fasting)states. Under such conditions, protein utilization is negligible, and carbohydrate utilization is minor, so the animal is considered to be metabolizing primarily fat. From Table 16-1, it can be seen that the oxidation of 1 g of mixed carbohydrate releases about 17.1 kJ (4.1 kcal) as heat. When 1liter of 0, is used to oxidize carbohydrate, 21.1 kJ (5.05 kcal) of heat is obtained; the value for fats is 19.87 kJ (4.7 kcal) and for protein (metabolized to urea), 18.6 kJ (4.46 kcal). A fasting aerobic animal presumed to be metabolizing mainly fats produces about 20.1 kJ (4.80 kcal) of heat for every liter of oxygen consumed. Another term often used to describe the ratio of MO2 to MCo2is the respiratory exchange ratio (R,), which is a measure of the instantaneous relation between Mo2 and Mco2as measured from gas exiting the respirometer or face mask. When CO,, for example, is temporarily being stored in body tissues rather than eliminated from the body (as during a period of submergence in a diving animal), the apparentMco2 is lower than is actually occurring at the tissue level. Under these conditions, the REwill be lower than the RQuntil a new CO, steady state isreached in body tissues and CO, is once again eliminated at the same rate at which it is produced by cellular respiration. Energy Storage Although animals continually expend metabolic energy, most do not ingest food steadily. cbnsequently, they do not strike a moment-to-moment balance between food intake and energy expenditure. As food is taken in bursts (i.e., in discrete meals), the animals immediate energy requirements are exceeded. However, the excess is stored, for later use, primarily as fats and carbohydrates. Protein is not an ideal storage material for energy reserves because nitrogen is a relatively scarce commodity and is generally the limiting factor in growth and reproduction; it would be wasteful to tie up valuable nitrogen in energy reserves. Fat is the most effective form of energy storage, because oxidation of fat yields 38.9 kJ. g 1 (9.3 k c a l - g l ) ,nearly twice the yield per gram for carbohydrate or protein (Table 16-1).This efficiency is of great importance in animals such as migrating birds or insects, in which economy of weight and volume is of the essence. Not only is the energy yield per gram of carbohydrate lower than that of fats, but carbohydrates are stored in a bulky hydrated form, with as much as 4 to 5 g of water required per gram of carbohydrate, whereas fats are stored in a dehydrated state. Nonetheless, some carbohydrates are important in energy storage. Glycogen, a branched, starchlike carbohydrate polymer, is stored as granules in skeletal muscle fibers and liver cells of vertebrates. Muscle glycogen can be rapidly converted into glucose for oxidation within the muscle cells during intense activity, and liver glycogen is used to maintain blood glucose levels. Glycogen is broken down directly into glucose 6-phosphate, providing fuel for carbohydrate metabolism more directly and rapidly than does fat. Thus, carbohydrates tend to be used to power short-term increases in metabolism-during activity, for

672


.........................................

example. Fats, which cannot be directly metabolized anaerobically, are metabolized aerobically in response to longerterm demands for energy and during fasting when carbohydrate stores have been depleted. Specific Dynamic Action Max Rubner reported in 1885 that a marked increase in metabolism accompanies the processes of digestion and assimilation of food independently of other activities. He gave this phenomenon the rather awkward name specific dynamic action (SDA). Since then, SDA has been documented in all five vertebrate classes, as well as in invertebrates including crustaceans, insects, and mollusks. Generally, an animal's oxygen consumption and heat production increases within about 1hour after a meal is eaten, reaching a peak some 3-6 hours later and remaining elevated above the basal value for several hours (Figure 16-4). In fish, amphibians, and reptiles with an SDA equivalent to a doubling or tripling of metabolic rate, there are also attendant large increases in heart rate and cardiac output and a temporary redistribution of blood toward the gut. Similar cardiovascular changes of lesser magnitude occur in animals with less prominent SDA responses (e.g., in humans). The mechanism of SDA is not clearly understood, but apparently the work of digestion (and the concomitant increase in metabolism of the tissues of the gastrointestinal tract) is responsible for only a small part of the elevated metabolism. A more likely explanation for this rise in metabolic rate may be that certain organs, such as the liver, expend extra energy processing recently absorbed nutrients for entry into metabolic pathways. The extra energy consumed by such processes is lost as heat. The increase in heat

01 ' -5

I

0

I

5

I

10

I

15

I

20

I

25

Time (hours from peptone injection) Figure 16-4 Specific dynamic artion occurs after feeding In the toad Bufo marinus. Specific dynamic action was induced by injecting peptone (a mixture of amino acids produced from chemically digested meat protein) into the animal's stomach. [Adapted from Wang et al., 1995.1

production differs, depending on the ingested food materials. The magnitude of the increased metabolic rate ranges from 5% to 10% of total energy of ingested carbohydrates and fats and from 25% to 30% of that of proteins. Specific dynamic action probably accounts for some of the variation in metabolic rate reported by different researchers for a single species. Very different metabolic rates could be obtained, depending on whether the animals measured were in a postabsorptive state or were in some part of the SDA response. Consequently, basal metabolism must be measured only during the postabsorptive state so as to minimize any contribution of SDA.

BODY SIZE AND METABOLIC RATE Body size is one of the more important physical characteristics that affects an animal's physiology. The study of how both anatomical and physiological characteristics change with body mass is called scaling. Changes in body size introduce changes that are not always simple and proportional (i.e., geometric). For example, the doubling of the height of an animal while retaining the same body proportions is accompanied by a fourfold increase in surface area and an eightfold increase in mass. The consequences of nongeometric scaling for the functional anatomy and physiology of the animal are immediately evident. You can carry out a thought experiment by imagining a mouse scaled up to the size of an elephant while retaining its mouselike body proportions. Clearly, the imaginary enlarged mouse has different proportions from those of an elephant, and its relatively slender legs would probably collapse under the weight of the very massive body. After all, for each doubling in height of the imaginary mouse, the mass increases by a factor of eight (height cubed) while the cross-sectional area of leg bone increases by a factor of only four (height squared). These same scaling factors make a small mouse capable of jumping many times its own body length without harm, whereas an elephant is essentially "earthbound." Changes in body mass have great effects on an animal's metabolic rate. Consider, for example, the respiratory and metabolic requirements of a tiny water shrew during diving compared with those of a submerged whale. Although both whales and water shrews normally dive, a whale can hold its breath and remain under water far longer than a shrew. The reason stems from the general prlnc~plethat small animals must respire at higher rates per unit body mass than large animals. In fact, there is an inverse relation between the rate of 0, consumption per gram of body mass and the total mass of the animal. Thus, a 100 g mammal consumes far more energy per unit mass per unit time than does a 1000 g mammal. The nonproportionality of the basal metabolic rates of mammals ranging from very small to very large is illustrated by the well known "mouse to elephant" curve (Figure 16-5A).A similar relation holds not only for other vertebrate groups, but throughout the animal and plant kingdom. Few biological principles are so widely applicable.

Kangaroo mouse Cactus mouse

0.01

0.1

1

10

100

1000

Mass (kg)

Figure 16-5 Mass-specific metabolic rate (MR) in mammals declines as body mass (M) Increases. (A) The mouse-to-elephant curve, with rnetabolic intensity (mass-specificmetabolic rate) given as O2consumption per unit mass plotted against body mass. Note the logarithmic scale of

body mass. (6) Generalizedrelationsbetween overall metabolic rate and body mass (blackcurve)and between metabolic intensity and body mass (red curve). (C) Log-log plots of part B. The MR and log MR plots in parts B andccross at M - 1 kg. [Part Aadaptedfrorn Schmidt-Nielsen, 1975.1

The inverse relation between metabolic rate and body mass applies within species as well as between species. Thus, a small human being, cockroach, or fish tends to have a higher metabolic rate per unit mass per unit time than does a larger member of that species. However, this relation is often difficult to demonstrate within a species, where the overall range of body mass may be quite small compared with that between species and where other factors such as sex, nutrition, and season may exert compounding effects. Metabolic rate is a power function of body mass, as described by the simple relation

line (and differs between species), and 6 is an empirically determined exponent that expresses the rate of change of MR with change in body mass. Mass-specific metabolic rate, also termed metabolic intensity, is the metabolic rate of a unit mass of tissue (i.e., amount of 0, consumed per kilogram per hour). It is determined by dividing both sides of equation 16-3 by M:

in which MR is the basal or standard metabolic rate, M is the body mass, a is the intercept of the log-log regression

The relation described by equation 16-3 is shown in color in Figure 16-5B. Because it is often more convenient to work with straight-line rather than curved plots (e.g., for statistical analysis) equations 16-3 and 16-4 are often put into their logarithmic form. Thus, equation 16-3 becomes

674


............................................................................ log MR = log a

+ b(1og M)

(16-5)

and equation 16-4 becomes log MR M

=

log a

+ (b

-

1)log M

(16-6)

These logarithmic equations are plotted in Figure 16-5C. See Appendix 2 for a discussion of logarithmic equations. Notice the difference in how whole-animal metabolic rate (black plots) and mass-specific metabolic rate (red plots) change with changes in body mass. These graphs show that overall metabolic rate rises with increasing body mass, whereas the mass-specific metabolic rate (metabolic rate of a unit mass of tissue) decreases with increasing body mass. This principle first emerges in the mouse-to-elephant plot in Figure 16-SA. The value of exponent b lies close to 0.75 for many different taxonomic groups of vertebrates and invertebrates and even holds true for various unicellular taxa (Figure 16-6).The exponential relation between body size and metabolic rate has been attracting the attention of physiologists since it was first recognized more than a century ago. There have been many attempts at giving a rational explanation for this nearly universal logarithmic relation between body mass and metabolism. In 1883, Max Rubner proposed an attractive theory known as the surface ~

-

Figure 16-6 A wide variety of groups of animals (including unicellular organisms) show the same general relation between metabolic rate and body mass. Metabolic rate is related to body mass by similar exponents in all three groupsforwhich data are presented here. All three solid lines

hypothesis. Rubner reasoned that the metabolic rate of birds and mammals that maintain a more or less constant body temperature should be proportional to body surface area because the rate of heat transfer between two compartments (i.e., warm animal body and cool environment) is proportional, all else being equal, to their area of mutual contact (Spotlight 16-2).The surface area of an object of isometric shape (i.e., nonvarying proportions) and uniform density varies as the 0.67 (or f ) power of its mass. This is because mass increases as the cube of linear dimension, whereas surface area increases only as the square. As noted, this relation holds true for a series of animals of different mass only if body proportions remain constant. This provision is generally satisfied only by adult individuals of different size within a species, because they tend to obey the principle of isometry -namely, proportionality of shape regardless of size. In this case, it follows that the surface area must vary as the 0.67 power of body mass. However, the principle of isometry is not followed in individuals of different size belonging to related but different species. Instead, they tend to follow the principle of allometrynamely, systematic changes in body proportions with increasing species size. An example of allometry was alluded to earlier when we compared the proportions of an elephant with those of a mouse. In a comparison of surfaceto-mass relations in mammals of different species ranging from mice to whales, surface areas were found to be proportional to the 0.63 power of body mass (Figure 16-7).

represent slopes (exponents in the allometric equation) of 0.75. The vertical position of each group on the graph is related to coefficient a in equation 16-3. [From Hernrningsen, 1969.1

M a s s (g) Figure 16-7 Body surface area of mammals ranging from mice to whale* is very closely correlated with body mass. The slope of the line gives an exponent of 0.63 rather than the 0.67 predicted by the isometric (i.e., proportional) scaling. The allometric (i.e., disproportional) scaling arises from the fact that, with increasing species size, there is a progressive relative thickening of body structures (i.e., bones, muscles, etc.), so a large species has relatively less surface area than would be predicted from isometric scaling. Recall the relat~veproportions of mouse and elephant. [From McMahon and Bonner, 1983.1

The surface hypothesis of Rubner gained support over the years from numerous findings that metabolic rate in animals maintaining a constant body temperature is approximately proportional to body surface area. An especially close correlation can be seen in comparing the metabolic rates of adult guinea pigs (all of the same species), which were found to be proportional to body mass raised to the 0.67 power (Figure 16-8A) or, assuming isometry of shape,

proportional to the surface area of the individual. Recall that isometry-and, hence a 0.67, power relating surface area to body mass-is characteristic of adult individuals of the same species. In spite of the logical attractiveness of the surface hypothesis, it is not without flaws. True, the difference in metabolic intensity between large and small homeotherms may indeed be an adaptation to the more rapid loss of heat from a smaller animal owing to surface-to-volume relations, the small animal having more surface area per unit mass. Nonetheless, several contradictory observations raisc serious concerns about the surface hypothesis. First, when metabolic rates of individuals of different species of mammals are plotted against body mass, the exponent relating metabolic rate to body mass is found to be approximately 0.75 (Figure 16-8B). The 0.75 power relating metabolic rate to body mass was first discovered by Max Kleiber (1932)and is often referred to as Kleiber's law. The exponent 0.75 is significantly higher than that predicted by the surface hypothesis (see Figure 16-8B);recall that the surface area of mammalian individuals taken from various species Of differing size is proportional to the body mass raised to the 0.63 power (see Figure 16-7). Thus, in comparing different species, the differences in metabolic rate clearly cannot be predicted simply on the basis of differences in body surface area. Another flaw of the surface hypothesis arises from the simple observation that the metabolic rates of animals whose body temperatures vary with that of their surroundings (such as fish, amphibians, reptiles and most invertebrates) exhibit nearly the same relation to body mass as the metabolic rates of animals that actively maintain a constant, high body temperature (i.e., birds and mammals;

Elephant

Mass (kg) Figure 16-8 Basal metabolic rate of animals is closely correlated with body mass. (A) Basal metabolic rate of individuals of the same species of various sizes plotted against individual body mass. The slope of the line indicates that the basal metabolic rate is proportional to the body mass raised to the 0.67 power. (B) Basal metabolic rate of animals of different

/

Mass (kg) species plotted against body mass. The black line through the points has a slope of 0.75. The red line has the slope of 0.63 predicted by the surface law. The discrepancy is statistically significant, indicating that metabolic rate is not strictly related to surface area in mammals. [Part Afrom Wilkie, 1977; black line in part B from Kleiber, 1932.1

676

I N T E G R A T I O N OF P H Y S I O L O G I C A L SYSTEMS

....................................... SPOTLIGHT 1 6 - 2

THE REYNOLDS NUMBER: IMPLICATIONS FOR BIG AND SMALL ANIMALS The energy expended in propelling an animal through a fluid medium (water or air) depends in part on the flow pattern set up in the medium. The flow pattern is determined not only by the density and viscosity of the medium, but also by the dimensions and velocity of the animal. Osborn Reynolds combined these four factors in a dimensionless ratio that relates internal forces (proportional to density, size, and velocity) to viscous forces. This is the so-called Reynolds number (Re), calculated as

The velocity at which turbulence appears is higher for a streamlined object such as a dolphin than for an unstreamlined object such as a human scuba diver with protruding tanks, and so forth. Because the value of a streamlined form is in reducing turbulence, it has no advantage for small organisms operating at very low Reynolds numbers, in that such organisms do not experience turbulence. To a small organism such as a bacterium, spermatozoan, or ciliate, the watery medium appears far more viscous than it does to a human being. The viscosity encountered by a paramecium swimming through water has been compared to the viscosity that would be experienced by a person swimming through a pool of honey (which, indeed, is difficult to imagine). This is another example of an allometric scaling effect. Viscous effects are proportional to surface area, which rises with the square of animal length. Inertial effects, due to momentum of the moving animal, are proportional to mass, which rises with the cube of length. Thus, the movements of small organisms are dominated by viscous affects, whereas those of large animals are dominated

where p is the density of the medium, V is the velocity of the

by inertial effects.

body, L is an appropriate linear dimension, and p is the viscos-

The relative importance of these two factors in the "coasting" of objects of different size can be illustrated by forcing a tiny

ity ofthe medium. Thus, when a body moves through a fluid such as water or air, the flow pattern depends on its Re. The larger the object or the higher its speed in water, the higher is its Re. The same object moving at the same speed in air as in water would

toothpick (low Re) floating on water t o a given velocity, say 0.1 m .s-', and then doing the same to a large floating log (high Re) of similar physical proportions but larger size. When it is let

be characterized by a lower Re in air (about 15 times lower) be-

go, the little toothpick abruptly comes to rest due to the drag ex-

cause of the much lower density of air. An Re below about 1.0characterizes movement in which the

erted on it by the viscosity and cohesion of the water. In contrast, the massive log coasts for many seconds after its release be-

object produces a purely laminar pattern of flow in the fluid passing over its surface. If Re is above about 40, turbulence begins to

cause its far greater momentum (based on its mass) overcomes the drag (based on its surface area). Similarly, a paramecium

appear in the wake of the object. As Re exceeds about lo6, the

comes to an abrupt halt if it stops its rap~dlybeating cilia,

fluid in contact with the object becomes turbulent. At this point, the energy needed to increase the velocity further rises steeply.

whereas a whale coasts with little loss of velocity between the slow thrusts of its flukes.

see Figure 16-6). There is no self-evident reason why the metabolic rate of animals with variable body temperature should be causally related through heat loss to body surface area. Relatively little or no metabolic energy is expended to warm animals when they are in temperature equilibrium with the environment. Scaling effects are also evident at the cellular level. There is a correlation between differences in metabolic intensity of animals of differing sizes and the number of mitochondria per unit volume of tissue. The cells of a small mammal contain more mitochondria and mitochondria1 enzymes in a given volume of tissue than do the cells of a large mammal. Because mitochondria are sites of oxidative respiration, this correlation comes as no surprise. However, we are still left with the problem of how metabolic intensity is functionally related to body size. The question of why large animals have lower metabolic rates per volume of tissue than those of small animals and the functional reasons for the allometric relations that exist between metabolic rate (as well as other variables) and ani-

ma1 size have been considered extensively. McMahon and Bonner (1983) pointed out that the cross-sectional area rather than the surface area of the body (or rather of its parts) more closely resembles the scaling of metabolic rate to body mass, because the cross-sectionalarea of any body part in a series of animals of increasing size should be proportional to the 0.75 power of body mass, owing to allometric principles that require an elephant's leg to be proportionately thicker than a mouse's leg. Remember that metabolic rate bears the same (0.75) power relation to body mass in a wide range of animals (see Figures 16-6 and 16-8B). Although the allometry of metabolic rate is well documented, comparative physiologists have yet to "prove" definitively why this relation exists, and both experiments and thought on the subject continue. However, there is no doubt of the physiological implications of allometry to animals. Small animals with proportionately higher metabolic rates must spend more of their time looking for resources and may also be more susceptible to temporary shortages of metabolic substrates or oxygen.

TEMPERATURE AND ANIMAL ENERGETICS Few environmental factors have a larger influence on animal energetics than temperature. Those animals whose body temperature fluctuates with that of the environment experience corresponding temperature-induced changes in metabolic rate, whereas those that can maintain a constant body temperature in fluctuating environmental temperatures have to expend metabolic energy to do so. Temperature Dependence of Metabolic Rate

Chemical reaction rates, especially those of enzymatic reactions, are highly temperature dependent. Therefore, tissue metabolism and, ultimately, the life of an organism depend on maintenance of the internal environment at temperatures compatible with metabolic reactions facilitated by enzymes. When we consider the effect of temperature on the rate of a reaction, it is useful to obtain a temperature quotient by comparing the rate at two different temperatures. A temperature difference of 10 Celsius degrees has become a standard (if arbitrary) span over which to determine the temperature sensitivity of a biological function. The so-called Qlo is calculated by using the van't Hoff equation:

where k, and k, are rates of reaction (rate constants) at temperatures t, and t2, respectively. The beauty of the Qlo concept is that it can be applied both to simple processes such as single enzymatic reactions and to complex processes such as running and growing. To relate the van't Hoff equation to metabolic rate, consider the following form of the van't Hoff equation:

in which MR, and MR, are the metabolic rates at temperature t, and t2, respectively. For temperature intervals of precisely 10 degrees, the following simpler form of equation 6-8 can be used:

where MR, is the metabolic rate at the lower temperature, and MR(,+ is the metabolic rate at the higher temperature. The Qlo of a given enzymatic reaction depends on the particular temperature range being considered, so it is important, when citing a Qlo value, to clearly indicate the range of temperatures (i.e., t, and t2)for which it was determined. As a rule of thumb, chemical reactions (and such physiological . -processes as metabolism, growth, locomotion, etc.) have Q,, values of about 2 to 3, whereas purely physical processes (such as diffusion) have lower temperature sensitivities (i.e., closer to 1.0). The temperature effect on enzymes results in the metabolic rate of an animal increasing exponentially with body temperature, as described by the equation

where MR is metabolic rate and M is body mass (making MRIM the metabolic intensity in kilocalories per kilogram per hour), k and b, are constants, and t is temperature in degrees Celsius. Because enzyme action largely dictates metabolic rate, we can see this same relation when looking at the effect of body temperature on oxygen consumption in animals that do not maintain body temperature at a constant value (Figure 16-9A).Again, it is useful to transform the relation into a logarithmic one to produce a linear plot. Thus, equation 16-10 becomes log MR M

=

log k

+ blt

Now the coefficient b, gives the slope of the line-that is, the rate of increase in log MRIM per degree (Figure 16-9B). The metabolic rates in most animals with variable body temperature increase two- to threefold for every 10 degree (Celsius) increase in ambient temperature, in accordance with what would be predicted for the Qloof enzymes. Yet, the metabolic rates of some ectotherms exhibit a remarkable temperature independence. For example, some intertidal invertebrates that experience large swings in ambient temperature with the ebb and flow of the tides have metabolic rates with a Qlovery close to 1.0, so the rate of metabolism changes very little with temperature changes as large as 20 degrees. These animals appear to possess enzyme systems with extremely broad temperature optima, which prevents their inactivation during environmental temperature swings. Such enzyme systems may be due to a staggering of the temperature optima of sequentialenzymes in a reaction, such that a drop in the rate of one step in a sequence of reactions "compensates" for an increased rate of other steps in the sequence. The "biological clocks" of animals also are temperature insensitive with Qlosof 1.Otherwise, the "time" kept by these "clocks" would be utterly dependent on an animal's body temperature, and even a fever in a mammal or bird would throw off its body rhythms.

678


......................................... a single environmental condition such as temperature. (Recall that evolutionary adaptation refers to evolutionary changes over thousands of generations of a species). Enzymatic acclimation Acclimation occurs in individual tissues as well as in whole animals. For instance, at a given experimental temperature, winter acclimated frogs and summer acclimated frogs have different contractile properties of skeletal muscles and different heart rates. Similarly, nerve conduction persists at low temperatures in cold-acclimated fishes, but it is blocked at these same temperatures in warm-acclimated ones. How can this be explained? It is reasonable to suppose that enzymatic reactions have been affected. In Figure 16-10, the plots of 0, consumption against temperature for frogs acclimated at 5°C and 25°C show differing slopes. That is, the net respiratory processes in the two acclimation groups exhibit different Qlos, suggesting that there has been a modification in the temperature sensitivity of enzyme activity. A change in the rate of enzymatically controlled reactions can indicate a change either in the molecular structure of one or more enzymes or in some other factor that affects enzyme kinetics. In some instances of acclimation, however, thermal compensation appears to result simply from a change in the quantity of an enzyme rather than its characteristics. This is indicated in experiments in which the plot relating a metabolic function to the test temperature exhibits displacement without a change in slope (Figure 16-11). Because the Q,, of the process remains unchanged but the activity is higher at every temperature in the cold-acclimated group, the acclimation appears to have led to an increase in the number of enzyme molecules without any change in the kinetics of the enzymes. The particular time course of acclimation depends on the rate at which enzyme type or concentration can be modified. -

L

.-0

Ambient temperature ( ' C )

(1

Ambient temperature ( " C ) Figure 16-9 The oxygen consumption of the tiger moth caterpillar increases sharply as its body temperature increases. (A) Geometric coordinates. (B) Semilog coordinates. The generalized ordinates are shown in color at the right in reference to equations 16-11 and 16-12. The constant k is obtained by extrapolating the metabolic rate to a body temperature of 0°C and is the proportionality factor in equations 16-11 and 16-12. [From Scholander et al., 1953.1

Thermal acclimation-enzymatic mechanisms Environmental heat or cold elicits compensatory changes in physiology and, in some cases, morphology in many species. These changes help an individual organism to cope with the temperature stress. An animal that cannot escape the winter cold (e.g., a pond-dwelling teleost fish in a temperate environment) will gradually undergo, in the course of several weeks, a whole suite of compensatory biochemical adaptations to low temperature. As noted in Chapter 1, the overall change that the animal undergoes in the natural setting is termed acclimatization. We will confine ourselves here to a more restricted concept, acclimation, which refers to the specific physiological change(s)developed over time in the laboratory in response to varying

0

10

Temperature

20

-

30

("C)

Figure 16-10 At any given measurement temperature, oxygen consumption in frogs acclimated at 5°C is greater than oxygen consumption in frogs acclimated at 25°C. This phenomenon minimizes the disruptive effects of temperature change in these and other ectotherms.

USING ENERGY: MEETING ENVIRONMENTAL CHALLENGES

679

...............................................................................

Warm-accl~mated

Test temperature ( " C ) Figure 16-11 Temperature acclimation greatly influences temperature effects on metabolic rate. Generalized plot of log metabolic rate againsttest temperature in a cold-acclimated individual and in a warmacclimated one. The similarity of slope in the two plots indicates identical Q,,s.

Homeoviscous membrane adaptations The cell membrane, which is composed largely of a bilayer of lipids with embedded proteins, is very sensitive to temperature change. Low temperatures can cause the membrane to enter a gel-like phase with very high membrane lipid viscosity, whereas high temperatures can cause the membrane to become "hyperfluid" with very little viscosity. Either situation can cause increasingly disruptive changes in physical properties as temperatures move away from optimal values for a particular animal. The many functions of the cell membrane, which range from forming a physical barrier to general solute diffusion to facilitating transmembrane movement of specific solutes, can be in jeopardy if the membrane lipid viscosity becomes too high or too low. We can imagine the effect of temperature on lipid viscosity by recalling that room temperatures lie below the melting point of a cooking grease but above the melting point of a cooking oil. The difference between the oil and the grease lies in the degree of hydrogenation of the carbon backbone. The greater the proportion of unsaturated (i.e., double unhydrogenated) carbon-carbon bonds of a lipid's fatty acid molecules, the lower its melting point. At temperatures above the melting point, the lipid is less viscous, or "oily"; below the melting point, it is more viscous, or "waxy." Part of the acclimatization of ectothermic animals to cold or hot environments is that the membrane lipids become more saturated during acclimatization to warmth and less saturated during acclimatization to cold, helping to stabilize the form of the lipids and thus the cellular functions that spring from them. This phenomenon is called homeoviscous adaptation, referring to adaptations at the molecular level through natural selection that help rninimize temperature-induced differences in viscosity. Unfortunately, there is no simple measure of membrane fluidity. Most often used as an index is the steady-state fluorescence anisotropy (a measure of the lack of symmetry of a molecule or structure). 1,6-Diphenyl-l,3,5-hexatriene

(DPH) is a commonly used probe of membrane fluidity. A high fluorescenceanisotropy indicates a high degree of lipid polarization and membrane order attendant with a low membrane viscosity. Figure 16-12shows changes in DPH polarization of the basolateral membranes of enterocytes isolated from rainbow trout. Initially, acute temperature changes are accompanied by changes in membrane polarization and fluidity. However, with time, homeoviscous adaptation of the membrane lip~dsresults in a lipid polarization and membrane viscosity after acclimation at 5°C that are similar to those after acclimation at 20°C. As Hazel (1995) argues, homeoviscous adaptation is a powerful paradigm for explaining acclimation and adaptation in animals with variable body temperatures but cannot be the only explanation. Some animals become fully acclimated to temperature change with moderate or even no homeoviscous adaptation in lipid membrane properties. Altered expression of membrane proteins and proliferation of mitochondria1 and sarcoplasmic reticular membranes, along with homeoviscous adaptation of membrane lipids, present a picture of cell membranes as dynamic structures that change in complex ways to retain their function despite temperature change. Finally, there are also regional differences in lipid properties, including melting point, in some mammals. In the limbs, which may be subjected to near-freezing temperatures, the tissue lipids are less saturated and so have a lower melting point than the fats in the body core. At 37"C, the fats in the limbs are much "oilier" than the waxier fats of

Degree of DPH polarizatio~ (degree of membrane viscosity)

Acute measurement temperature ( O C ) Figure 16-12 Homeoviscous adaptation maintains relatively constant membrane lipid properties in rainbow trout enterocytes. After an initial measurement at 25'C (point I), a warm-acclimated(20°C) trout is rapidly cooled to 5°C. Initially, the membranes of its enterocytes become more polarized and more viscous (point 2), but as homeoviscous adaptation occurs the lipid membranes become less polarized and regain their fluidity (point 3). Similarly, if a cold-acclimated trout at a measurement temperature of 5°C is rapidly warmed to 25°C and measured, the lipid membranes are initially highly depolarized (point 4) but become more polarized with acclimation (point 1).[Adapted from Hazel, 1995.1

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...............................................................................

the warmer body regions. The low-viscosity oils extracted from the limbs of slaughtered cattle are marketed as neat'sfoot oil, a penetrating leather preservative and softener.

Determinants of Body Heat and Temperature The temperature of an animal depends on the amount of heat (calories) contained per unit mass of tissue (see Spotlight 3 6-2). Because tissues consist primarily of water, the heat capacity of tissues between 0°C and 40°C approximates 1.0 cal ."C-' - g-'. It follows that the larger the animal, the greater its body heat content at a given temperature. The rate of change of body heat depends on (1)the rate of heat production through metabolic means, (2)the rate of external heat gain, and (3)the rate of heat loss to the environment (Figure 16-13). We can state that

body heat = heat produced + (heat gained - heat lost) = heat produced + heat transferred Thus, body heat, and hence the body temperature of an animal, can be regulated by changes in the rate of heat production and heat transfer or exchange (i.e., heat gained minus heat lost). Numerous factors affect the rate of body heat production. Behavioral mechanisms such as simple exercise cause an increase in heat production by elevating metabolism. The activation of autonomic mechanisms leading to release of hormones can produce accelerated metabolism of energy reserves. Acclimatization mechanisms, which are slower than the other two processes, often lead to an elevation in basal metabolism and the associated heat production. The total heat content of an animal is determined by the metabolic production of heat and the thermal flux between the animal and its terrestrial surroundings, as shown in Figure 16-13. The relation between these factors can be represented as

in which Htotis the total heat, Hv is the heat produced metabolically, Hc is the heat lost or gained by conduction and convection, y is the net heat transfer by radiation, He is the heat lost by evaporation, and Hs is the heat stored in the body. Heat leaving the animal has negative ( - ) value,

lnfrared thermal

lnfrared thermal

a

Scattered sunlight

-

Wind

radiation from ground Figure 16-13 Heat IS transferredbetween an antmal and ~tsenvlronment In numerous ways lnfrared thermal radtatlon and d~rectand reflected sunllght transfer heat Into the anlrnal, whereas rad~atlonand evapora-

tlon transfer heat out to the envlronment [Adapted from Porter and Gates, 19691

'

USING ENERGY: M E E T I N G ENVIRONMENTAL CHALLENGES

681

............................................................................... whereas heat entering the body from the environment has positive (+)value. Animals can lose heat by conduction, convection, radiation and evaporation. Let us now consider each of these key terms. Conduction The transfer of heat between objects and substances that are in contact with each other is conduction. It results from the direct transfer of kinetic energy of the motion from molecule to molecule, with the net flow of energy being from the warmer to the cooler region. The rate of heat transfer through a solid conductor of uniform properties can be expressed as

in which Q is the rate of heat transfer (in joules per centimeter per second) by conduction; k is the thermal conductivity of the conductor; A is the cross-sectional area (in square centimeters); and 1 is the distance (in centimeters) between points 1 and 2, which are at temperatures t, and t,, respectively. Conduction is not limited to heat flow within a given substance; it may also be between two phases, such as the flow of heat from skin into the air or water in contact with the body surface. Convection The transfer of heat contained in a mass of a gas or liquid by the movement of that mass is convection. Convection may result from an externally imposed flow (e.g., wind) or from the changes in density of the mass produced by heating or cooling of the gas or fluid. Convection can accelerate heat transfer by conduction between a solid and a fluid, because continuous replacement of the fluid (e.g., air, water, or blood) in contact with a solid of a different temperature maximizes the temperature difference between the two phases and thus facilitates the conductive transfer of heat between the solid and the fluid. Radiation The transfer of heat by electromagnetic radiation takes place without direct contact between objects. All physical bodies at a temperature above absolute zero emit electromagnetic radiation in proportion to the fourth power of the absolute temperature of the surface. As an example of how radiation works, the sun's rays may warm a black body to a temperature well above the temperature of the air surrounding the body. A dark body both radiates and absorbs more strongly than does a more reflective body having a lower emissivity. For temperature differences between the surfaces of two bodies of about 20 Celsius degrees or less, the net radiant heat exchange is approximately proportional to the temperature difference. Evaporation Every liquid has its own latent heat of uaporization, which is the amount of energy required to change it from a liquid to a gas of the same temperature-that is, to evaporate. The energy required to convert 1g of water into water vapor is relatively high, approximately 585 cal.

Many animals dissipate heat by allowing water to be evaporated from body surfaces. Heat storage Heat storage leads to an increase in temperature of the heat-storing mass. The larger the mass, or the higher its specific heat, the smaller its rise in temperature (in "C) for a given quantity of heat (in joules) absorbed. Thus, a large animal that has a small surface-to-massratio tends to heat up more slowly in response to an environmental heat load than does a small animal that has a relatively high surface-to-mass ratio. This follows from the simple fact that heat exchange with the environment must take dace through the bodv surface. The rate of heat transfer (kilocalories per hour) into or out of an animal also depends on several factors. Changing the value of any one of them alters heat flow across the body surface in the direction of the temperature gradient:

Surface area per gram of tissue decreases with increases in body mass, providing small animals with a high heat flux per unit of body weight (as already noted). Animals can sometimes control their apparenc surface area by changing posture (e.g., by extending limbs or drawing them close to the body). Temperature difference between the environment and the animal's body has a large effect by altering the temperature gradient (i.e., change in temperature per unit distance) for heat transfer. The closer an animal maintains its temperature to the ambient temperature, the less heat will flow into or out of its body. Specific heat conductance of the animal's surface varies with the nature of the body surface. Animals with high heat conductances in surface tissues are typically close to the temperature of their surroundings, with some exceptions, such as the elevation of body temperature when an animal basks in sunlight. Animals that actively maintain a constant body temperature (birds, mammals) have feathers, fur, or blubber that decrease the heat conductance of their body surfaces. An important feature of fur and feathers is that they trap and hold air, which has a very low thermal conductivity and therefore further retards the transfer of heat. Such insulation spreads out the temperature difference between the body core and the animal's surroundings over a distance of several millimeters or centimeters so that the temperature gradient is less steep and thus the rate of heat flow is reduced. Most animals have body temperatures similar to those of their environments. Animals breathing water can maintain only parts of the body above ambient temperature because oxygen transfer is slower than heat transfer and water contains little oxygen but has a high specific heat, and so delivery of oxygen to the respiratory surface inevitably removes all heat produced by metabolism. Air-breathing

animals, on the other hand, can obtain a sufficient amount of oxygen from a small volume of air and can heat that air to high temperature. They have heat "to spare" to raise body temperature. Air, unlike water, has a high 0, content and a low specific heat. Thus, air-breathing animals can raise body temperature above ambient temperature, whereas water-breathing animals cannot. Some animals can keep parts of their bodies (e.g., dog legs, bird feet, tuna muscle, etc.) at different temperatures because of countercurrent heat exchangers (see next section, Temperature classifications of animals). Thus, water-breathing animals such as tuna can maintain regions of their bodies above ambient temperatures; but, each time the blood perfuses the respiratory surface, its temperature approaches ambient temperature because heat loss exceeds heat production. Air-breathing animals can have an elevated body temperature because it is theoretically possible for heat production to exceed heat loss. To maintain high body temperature requires a high rate of heat production because of obligatory heat loss through respiration. Animals use several different mechanisms to regulate the exchange of heat between themselves and the environment: Behavioral control includes moving to a part of the environment where heat exchange with the environment favors attaining optimal body temperature. For instance, a desert ground squirrel retires to its burrow during the midday heat; a lizard suns itself to gain heat by radiation from its surroundings, raising its body temperature well above ambient temperature. Animals also control the amount of surface area available for heat exchange by adjusting their postures. Autonomic control of blood flow to the vertebrate skin affects the temperature gradient and, hence, the heat flux at the body surface (Figure 16-14). For example, the activation of piloerector muscles increases the extent of fluffing of pelage and plumage, which increases the effectiveness of insulation by increasing the amount of trapped, unstirred air (Figure 16-15A). Sweating and salivation during panting cause evaporative cooling. Acclimatization includes long-term changes in pelage or subdermal, fatty-layer insulation, as well as changes in * the capacity for autonomic control of evaporative heat loss through sweating. Acclimatization can also include the capacity for metabolic heat generation, as in finches, for example.

Temperature Classifications of Animals It should be clear to you now that animals deal with variation in the thermal characteristics of their environment in a variety of ways. The "traditional" scheme used by comparative physiologists to classify thermoregulatory modes of animals is based on the stability of body temperatures. When exposed to changing air or water temperatures in the laboratory, homeotherms (or homoiotherms) maintain

Vasoconstr~ct~on Shunt L o w conductance

urface essels

Artery Vein

Air

High c o n d u c t a n c e

~asodilatio

-+

Blood flow

I , Heat

transfer

Figure 16-14 Blood flow to the skin helps regulate the heat conductance of the body surface. Vasomotor control of peripheral arterioles shunts the arterial blood either to the skin or away from it. In response to environmental cold, peripheral blood vessels vasoconstrict, shunting blood away from the surface of an endotherm. In response to high temperatures, the blood is diverted to the skin, where it approaches temperature equilibrium with the environment. In ectotherms, cutaneous blood flow is often increased through peripheral vasodilation to absorb heat from the environment. --

body temperatures above ambient temperatures and regulate their body temperatures within a narrow physiological range by controlling heat production and heat loss (Figure 16-16).In most mammals, the normal physiological range for core body temperature is typically from 37°C to 38°C; whereas, in birds, it is closer to 40°C. Some vertebrates other than birds and mammals and some invertebrates also can control their body temperatures in this manner, although such control is often limited to periods of activity or rapid growth in these organisms. Poikilotherms are those animals in which body temperature tends to fluctuate more or less with the ambient temperature when air or water temperatures are varied experimentally. The colloquial terms "warm blooded" for homeotherms and "cold blooded" for poikilotherms are unsatisfactory because many poikilotherms can become quite warm. For example, a locust sustaining flight in the equatorial sun or a lizard running across the sand at midday in a hot desert may have blood temperatures exceeding those of warm blooded mammals.

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.......................................

Artery Vein

Body core

Surface vessels

/ Shunt vessels

Blubber

Figure 16-15 Fur and blubber act as heat insulation. (A) Fur is outs~de the skin and circulation, and its insulating properties can be changed rapidly only by flattening or fluffing through pilomotor control. (B) Because blubber is located under the skin and is supplied with blood vessels, its insulating value can be regulated by shunting the blood through vasomotor control to the surface or away from the surface below the blubber.

Early comparative physiologists considered all fishes, amphibians, reptiles, and invertebrates to be poikilotherms, because all of these animals were thought to lack the high rates of heat production found in birds and mammals. Several difficulties with the homeotherm-poikilotherm classification scheme became apparent with the completion of

Ambient temperature ("C)

Figure 16-16 Homeotherms maintain body temperature as amb~ent temperature changes, whereas the body temperatures of po~kllotherms more closely track amb~enttemperature

more field studies (especially with the use of radio telemetry of body temperature). For example, some deep-sea fishes have more stable body temperatures than do many higher vertebrates because these fishes live in specialized environments that are thermally very stable. Many so-ca!led poikilotherms (e.g., lizards) are able to regulate their body temperatures quite well in their natural surroundings by controlling heat exchange with their environment, although this ability is ultimately limited by the availability of heat in the environment. Moreover, numerous birds and mammals are now known to allow their body temperatures to vary widely, either regionally in the body or in the whole body over time. These inconsistencies have led to a more widely applicable temperature classification scheme based on the source of body heat. In this scheme, endothermic animals generate their own heat, and ectothermic animals rely almost entirely on environmental sources of heat. (It should be emphasized that the concepts of homeothermy versus poikilothermy as well as endothermy versus ectothermy are idealized extremes, and most organisms are not at these extremes.) Endotherms are animals that generate their own body heat through heat production as a by-product of metabolism, typically elevating their body temperatures considerably above ambient temperatures. Most produce heat metabolically at high rates, and many have relatively low thermal conductivity because of good insulation (fur, feathers, fat), which enables them to conserve heat in spite of a high temperature gradient between body and environment. Mammals and birds exemplify animals that regulate their temperatures within relatively narrow limits and are therefore said to be homeothermic endotherms. A few large fishes (sharks and larger tuna) and some flying insects are termed regional heterothermic endotherms because they maintain regions of their body above ambient temperatures, sometimes for short periods of time under specific circumstances, as in flying insects. Because endotherms (all birds and mammals plus many terrestrial reptiles and a number of insects) maintain their body temperatures well above ambient temperatures in cold climates, thsy have been able to invade habitats that are too cold for most ectotherms. Endotherms keep warm at considerable metabolic cost: the metabolic rate of an endotherm at rest is usually at least five times that of an ectotherm of equal size and body temperature. Ectotherms produce metabolic heat at comparatively low rates-rates normally too low to allow for endothermy. Often, ectotherms have low rates of metabolic heat production and high thermal conductances-that is, they are poorly insulated. As a result, heat derived from metabolic processes is quickly lost to cooler surroundings. Accordingly, heat exchange with the environment is much more important than metabolic heat production in determining an ectotherm's body temperature. On the other hand, the high thermal conductance allows ectotherms to absorb heat readily from their surroundings. Behavioral temperature regulation is the principal means by which ectotherms regulate their body temperatures. (That reptiles

regulate body temperature is well known). Behavioral temperature regulation can be demonstrated in the laboratory by placing animals in thermal gradients and monitoring the preferred body temperatures. Alternatively, animals can be placed in a "shuttle box," which consists of one chamber that is well below the preferred body temperature and a connected chamber that is well above the preferred body temperature. The animal will "shuttle" back and forth, keeping its body temperature at a level between those of the two thermal environments. Field observations combined with radio telemetry of body temperature indicate the considerable thermoregulatory ability of reptiles through behavioral as well as physiological means (e.g., shunting blood to the skin to cool or warm). Many ectotherms needing to change body temperature behave in a way that facilitates heat absorption from the environment or helps the animal unload heat to the environment (or minimizes heat uptake from the environment). A lizard or a snake may bask in the sun with its body oriented for maximal warming until it achieves a temperature suitable for efficient muscular function. Small ectotherms in hot environments (lizards, ants) often elevate their bodies to the extent that their legs allow to avoid the hottest temperatures immediately adjacent to the sand or rock over which they are moving. In general, the most effective thermoregulatory action taken by ectotherms is movement into a suitable microclimate in the environment. A burrow under a rock, for example, is often much more moderate in temperature than the surface temperature (Figure 16-17).Intertidal zones in the tropics often appear to be devoid of invertebrate life during the heat of the day, but that same habitat may be teeming with life at night as animals emerge from their microclimates underneath rocks and in burrows. Immersed

Emersed

Heterotherms are those animals capable of varying degrees of endothermic heat production, but they generally do not regulate body temperature within a narrow range. They may be divided into two groups, regional and temporal heterotherms. Temporal heterotherms constitute a broad category of animals whose temperatures vary widely over time. Monotremes (egg-laying mammals) such as the echidna are temporal heterotherms (Figure 16-18),as are other mammals and birds in torpor and hibernation. Temporal heterothermy is also shown by many flying insects, pythons, and some fishes, which can raise the temperature of their bodies (or regions of their bodies, as in the fishes) well above ambient temperature by virtue of heat generated as a by-product of intense muscular activity. Some insects prepare for flight by exercising their flight muscles for a time to raise their temperatures before takeoff. Some species of small mammals and birds have accurate temperature control mechanisms and so are basically homeothermic. Yet, they behave like temporal heterotherms because they allow their body temperatures to undergo daily cyclical fluctuations, having endothermic temperatures during periods of activity and lower temperatures during periods of rest. In hot environments, this flexibility gives certain large animals, such as camels, the ability to absorb great quantities of heat during the day and to give it off again during the cooler night. Certain tiny endotherms, such as hummingbirds, must eat frequently to support their

Reimmersed

Exposed rock face

0

1

2

3

4

5

Time (hours) Figure 16-17 Microclimates under rocks afford ectotherms protection from harsh thermal environments. The tropical ch~tonChiton stokesiican live in an intertidal zone that reaches lethally high temperatures during the day by seeking much cooler microclimates under rocks. Temperatures in this illustration were recorded from the exwosed face of the chiton-bearing rock, in shaded air, and in the space beneath the attached foot of a ch~tonhidden under a rock [Adapted from McMahon e t a l , 1991 ]

Ambient temperature ("C) Figure 16-18 The relation between body temperature and ambient temperature differs in various animals. The cat is a strict homeotherm, maintaining body temperature independent of ambient temperature, whereas monotremes (platypus and echidna) are temporal heterotherms. The lizard is a strict heterotherm. [Adapted from Marshall and Hughes, 1980.1

U S I N G ENER

........................................ high daytime metabolic rate. To avoid running out of energy stores at night when they cannot feed, they enter into a state of torpor during which they allow metabolic rate to decline and body temperature to drop toward ambient temperature. Even some large endotherms resort to a long winter torpor with reduced body temperature to save energy (see Hibernation and winter sleep later in this chapter). Regional heterotherms are generally ectotherms that can achieve high core (i.e., deep-tissue) temperatures through muscular activity, while their peripheral tissues and extremities approach the ambient temperature. As mentioned earlier, examples include mako sharks, tuna, and many flying insects. Elevated temperatures generally allow higher metabolic rates than would be achieved at ambient environmental temperatures. Fishes that are regional heterotherms depend on countercurrent heat exchangers. Heat is conserved in the body core by a specialized parallel arrangement of incoming arteries and outgoing veins (see Temperature relations of heterotherms) that, in the case of heat exchangers, facilitates heat transfer between blood vessels and retains heat in the body core. Some large billfin fishes (e.g., marlin) use specialized ocular muscle called "heater tissue" to elevate brain temperature (see Thermogenesis later in this chapter). Another special example of regional heterothermy is seen in the scrotums of some mammals, including canines, cattle, and human beings, which hold the testes outside the body core to keep them at a slightly lower temperature. The scrotum shortens in cool air, drawing the testes against the warmer body, and lengthens as its temperature rises. These actions regulate testicular temperature and in particular prevent overheating of the testes, which has a harmful effect on sperm production.

TEMPERATURE RELATIONS OF ECTOTHERMS Ectotherms occupy a wide variety of environments- both hot and cold. A few very specialized environments have highly stable temperatures, varying no more than a degree or two throughout the year. Examples are the shallow marine waters under the Arctic and Antarctic ice, the deep regions of the seas, the air deep within the interior of many caves, and the microenvironments within deep groundwater. Normally, however, almost all environments show significant long- or short-term variation in temperature. Such variation is maximized in terrestrial temperate environments, some of which may have daytime summer surface temperatures of nearly 40°C and nighttime winter temperatures of -40°C. Most animals occupy microhabitats with less extreme temperature fluctuations. However, some degree of thermal stress is inherent in living in most environments, and a wide variety of mechanisms have evolved through natural selection to sustain animal life. Ectotherms in Freezing and Cold Environments

Because the body temperature of many ectotherms depends to a considerable extent on the ambient temperature, freez-

ing is a threat to those species living in environments with ambient temperatures below freezing. The formation of ice crystals within cells is usually lethal because, as the crystals grow in size, they rupture and destroy the cells. No animal is known to survive complete freezing of its tissue water, but some come close. Certain beetles can withstand freezing temperature because the extracellular fluid contains a substance that accelerates nucleation (the process of crystal formation). Consequently, the extracellular fluids freeze more readily than the intracellular fluids. As ice forms in the extracellular fluid, the unfrozen extracellular fluid becomes more concentrated with solutes. This process draws water out of the cells, lowering the intracellular freezing point. As the temperature drops further, the process continues and produces further depression of the freezing point of the remaining intracellular water. The freshwater larvae of the midge Chironomus, which survive repeated freezing, yield some unfrozen liquid at temperatures as low as -32°C. If ice crystals form and grow within cells, they damage the tissue by breaking the cells. In contrast, ice crystals that form outside the cells do little damage. The adaptiveness of this process therefore lies in crystal formation in the extracellular space where little tissue damage is caused. Red blood cells, yeast, sperm, and other cell types also can withstand freezing damage, provided intracellular ion concentrations do not rise above those levels that cause damage to cell organelles. As K. B. Storey and J. M. Storey and their colleagues (1992) have found, a few vertebrates, primarily anuran amphibians, also can withstand freezing. Both icenucleating proteins that initiate and control the formation of extracellular ice and cryoprotectants ("antifreezes") are employed to survive freezing environments. Some animals can undergo supercooling, in which the body fluids can be cooled to below their freezing temperature, yet remain unfrozen because ice crystals fail to form. Ice crystals will not form if they have no nuclei (mechanical "seeds," so to speak) to initiate crystal formation. Thus, certain fishes dwelling at the bottom of Arctic fjords live in a continually supercooled state and normally do not freeze. If they brush against frozen ice on the water surface, however, ice crystallizes rapidly throughout their bodies and they die immediately. Thus, survival for these fish depends on their remaining well below the surface where ice is absent. The body fluids of some cold-climate ectotherms contain antifreeze substances. For example, the body fluids of a number of arthropods, including mites and various insects, contain glycerol, the concentration of which typically increases in the winter. Glycerol, acting as an antifreeze solute, lowers the freezing point to as low as - 17°C. The tissues of larvae of the parasitic wasp Brachon cephi can withstand even lower temperatures; they have been supercooled to -47°C without ice crystal formation. The blood of the antarctic ice fish Trematomus contains a glycoprotein antifreeze that is from 200 to 500 times as effective as an equivalent concentration of sodium chloride in preventing ice crystal formation. The glycoprotein lowers the

temperature at which the ice crystals enlarge, but it does not lower the temperature at which they melt. For many animals living in cool (but not freezing) environments, survival requires maintaining adequate metabolism at the very low levels of enzyme activity characteristic of low temperatures. Many animals living in cold environments have enzymes that show maximal activity at temperatures many degrees below those of homologous enzymes in animals living in warmer environments. Figure 16-19 shows strong evidence of thermal adaptation in the Michaelis-Menten constant (K,) of pyruvate for A,-lactate dehydrogenase. The Kmvalue in Termatomus centronotus, a fish that lives in water that is almost always at -1.9"C, is far higher than that in fishes and other vertebrates that inhabit more eurythermic environments. Even within a single species (barracuda),fish that inhabit cooler waters have a Kmfor pyruvate that is higher than that in individuals of the same species that inhabit warmer waters, allowing the cooler fish to maintain a relatively higher metabolism for their body temperature than that of the warmer fish.

Ectotherms in Warm and Hot Environments Heat exchange with the environment is closely related to body surface area, so the temperature of a small ectotherm (which has a relatively large surface area) rises and falls rapidly as environmental temperatures undergo daily fluctuations. All ectotherms have a critical thermal maximum, a temperature above which long-term survival is not possible. Generally, this is determined by measuring the temperature at which 50% mortality occurs in a population. The critical thermal maximum varies enormously, depending on the organism. Some thermophilic bacteria can thrive at temperatures above 9O0C, although almost all metazoans have critical thermal maxima below 45°C. The physiological causes for a critical thermal maximum are varied. An ultimate upper limit is the temperature at which proteins are denatured, though proteins such as enzymes usually fail to function at levels well below the temperature

\

-

1

.aw

Gillichthys mirabillis (9-35°C) \

. -----

Barracuda congeners: S. idiastes (14-22°C) S. lucasana (16-28°C)

\.**.*'-

-

0.00 -5

..-•

...*. ..

Trematornus centronotus (-1 .g°C)

-

of denaturation. Often, critical thermal maxima relate to a breakdown in some critical physiological process. For example, most tissue functions in many ectotherms are handicapped by a decreased affinity of the respiratory pigment for oxygen at the upper limit of body temperature. At 5O0C, the blood of a chuckwalla (Sauromalus) cannot achieve more than 50% 0, saturation of arterial blood, which prevents vigorous activity by the animal. At slightly lower temperatures (47-48°C) that prevent higher arterial oxygen saturation levels, the desert iguana Dipsosaurus continues to be active. Above 43"C, the iguana pants, much as a dog does, to increase heat loss through evaporative cooling. Most ectotherms never experience such extreme temperatures. Yet, even in temperate climates, many experience general environmental temperatures that are high enough to require active responses to prevent unacceptably elevated body temperatures. Many ectotherms expose their bodies to sun or to shade to absorb more or less heat, respectively, from the environment. The effectiveness of such behavioral thermoregulation is enhanced by the high heat conductance of ectotherms. Certain reptiles, however, additionally use nonbehavioral (i.e., physiological) means to control the rates at which their bodies heat and cool. For example, the diving Galapagos marine iguana Amblyrhynchus (Figure 14-20) can permit its body temperature to rise at about twice the rate at which it drops by regulating both heart rate and flow of blood to its surface tissues. When the iguana wishes to warm up, it basks in the sun and simultaneously diverts cooler core blood to the surface (see Figure 16-20A).The net effect is a large difference between body and environmental temperature. The increased blood flow increases the skin's heat conductance and speeds absorption of heat into the animal. Increased pumping of blood accelerates the removal of heat from surface tissues to deeper tissues. During the iguana's prolonged feeding dives in the cool ocean, loss of body heat is slowed by a reduction in blood flow to the surface tissues and by a general

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Figure 16-19 Higher Kmsof pyruvate for A,lactate dehydrogenase have been selected for animals living in colder environments.This relation holds both between species (Trernatornus vs. Gillichthys) and within species (barracuda).[Adapted from Sornero,

1995.1

I 40

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Hlgher body temperature, r a p ~ dheartbeat, and vasodilation

Figure 16-20 The Galapagos marine iguana heats and cools at different rates, indicating an active regulation of heat exchange with its environments. (A) On land, the basking iguana absorbs heat from the sunk rays. Vasodilation of cutaneous blood vessels and rapid heartbeat (as recorded in the electrocardiogram, ECG) assure heating of the blood and efficient

c~rculat~on, whlch qu~cklydlstrlbutes the heat throughout the body Underwater heat loss 1s retarded by a slowed heartbeat and vasoconstrldlon In cutaneous blood vessels, both of whlch mlnlmlze the flow of blood to the skln

slowing of the circulation. This is apparent in experiments that demonstrate a hysteresis (asymmetrical response) of the heart rate relative to body temperature during a rise and fall in body temperature (Figure 16-21).Essential physical concepts underlying this tactic include not only the difference between the rate of heat convection and the rate of heat conduction, but also the differing heat capacities of air and water. Because of its far higher heat capacity, water can remove heat by conduction from the surface of the iguana much faster than air can; thus, it is especially important that circulation ~othe skin be slowed during diving. Similar dissimilarities between rates of heating and cooling, which indicate active regulatory processes, have been observed in amphibians and arthropods.

birds and mammals for theirs. Indeed, the endotherms and ectotherms do represent different life-styles, the former representing a fast, high-energy way of life and the latter representing a slower, low-energy approach. Many of the

Costs and Benefits of Ectothermy: A Comparison with Endothermy

Early comparative physiologists assumed that ectothermy was inferior to endothermy as a way of life. The endothermal vertebrates (primarily birds and mammals) were viewed as more complex recent evolutionary arrivals than the primarily ectothermal "lower vertebrates" (fishes, amphibians, and lizards). More recently, however, the term "lower vertebrates" has fallen out of favor as we realize that they are as highly adapted for their way of life as are

90

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o Heating

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Body temperature ( " C ) Figure 16-21 A hysteresis In the relatlon of heart rate to body temperature durlng heatlng, and then coollng, In water can be demonstratedIn the marlne Iguana The heart rate rlses steeply durlng heatlng but drops back st111more steeply w~thcoollng [From Bartholomew and Laslewskl, 1965 ]

anatomical and functional properties of ectothermic vertebrates are adaptations facilitating a life of modest energy requirements. Those modest requirements allow some reptiles, amphibians, and fishes, for example, to exploit terrestrial niches unavailable to birds and mammals. Small salamanders with low metabolic rates live in great abundance in the cool litter on forest floors of New England. The total biomass of such salamanders is estimated to exceed that of the forest's birds and mammals. Body size is often a critical factor in the advantages of ectothermy in certain environments. Because few ectotherms elevate their body temperatures above ambient temperatures, they do not experience the increased loss of body heat that occurs with decreasing body size (resulting from increased surfaceto-volume ratio). Thus, ectotherms can function with much smaller body masses than endotherms. Shrews and hummingbirds are unusually small endotherms, but many ectotherms, such as frogs and salamanders and most invertebrates, are much smaller. Thus, in considering the "costs and benefits" of ectothermy relative to endothermy in animals, the following generalizations can be made:

1. Because their body temperatures are generally closer to ambient temperatures, ectotherms generally live at a lower metabolic rate. As a consequence, ectotherms can "invest" a larger proportion of their "energy budget" in growth and reproduction. Ectotherms require less food, and so they can spend less time foraging and more time quietly avoiding predators. They also need less water, because they lose less by evaporation from their typically cooler surfaces, and they need not be massive for the purpose of reducing surface-to-volume ratio. 2. The benefits in item 1 are balanced by certain costs, among which is the inability of ectotherms to regulate their body temperatures (unless their environments permit behavioral thermoregulation). For example, a lizard can elevate its temperature by basking only if there is sufficient solar radiation, which limits the times of day and seasons of the year when such activity is possible. Other costs of ectothermy are that a low rate of aerobic metabolism limits the duration of bursts of high activity and an oxygen debt develops during anaerobic respiration. Such factors have been evoked to argue that large dinosaurs must have been endotherms. 3. The biological costs and benefits of being an endotherm are the inverse of those of being an ectotherm. Because of their high rates of aerobic respiration and their elevated temperatures, endotherms maintain elevated body temperatures and can generally sustain longer periods of intense activity. Thus, the endotherms can be thought of as "high-rolling big spenders" in energetic terms compared with the energetically more modest ectotherms, which are characterized by lower energy intake and lower energy expenditure. Another advantage of endothermy is that the con-

4.

stancy of body temperature allows enzymes to function more efficiently over a relatively narrow range. Endothermic animals can do certain things on a bigger, faster scale, but they do so at a price. The field metabolic rates (daily costs of survival in their natural habitats) of endotherms are as much as 17 times as high as the field metabolic rates of ectotherms. The price paid by the endotherms for their high metabolic rate includes the requirement that they take in much larger amounts of food and water daily. Thus, a 300 g rodent needs 17 times as much food per day as does a 300 g lizard living in the same habitat and having the same diet of insects. The high rate of respiratory gas exchange makes endotherms susceptible to dehydration in hot, dry climates. High body temperature relative to ambient temperature makes very small body mass problematic because of surface-to-volume considerations that cause a small animal to lose heat faster than a larger one. Because such a large quantity of energy is consumed by an endotherm to elevate and maintain body temperature, only a relatively small percentage of energy can be budgeted for growth and reproduction.

It is apparent, then, that ectothermy and endothermy constitute a metabolic dichotomy affecting far more than just body temperature. Indeed, the implications of these two types of energy economies also extend to such areas as activity, physiology, behavior, and evolution. Both ectothermy and endothermy have their respective advantages and disadvantages. Ectothermy is always mechanistically less complex than endothermy. Some thermoregulating terrestrial ectotherms are capable of regulating their body temperatures with precision and at levels as much as 30 Celsius degrees higher than the air temperature. Whereas endotherms typically maintain a relatively constant temperature set point, some thermoregulating ectotherms can vary the temperature set point, depending on activity requirements; such regulation allows body temperature to fall during periods of rest and rise prior to activity, as in the basking behavior of lizards. This has the advantage of fuel economy, very much like lowering and raising the thermostatic temperature setting In a home according to the temperature needs of its inhabitants. Endothermy and ectothermy also offer animals differing advantages in different climates. In the tropics, ectotherms such as reptiles compete successfullywith, or even outcompete, mammals both in the abundance of species and in numbers of individuals. This competitive success is thought to be due in part to (1)the warm, relatively even tropical climate, allowing reptiles to expand into nocturnal activity rhythms, whereas tropical mammals tend to be diurnal in habit; and (2)the greater energy economy enjoyed by the ectotherms, because they need not expend energy to elevate body temperature. The metabolic energy thus saved by tropical ectotherms can be diverted to reproduction and to other uses that promote species survival. In moderate


689

............................................................................... and cold climates, ectotherms are necessarily more sluggish, are thus less competent as predators, and are generally less abundant than mammals are in those climates. Endotherms have a significant competitive edge over ectotherms in the cold because their tissues are kept warm. Generally speaking, the farther from the equator, the higher the prevalence of terrestrial endothermy. In polar regions, for example, there are no reptiles and almost no insects, and only a few genera of amphibians and insects occupy subpolar arctic environments.

Resting

Warmup

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Time from onset of warmup (min) Figure 16-22 The sph~nxmoth Manduca sexta undergoes a pre-

flight thermogenesls Shlverlng of the thoraclc flight muscles causes a steep Increase In thorac~ctemperature prlor to flight [Adapted from Heinrich, 1974.1

TEMPERATURE RELATIONS OF HETEROTHERMS Heterotherms are animals intermediate between pure ectotherms and endotherms. As mentioned earlier in this chapter (in Temperature classificationsof animals), certain insects and fishes are heterotherms. Some flying insects, including locusts, beetles, cicadas, and arctic flies, can be considered both temporal and regional heterotherms because, when preparing to fly, they raise the core temperatures in their thoracic parts to more or less regulated levels. At moderate ambient temperatures, these insects are unable to take off and fly without prior warmup because their flight muscles contract too slowly to produce sufficient power for flight at temperatures much below 40°C. When inactive, however, these insects behave strictly as ectotherms. After such an insect is aloft, its flight muscles produce enough heat to maintain elevated muscle temperatures, and the insect even employs heat-dissipating mechanisms to prevent overheating. These flying insects generally have quite a large mass; and some, such as bumblebees, butterflies, and moths, are covered with heat-insulating "hairs" or scales. To warm up, these insects activate their large thoracic flight muscles, which are among the most metabolically active tissues known. The activation is such that antagonistic muscles work against one another, producing heat without much wing movement other than small, rapid vibrations akin to shivering. Flight is finally initiated when the thoracic temperature has reached the temperature that is maintained during flight, about 40°C (Figure 16-22). Like endotherms in general, endothermic flying insects face problems of regulating their body temperature when their environmentshave large temperature gradients. At am-

.

bient temperatures approaching O°C, convective heat loss is generally so rapid that flight temperatures cannot be maintained. High ambient temperatures, on the other hand, place the insect in danger of overheating. Thus, at ambient temperatures above 20°C, the hovering sphinx moth Manduca sexta prevents thoracic overheating by regulating the flow of warm blood to the abdomen. The flow of heat from the active thorax to the relatively inactive and poorly insulated abdomen increases the loss of heat to the environment through the body surface and especially through the tracheal system. An interesting and somewhat unusual example of true shivering thermogenesis in an insect is found in honeybee swarms, in which individual honeybees regulate the swarm's core temperature by muscle contraction in the form of shivering movements together with alterations in swarm structure. At low ambient temperatures (e.g., joC), the swarm packs more tightly, restricting the free flow of air into and out of the swarm to that needed for respiration. Through shivering activity, the core of the swarm can be maintained as high as 35°C. In warm weather, in contrast, the swarm loosens, providing ventilatory passages for air flow so that the core temperature exceeds the outside temperature by only a few degrees. Another example of muscle-generated heat in a heterothermal species is found in the brooding female Indian python as it elevates its body temperature with shivering thermogenesis so as to provide warmth for the group of eggs around which it coils itself. In the laboratory, the rate of occurrence of muscle contractions was found to increase with declining ambient temperature, and the increase in contractions was accompanied by an increased difference between the ambient and body temperatures.

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Unlike terrestrial ectothermic vertebrates, which can bask in the sun to warm up, marine ectotherms cannot obtain radiant energy as a source of underwater heat, owing to the high infrared absorption of water. As a result, fishes can rise above ambient temperature only through intensive metabolic activity. Many teleosts are strictly ectothermic, operating at core temperatures close to ambient temperatures. However, as already mentioned, some fishes, such as tunas, have specializations for generating and retaining sufficient heat to raise the temperature of body

A

muscle, brain, and eyes some 10 degrees or more above their surroundings. These fishes can therefore be classified as regional heterotherms. The large mass (and hence small surface-to-volume ratio) of some of these fishes helps them attain a relatively constant muscle temperature. In these fishes, the retention of heat in the body core depends crucially on the organization of the vascular system. Unlike ectothermic fishes, which have a centrally located aorta and postcardinal vein (Figure 16-23A), heterothermic fishes, (e.g., tunas and lamnid sharks such as the mako) have

Ectothermic

Dorsal

B

Heterothermic

cutaneous artery

Rete rnirab~le

Cutaneous vein

Figure 16-23 Differences In vascular anatomy of a typlcal coldbodled flsh and a warm-bodled f~sh,the bluefin tuna Tunnus thynnus, account for the ablllty of the bluefln tuna to show reglonal heterothermy The ectotherm~cflsh (A) has ~ t smajor blood vessels located centrally, whereas the heterothermlc flsh (B) has ~ t s major blood vessels located underthe skln and uses retla to conserve deep body heat by countercurrent exchange The advantage of the arrangement of vessels In the heterotherm 1s that no body heat 1s lost In warmlng arterial blood that 1s unavoidably cooled whlle passlng through the gills [Adapted from F G Carey, 1973 ]

major blood vessels (lateral cutaneous arteries and veins) located under the skin (Figure 16-23B). Blood is delivered to the deep red muscles through a rete mirabile that acts as a heat-exchange system (Figure 16-24). Arterial blood, which is rapidly and unavoidably cooled during passage through the extensively perfused respiratory tissues of the gills and through the surface vessels, passes from the cool periphery into the warm deeper muscle tissue through a rete of fine arteries that intermingle with small veins carrying warm blood away from the muscles. This constitutes a countercurrent heat-exchange system such that the cool arterial blood passing from the surface toward the core picks up heat from the warm

venous blood leaving the muscle tissue and passing toward the periphery. This permits the retention of heat in the deep red-muscle tissue and minimizes heat loss to the surroundings. Two anatomical features permit heterothermic fishes to maintain their swimming muscles at a temperature suitable for vigorous muscular activity while the temperature of the surface tissues approaches that of the surrounding water. First, the red (dark) aerobic swimming muscles are located relatively deep in the core of a fish's body. Second, escape to the periphery (skin, gills, etc.) of the heat produced during muscular activity is retarded by the countercurrent arrangement of the vessels composing the rete.

Water 19.3"

Y ..

C

35 -

/

.,-

-0 2 0)

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20-

E Figure 16-24 The bluef~n tuna controls reg~onalbody temperature w ~ t h a countercurrent arter~al-venousheat-exchange rete The rete (shown In color) helps the tuna retaln heat produced In act~vedeep muscles (A) Enlargement of the rete area (B) Isotherms (left), plotted at 2 Cels~usdegree Intervals, show temperature d~str~but~on In cross sectlon [rrght) (C)Maxlmum muscle temperatures of bluef~nscaught In waters of d~fferenttemperatures The dashed Ihne nd~catesequalty between body temperatures and water temperatures [From Carey and Teal, 1966.1

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Another important factor is that these regional heterotherms swim continuously, so the red muscle never cools down to the ambient temperature. One of the implications of regional heterothern~yis the energy savings in the cool tissues while the temperatures of only certain tissues, such as the swimming muscles, are being elevated.

TEMPERATURE RELATIONS OF ENDOTHERMS In homeothermic endotherms (most mammals and birds), body temperature is closely regulated by homeostatic mechanisms that regulate rates of heat production and heat loss so as to maintain a relatively constant body temperature independent of environmental temperatures. As mentioned earlier, core temperatures are maintained nearly constant between 37°C and 38°C in mammals and about 40°C in birds. The temperatures of peripheral tissues and extremities are held less constant and are sometimes allowed to approach environmental temperatures. Basal heat production for different homeotherms of a given size is about the same, and the basal metabolic rate can be 10 times as high as the standard metabolic rate of ectotherms of comparable size measured at similar body temperatures. This elevated basal metabolism, in conjunction with heatconserving and heat-dissipating mechanisms, allows homeotherms to maintain constant body temperatures as much as 30 Celsius degrees or more above ambient temperatures.

man beings are a vestige of the pilomotor control of a longlost pelage. As the ambient temperature decreases, an endotherm will eventually reach its lower critical temperature (LCT; see Figure 16-25),below which the basal metabolic rate becomes insufficient to balance heat loss despite these manifold adjustments in thermal conductance. Below this temperature, an endotherm must increase heat production above basal levels to offset heat loss (i.e., by thermogenesis, described in the next subsection). Heat production then rises linearly with decreasing temperature below the lower critical temperature, in what is termed the zone of metabolzc regulation (see Figure 16-25). If the environmental temperature drops below the zone of metabolic regulation, compensating mechanisms fail, the body cools, and the metabolic rate drops. Many animals tolerate varying degrees of hypothermia during their normal rest period (including human beings during sleep). However, if an animal's body temperature falls below its normal values, the animal enters a state of hypothermia (see Figure 16-25).If this condition persists, the animal grows progressively cooler and, because cooling only further lowers the metabolic rate, the animal soon dies. The thermal neutral zone lies entirely below the normal body temperature, Tb(37-40°C), as shown in Figure 16-25. Thermal neutral zone

Mechanisms for Body Temperature Regulation

Endotherms use a wide variety of both physiological and behavioral mechanisms to maintain body temperature within a narrow range. Before these mechanisms are considered, however, the concept of thermal neutral zone must be introduced. Thermal neutral zone The degree of thermoregulatory activity that homeotherms require to maintain a constant core temperature increases with increasing extremes of environmental temperature. At moderate temperatures, the basal rate of heat production balances heat loss to the environment. Within this range of temperatures, termed the thermal neutral zone (Figure 16-25), an endotherm does not need to expend energy to maintain its body temperature; it can regulate its body temperature by adjusting the rate of heat loss through alterations in the thermal conductance of the body surface. These adjustments include vasomotor responses (see Figures 16-14 and 16-15), postural changes to alter exposed areas of surface, and regulation of the insulating effectiveness of the pelage by raising or lowering hairs or feathers. Thus, within this range, fur or feathers are fluffed by pilomotor muscles in the skin to provide a thicker layer of stagnant air; at the upper end of this range, fur or feathers are held closer to the skin. The "goose bumps" of hu-

LCT

UCT

Ambient temperature, T, ("C) Figure 16-25 The resting metabolic rate of an endothermic homeotherm (red plot) is higher at extremes of ambient temperature. The thermal neutral zone extends from the lower (LCT) to the upper critical temperature (UCT). Above and below this range, the metabolic rate must rise to either increase thermogenesis in the zone of metabolic regulation or increase active dissipation of heat by evaporative cooling if body temperature, Tb(blackplot) is to remain essentially constant.Within the thermal neutral zone, body temperature is regulated entirely by changing the heat conductance of the body surface, which requires essentially no change in metabolic effort. At ambient temperatures below the LCT, thermogenesis is unable to replace body heat at the rate at which it is lost to the environment, and hypothermia sets in. At ambient temperatures above the UCT, heat production and gain exceed the rate of heat loss, and hyperthermia occurs.


693

.............................................................................. To see why, consider that heat loss by passive mechanisms cannot be increased further beyond the upper crztzcal temperature, because the surface insulation is minimal (i.e., it cannot be made lower) at that temperature. Any further increase in ambient temperature, Ta, above that temperature will therefore cause a rise in body temperature, unless active heat-dissipating mechanisms such as sweating or panting are brought into play. Without evaporative heat loss, temperatures above the thermal neutral zone lead to hyperthermia, because the heat produced by basal metabolism cannot escape passively from the body as fast as it is being produced. Regardless of ambient temperature, a living animal is continuously producing some heat and, unless this heat is dissipated, the body temperature must continue to rise. (Sauna and hot-tub enthusiasts should bear this in mind.) Why does the metabolic rate rise linearly with temperature below the lower critical temperature, along a line that extrapolates to zero at an ambient temperature equal to body temperature, as shown in Figure 16-25? This is explained by considering Fourier's law of heat flow:

in which Q is the rate of heat loss from the body (in calories per minute) and C is the thermal conductance (see Spotlight 16-2). Because Tb is constant, Q varies linearly with the ambient temperature. The thermal conductance determines the slope of the plot below the neutral zone; the better the insulation (i.e., the lower Cis), the shallower the slope and the less heat must be produced metabolically at low temperatures. The extrapolated intercept with zero is at Tb because, if Ta = Tb, C(Tb- Ta)= 0. With Q = 0, there is no net heat loss. We know that the metabolic rate does not normally drop below the basal metabolic rate. When Ta = Tb, body temperature must be above the neutral zone because there

is no gradient for heat loss, so the animal will tend to warm up. The animal must cool by some means other than heat conduction. The only means of cooling when Ta lies above the upper critical temperature is by evaporation. Thermogenesis When the ambient temperature drops below the lower critical temperature, an endothermic animal responds by generating large amounts of additional heat from energy stores, thereby preventing a decrease in the core temperature. There are two primary means of extra heat production other than exercise: shivering and nonshivering thermogenesis. Both processes convert chemical energy into heat by a normal energy-converting metabolic mechanism that is adapted to primarily produce heat. Essentially all the chemical-bond energy released in this process is fully degraded to heat rather than to chemical or mechanical work. Shivering is a means of using muscle contraction to liberate heat. Shivering thermogenesis occurs in some insects as well as in endothermic vertebrates. The nervous system activates groups of antagonistic skeletal muscles so that there is little net muscle movement other than shivering. The activation of muscle causes ATP to be hydrolyzed to provide energy for contraction. Because the muscle contractions are inefficiently timed and mutually opposed, they produce no useful physical work, but the chemical energy released during contraction appears as heat. In nonshivering thermogenesis, enzyme systems for the metabolism of fats are activated throughout the body, so conventional fats are broken down and oxidized to produce heat. Very little of the energy released is conserved in the form of newly synthesized ATP. A specialization found in a few mammals for fat-fueled thermogenesis is brown fat, also called brown adipose tissue (BAT). Generally found as small deposits in the neck and between the shoulders (Figure 16-26),brown fat is an adaptation for rapid, Blood flow in

Figure 16-26 Brown-fat deposits are found between the scapulae in bats and many other mammals. The detail shows the special vasculariza-

tion of this tissue. During brown-fat oxidation, this tissue is detectable as a warm region by its infrared emission.

massive heat production. This fat contains such extensive vascularization and so many mitochondria that it is brown (owing largely to mitochondrial cytochrome oxidase) rather than white. In brown fat, oxidation takes place within the fat cells themselves, which are richly endowed with fat-metabolizing enzyme systems. In ordinary body fat, deposits must first be reduced to fatty acids, which enter the circulation and eventually are taken up by other tissues, where they are oxidized. Nonshvering thermogenesisin fat (includingbrown fat) is activated by the sympathetic nervous system through the release of norepinephrine, which binds to receptors on the adipose cells of brown-fat tissue. Through a second-messenger mechanism, described in Chapter 9, this signal leads to thermogenesis by two mechanisms. In the first of these mechanisms, normal ATP utilization for cellular processes rises in these fat cells in response to the sympathetic signal, accounting for part of the increased heat production. Through processes such as ion pumping by the plasma membrane, ATP is hydrolyzed to produce work and heat. In the second mechanism, ATP production is uncoupled during respiratory chain oxidation. The resynthesis of ATP from ADP and Pi is normally coupled to the movement of protons (H+)down their electrochemical gradient from intermembrane space into mitochondria across the inner mitochondrial membrane. Thermogenesisin brown fat is characterized by the appearance in the inner mitochondrial membrane of uncoupling proteins, which provide a pathway for protons to leak across this membrane without the energy of their downhill movement being harnessed for the phosphorylation of ADP to ATP. Once inside the mitochondrion, the protons oxidize substrate oxygen to produce water and heat, or else further utilization of metabolic energy is required to subsequently pump them into the intermembrane space and eventually out of the mitochondria.

Brown fat heats up significantly during thermogenesis. This newly produced heat is rapidly dispersed to other parts of the body by blood flowing through the extensive vasculature of the brown fat tissue. Nonshivering thermogenesis is especially pronounced during arousal of hibernating or torpid mammals, when it supplements shivering to facilitate rapid warming. One consequence of acclimation to cold by mammals is an increase in brown fat deposits, which allows for a gradual changeover from shivering to nonshivering thermogenesis at low ambient temperatures. The acclimatory increase in brown-fat thermogenesis is mediated by the thyroid hormones. Brown fat is also present in some mammalian infants, includinghuman infants, where it is generally located in the region of the neck and shoulders, the spine, and the chest. Because an infant is relatively small and inactive at birth, deposits of brown fat provide an important and rapid means of warming if the infant is threatened with temperature reduction. Another example of tissues specialized for heat production are the heater tissues formed from modified eye muscles in billfishes. B. A. Block and her colleagues (1994)have extensively investigated these tissues, which have an enormous capacity to generate heat (as high as 250 W . kg-'). Heater cells, which lack myofibrils and sarcomeres, produce heat through the release of Ca2+ from internal cytoplasmic stores. The released Ca2+ ions then stimulate catabolic processes and mitochondrial respiration (Figure 16-27).

Figure 16-27 The metabolism of heater tissues in billfish is specialized for heat production. This model for nonshivering thermogenesis in the skeletal muscle of billfish heater tissue shows how exogenously stimulated release of Ca2+into the cytoplasm stimulates mitochondrial respiration and the associated release of heat. After stimulation, a T-tubule re-

ceptor activates a sarcoplasmic reticulum (SR) Ca2+channel, releasing Ca2+stored in the sarcoplasmic reticulum. The increased cytoplasmic Ca2+ions then triggerthe release of energy in the form of heat from ATP previously manufactured in the mitochondria. [Adapted from Block, 1994.1

Endothermy in cold environments Endotherms adapted to cold environments have necessarily evolved a number of mechanisms, both temporary and permanent, that help them retain body heat. For example, an animal sensing heat loss in a windy place will fluff its fur or feathers and move to a more sheltered area. This reduces

U S I N G ENE

....................................... convection and the dissipation of body heat by the wind. More enduring responses to cold include the thick layers of insulation in many arctic animals, in the form of subcutaneous fat or a thicker pelage or plumage. The insulating effectiveness of pelages in arctic and subarctic animals changes with both season and latitude to match insulation qualities with insulation needs. In addition, animals living in the temperate zone exhibit seasonal variations by shedding old fur or feathers and growing new bodily cover, thereby providing thick insulation during the winter, yet preventing overheating during the summer. The specific conductances of homeotherms vary over a large range and decrease with body size (Figure 16-28). Larger animals have lower specific heat conductances owing to their generally thicker coats of fur or feathers. In addition, they face smaller heat loss in cold climates because of their relatively smaller surface areas. Thus, one adaptation of endotherms to cold latitudes is an increase in body size. As the surface-to-volme ratio becomes smaller, pelage becomes thicker and conductance decreases. With increased insulation, the lower critical temperature of a homeotherm decreases and the thermal neutral zone extends to lower temperatures (Figure 16-29). An exception is that small animals and immature animals often have feathers or fur that is less conductive per unit thickness, as evident in the fluffy feathers of young chicks, for example. Blubber, a fatty tissue typically found under the skin in cetaceans, is a good insulator because, like air, blubber has a lower thermal conductivity than does water, which is the main constituent of nonfatty tissues. In addition, fatty tissues are metabolically very inactive and require little perfusion by blood, which would ordinarily carry heat to be lost at the body surface. In whales, the outermost regions of the thick blubber layer are always at a temperature near that of the surrounding water. An important means of controlling heat loss from the surface is the diversion of blood flow to or away from the skin (see Figure 16-14).Vasoconstriction of arterioles leading to the skin keeps warm blood from perfusing cold skin and conserves the heat of the body core. An interesting advantage of blubber over pelage in the control of heat loss is illustrated in Figure 16-15B, which reminds us that fur is located outside the body proper whereas blubber is contained

Body mass (g) Figure 16-28 Thermal conductance decreases exponentially as body mass Increases.

Ambient temperature ("C)

1,

Figure 16-29 The decline in metabolic rate in endotherms as ambienttemperaturesfall depends on the extent of the animal's insulation.Adecrease in insulation (i.e., an increase in conductance) raises the lower critical temperature and makes the slope of increasing metabolism steeper. The slope, however, still extrapolates to body temperature at zero metabolic rate.

within the body and is supplied with blood vessels. Thus, whereas the insulating properties of fur remain unaffected by circulatory adjustments to the area, the insulating properties of blubber depend on whether blood flow to the surface is restricted or not. Hence, the more blood flow is diverted away from the vessels within the blubber, the higher the effective thickness of the insulating layer. Conversely, the greater the blood flow into the blubber, the lower the effective thickness of the insulating layer. This ability to regulate heat transfer through blubber allows a marine mammal to facilitate the loss of excessive body heat by shunting its surface blood effectively to the outer regions of the insulating layer of blubber during periods of intense activity in warmer waters or when lying on land in warm air. Countercurrent heat exchange Effective locomotion requires that the limbs of endotherms not be mechanically hindered by a massive layer of insulation. The flukes and flippers of cetaceans and seals and the legs of wading birds, arctic wolves, caribou, and other coldweather homeotherms require blood to nourish cutaneous tissue and limb muscles used in locomotion. The well vascularized limbs are potential major avenues of body heat loss because they are thin and have large surface areas. Excessive heat loss from these appendages can be reduced drastically by countercurrent heat exchange. Countercurrent exchange systems have already been discussed in the context of oxygen and carbon dioxide exchange (Chapter 13). Arterial blood, originating in the animal's core, is warm. Conversely, the venous blood returning from peripheral tissues may be very cold. As blood flows from the core, it enters arteries in the limb that lie next to veins that carry blood returning from the extremity. As the arteries and veins pass each other, the warm arterial blood gives up heat to the returning venous blood and thus becomes successively cooler as it enters the extremity. By the time it reaches the periphery, the arterial blood is precooled to

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within a few degrees of the ambient temperature, and little heat is lost. Conversely, the returning venous blood is warmed by the arterial blood, so it is nearly at core temperature as it flows into the core. The advantage of such a system is that heat exchange with the environment is restricted without a reduction in blood flow and the oxygen and nutrients that the blood carries. A similar situation was described for heterothermic fishes (see Figures 16-23 and 16-24).Another highly evolved example of countercurrent heat exchange is found in the mirabile of the flipper of the porpoise. Here, the artery carrying warm blood flowing toward the extremity is completely encased in a circlet of veins carrying cold blood back from the extremity. Birds and arctic land mammals also use countercurrent exchange to minimize heat loss from their extremities in cold climates, and to some extent this mechanism is also present in the extremities of human beings. As a result, the extremities of cold-climate endotherms are maintained at temperatures that are far below the core temperature and often approach ambient temperature (Figure 16-30). The efficiency of the countercurrent heat exchangers can generally be regulated by vasomotor control in which blood flow is shunted past the heat exchanger network by parallel vessels. Endothermy in hot environments-dissipation of body heat In very hot, dry climates, large animals have the advantages of relatively low surface-to-mass ratios and large heat capacities. Camels, well known for their ability to tolerate heat, have not only a large body mass, but also a thick pelage that helps insulate them from external heat. Low surface-area-tovolume ratio and thick pelage retard the absorption of heat from the surroundings. Furthermore, because of its large mass and the high specific heat of tissue watel; the camel, as

Air =

well as other large mammals, can absorb relatively large quantities of heat for a given rise in body temperature. These features also result in a slow loss of heat during the cool hours of the night. Thus, the large mass acts as a heat buffer that, by reducing rates of both absorption and loss of heat, minimizes extreme temperature fluctuations. A dehydrated camel can also tolerate an elevation of its core temperature by several degrees, further increasing its heat-absorbing capacity. Large amounts of heat gradually accumulated during daytime hours are subsequently dissipated in the cool of the night. In preparation for the next onslaught of daytime heat, the dehydrated camel allows its core temperature to drop several degrees below normal during the night. As a consequence, the camel starts the day with a heat deficit, which allows it to absorb an equivalent amount of additional heat during the hot part of the day without reaching harmful temperatures. This practice, called limited heterothermy, allows the camel to tolerate the extreme daytime desert heat without using much water for evaporative cooling. Limited temporal heterothermy is also practiced by the antelope ground squirrel (Ammospermophilus leucurus), a diurnal desert mammal. Because of its small mass, the antelope ground squirrel cannot continuously gain heat for several hours in the hot sun, and its small surface-to-mass ratio would lead to rapid heating. Instead, this desert mammal exposes itself to high environmental temperatures for only about 8 minutes at a time. It then returns to its burrow, where its stored heat escapes into the cool underground air. By allowing its temperature to drop a bit below normal before returning to the hot desert floor, it is able to extend its stay a few minutes without lethal overheating. The temperature of the body surface is an important factor affecting heat loss to the environment, because it de-

- 16°C

Air =

b

Figure 16-30 Endotherms can be regionally heterothermlc Temperatures In the extremltles of arctlc birds and mammals are much lower than the core temperature of about 38°C [From I ~ l n g1966 , ]

-31°C

U S I N G ENERGY: M E E T I N G E N V I R O N M E N T A L C H A L L E N G E S

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............................................................................... termines the temperature gradient, Tb - T,, along which heat will flow. Heat can be lost by conduction, convection, and radiation (see Spotlight 16-2) as long as the ambient temperature is below the body surface temperature. The closer the surface temperature is to the core temperature in an endotherm, the higher the rate of heat loss through the surface to cooler surroundings. Heat is transferred from the core to the surface primarily by the circulation; the rate of heat loss to the environment is regulated by the flow of blood to surface vessels (see Figures 16-14 and 16-15). Endotherms thus use various "heat windows" to regulate the loss of body heat, opening or shutting them by regulating blood flow. These heat windows permit the loss of heat by radiation, conduction, and, in some cases, evaporative cooling. An example of such temperature-regulating windows can be seen in the thin, membranous, and lightly furred ears of rabbits, with their extensively interconnecting arterioles and venules. Another example is seen in the horns of various mammals; in goats and cattle, the horns are highly vascularized by a network of blood vessels that, under conditions of heat load, vasodilate and act as radiators of heat. Similarly, the legs and snout, having large surfaceto-volume ratios, are used as thermal windows for the dissipation of heat by regulation of the rate of blood flow through the arterioles serving the skin of the appendages. Some mammals living under conditions of intense solar radiation or high temperatures have certain areas of the body surface exceptionally lightly furred or even naked to facilitate heat loss by radiant, evaporative, or conductive means. Such areas generally include the axilla (armpit),groin, scrotum, and parts of the ventral surface. Some of these areas, such as the udder and scrotum, carry additional temperature sensors that are used to detect changes in air temperature with minimal interference from the core temperature. By this means, the animal can anticipate changing temperature loads and make the corresponding adjustments in advance. Variations in posture or body orientation also can affect rate of heat absorbance or loss. For example, the guanaco, a medium-sized camel-like inhabitant of the Andes, has very densely matted hair on its back and a lighter covering of fur on its head and neck and the outer sides of its legs. The inner sides of the upper thighs and the underside are nearly naked, acting as thermal windows covering nearly 20% of the body surface. By adjusting the posture ' and orientation of its body with respect to solar radiation and cooling breezes, the guanaco can adjust the degree to which its thermal windows are open or shut, permitting a fivefold change in thermal conductance. This posturally controlled flexibility in surface insulation permits a variability in heat transfer across the surface of endotherms that is independent of surface-to-mass ratio.

and birds and some mammals take available body water (saliva and urine) or standing water from the environment and spread it on various body surfaces, allowing it to evaporate at the expense of body heat. Animals with naturally moist skin, such as amphibians, may have a body temperature lower than ambient temperature because of evaporative cooling, though this is not an effect that has been selected for. Some vertebrates use sweating or panting to produce evaporative cooling. In sweating, found in some mammals, sweat glands in the skin actively extrude water through pores onto the surface of the skin (see Chapter 8).Sweating is under autonomic control. Although it is a mechanism for evaporative cooling, sweating can persist in the absence of evaporation when the relative humidity of air is very high. Water will continue to be secreted from sweat glands even if the humidity is too high for evaporation to keep up with the rate of sweating, leading not only to elevated body temperature, but also to elevated water (and salt) loss. In panting, mammals and birds use the respiratory system to lose heat by evaporative cooling (seealso Chapter 14 on osmoregulation in desert environments). Panting mammals breathe through the mouth instead of through the nose. Heat is carried away in exhalant air because the dimensions of the mouth are such that exhalant air retains the heat absorbed in the lungs. As noted earlier, the nasal passages and their vascularization are effective in many mammalian species in retaining both water and body heat. Mammals also hyperventilate to increase heat loss. A change in alveolar ventilation, however, will result in a change in blood PCo2and blood pH. This situation is avoided during panting by a disproportionate increase in dead-space ventilation (i.e., flow through the mouth and trachea) without an increase in ventilation of the alveolar respiratory surface (Figure 16-31). Breathing rate is increased,

-

Evaporative cooling The evaporation of 1g of water requires 2448 J (585 cal) of energy. Consequently,evaporation is the most effective means of removing excess body heat, assumingthat there is sufficient water available to "waste" in thls fashion. Certain reptiles

100

200

300

Total respiratory ventilation (L . min-') Figure 16-31 Panting induces a shift from alveolar ventilation t o alveolar and dead-space ventilation. As the total respiratory vent~lation(abscissa) increases in the panting ox, the dead-space vent~lation(flow through the mouth and trachea) increases steadily. The alveolar ventilation, however, does not increase until the total ventilation exceeds about 200L.min-'. In extreme panting, the respiratoryfrequency (f) decreases as tidal volume (V,) increases (figures at top o f graph). [Adapted from Hales, 1966.1

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but tidal volume is reduced. Overheated canines and birds pant by inhaling through the nose and exhaling through the mouth, exposing the tongue and other mouth structures to encourage further water evaporation and therefore heat loss (Figure 16-32).Panting produces a one-way air flow over the nonrespiratory surfaces of the nose, trachea, bronchi, and mouth, causing evaporation without causing stagnation of saturated air in these passages. The amount of respiratory work required in panting is lower than it might seem, because a panting animal causes its respiratory system to oscillate at its resonant frequency, thereby minimizing muscular effort. Panting is accompanied by increased secretions from the salivary glands of the nose, secretions that are under autonomic control. Most of the water that is not evaporated by panting is swallowed and conserved. Because evaporation from skin or respiratory epithelium is the most effective means of ridding the body of excess heat, there is a close link between water balance and temperature control in hot environments (see Chapter 14). In hot, dry, desertlike environments, animals can be faced with the choice of either overheating or desiccating. Dehydrated mammals conserve water by reducing evaporation caused by panting or sweating and thus allow the body temperature to rise. Having a small heat capacity, a small mammal exposed to the desert heat will, in the absence of thermoregulatory water, undergo a rise in temperature that is far more rapid and more threatening than it would be for

Nose

a larger animal. To survive, small mammals must either drink water or stay out of the heat. The reciprocity of water conservation and heat dissipation in a small desert animal can be illustrated by considering the water balance and temperature control in the kangaroo rat. To conserve water, this animal uses a temporal countercurrent heat-exchange system in which the nasal epithelium is cooled during inspiration by the inhalant air. During exhalation, most of the moisture picked up by the air in the warm, humid respiratory passages is conserved by its condensation on the cool nasal epithelium. However, this mechanism also recycles body heat and requires that the inhaled air be cooler than the body core. As a consequence, the kangaroo rat is confined to its cool burrow during the hot times of day. If inhaled air were at or above body temperature, the kangaroo rat's loss of respiratory moisture would increase. Although the evaporative loss of water would help cool the animal, it would also seriously alter its water balance. The importance of water in the control of body temperature in a large desert mammal has been long known, as illustrated by simple and now classic observations made of camels. The camels were either allowed to drink freely or were subjected to periods of dehydration during which water was withheld for several days. Rectal temperatures were highest in daytime and lowest at nighttime. These fluctuations were minimal when the camels were allowed to drink but still large in comparison with a water-drinking human being. The temperature swings became even more exaggerated during periods of dehydration, when reserves of body water dwindled, leaving less for heat storage and for thermoregulation by sweating. Thermostatic Regulation of Body Temperature

Mouth

--

lnspiration

Expiration

Figure 16-32 The pathway of respiratorygas flow varies with the extent of panting in the dog. (Top) Air flow through the nose of a panting dog. Horizontal lines extending to the left of the vertical midline indicate inspiration; to the right, expiration. Mean inhaled and exhaled volumes are indicated by vectors placed adjacent to the dog's nose. (6ottom)Air flow through the mouth of a panting dog. lnspirationthrough the mouth isvirtually zero; expiration through the mouth carries most of the air taken in through the nose. [From Schmidt-N~elsenet al., 1970.1

Homeothermic endotherms use a system of body temperature control that is similar to the mechanized thermostatic control found in a laboratory temperature bath (see Figure 1-4) or a home heating system. In the water bath, a temperature comparator compares the water temperature, Tw,detected by a temperature sensor with a set-point temperature, T,,, . If Twis below T,,,, the thermostat closes the circuit that activates the production of additional heat until Tw = Tset,after which the thermostat contacts open and heat production ceases. The cycle is repeated as Twdrops again. This analogy is especially apt in the zone of metabolic regulation (see Figure 16-25),in which heat production increases with decreasing ambient temperature. Both homeothermic endotherms and homeothermic ectotherms also use nonmetabolic means to regulate body temperature. The regulation of body temperature, Tb, is not fully understood even after decades of research on the topic but appears to work along principles of negative feedback (see Spotlight 1-1). Most animals have not one but many temperature sensors in various regions of the body. Furthermore, to maintain Tb at about T,,,, homeothermic animals can call on several heat-producing and heat-exchanging mechanisms, so the thermostat controls heat-

The hypothalamus-the mammal's "thermostat" Mammalian body temperature can vary widely (as much as 30 Celsius degrees) between the periphery and the body core, with the extremities undergoing far more variation than the core. Temperature-sensitive neurons and nerve

endings exist in the brain, the spinal cord, the skin, and sites in the body core, providing input to thermostatic centers in the brain. Although a mammal may have several thermoregulator~centers, the most important one, considered to be the body's "thermostat," is located in the hypothalamus (see Figure 9-5). It was discovered by Henry G. Barbour in 1912 in the course of a series of experiments in which a small temperature-controlledprobe was implanted in different parts of the rabbit brain. The probe evoked strong thermoresponses only when it was used to heat or cool the hypothalamus. Cooling the hypothalamus produced an increase in metabolic rate and a rise in Tb, whereas heating it evoked panting and a drop in Tb. This experiment is analogous to changing the temperature of a thermostat in your home by holding a lighted match nearby. As the thermostat is warmed above its set-point temperature, it shuts down the furnace, allowing the room temperature to drop below the set point. An apparatus for controlling hypothalamic temperature and measuring a homeotherm's response to changes in that temperature is shown in Figure 16-33.

Figure 16-33 Temperature regulation by the hypothalamus can be measured by experimentally changing hypothalamic temperature. The apparatus measures the temperature sensitivity of the hypothalamus and thermoregulatory response to changes in hypothalamic temperature, which is altered by means of a water-perfused thermode implanted in the

hypothalamus.Metabolic rate and evaporative water loss are measured by analyzing the effluent air for water, O, and CO, content. The metabolic chamber is at constanttemperature [From "The Thermostat of Vertebrate Animals" by H. C. Heller, L. I. Crawshaw, and H. T. Hammel. Copyright 0 1978 by Scientific American, Inc. All rights resewed.]

conserving and heat-loss mechanisms as well as heat production. This means of control is analogous to the microprocessor-controlled heating and cooling system of the futuristic "smart house" in which the thermostat, in addition to cycling the furnace and air conditioner, controls the position of window shades, window opening and closing, the conductance of the wall and roof insulation, and so forth. Furthermore, control of thermogenesis in the homeotherm is not all-or-none, like the turning on and off of a furnace. Instead, the rate of heat production by metabolic means is graded according to need. The colder the temperature sensors become (within limits), the higher the rate of therrnogenesis. Engineers call this proportional control, because heat production and conservation are more or less proportional to the difference of Tb - Ts,.

Experimental procedures like Barbour's have shown that the mammalian hypothalamic thermostat is highly sensitive to temperature. Variation of mammalian brain temperature of only a few Celsius degrees seriously affects the functioning of the brain, so it is not surprising to find the major thermoregulatory center of mammals located there. Neurons that are highly temperature sensitive are located in the anterior part of the hypothalamic thermostat. Some of these neurons show a sharply defined increase in firing frequency with increased hypothalamic temperature (Figure 16-34).These neurons are believed to activate heatdissipating responses such as vasodilation and sweating. Others show a decrease in firing frequency with increase in temperature above a certain value. Still other neurons increase their firing frequency when the brain temperature drops below the set-point temperature. They appear to control the activation of heat-producing responses (e.g., shivering, nonshivering thermogenesis, brown-fat metabolism) and heat-conserving (e.g., pilomotor) responses. In addition to the information about its own temperature generated by these thermosensitiveneurons, the hypothalamus receives neural input from thermoreceptors in other parts of the body. All this thermal information is integrated and used to control the output of the thermostat. Neural pathways leaving the hypothalamus make connections with other parts of the nervous system that regulate heat production and heat loss. Some of these pathways are activated by high temperatures signaled from peripheral and spinal thermoreceptors and by the hypothalamic temperature-sensitiveneurons. The efferent pathways activate increased sweating and panting, as well as a lowered peripheral vasomotor tone,that produces increased blood flow to the skin. Conversely, body cooling leads to thermogenesis and increased peripheral vasomotor tone. These same responses can be elicited without cooling the whole body by simply cooling the neurons of the hypothalamus.

Thus, experimentally lowering hypothalamic temperature in a dog leads to elevated metabolic heat production by shivering. On the other hand, warming the dog's hypothalamus elicits the heat-dissipating response of panting. A rise in core temperature of only 0.5 Celsius degrees in most mammals causes such extreme peripheral vasodilation that the blood flow to the skin can increase several times above normal. In human beings, this response produces a flushed appearance to the skin. The effect of elevated core temperature on peripheral vasodilation and, hence, skin temperature is illustrated in Figure 16-35, which shows that the skin temperature of a rabbit's ear rose very sharply from less than 15°C to about 35°C at the point at which the rabbit's core temperature exceeded 39.4"C. Because the temperature of the ear reached a maximum, it can be assumedthat the vessels of the ear dilated fully as soon as the core temperature exceeded this limit. The effect of the hypothalamic thermostat on such peripheral heat-exchange mechanisms is about 20 times as great in some mammals as reflexive adjustments initiated by peripheral temperature sensors. This hypothalamic "override" is significant in light of the importance of carefully regulated brain temperature. Without dominance of the hypothalamic thermostat, an internally overheated animal exercising in a cold environment would fail to activate heat-dissipating blood flow to the surface capillaries, and its core temperature would continue to rise to dangerous levels. In some homeotherms, especially small animals subject to rapid cooling at low ambient temperatures, the set-point temperature of the hypothalamic thermostat changes with ambient temperature, presumably because ambient deviations are sensed by peripheral receptors. Thus, in the kangaroo rat, a sudden drop in ambient temperature is quickly followed by a rise in set-point temperature. This rise causes an increase in metabolic heat production in anticipation of increased heat loss to the environment.

Core temperature (OC)

Neuron temperature ("C) Figure 16-34 Different hypothalamic neurons show different temperature-activity patterns in a rabbit. Neuron 1 exhibits a linear decrease at a temperature above 38.4"C, whereas neuron 2 shows a steep linear increase at a temperature above 38.7"C. [Adapted from Hellon, 1967.1

Figure 16-35 Ear heat loss rises suddenly as core temperature increases in a rabbit at an ambient temperature of 10°C. The core temperature was raised by forcing the rabbits to run on a treadmill. As temperature rose to above 39.5"C, blood flow t o the ears increased, raising ear temperature and heat loss (given in watts). [From Kluger, 1979.1

The relations between thermoregulatory responses controlled by the hypothalamic centers and those controlled by the core temperature are illustrated in Figure 16-36. Small deviations in core temperature from the set point produce only peripheral vasomotor and pilomotor responses (black plot) that in effect alter the thermal conductance of the body. These small deviations in core temperature usually result from moderate variations within a range of ambient temperatures corresponding to the thermal neutral zone (see Figure 16-25).When the core temperature is forced out of this range by more extreme ambient-temperature deviations or by exercise, passive thermoregulatory responses no longer suffice, and the hypothalamic centers institute active measures-that is, thermogenesis or evaporative heat loss (red plots in Figure 16-36). Nonmammalian thermoregulatory centers Thermostatic control of body temperature has received less attention in birds than in mammals, perhaps because the manner of control seems to be more complex in birds. The region of the hypothalamus that serves as the thermoregulatory center in mammals is virtually insensitive to temperature changes in those birds tested (mainly pigeons). The spinal cord was found to be a site of central temperature sensing in pigeons, penguins, and ducks, but core receptors outside the central nervous system are the dominant temperature receptors in birds. The temperature sensors in the core presumably signal the avian hypothalamic thermostat, which in turn integrates the input and activates the thermoregulatory effectors. Fishes and reptiles, like birds and mammals, have a temperature-sensitivecenter in the hypothalamus. Heating the hypothalamus with an implanted thermode leads to hyperventilation in the scorpion fish; cooling leads to slower ventilatory movements. Peripheral cooling also produces similar ventilatory responses. Because the fish's rate of metabolism varies with body temperature, a rise in temperature leads to an increased need for oxygen. The temperature-determined adjustment in the rate of respiration is adaptive in that it anticipates changes in respiratory need and serves to minimize fluctuations in blood oxygen. The

Thermogenesis

t

reptilian response to cooling of the hypothalamus is to engage in thermophilic (i.e., heat-seeking) behavior, whereas heating of the hypothalamus elicits thermopbobic (i.e., heat-avoiding) behavior. Experiments by S. C. Wood and his colleagues (1991) have drawn some intriguing links between behavioral thermoregulation and hypoxic exposure. As an alternative to increasing convective 0, supply to tissues by increasing ventilation and cardiac output during hypoxic exposure, a wide variety of both vertebrates and invertebrates respond to hypoxia by reducing oxygen demand by temporarily selecting a lower preferred body temperature. Thus, vertebrates such as mice, toads, fishes, and lizards in an experimental thermal gradient will move toward a cooler region when made hypoxic (Figure 16-37). Invertebrates such as spiders and crayfish and even unicellular organisms such as Amoeba also respond in this way.

Fever An interesting feature of the hypothalamic thermoregulatory center is its sensitivity to certain chemicals collectively termed pyrogens (fever-producing substances). There are two general categories of pyrogens, based on their origins. Exogenous pyrogens are endotoxins produced by gramnegative bacteria. These heat-stable, high-molecular-weight g of purified polysaccharides are so potent that a mere endotoxin injected into a large mammal causes an elevation of body temperature. Endogenous pyrogens, on the other hand, arise from the animal's own tissues and, unlike those of bacterial origin, are heat-labile proteins. Leukocytes release endogenous pyrogens in response to circulating exogenous pyrogens produced by infectious bacteria. Thus, it appears that exogenous pyrogens cause a rise in body temperature indirectly by stimulating the release of endogenous pyrogens that act directly on the hypothalamic center. This idea is supported by evidence that the hypothalamus is more sensitive to direct application of endogenous pyrogens than to exogenous ones. The sensitivity of the hypothalamic temperaturesensing neurons to these pyrogenic molecules leads to an

Set-point temperature

vaporative heat (sweating or

Active processes

ing)

Intensity

response

Passive processes

0

Core temperature ("C)

Figure 16-36 The degree of an animal'sthermoregulatory response is greatest at body temperatures above or below normal core temperature. Within a range (light gray area) of the set-point temperature (dark gray area), regulation of body temperature is only through control of heat conductance to the environment by varying the peripheral blood flow or the insulating effectiveness of fur or feathers (black plot). Above and below this range, these passive measures are exhausted, and active thermogenesis (left) or evaporative heat loss (right)develops (red plots).

10

0 10 15 20 25

5

Oxygen partial pressure (mm Hg)

Inspired oxygen (%)

Figure 16-37 Hypoxla Induceschanges In selected body temperature In goldflsh (Carassfus auratus) and toads (Bufo mannus). [Adapted from Wood, 1991.]

elevation in the set point to a higher temperature than normal. The result is that the body temperature rises several degrees, and the animal experiences a fever. Anesthetics and opiates such as morphine, in contrast with pyrogens, cause a lowering of the set-point temperature and hence a drop in body temperature. The adaptive significance of endogenous pyrogens and of their production of fever in homeotherms may relate to the bacteriostatic effects of elevated body temperature. Pyrogenic bacteria elevate body temperature in some ectotherms as well as in endotherms. In a classic experiment by H. A. Bernheim and M. G. Kluger (1976), body temperatures were monitored in a desert iguana under

laboratory conditions simulating a desert environment before and after administration of pyrogenic bacteria (Figure 16-38).In response to the fever-producing bacteria, the lizards positioned themselves more frequently in radiantly heated zones of the artificial environment, effectively raising their temperatures to unusually high levels (i.e., fever). This behavioral response and the fever temperatures it produced conferred protection against bacterial infection. This protection is thought to take two forms: (1)the antiviral and antitumor agent interferon is more effective at higher temperatures, and (2) elevated temperatures also diminish the growth of some microbes.

Fever resulting

Figure 16-38 An ectotherm responds to Injedlons of pyrogenlc bacteriawlth a fever Llke other Ilzards, the desert Iguana, D~psosaurus dorsal~s,regulates ~tsbody temperature behavlorally by adjusting lts locatlon and posture wlth respect to radlant heat from the sun or hot objects such as dark rocks After ~nfect~on by pyrogenlc bacteria, the llzards ralsed thelr

44

42

-

Day 3

9 $?

a

40

from

body temperatures above normal levels by ~ n -

Day 2

increased bask~ng

creased basklng behavlor The graph shows the Increase In body temperature wlth successlve days after lnjectlon of the bacteria [Adapted from Bernhelm and Kluger, 19761

Day 1

bacteria injected

C

$Q

E

C

g

38

f)

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36

Purogenic 34

10

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Time of day (hour)

4

USING ENE RGY: M E E T I N G E N V I R O N M E N T A L CHALLENGES

703

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thevrnovegulation during activity The energy efficiency of muscle contraction is only about 25%. For every joule of chemical energy converted into mechanical work, 3 J of energy is degraded to heat. During exercise, this extra heat, added to the heat produced by basal metabolism, will cause a rise in body temperature above the set-point temperature unless it can be dissipated to the environment at the same rate at which it is produced. Most of the excess heat manages to be transferred to the environment, but a rise in core temperature of homeotherms does occur during exercise, indicating incomplete removal of the excess heat. The rise in temperature is moderately useful in two respects: (1)it increases the difference Tb - Taand thereby increases the effectiveness of the heatloss processes by increasing the gradient for heat loss, and (2)it leads to an increased rate of metabolic reactions, including those that support physical activity. However, the core temperature can rise to dangerously high levels during heavy exercise in warm environments, and this excess heat has to be dealt with. The level to which the core temperature rises in homeotherms is proportional to the rate of muscular work. During light or moderate exercise in cool environments, body temperature rises to a new level and is regulated at that level as long as the exercise continues. Thus, temperature appears to remain under the control of the body's thermostat. The rise in temperature proportional to the level of exercise appears to be a consequence primarily of an increase in the error signal, Tb - T,,,, of the thermostatic feedback control by the hypothalamus. The error signal is the difference between the thermostat's set point and the actual core temperature. The greater this difference (i.e., the greater the error signal), the greater the activation of heat-loss mechanisms. Thus, the rate of heat dissipation increases as the core temperature rises above the set point, and a new equilibrium becomes established between heat production and heat loss. During heavy exercise, especially in warm environments, the heat-dissipating mechanisms are not able to balance heat production until body temperature rises several degrees, increasing the T, - Ta difference. Thus, elevations of 4 to S Celsius degrees in core temperature are commonly observed in human beings after strenuous, sustained running and in race horses, greyhounds, and sled dogs after racing. The rise in Tb(and in the error signal as Tb rises above Ts,,) is kept small by the high sensitivity of the feedback control of heat-dissipating mechanisms. For example, a small increase in T,,above the set-point temperature produces a strong and steep increase in the rate of sweating (Figure 16-39).The effectiveness of heat loss is affected by humidity of the surrounding air-the higher the humidity, the less effective the heat loss (as any person familiar with either desert or high-humidity environments can testify). The heat-loss mechanisms are initiated by vigorous exercise even before peripheral body temperature has undergone any significant increase. For example, in human beings, an increased rate of sweating begins within 2 seconds after on-

Internal temperature ("C) Figure 16-39 Rate of sweating In humans increasessharply as body temperature approaches about 37°C. Core temperature was elevated by exercise or by elevating the ambient temperature. [Adapted from Benzinger, 1961.1

set of heavy physical work, even though there is no detectable increase in skin temperature in that time. However, core blood temperature shows a detectable rise in temperature within 1 second after exercise has begun. Apparently, the onset of sweating, nearly concurrent with the onset of neural activity underlying exercise, results from the reflex activation of sweating by central temperature receptors. The set points for heat loss are lower in well trained athletes, especially in warm weather. A special countercurrent heat exchanger to prevent overheating of the brain during such strenuous exercise as running is employed by certain groups of hoofed mammals (e.g., sheep, goats, and gazelles) and carnivores (e.g., cats and dogs). This system, the carotid rete (Figure 16-40),uses cool venous blood returning from respiratory passages to remove heat from hot arterial blood traveling toward the brain. In these animals, most of the blood to the brain flows through the external carotid artery. At the base of the skull, the carotid anastomoses into hundreds of small arteries that form a vascular rete, the vessels of which rejoin just before passage into the brain (see Figure 16-40). These arteries pass through a large sinus of venous blood, the cavernous sinus. This venous blood is significantly cooler than the arterial blood because it has come from the walls of the nasal passages, where it was cooled by respiratory air flow. Thus, the hot arterial blood flowing through the rete gives up some of its heat to the cooler venous blood before it enters the skull. As a result, brain temperature may be from 2 to 3 Celsius degrees lower than the core temperature of the

704


............................................................................... Figure 16-40 The sheep has a carotid rete for countercurrent cooling of carotid blood. The carotid rete, found in sheep and some other mammals, is shown in red. A network of small arteries acts as a heat exchanger for blood supplying the brain. Cool venous blood returning from the nasal cavity bathes the carotid rete contained in the cavernous sinus, removing heat from arterial blood flowing t o the circle of Willis and then t o the brain. [Adapted from Hayward and Baker, 1969.1

,, ,, /

Circle of

Bin

,Willis

,/

Cavernous

sinus \

/

,

Carotid artery

body. Although sustained running in hot surroundings inevitably places a heat load on these animals, the most serious and acute consequence of overheating-spastic brain function-is thereby prevented. This system of cooling is most effective when the animal is breathing hard during strenuous exercise.

DORMANCY SPECIALIZED METABOLIC STATES Dormancy is a general term for reduced body activities, including reduced metabolic rate. It often includes heterothermy. Dormancy can be variously classified according to its depth (in reference both to ability for arousal and to decrease in Tb)and its duration, and it includes sleep, torpor, hibernation, winter sleep, and estivation. Sleep has been the most thoroughly investigated (probably because it is the only state of dormancy experienced by people). The remaining four categories are less well understood than sleep; however, in homeotherms, all appear to be manifestations of physiologically related processes. Sleep Studied intensively in human beings and other mammals, sleep entails extensive adjustments in brain function. In mammals, slow wave sleep is associated with a drop in both hypothalamic temperature sensitivity and body temperature, as well as changes in respiratory and cardiovascular reflexes. During rapid-eye-movement (REM) sleep, hypothalamic temperature control is suspended. Although there may be a variety of triggers of sleep, in mammals there is evidence of sleep-inducing substances that build up during wakefulness, accumulating in extracellular fluids of the central nervous system. The identity and mode of action of these substances is under investigation. The time course and extent of sleep varies greatly among animals. Seals resting on pack ice sleep for only a few minutes at a time before

rousing to scan the ice for approaching polar bears. Human beings and many other mammals sleep for hours at a time. Many of the big carnivores (e.g., lions and tigers) sleep for as long as 20 hours a day, especially after a meal. Torpor The lower the Tb, the lower the basal metabolism and the lower the rate of conversion of energy stores, such as fatty tissues, into body heat. Thus, it is generally advantageous to allow body temperature to decrease during periods of nonfeeding and inactivity. Small endotherms, because of their high rates of metabolism, are subject to starvation during periods of inactivity when they are not feeding. During those periods, some animals enter a state of torpor, in which temperature and metabolic rate subside. Then, before the animal becomes active, its body temperature rises as a result of a burst of metabolic activity, especially through shivering or oxidation of brown-fat stores or both (if a mammal). Daily torpor is practiced by many terrestrial birds. The hummingbird is a classic example, allowing its body temperature to fall from a daytime level of about 40°C to a nighttime level as low as 13°C (in the rufous hummingbird when a low ambient temperature permits). Several species of small mammals also undergo torpor (e.g., shrews), but large mammals have too much thermal mass to cool down quickly for short periods of torpor. Hibernation and Winter Sleep

A period of deep torpor, or winter dormancy, hibernation lasts for weeks or even several months in cold climates. It is entered into through slow wave sleep and is devoid of rapid-eye-movement sleep. Hibernation is common in mammals of the orders Rodentia, Insectivora, and Chiroptera, which can store sufficient energy reserves to survive the periods of nonfeeding. Many hibernators arouse periodically (as often as once a week or as infrequently as every 4-6 weeks) to empty their bladders and defecate.

USING EN

....................................... During hibernation, the hypothalamic thermostat is reset to as low as 20 Celsius degrees or more below normal. At ambient temperatures between 5°C and 1S0C, many hibernators keep their temperatures as little as 1degree above ambient temperature. If the air temperature falls to dangerously low levels, the animal increases its metabolic rate to maintain a constant low Tbor becomes aroused. Thermoregulatory control is not suspended during torpor and hibernation-it merely continues with a lowered set point and reduced sensitivity (gain), as in slow wave sleep. In the hibernating marmot, for example, experimental cooling of the anterior hypothalamus with an electronically controlled, implanted probe increases metabolic production of body heat. The increase in heat production is proportional to the difference between the set-point temperature and the actual hypothalamic temperature. The set-point temperature drops about 2.5 Celsius degrees within a day or two as the animal enters a deeper state of hibernation. As might be expected, body functions are greatly slowed with the lowered body temperature characteristic of torpor and hibernation. The effect of reduced body temperature on the metabolic rate (expressed asvCo,) of golden-mantled ground squirrels is shown in Figure 16-41. In conjunction with a decrease in metabolism, blood flow in hibernating mammals is typically reduced to about 10% of prehibernation values, although the head and brown-fat tissue receive a much higher blood flow than do other tissues. The cardiac output decreases to only a small percentage of the normal rate. This retardation is accomplished by a drastic slowing of the rate of heartbeat, with stroke volume remaining essentially unchanged. As a result of reduced respiratory exchange, the blood of many hibernators becomes more acidic. This acidosis may further lower enzyme activity because of the departure from the pH optimum of metabolic enzymes. The rate of arousal from hibernation is often much higher than the rate of entry into hibernation. Thus, in the ground squirrel, the transition to the torpid state is completed within 12 to 18 hours (Figure 16-42), whereas

arousal requires less than 3 hours. The speed at which this midsized mammal arouses depends on rapid heating initiated by intensive oxidation of brown fat, accompanied by shivering. This frequently leads to a large surge in metabolic rate, as evident in Figure 16-42. Although many small endotherms undergo a daily cycle of torpor, their high rates of metabolism preclude extended periods of torpor in the form of hibernation because, even in the hibernating state, they would quickly consume stored energy reserves with little remaining for the metabolically expensive process of arousal. All true hibernators are midsized mammals weighing at least several hundred grams and large enough to store sufficient reserves for extended hibernation. There are no true hibernators among large mammals. Bears, which were once thought to hibernate, in fact simply enter a "winter sleep" in which body temperature drops only a few degrees, and they remain curled up in a protected microhabitat such as a cave or hollow log. With its large body mass and low rate of heat loss, a bear can store sufficient energy reserves to enter winter sleep without dropping body temperature. Bears are able to wake and become active quickly at any point during the winter, making it dangerous to encounter a bear even if it is in winter sleep. Typically, however, bears stay in winter sleep for long periods, retaining metabolic wastes in their bodies and even bearing their cubs. Winter sleep, with its relatively high body temperature, does not offer the same degree of energy savings as deep hibernation, but a fall in body temperature of even a few degrees saves energy. Why are there no large hibernators? First, they have less need to save fuel, because their normal basal metabolic rates are low relative to their fuel stores owing to the allometry of metabolism and fuel storage. Second, because of the large mass and relatively low rate of metabolism, a prolonged metabolic effort would be required to raise body temperature from a low level near ambient temperature to normal body temperature. It has been calculated, for example, that a large bear would require at least 24-48 hours to warm up to 37°C from a hibernating temperature of 5°C. Warming of such a large mass would also be energetically very expensive. Estivation

Temperature ("C) Figure 16-41 Both experimentally induced hypothermia and natural hibernation reduce metabolism in the golden-mantled squirrel. Open symbols, experimentally induced hypothermia; asterisk, unanesthetised animals awake and hibernating. [From Milsorn, 1992.1

The poorly defined term estivation, which has been called "summer sleep," refers to a dormancy that some species of both vertebrates and invertebrates enter in response to high ambient temperatures or danger of dehydration or both. Land snails such as Helix and Otala become dormant during long periods of low humidity after sealing the entrance to the shell by secreting a diaphragm-like operculum that retards loss of water by evaporation. Many land crabs similarly spend dry seasons in an inactive state at the bottom of their burrows. Well known as estivators are African lungfish, Protopterus. These air-breathingfish survive periods of drought in which their ponds dry up by estivating in the semidry bottom until the next rainy season floods the area. The lungfish seals itself inside a "cocoon," in which a

Hours

Weeks

Hours

Figure 16-42 Metabolism increases briefly during an episode of arousal from hibernation in a ground squirrel. The squirrel was kept in a chamber having a temperature of 4°C. The period of steady-state hibernation is shaded in color, and the body temperature, Tb,is graphed in red. Metabolism is graphed in black. At onset of hibernation, the set point

for body temperature is depressed. Metabolism decreases, allowing T, to drop to 1-3 Celsius degrees above T,throughout hibernation.Arousal occurs when the set-point temperature climbs to 38'C, and a strong surge of metabolic heat production raises Tb to the new set-point level. Abbreviation: RAMR, resting average metabolic rate. [From Swan, 1974.1

small tube leads from the fish's mouth to the exterior to allow ventilation of the lungs. Interestingly, chemical estivation-inducing factors in the plasma of estivating lungfish produce a torporlike state when injected into mammals. Some small mammals, such as the Columbian ground squirrel, spend the hot late summer inactive in their burrows, with their core temperatures approaching the ambient temperature. This state is probably similar physiologically to hibernation, but it differs in seasonal timing.

is usually expressed in units of kilocalories per kilogram per kilometer. This energy is taken as that expended above and beyond that which is expended under basal conditions of rest. Measurements of 0, consumption and CO, production associated with locomotion are generally made while the animal is running on a treadmill, swimming in a flow tank, or flying in a wind tunnel. The measured rate of gas exchange is then translated into rate of energy conversion. Relations between the net work done in the locomotion of an animal and the gross energy conversion powering the underlying muscle activity are complicated by several factors, not all of which are sufficiently well understood to be discussed here. Nonetheless, we know that a significant percentage of muscular effort during locomotion does not contribute directly to the production of forward motion. Some muscle contraction holds limb joints in their proper articulating positions. Another large percentage of muscle work is performed in an elingating muscle to counteract gravity, to absorb shocks, and to finely tune the movements of limbs during contraction of antagonists. The comparative energetics of animal locomotion are further complicated by the well established inverse relation between the force produced by a contracting muscle and its rate of shortening (i.e., muscle length or sarcomere length per second; see Figure 10-13).The higher the rate of cross-bridge cycling, the higher the metabolic cost of shortening the muscle by a given distance. Small animals show higher rates of limb stride, tail beat, or wing motion. Thus, small animals employ higher rates of muscle shortening (and hence crossbridge cycling) to achieve a given velocity of locomotion than do larger animals. For that.reason, they must convert correspondingly larger amounts of metabolic energy to

ENERGETICS OF LOCOMOTION Early in this chapter, we considered the basal rate of metabolism characteristicof the resting animal. Additional energy over and above the basal rate is expended when the animal is active-that is, producing movement with its muscles. The most readily quantified type of muscle activity in most animals is simple locomotion. Because it is required for finding food and mates and for escaping predators, locomotion is also one of the more important types of routine activity. We now turn to an examination of the metabolic cost of animal locomotion. Animal Size, Velocity, and Cost of Locomotion

The metabolic cost of locomotion is the amount of energy required to move a unit mass of animal a unit distance and

USING ENERGY: MEETING ENVIRONMENTAL CHALLENGES

707

............................................................................... produce a given amount of force per unit cross section of contractile tissue in moving their limbs. Several generalizations can be made in relating the overall energy cost of locomotion to the size and velocity of an animal. Locomotion is a metabolically expensive process. The rate of oxygen consumption in excess of the basal metabolic rate increases linearly with the velocity (Figure 16-43A). It is noteworthy, however, that the increase in energy utilization per unit weight for a given increment in speed is less for larger animals than for smaller ones. This can be seen in the different slopes of the plots in Figure 16-43A. When the cost of locomotion is plotted as energy utilization per gram of tissue per kilometer against body mass, it is again apparent that larger animals expend less energy to moue a given mass a given distance (Figure 16-43B).The lower energy efficiency of small animals during locomotion may, to a limited degree, be due to the greater drag that they experience (to be discussed shortly), but this explanation certainly does not suffice for A

Wh~temouse (21 g)

Kangaroo rat (41 g)

C

0 a

Kangaroo rat (1 00 g)

g=?:

8 ;m

-0.c 0"

-

a,

d

2-

R u n n ~ n speed g (km/ h)

Weight

terrestrial animals moving at low and moderate speeds through air, where drag is negligible. More likely, the lower energy efficiency is related to the lower efficiency of rapidly contracting muscle. The relation between velocity and the cost of locomotion is complex. As velocity of running increases in quadripedal mammals, for example, the metabolic cost of traveling a given distance initially decreases (Figure 16-44). This is because nonlocomotory expenses account for a progressively smaller fraction of the total energy expended. However, as velocity continues to increase, animals that swim, fly, or run all begin to experience an increase in the cost of locomotion as they generate near maximal locomotory velocities. Figure 16-45 illustrates this phenomenon in cephalopods (e.g., squids and nautilus), for which a typical U-shaped curve describes the cost of locomotion at various locomotory velocities. The cost of locomotion initially decreases sharply as velocity increases but then begins to rise at higher velocities. Figure 16-43 Metabol~crate durlng locomot~on depends both on body slze and on runnlng speed (A) Relat~onbetween rate of oxygen consumptlon and veloc~tyof runnlng In mammals of d~fferentslzes The slope of each plot represents the cost of transportlng a unlt mass over a unit d~stance(B) Log-log plot of metabol~ccost of transportlng 1 g a dlstance of 1 km In runnlng mammals of d~fferentslzes The cost of basal metabol~smwas subtracted before plottlng the values Data are from slopes of plots In part A Values for tetrapods Ile close to a stra~ghtllne [FromTaylor et a1 ,19701

708


.............................................................................. I Wh~temouse (21 g) 2 Kangaroo rat (41 g) 3 Kangaroo rat (I00 g) 4 White rat (384 g) 5 Ground squlrrel (236 g) 6 Dog (2 6 kg) 7 Dog (18 kg)

.

Speed (m s-')

2

4

6

8

10

Running velocity (km . h-') Figure 16-44 The energetic cost of transporting a unit of body mass by running decreases as body size increases in mammals. The cost of running 1 km drops and levels off with increasingvelocity. Dashed proportions are extrapolated. [From Taylor et al., 1970.1

Figure 16-46 Hopping, bipedal animals such as wallabys and kangaroos can increase velocity with no increase in oxygen consumption. Similarsized quadrapeds and wallabys initially show a linear increase in oxygen consumption as velocity increases. As wallabys switch t o bipedal locomotion, however, their rate of oxygen consumption does not increase with further increases in velocity. That blood lactate similarly stays level as velocity increases indicates that the higher speeds are not achieved by an increase in anaerobic metabolism. [Adapted from Baudinette, 1991.1

Physical Factors Affecting Locomotion

A noted exception to the U-shaped, cost-velocity relation typical of many running animals is found in hopping bipedal animals, especially kangaroos and wallabies. At slow velocities, oxygen consumption increases linearly in both a wallaby and a typical quadriped of similar size (Figure 16-46). At moderate to high velocities, however, wallabies steadily increase velocity without increasing oxygen consumption-a seemingly impossible achievement. They do so by using their powerful hind legs as springs; the hind legs store much of the kinetic energy expended in elevating the animal's body mass during leg extension.

Loligo (long-finned squid) 1

(short-finned squid)

Velocity (m s-') Figure 16-45 The metabolic cost of transport In cephalopods shows the "U" shape typlcal of many fly~ng,swlmmlng, and runnlng an~mals. Both very slow and very rap~dlocomot~onare relatively expenslve All cephalopods plotted we~ghapproximately 0 6 kg [From O'Dor and Webber, 1991.]

The metabolic cost of moving a given mass of animal tissue over a given distance also depends on the purely physical factors of inertia and drag. Inertia is the tendency of a mass to resist acceleration, whereas momentum refers to the tendency of a moving mass to sustain its velocity. These concepts are closely related, and effects due to both properties are often lumped under the term inertial effects. Every object possesses both inertia and momentum proportional to its mass. The larger the animal, the greater its inertia, and the greater its momentum when it is in motion. The high inertial forces that must be overcome during the acceleration of a large animal account for a significant utilization of energy during the period of acceleration (Figure 16-47A. Small animals, like small cars or small airplanes, require less energy to accelerate to a given velocity. Likewise, they need less energy to decelerate. Therefore, a small animal starts and stops abruptly at the beginning and end of a locomotory effort, whereas a large animal accelerates more slowly after locomotion begins and slows down more gradually as locomotion ends (Figure 16-47B). Similarly, in terrestrial animals, the limbs are engaged in backand-forth movements during running. The limbs are subject to inertial forces related to their mass as they accelerate and decelerate during locomotion. The limbs of a large animal exhibit greater inertia and momentum than do those of a small animal. Because animals do not move in a vacuum, the energetics of sustained locomotion are affected by the physical properties of the gas or liquid through which they move. Drag is the force exerted in the opposite direction of an animal's movement and is caused by the viscosity and density of the gas or liquid environment through which the animal

A

motion is similarly constrained by the environment in which it is employed and by the laws of physics.

Time

Time

Figure 16-47 Body mass affects both rate of energy expenditure and acceleration during locomotion. (A) Rate at which energy is used per unit mass during onset and maintenance of locomotion (shaded region) in a large and a small animal of similar type. (6)Velocity of a small and a large animal during acceleration and deceleration atthe beginning and end of a period of locomotion (shaded region).

h

moves. The drag produced in a given medium depends on the velocity, surface area, and shape of an object. For an object of a given shape, drag is proportional to the surface area. Because larger animals have lower surface-to-mass ratios, they experience less fluid drag per unit mass than do smaller animals, for whom overcoming drag is energetically more costly. Once it is under way, a larger animal expends less energy per unit mass to propel itself at a given velocity than does a smaller animal of similar type (see Figure 16-47A).Drag is also proportional to the square of an animal's velocity, meaning that the energy required to overcome drag and propel the animal at faster speeds increases with velocity. These effects are far more pronounced in water than in air because water, having the higher viscosity and density, produces far more drag on a moving object than air does. Drag is of major importance in swimming and flying because of the high viscosity of water faced by swimmers and the high velocity experienced by flyers. Drag is of little importance in running, because the velocities attained in running are low, as is the viscosity of air. These relations are quantified in the Reynolds number (see Spotlight 16-2). Aquatic, Aerial, and Terrestrial Locomotion Animals have evolved myriad ways of moving in water, on land, and in air. Despite this diversity, each mode of loco-

Swimming Animals that swim in water need to support little or none of their own weight. Many have flotation bladders or large amounts of body fat that enable them to suspend themselves at a given depth with little expenditure of energy. However, although the high dellsity of water allows them to be neutrally buoyant, it also produces high drag. This hindrance to objects moving through a fluid has led to a convergence of body forms among marine mammals and fishes. The streamlined, fusiform body shape is wonderfully developed in most sharks, teleost fishes, and dolphins. The reasons are evident enough on intuitive grounds, but they can be understood more clearly in relation to flow pattern. The ease with which an object moves through water depends in part on the flow pattern of the water. The fluid at the immediate surface of the object moves at the same velocity as the object, whereas the fluid at a great distance is undisturbed. If the transition in fluid velocity is smoothly continuous as the fluid progresses away from the object's surface, then laminar flow (Chapter 12) occurs at the boundary layer-the layer of unstirred fluid immediately adjacent to the object's surface (Figure 16-48A). In contrast, turbulent flow results when there are sharp gradients and inconsistencies in the velocity of flow. Because of conservation of energy, pressure and velocity are reciprocally related in a given fluid system, and the higher the velocity of fluid at a given site, the lower the pressure. Thus, strongly differing flow rates around an object cause eddy currents owing to secondary flow patterns set up between regions of high and regions of low pressure. Moreover, the higher the viscosity of the medium or the higher the relative motion between the object and the surrounding fluid, the greater the shear forces produced and, hence, the greater the tendency toward turbulence. Because its production dissipates energy as heat, turbulence retards the efficient conversion of metabolic energy into propulsive movement. Long, streamlined shapes promote laminar flow with minimal eddy current formation. Fishes and marine mammals such as seals, porpoises, and whales are admirably streamlined, exhibiting nearly turbulence-free passage through water even at high speeds. Flying birds are similarly streamlined in flight. An additional factor reducing turbulence in these animals is the compliance (deformability) of the body surface. A high body compliance damps small perturbations in the pressure of the water flowing over the body surface and thereby lessens the local variations in water pressure that give rise to energy-dissipating turbulence. The speed of a swimming animal is proportional to the power-to-drag (thrust-to-drag) ratio. Power developed by contracting muscle is directly proportional to muscle mass, and, if we assume that muscle mass increases in proportion to total body mass, power (thrust) rises in proportion to body mass. On the other hand, for a large swimming animal, total drag increases but drag per unit mass decreases,

Reynolds number Viscosity dominates

Both effects

Inertia

important

dominates

Figure 16-48 Velocity and fluid dynamics are highly dependent on body mass in both flying and swimming animals. (A) Fluid flow around a body moving through water. Movement through a fluid can create turbulence owing to uneven fluid pressures. Laminar flow occurs where pressure gradients are minimal.The larger the body and the less viscous the fluid, the higher the velocity before turbulence occurs. (B) Log of animal size plot-

ted against log of the respective Reynolds numbers (Res) at cruising velocities. Small animals that move slowly have small Res because viscous forces predominate at small dimensions. Larger animals move rapidly with high Res because inertial forces predominate at large dimensions. [Part B from Nachtigall, 1977.1

for a given velocity, as body mass increases. This is because, if the shape remains constant, the surface and cross-sectional areas (which determine the drag) increase as a function of some linear dimension squared, whereas body mass (which determines the power available) increases as a function of that linear dimension cubed. Thus, a large aquatic animal can develop power out of proportion to its drag forces; therefore, it is able to attain higher swimming velocities than thosi of a smaller animal of the same shape. Large fishes and mammals swim faster than their smaller counterparts. Because of the high drag forces developed in water and because drag increases as the square of velocity, aquatic animals can reach the speeds of a bird in flight only if they are much larger and more powerful than the bird.

to the relatively low drag forces developed, birds can achieve much higher speeds than those of fishes. The production of propulsive force, which drives the bird forward, and lift, which keeps it aloft, is accomplished simultaneously during the downstroke of the bird wing (Figure 16-49). The wing is driven downward and forward with an angle of attack that pushes the air both downward and backward, creating an upward and forward thrust. The components of lift and forward propulsion overcome the bird's weight and drag, respectively. The body shapes of fishes and birds demonstrate the great differences that exist between the physical properties of water and those of air, as well as the biological divergence that results from adaptation to these two dissimilar media. When a bird is gliding, its elongated, extended wings that take the form of an airfoil produce excellent lift; but, if the bird were in water, they would obviously generate far too much drag. Thus, the wings of penguins are modified to serve as short paddles and are folded against the body while the birds are coasting under water. Because drag forces are much higher in water than in air, only

Flying Unlike water, air offers little buoyant support, so all flyers must overcome gravity by utilizing principles of aerodynamic lift. Although the effects of drag increase with speed, there is still less need for streamlining among birds than among fishes because of the low density of air. Thus, thanks

N

Angle of attack

DL Local drag T, Local thrust

Figure 16-49 The downstroke of a bird wing develops forces in several directions. (Top) the wing in stage 3 of the flight cycle shown at the bottom. Red arrows illustrate forces relating to the wingbeat; black arrow illustrates force relating to the body. lnduced drag equals the drag produced as a consequence of lift production. Induced thrust is corn~lementary to induced drag. [From Nachtigall, 1977.1

medium-sized to large animals can coast in water. In contrast, only very small flying animals, smaller than a dragonfly, are unable to coast (glide) in air. Small insects such as flies and mosquitoes must continually beat their wings to maintain headway, because they have very little momentum. Running When swimming, flying, and running are compared with respect to the energy cost of moving a given body mass a given distance (Figure 16-50), it is apparent that terrestrial locomotion (i.e., running) is the most costly, whereas swimming is the least expensive. A swimming fish expends less energy in locomotion than a bird does in flying through the air because, as already noted, the fish is close to neutral buoyancy, whereas a bird must expend energy to stay aloft. But why is running less efficient than either flying or swimming? Running differs from swimming and flying in the way limb muscles are used, and this difference accounts for the low work efficiency of running. When a biped or quadruped animal runs, its center of mass (CM)rises and falls cyclically with the gait. The rise in the CM occurs when the foot and leg extensors push the body up and forward, and the fall occurs as gravity inexorably tugs at the body, bringing it back to earth between locomotory extensions. Effi-

ciency is lost because the antigravity extensor muscles that contract to propel the CM upward and forward must also break the fall of the CM that occurs before the next stride. To control the fall, the extensor muscles must expend energy to resist lengthening as they slow the rate of descent of the body in preparation for the next cycle. This technically unproductive use of muscle energy to counteract the pull of gravity is said to produce "negative work." You may be familiar with such negative work carried out by your legs' extensor muscles while hiking down a steep trail. In short, running or walking is less efficient than flying, swimming, or bicycling because the muscles must be used for deceleration (negative work) as well as acceleration (positive work). One reason that a person riding a bicycle is so efficient (and thus why people can cycle many times faster and farther than they can run) is that the CM does not rise and fall, which means that more muscular energy can be transferred into forward velocity. Elastic energy storage in elastic elements of the limbs appears to be especially important in running and hopping animals. Consider the bounding of a kangaroo. The greater the height achieved during the hop, the greater is the speed of descent and, when the legs strike the ground, the more energy is transferred to the elastic elements in the limbs and the greater the force of elastic recoil ofthe limbs when they subsequently extend at the beginning of the next hop. Not but the concept of many elastic energy storage is important when considering changes in gait (e.g., walking to trotting to galloping in a horse). By changing gait at appropriate speeds, land animals enhance their locomotory efficiency and avoid potentially injurious forces on legs. For example, consider a pony made to trot on a treadmill at a speed at which it would normally gallop, or to gallop when it would normally trot, or to trot when it would normally walk. In all cases, it expends more energy than if allowed to change its gait naturally. Optimum gaits result from the relative amounts of energy stored in the elastic elements of the body, such as tendons, when performing the different gaits. For instance, little energy is stored when an animal walks; somewhat more is stored when it trots. When the animal is galloping, its entire trunk is involved in elastic storage. At least half the negative work done in absorbing kinetic energy during the stretching of an active muscle appears as heat; the remainder is stored in stretched elastic elements such as muscle cross-bridges, sarcoplasmic reticulum, and Z-lines of muscle cells and tendons. Only the elastically stored energy is available for recovery on the rebound, and only from 60% to 80% of that is recovered on its release. The energy converted into heat is not available for conversion into mechanical work in living tissue. Locomotory energetics of ectotherms versus endothewns You might think that, on simple energetic grounds, terrestrial endotherms and ectotherms of the same size will expend the same net metabolic energy to run at a given velocity. This reasonable assumption is almost, but not

712


............................................................................... Crawlers, runners

F l ~ e r s Sw~mmers

V

Insects

V-

Mosqu~to F~shes Reptiles

o

B~rds

0

A

V

Mammals

o

A

V

Mach~nes

F105 ftghter alrplane

Dtr~g~ble Bicycl~st W

I

Tractor traller/truck

Fre~qhtsteamer

Log mass (kg) Figure 16-50 Cost of transport is more closely related to the kind of locomotion than it is t o the kind of organism. Cost is given in kilo-

calories per gram per kilometer for animals as well as machines. [From Tucker, 1975.1

entirely, correct. When 0, consumption is plotted against running velocity for a lizard and a mammal of similar size, the aerobic parts of the plots for both exhibit rather similar slopes. Thus, when movement begins, a similar increment in metabolic energy expenditure is required for a similar incremental increase in velocity for both the mammal and the lizard. The difference between the two animals lies in the lower y-intercept of the lizard's plot relative to its standard metabolic rate while at rest. The reason for the differences between the resting metabolic rates and the y-intercepts is not completely certain, but these differences may represent the "postural cost" of locomotion-this cost being higher for a mammal than for a lizard. As noted earlier, the rate of 0, consumption rises linearly with increasing velocity of locomotion. This relation is true for ectotherms as well as for endotherms. An endotherm of a given mass typically has a basal metabolic rate about 6 to 10 times that of an ectotherm of similar mass. In both groups, a similar relation exists between the basal rate and the maximum metabolic rate that can be achieved with intense exercise. That is, the factorial scope for locomotion exhibited by both groups is about the same. Thus, in response to intense exercise, endotherms can achieve a maximum rate of 0, consumption of as much as tenfold that of ectotherms of similar size, and so an en-

dotherm of a given size can achieve a higher rate of activity while undergoing aerobic metabolism than a similar-sized ectotherm can. The locomotor speed at which the maximum rate of aerobic respiration is reached is termed the maximum aerobic velocity (MAV).As an animal exceeds its maximum aerobic velocity, the additional activity is supported entirely by anaerobic metabolism, which leads to glycolytic production of lactic acid. As lactic acid production progresses, an oxygen debt develops (see Metabolic scope, earlier in this chapter).Anaerobic metabolism is also associated with muscle fatigue (due to progressive depletion of chemical energy stores) and metabolic acidosis, which if extreme can disrupt tissue metabolism. Because of these two consequences, anaerobic metabolism is unsuitable for sustained activity. Thus, only locomotion below the maximum aerobic speed can be sustained by animals of either group. Because endotherms are capable of far higher rates of aerobic metabolism than are ectotherms, they are generally capable of higher rates of sustained locomotor activity. Clearly, the implications for ectothermy and endothermy are not limited to mechanisms of temperature control but are also of great importance to the kinds of activity that an animal can undertake. The metabolic differences between ectotherms and endotherms determine, for

U S I N G ENERGY: M E E T I N G E N V I R O N M E N T A L C H A L L E N G E S

713

............................................................................... example, how far and how fast they can travel. This is not to say that ectotherms cannot achieve rates of activity and speeds of locomotion as high as those of endotherms. However, because locomotor activity in excess of the maximum aerobic velocity relies on prodigious rates of anaerobic metabolism, high rates of locomotion can be sustained by ectotherms for only brief periods. This limitation can be observed in ectothermic vertebrates such as some species of lizards and frogs, in which a rapid burst of activity, seldom lasting more than a few seconds, takes the animal quickly to a new resting or hiding place when disturbed. In some fishes, more than 50% of body mass is glycolytic white muscle fibers specialized for very short bursts of locomotor activity. The disadvantage that ectotherms have in sustaining high rates of activity is offset by their more modest energy requirements, which enable them to spend more time hiding and less time looking for a meal.

Why can fleas leap hundreds of times their own body length, whereas most medium-sizedmammals can jump only a few body lengths, and large mammals do not even attempt to leap? Consider both the physical surroundings of the animals and their own structural makeup.

Circadian Rhythms

Biological rhythms lasting from milliseconds (at the cellular level) to years (at the whole-animal level) have been identified in a wide variety of animals. Most rhythms (and certainly many of the most prominent and well studied rhythms) relate to daily cycles, which are called circadian rhythms. A true circadian rhythm, which is endogenously generated, can be distinguished from a physiological or other variable that just happens to track daily changes in environment by the use of four different criteria. First, a circadian rhythm shows persistence, remaining for at least several days or weeks in an animal that has been removed from the natural environment and placed in a laboratory setting with constant environmental conditions (constant temperature, constant light or constant dark, etc.). A true circadian rhythm will persist in the animal, manifest most often as a continuation of that animal's normal daily cycles, the innate period being near 24 hours. Although anv of a variety of physiological or behavioral characteristics can be measured, one of the most frequently measured is general activity. Figure 16-51 shows a typical apparatus for measuring the locomotor activity of a rodent. When the animal runs ~n the exercise ivheel, the ~ctivityis recorded, either directly on a chart recorder or on a

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BODY RHYTHMS AND ENERGETICS Most animals strive to maintain some sort of constancy in their interior milieu. Although variations in body temperature, metabolic rate, intracellular pH, and body energy content, for example, may of necessity fluctuate widely in response to environmental constraints and demands, most animals have a preferred range for these and other physiological variables. Despite the evolution of mechanisms that help achieve this relative constancy, almost all animals also show an innate, usually subtle, rhythmic variation in these variables. These variations occur on a daily, tidal, lunar, or other basis and can usually be linked to a rhythmic change in the animal's environment. Early experiments in biological rhythms by chronobiologists concentrated on endotherms, where variations were found in body temperature and metabolic rate. It has been known for centuries, for example, that the body temperature of human beings living on typical activity-sleepcycles falls by a half degree or so during the early morning hours (about 3:OO-5:00 A.M.), only to rise again at about normal waking time. In fact, virtually all animals and plants show some type of rhythmic variation in metabolism or some other physiological variable. Biological rhythms are so innate to animals that even individual cells in cell culture will show rhythms in rate of cell division. The cell does not have to be a particularly complex cell-a daily rhythm occurs, for example, in prokaryotic cells such as nitrogenfixing cyanobacteria.

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Figure 16-51 Circadian rhythms can b e recorded by monitoring spontaneous activity. In this example, a rodent runs in an exercise wheel, which activates a switch closing an electric circuit every time it turns. The resulting electrical record of activity can b e written direaly on a chart recorder or, more conveniently, logged in a computer for later analysis.

computer. The apparatus can be modified by replacement of the exercise wheel with some other form of activitymeasuring device to record activity in birds, fishes, or virtually any other animal. Figure 16-52 shows activity levels in a blinded house sparrow. For the first two weeks of the record, the sparrow was in a light-dark cycle. Even though the bird was unable to see, its circadian rhythm nonetheless persisted, with a free-running period several minutes longer than 24 hours. The second characteristic feature of circadian rhythms is that they tend to be largely body-temperature independent. We have seen that metabolism and physiological processes that derive from and contribute to metabolism have a temperature quotient, Q,,, - of between 2 and 3. Yet, a rise in temperature typically causes very little or no increase in the cycling of the circadian rhythm; in some animals, an increase in body temperature may actually slow down the circadian rhythm. Circadian rhythms are also characterized by the fact that they can be made conditionally arrhythmic-that is, a certain set of environmental temperatures, lighting regimes, A

One day'sactivity

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oxygen levels, and so forth, can disrupt the normal circadian rhythm. Often, there is a threshold level of temperature below which the rhythm is finally disrupted. The effects of light are more graded. In the mosquito, forexample, a circadian activity rhythm is evident when the light phase of the light-dark cycle consists only of low light, but this rhythm gradually diminishes as light intensity increases. A final characteristic feature of circadian rhythms is that they can be entrained. If an animal is placed in total darkness, for example, the length of its circadian rhythm remains close to 24 hours but is generally slightly shorter or longer, resulting in a progressive "creep" in activity cycles that is quite obvious in a long-term record of an animal's activity (see Figure 16-52). However, if the animal in darkness is then given a new lighting regime with a periodicity of slightly longer or shorter than 24 hours, the animal's activity will be entrained to the new lighting regime. Reentrainment is not instantaneous but takes place in a series of transients that manifest the inability of the internal clock to be shifted more than a certain amount each cycle. Through the use of new light cues, activity cycles can be advanced or delayed to the point of being in a completely opposite phase to that of the original circadian rhythm. In the experiment with the blinded house sparrow (see Figure 16-52), the feathers were removed from the top of its head. This allowed light to penetrate through the skull to the brain, parts of which are light sensitive, and the sparrow's activity was then entrained to the light-dark cycle. As the feathers grew in, the entrainment began to be lost and the circadian rhythm lengthened, but removal of the feathers for a second time returned the entrained activity rhythm. In a final experiment, injection of dye under the scalp on top of the skull blocked light penetration to the brain, and the circadian activity rhythm began to drift once again. Light is the most commonly effective zeitgeber, or environmental entrainment factor. However, environmental temperature, food availability, and interactions with other animals of the same or different species also may be zeitgebers that exert an effect on metabolism, activity, and other basic facets of animal life.

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Noncircadian Endogenous Rhythms 24

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Light-dark cycle (hours) Figure 16-52 C~rcad~an rhythms can be entra~nedby l~ghtor other cues In th~sexperiment w~tha bllnded house sparrow In a Ihght-darkcycle of

I~ght~ng, even the small amount of l~ghtreach~ngthe bra~nthrough the top of the skull is suffic~entto entrain a dally actlvity rhythm [Adapted from Menaker, 1968.1

With the circadian rhythm as the "standard," endogenous biological rhythms can be classified into infradian rhythms, less than a day in length, and ultradian rhythms, greater than a day in length. Infradian cycles are usually related to aspects of cell function. In fact, some 400 distinct infradian rhythms in cell function have been identhed to date. These infradian

cycles greatly affect anlmal energetics, but the effects are more difficult to measure than are changes occurring on a daily or longer basls. Many infradian rhythms, such as those related to certain aspects of cell division, have not yet been correlated with any type of rhythmic environmental change. In some cases, external environment may have little or no role; in other cases, we have probably just failed to identify the environmental factor that entrains the rhythm. Ultradian rhythms are very common in anlmals. The influences of the moon through and its - both its llght production of ocean tides greatly affects the physiology of many intertidal animals. Circatidal rhythms, which correlate with tidal cycles, are generally 12.4 hours in length. Many intertidal animals show such rhythms, which have many of the same characteristics (except for their length) as circadian rhythms (Figure 16-53). Circalunar rhythms correlate with the 29.5 day lunar cycle and affect reproduction in many animals. Circannual cycles correlate with the 365 day earth year and are most evident in the often strong, seasonal cycles that affect everything from fur color to hibernation to migration of many animals. Note that all of the circ- rhythms are endogenous rhythmsthey persist if external cues are removed, and they are entrainable.

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Temperature Regulation, Metabolism, and Biological Rhythms

Many animals show circadian or other rhythms in body temperature. In endotherms, considerable amounts of energy are used in maintaining constant body temperature, either directly through thermogenesis or indirectly by powering physiological mechanisms that regulate heat loss or gain or both. In ectotherms, body temperature directly affects the animal's metabolism. Thus, for both endoderms and ectotherms, circadian and other rhythms affecting body temperature also affect an animal's energy metabolism. Because the consequences of rhythms on temperature regulation and metabolism are inseparable, we will consider them together.

Vertebrate endotherms Circadian rhythms in body temperature have been identified in most birds and mammals. There is a relatively strong scaling effect, with smaller animals showing larger circadian variation in body temperature. Thus, human beings weighing from 50 to 80 kg show a daily variation of about 0.6 Celsius degree, whereas the far smaller shrews, deer mice, and hummingbirds, each weighing only a few grams, may show daily temperature fluctuations of as much as 20 degrees. The large daily variation in these small endotherms is probably due to the fact that they frequently enter into a nightly (or, in some cases, daily) torpor. The large daily swing in body temperature probably relates to the greater metabolic cost of maintaining body temperature in very small endotherms. Consequently, the smaller the animal, the greater will be the energy savings from allowing body temperature to fall by several degrees.

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Time of day Figure 16-53 Afiddlercrab kept in constant darkwith no tide cont~nues t o show a tidal act~vityrhythm. There are two low t ~ d e seach day, which occur on average 50 minutes later each day. The crab's activity continues t o coincide with these tide cycles. [Adapted from Palmer, 1973.1

What is the root cause of a circadian body temperature rhythm in endotherms? Because body temperature in an endotherm is a function of its heat production and heat loss and gain from the environment, it follows that one or more of these factors must show rhythmic change to account for daily or other variations in body temperature. Relatively few studies have examined the total heat production and heat conduction budgets of an endotherm in the context of circadian or other rhythms. However, experiments in human subjects have simultaneously measured the circadian rhythms in body temperature, heat conductance, and heat production. These data show that changes in heat production (i.e., changes in metabolic rate) account for about one-

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quarter of the 0.6 degree daily swing in core temperature, with about three-quarters of the change resulting from changes in heat conductance between the core and the environment. Some endotherms modify the amplitude, but not the periodicity, of circadian body temperature rhythms when exposed to stresses ranging from temperature extremes to inadequate food or water or both. In a classic, late-1950s study on endotherm thermoregulation, K. Schmidt-Nielson and his colleagues studied African camels (Camelus dromedarius) in the Sahara desert of Algeria. Well hydrated and fed camels showed a circadian body temperature rhythm with an amplitude of about 2 Celsius degrees, but this increased to about 6 degrees when the camels were unable to drink water. The maximum core temperature (in late afternoon) was increased, whereas the minimum core temperature (in early morning) decreased. These changes are presumably to reduce water loss through evaporative cooling in the day and to decrease heat loss because of a reduced thermal gradient to the cooler nighttime surroundings. Both reduce the need for metabolic heat production. Birds such as kestrels and pigeons, which also show circadian body temperature rhythms, similarly showed a larger daily core temperature range, mainly owing to a lower nighttime core temperature. Field observations alone may be insufficient to identify circadian rhythms in regulated body temperature because many endotherms also show strong daily rhythms in activity levels. Depending on the animal's ability to dissipate metabolically produced heat, a rhythmic rise in body temperature could be solely the result of increased locomotor activity (which itself is a manifestation of circadian rhythms). However, two lines of evidence indicate that there is usually a distinct intrinsic rhythm in body temperature independent of activity: (1)temperature rhythms persist in animals in the laboratory in which activity levels have been corrected for or controlled, and (2)temperature rhythms persist in human beings over several days of complete bed rest. In reality, circadian rhythms in body temperature are often imposed on activity rhythms with a similar time component, amplifying the daily range of body temperature. Ultradian rhythms in body temperature are best exemplified by the hibernators, which, as noted earlier, lower their core temperature by 20-35 Celsius degrees for periods of weeks or months, punctuated by brief periods of arousal. These rhythms can persist for at least four years in hibernating golden-mantled ground squirrels (Citellus) isolated at birth from any light or temperature zeitgebers. In hibernating bats, circadian rhythms in body temperature and metabolism can be measured at the new, much lower mean core temperature typical of hibernation. This emphasizes the generally temperature independent nature of the biological clock responsible for circadian rhythms. ~owever,ihecircadian rhythm eventually disappears as hibernation continues. Rodents such as the 13-lined ground squirrel (Spermophilus tridecemlineatus) show no evidence

of a continuing circadian rhythm in 0, consumption with the onset of hibernation. Some mammals show little or no evidence of a circadian rhythm in either body temperature or metabolic rate. Such animals tend to be those that live in environments with very stable conditions of temperature, light, food availability, and so forth. Fossorial (burrow-dwelling) pocket gophers and moles, for example, live in constant darkness with little temperature variation and show no metabolic circadian rhythms. It is unclear what advantage there would be to a significant body temperature and metabolic rhythm in such animals. Mammals such as voles, which have a herbivorous diet with high bulk, feed nearly constantly to derive sufficient energy. These animals similarly show little or no metabolic rhythmicity. Vertebrate ectotherms All ectotherms by definition rely on external sources of heat to elevate body temperature. However, both the behavioral and the physiological adjustments made to regulate body temperature are modified by circadian rhythms in preferred body temperatures. Because metabolic rate is closely related to body temperature, circadian rhythms in 0, consumption and CO, production are closely correlated with body temperature changes. Fishes have long been known to show circadian rhythms in activity, body temperature, and metabolic rate. In many cases, daily periods of activity correspond to the highest body temperatures and metabolic rates. In a classic study, J. R. Brett (1971)examined lake-dwelling sockeye salmon, Oncorhyncus nerka, monitoring position in the water column (Figure 16-54). During the day, these salmon stayed in deep, cold water, and presumably both body temperature and metabolism reflected this low wa-

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Time of day (hour) Figure 16-54 Vertical migration in sockeye salmon (Onchorhynchus nerka) shows a strong diurnal rhythm. These fish rise through the water column to feed at dusk. They remain near the surface to feed at dawn before descending to cold, deep wafer during the day. [Adapted from Brett, 1971.]

ter temperature. As dusk approached, the fish rose to the surface to feed, which brought them through the thermocline into water of about 17"C, where they remained for a bout of dawn feeding before descending to cold water for the day. This overall pattern of activity allows the sockeye salmon to conserve energy by having a cold-induced, low metabolic rate during periods of inactivity. Such circadian rhythms of vertical migration related to feeding are very common in pelagic fishes. When they take animals through temperature gradients, then metabolic rate will similarly show daily variations. But are there true circadian rhythms in metabolic rate or are these changes merely reflecting body temperature changes? In fact, daily rhythms in oxygen consumption persist in conditions of constant temperature and light in many fish species. Studies on circadian rhythms in temperature regulation and metabolism in amphibians are few. Circadian rhythms in preferred body temperature and activity have been found in the aquatic salamander Necturus maculosus but not in the toad Bufo boreas, the larvae of the frog Rana cascadae, or the salamander Plethodon cinereus. Many species of toads and frogs show pronounced activity patterns related to feeding, predation, and so forth, but we currently know very little about how many of these patterns are cued by the external environment and how many are due to innate circadian rhythms-that is, are controlled by "biological clocks." Many reptiles show a circadian rhythm in preferred body temperature. Because resting metabolism tracks body temperature in these animals, metabolic rate correspondingly ranges up and down in the course of a 24-hour period. Again, establishment of whether these thermoregulatory and metabolic rhythms are true innate rhythms requires monitoring animals in constant environmental conditions. In fact, in the lizard Sceloporus occidentalis and other species, a circadian rhythm in preferred body temperature persists for several days under constant light conditions in the laboratory. Similarly, circadian rhythms in oxygen consumption are found in some species of the lizard Lacerta. Invertebrates Invertebrates from many different phyla have been identified as having some degree of thermoregulatory capacity, primarily through behavioral means but also by physiological means (e.g., ;he metabolic heat production in flying insects considered earlier).Daily or other rhythms in preferred body temperatures have been observed in numerous Arthropoda-among them crayfish, shrimp, and a variety of insects, including silkmoths, bees, and wasps. Oxygen consumption tracks body temperature in these animals, so metabolic rate similarly shows daily cycles if an animal changes its body temperature. Daily rhythms in oxygen consumption have also been observed in earthworms, amphipods, seapens, and mollusks. Intertidal animals may show combinations of circadian, tidal, and lunar metabolic cycles. With few exceptions, it is unknown whether these daily rhythms are endogenous or are exogenously triggered.

Male American silkmoths, Hyalophora cecropia, show endogenous rhythms of endothermic warming in preparation for flight; these rhythms are unaffected by ambient temperature. Honey bees (Apis mellifera) show very prominent circadian rhythms in oxygen consumption when kept in constant darkness, with oxygen consumption increasing from 20 to 30 times over resting levels during the periods that correspond to daylight activity and foraging. The fruit fly Drosophila similarly shows an increase in metabolic rate during the period corresponding to daylight when kept in constant darkness. Unicellular organisms The presence of true circadian or other rhythms in unicellular organisms is of particular interest to chronobiologists.Although the study of more complex animals has implicated the brain, pineal gland, and other tissues as the site of a biological clock, the presence of true biological rhythms in unicellular organisms indicates that all the necessary components for a clock can be found among the cellular organelles. Unicellular organisms exhibit rhythms in rates of photosynthesis, oxidative metabolism, bioluminescence, cell division, growth, phototaxis, and vertical migration-to name but a few variables. The first definitive demonstration of an innate circadian rhythm was made in 1948 on the unicellular alga Euglena gracilis, which shows phototactic rhythms (migration to light). Since that time, a variety of other eukaryotic cells, including Paramecium, have been shown to exhibit true circadian rhythms that persist under constant conditions and can be entrained. More recently, circadian rhythms have been found in the much simpler prokaryotic cells of cyanobacteria. The knowledge that individual cells show circadian cycles in cell division has been put to use in designing more effective chemotherapy for human cancer patients. Different chemotherapeutic agents act on different ~hasesof the cell division cycle, which in human beings has a circadian rhythm. By timing the administration of the drug to the vulnerable period of the cell cycle (typically,two o'clock in the morning rather than during the usual business hours), as much as a 10-fold increase in effectiveness has been achieved. Moreover, undesirable and deleterious side effects of the treatment are also greatly reduced.

ENERGETICS OF REPRODUCTION Reproduction is the ultimate goal of all organisms, and the evolution of virtually all specializations can be linked directly or indirectly to improvement in an animal's reproductive fitness. Given the overall importance of reproduction, it is not surprising that this process accounts for a considerable proportion of an animal's energy budget. Exactly what proportion depends on many factors, including mode of reproduction, body size, whether an animal is ectothermic or endothermic, and so forth. We begin by considering the different patterns of energy investment in reproduction that have evolved.

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Patterns of Energetic Investment in Reproduction

Natural selection of reproductive structures and processes has resulted in a wide variety of reproductive patterns. The most favorable mode for a species is that which maximizes the reproductive value of the offspring by raising the largest number possible to sexual maturity. By the mid-1960s, several groups of ecologists and evolutionary biologists had recognized that, collectively, animals had evolved one of two general patterns of energy investment in reproduction. That is, they recognized that a given amount of energy to be invested in producing offspring can be partitioned, or "spent," in two different ways. These two patterns were called v-selection and K-selection, with the letters Y and K coming from the loglstlcs equation that models the growth rate of continually reproducing animal populations. (Consult a text In ecology or evolution for further details on the logistics equation and the growth of animal populations.) r Selection- "smaller and more" In the first pattern of energetic investment in reproduction, as exhibited by r-selected animals, an animal produces offspring that, at the start of their development, are very small. By virtue of each offspring having a very small energy content (and thus a small energy cost to the parents), the pare n t ( ~can ) produce much larger numbers of offspring. Females of some species of sea urchins, for example, release as many as 100,000,000 eggs in a spawning. Among vertebrates, pelagic fishes may similarly release huge numbers of fertilized eggs; for example, the mackerel Scomber scombrus releases tens of thousands of eggs in a spawning. These are extreme examples, but most invertebrates and many ectothermic vertebrates produce dozens or more offspring in a single breeding episode. The "trade off" in producing large numbers of small offspring is that the parent is less able to afford parental care to each of so many offspring. Most r-selected animals simply release their offspring into the environment to fend for themselves. Because they are small and vulnerable, relatively few survive to reproduce. The probability of a mackerel spawn surviving to reproductive age is 0.000006.

K Selection- "larger and fewer" The second pattern of energetic investment in reproduction is exhibited by K-selected animals. These animals produce relatively large bffspring-offspring that have a high energy content and represent a large energetic investment by the parents. As a consequence, the number of offspring produced is far smaller than that produced by r-selected animals. Mammals and birds, for example, tend to produce offspring that, at birth or hatching, are at least a few percent of the body mass of the mother and may be much larger. Litters or egg clutches rarely consist of more than 8 to 10 offspring, because of the huge energy cost of producing them. However, because numbers are small, the offspring are more manageable, and K-selected animals usually invest additional energy in parental care (see Parental care as an energy cost of reproduction later in this chapter).

Because the offspring of K-selected animals tend to be large and cared for during their early development, their chances of successfully reaching reproductive age are far greater. Contrast the six-in-a-million chance of a mackerel surviving with that of a bird or mammal, whose chances may approach 50% or higher. The classification of animals as r- or K-selected, however, is not absolute, and many species show characteristics of both. For example, many Cichlid fishes produce hundreds of small larvae (an "r" characteristic). However, they then show "K" characteristics by investing huge amounts of time and energy in parental care-females may even stop feeding themselves for weeks to protect the young.

Allometty and the energy cost of reproduction Like virtually all other aspects of an animal's physiology, allometric scaling affects the energy cost of reproduction. M. Reiss (1989) assembled data on the energy cost of reproduction within taxa that ranged from spiders to salamanders to mammals. These data show that, in general, larger animals Invest relatively less energy in their offspring than do smaller animals. The value of the exponent in the allometric equatlon relating energy cost of reproduction to body mass ranges from a low of 0.52 in ducks and geese to 0.95 in hoverflies, with mammals showing a range of about 0.69-0.83. If we consider both invertebrates and vertebrates, wzthin a specles, larger females devote relatively more energy to reproduction than do smaller females. One can see this clearly in mammals: a large, fat rodent, feline, or canine produces a larger litter than does a thin female wlth few energy reserves. Yet, in species with a sexual dimorphism in which males are larger than females, the data suggest that larger males expend relatively less energy on reproduction than do smaller males. The "Cost" of Gamete Production

Reproduction begins with the production of gametes (eggs and sperm). The energy cost of gamete production varies greatly. Gamete production is a costly business in most invertebrates, with half or even more of the total energy assimilated being diverted into the gametes. Usually, the female makes the largest energy expenditure in producing yolky eggs that will nurture the growing embryos. Sperm production usually requires a lower energy expenditure. However, some males expend disproportionately large amounts of energy on sperm production. The testes and associated accessory organs in the male cricket (Acheta

U S I N G EN

....................................... domesticus), for example, account for 25% of the animal's body mass. The spermatophore (the packet containing sperm that is passed to the female during copulation) that it produces is about 2.5% of its body mass, and it can produce two or more spermatophores per day. Although the energy cost of gamete production may be high, very few invertebrates expend energy on protecting and nurturing their offspring after they are produced. Notable exceptions are found among the arthropods. Female scorpions carry their newly hatched offspring on their backs. For insects such as ants and bees, the entire social structure is organized on the care and nurturing of the offspring (see next subsection). Ectothermic vertebrates, like invertebrates, spend a large proportion (as much as half) of their total energy budget on gamete production and reproduction. Sperm production is relatively inexpensive for males of all species, but egg production can be energetically costly. As already mentioned, many fishes produce large clutches of eggs. Different species of oviparous (egg-laying)amphibians produce egg clutches ranging from 4 to 15,000 eggs. Estimates of the energy costs are few; but, in the salamander Desrnognathusochrophaeus, about 48% of the female's energy is used in a combination of egg production and parental care. In the lizard Uta stansburiana, reproductive behavior of males consumes 32% of their energy during spring, and the females expend as much as 83% of their energy (26% for increased metabolic costs and 57% for producing eggs) on reproduction. Female crocodiles, alligators, and, especially, brooding snakes such as pythons similarly invest large amounts of energy in combined egg production and parental care. The energy cost of gamete production is highly variable among endothermic animals. In birds, some of which produce large numbers of relatively large, yolky eggs, estimates of the cost of egg laying range from less than 10% to more than 30% of the animal's total energy budget. Domestic chicken hens (white leghorn), which have been subjected to human selection to maximize efficiency of egg production, expend about 15% to 20% of their energy on egg production. The energy cost of producing sperm in roosters, however, is negligible. Similarly, the cost of sperm production in mammals is virtually negligible. Almost all mammals produce very small numbers of very small eggs and, again, their energy cost is negligible. However, as we will see next, almost all mammals invest a considerable amount of energy in protecting the developing offspring after fertilization.

which have been selected for copious milk production, as much as 50% of total energy may be used for milk production. Animals other than mammals also produce secretions for their developing offspring. One or both parents of many bird species regurgitate semidigested food into the mouths of offspring. Although this practice is not as energetically costly as producing the same weight of milk, it is a real cost in that the regurgitated food might otherwise be digested and assimilated into the parent's own body. Doves produce "crop milk," a viscous fluid formed from the initial digestion of material temporarily stored in the crop and then given to their offspring. Certain species of viviparous and ovoviviparous amphibians and reptiles produce uterine secretions ("uterine milk") that nourishes the embryos until birth. Embryos of viviparous caecilian (apodan) amphibians use specialized dentition to scrape away and ingest the lining of the oviduct. The female of the poison-dart frog Dendrobates pumilio returns to the small ponds holding her larval offspring and deposits unfertilized eggs for her larvae to eat. Among the invertebrates, ants, bees, and wasps can expend large amounts of their total energy in gathering raw materials and producing honey or equivalent substances for the nourishment of the developing animals in the colony. The second type of cost of reproduction associated with parenting is the metabolic cost of behaviors specifically associated with parenting. Complex parental care is evident in numerous invertebrate taxa, including mollusks (Octopus), polychaete worms, and the social insects (ants, bees, and wasps), which have elaborate behaviors of nest building, brooding, and so forth. Among the vertebrates, parental care is found in all classes and is widespread in birds and mammals. Unfortunately, hard data on the energy cost of parental care are difficult to obtain, because such behaviors often include such parental activities as brooding, foraging, grooming, and so forth. Although such behaviors clearly require metabolic energy expenditure on the part of the parent, they are often complex, not easily duplicated in the laboratory, and not easily separated from on-going energy costs not related to parenting. (Some might say that the estimated $200,000 that it takes to raise and educate a child in the United States is an indirect cost of human parenting.)

Parental Care as an Energy Cost of Reproduction

In the introduction to this book as well as the introduction to this part of the book, we describe the interdependency of numerous physiological systems. As Donald Jackson (1987) commented in considering the problems faced by interacting physiological systems, "A disturbance to one part reverberates throughout the organism, and produces responses, compromises and adaptations of various functions." The resolution of potential conflicts between the differing demands of networked-physiologicalsystems must be made in the context of space and time. A conflict between

Many animal species do not exhibit parental care but, in those species that do, it can be a major energy cost. Parental costs fall into two categories. In the first category is the cost of the actual transfer of material from a parent to the developing offspring. One of the best examples of this is mammalian lactation, in which large amounts of milk rich in lipids and carbohydrates are secreted to nourish the newborns. Typically, lactating mammals expend as much as 40% of their energy on producing milk. In dairy cattle,

ENERGY, ENVIRONMENT, AND EVOLUTION

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the demands of two physiological systems can be tolerated for short periods of time but must eventually be resolved by some other appropriate physiological action. Moreover, different environmental stresses carry with them very different senses of urgency. Figure 16-55 indicates the very different amounts of time during which an absence of oxygen, water, and food and an excess of body heat can be tolerated by an organism. In most animals, a physiological conflict that denies an animal food or water, for example, can be tolerated far longer than a physiological conflict that denies an animal oxygen. Often then, physiological conflicts are resolved on the basis of which of two resulting conditions is the least threat to homeostasis. These tolerances also vary greatly between species: a human being can survive without oxygen for only a few minutes; a turtle can survive for several hours; and some simple metazoans survive without oxygen indefinitely and may actually be killed by oxygen. The field of animal physiology is only now beginning to focus on interactions between different physiological systems, rather than on the isolated characters of the individual systems. This integrated approach will by necessity draw in systems-level physiology, ecological physiology, environmental physiology, and evolution.

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Figure 16-55 The rate of decline in cellular metabolic rate toward lethally low levels differs greatly among species after the abrupt removal of oxygen, food, or water or in failure to eliminate metabolicallyproduced heat. Note that the time scale is logarithmic (1000 hours is about 42 days). Different species have very different tolerances for oxygen deprivation, starvation, and dehydration. For example, the brain cells of human beings begin to die within minutes of oxygen deprivation, whereas some species of turtles can survive for days or even months in the complete a b sence of oxygen. [Adapted from Jackson, 1987.1

ater falls is sending large q

This concluding chapter has integrated the animal and its physiology into its environment. Environmental constraints place limitations and demands on design and function. Animals living in water have very different shapes from those of terrestrial animals. Drag forces are much greater in water than in air, and so aquatic animals are much more streamlined. Animals in water have a density that is similar to that of the environment, but this is not the case in air. Gravity has an important effect on the circulation in terrestrial animals not seen in aquatic animals. In terrestrial animals, blood tends to pool in veins, and there are many mechanisms to ensure adequate venous return to the heart. The giraffe must have a strong, fibrous skin around the lower part of its limb to prevent blood from pooling in veins in its legs. This problem does not occur in fishes and other aquatic animals. However, in these animals, motion results in strong forces on the body surface that could interfere with venous return; so, at least in fishes, most large veins travel through the center of a fish's body. Survival of an individual animal often depends on the allocation and rate of use of available energy. Different an-

imals adopt different strategies. Mammals, for example, have high rates of energy turnover that require them to seek food continually. Reptiles, on the other hand, have much lower energy turnover rates and can survive on much less energy. Different environments favor different strategies at different times. For example, reptiles seem to have the advantage in the water and in food-scarce desert environments during the day, but mammals seem to have the edge during the cooler nights. Mammals spend energy on maintaining a high body temperature and therefore need more food but can function in the cold nights. Success of an individual animal is measured by the genetic legacy left by that animal-that is, by surviving to reproduce. Reproduction occurs when the animal is mature and conditions are favorable for survival of the young. For example, during periods of decreased energy availability, unfavorable for reproduction, the animal may enter a state of suspended animation, as in insect diapause or mammalian hibernation. There is a marked reduction in energy expenditure during these periods. Essentially, the animal is cutting its energetic costs during the "bad times" to balance energy input and output. There is a marked increase in energy expenditure during exercise. Animals often migrate to avoid certain environments where, for example, food is short and temperatures are low. The cost of migration varies with the nature of the environment, with flying being much less costly per unit distance than walking and running on land or swimming in watel: The results of the process of an-

U S I N G ENER

........................................ imal evolution have given us numerous examples of adaptions for survival in a multitude of different habitats. These examples are variations in the organization of a series of basic component parts that make up the vast panoply of life.

SUMMARY The utilization of chemical energy in tissue metabolism is accompanied by the inexorable production of heat as a low-grade energy by-product. The total energy liberated in the conversion of a higher energy compound into a lower energy end product is independent of the chemical route taken. In addition, a given class of food molecules consistently liberates the same amount of heat and requires the same amount of 0, when oxidized to H,O and CO, . These characteristics of energy metabolism make it possible to use either the rate of heat production or the rate of 0, consumption (and CO, production) as a measure of metabolic rate. The respiratory quotient-the ratio of CO, production to 0, consumption-is useful in determining the proportions of carbohydrates, proteins, and fats metabolized, each of which has a different characteristic energy yield per liter of 0, consumed. The basal metabolic rate and the standard metabolic rate are related to body size-the smaller the animal, the higher the metabolic rate per unit mass of tissue (termed metabolic intensity). Although there is a fairly good correlation between metabolic intensity and body surface-to-volume ratio, suggesting that metabolic rate is determined by heat balance mechanisms, this correlation may be incidental. Similar correlations are seen in ectotherms that are in temperature equilibrium with the environment and in endotherms that steadily lose heat to the environment. Dependence of enzymatic reactions and metabolic rate on tissue temperature is described by the Q,, ,the ratio of 'metabolic rate at a given temperature to the metabolic rate at a temperature 10 Celsius degrees lower. This ratio typically lies between 2 and 3. Endotherms are animals that generate most of their own body heat, allowing them to elevate their core temperatures above that of the environment. Ectotherms obtain most of their body heat from their surroundings, and some elevate their temperatures by various behavioral means, such as basking. Poikilothermy, homeothermy, and heterothermy refer to var);ing degrees of body temperature control. Ectotherms show a variety of methods for survival in temperature extremes. Some species cope with subzero temperatures by using "antifreeze" substances or by supercooling without ice crystal formation, but no animals have been shown to survive freezing of water within cells. Other ectotherms elevate body temperature by shivering or nonshivering muscle contraction at certain times or in certain parts of their bodies. Such heat production is used by some insects and large fishes to warm locomotor muscles to optimal operating temperatures. Heat absorption or heat loss to the environment is regulated in some ectothermic species by changes in blood flow to the skin. In this way,

heat absorbed from the sun's rays can be quickly transferred by the blood from the body surface to the body core during heating or, conversely, core heat can be conserved in a cold environment by restricted circulation to the skin. Endotherms subjected to cold environments conserve body heat by increasing the effectiveness of their surface insulation. They do this by decreasing peripheral circulation, increasing fluffiness or thickness of pelage or plumage, or adding fatty insulating tissue. In cold-climate endotherms, heat is also conserved by countercurrent heat-exchange mechanisms in the circulation to the limbs. Within the thermal neutral zone of ambient temperatures, changes in surface conductance compensate for changes in ambient temperature. Below this temperature zone, thermogenesis compensates for increased heat loss to the environment. Thermogenesis occurs by shivering or by nonshivering oxidation of substrates, as well as by exercise, specific dynamic action, activity of the Na+-K+ ATPase, and other activities. At ambient temperatures above the thermal neutral zone, endotherms actively dissipate heat by means of evaporative cooling, either by sweating or by panting. The use of water places an osmotic burden on desert dwellers. Most small desert inhabitants, subject to rapid changes in body temperature, minimize such changes by usually remaining in cool microenvironments to avoid daytime heat. Large desert mammals, buffered against rapid temperature changes by more favorable surface-to-volume ratios and large thermal inertia, can conserve water that they would otherwise use for cooling by slowly absorbing heat during the day without reaching lethal body temperatures; they can then rid themselves of heat during the cool night. The brain is specially protected from overheating in some mammals by a highly developed carotid rete in which cool venous blood from the nasal epithelium removes heat from arterial blood heading toward the brain. Body temperature in endotherms and some ectotherms is regulated by a neural thermostat sensitive to differences between the actual temperature of neural sensors and the thermostatic set-point temperature. Differences result in a neural output to thermoregulatory effectors for corrective heat loss or heat gain. Fever develops when the set-point temperature is raised by the cellular action of endogenous pyrogens, which are protein molecules released by leukocytes in response to exogenous pyrogens produced by infectious bacteria. Ordinary sleep, torpor, hibernation, winter sleep, and estivation are all neurophysiologically and metabolically related forms of dormancy. During periods when food intake is necessarily absent or restricted, small- to medium-sized homeotherms allow their temperatures to drop in accord with a lowered set-point temperature of the body thermostat. By lowering body temperature to within a few degrees of ambient air, the homeotherm conserves energy stores. Oxidation of brown fat and shivering thermogenesis are used to produce rapid warming at the termination of torpor or hibernation.

722


.........................................

The energetics of locomotion is also related to body size. The smaller the animal, the higher the metabolic cost of transporting a unit mass of body tissue over a given distance. The Reynolds number (Re) of a body moving through a liquid or gaseous medium is the ratio of the relative importance of inertial and viscous forces in the medium. Small animals swim with a low Re and large animals with a high Re because, with increasing size, viscosity plays a lesser role and inertia a greater one. The rate of energy utilization during different kinds of locomotion typically increases with velocity. By changing gait from walking to running, hopping, or trotting, and so forth, terrestrial animals increase efficiency. Increased efficiency is achieved when the energy of falling at the end of the stride is elastically stored for release during the next stride, as in a hopping kangaroo. Metabolic rate in many animals shows distinct endogenous rhythms that may be manifest in locomotor activity or changes in body temperature (in ectotherms). These rhythms may be circadian (daily),infradian (shorter than a day), or ultradian (longer than a day). Circadian rhythms are characterized by their persistence in the absence of environmental cues, are temperature independent, are conditionally arrhythmic, and are entrainable by zeitgebers such as light. Reproduction requires a significant energy expenditure for many organisms. The two general patterns in which energy expended in the reproductive effort may be partitioned are r-selection and K-selection. r-Selected animals produce large numbers of very small offspring and offer no parental care. The low rate of survival is offset by the large number of offspring produced. K-Selected animals produce small numbers of large offspring. Owing in part to parental care, survival is high. Energy costs of reproduction to parents include the production of gametes; the cost of providing nutrition, as in mammalian lactation; and the cost of behaviors constituting parental care.

REVIEW QUESTIONS 1. Define ectotherm, endotherm, poikilotherm, homeotherm, basal metabolic rate, standard metabolic rate, and respiratory quotient. 2. Explain why the rate of heat production can be used to measure metabolic rate accurately. 3. Why tan respiratory gas exchange be used as a measure of metabolic rate? 4. Why is the surface hypothesis inadequate as an explanation for the high metabolic intensity of small animals? 5. Why is the locomotion of a small aquatic animal affected more strongly by the viscosity of the medium than is that of a large animal? 6. What factors affect the flow pattern of fluid around a swimming animal?What factors minimize turbulence? 7. Why does riding a bicycle for 10 km require less energy than running the same distance at the same speed?

Why are the giant ants and other photographically scaled up insect monsters of old grade-B science fiction movies anatomically untenable? Give examples of the effect of body size on the metabolism and locomotion of animals. The potency of some medications depends on metabolic factors. Explain why it might be risky to give a 100 kg person 100 times the dose of a drug proved effective in a 1.0 kg guinea pig. Give examples of low-temperature adaptations of some ectotherms and some endotherms. What are some of the factors that determine the limits of the thermal neutral zone of a homeotherm? What thermoregulatory mechanisms are available to a homeotherm at temperatures below and above the thermal neutral zone? Explain and give examples of the relations that exist between water balance and temperature regulation in a desert animal. Describe the integration of peripheral and core temperatures in the thermostatic control of temperature in a mammal. Describe two naturally occurring situations in which the set-point temperature of the hypothalamic thermostat is changed and the body temperature correspondingly changes. Explain the mechanism of heat production in two different kinds of thermogenesis. What is the role of countercurrent heat exchange in porpoises, arctic mammals, tuna fishes, and sheep? What are the sphinx moth's two major means of regulating thoracic temperature? What means does the marine iguana use to speed the elevation of its body temperature and then retard cooling during diving? How would you distinguish between (1)a true circadian rhythm, (2) a metabolic rhythm, and (3) a rhythm that is triggered by rhythmic changes in environmental cues? Compare and contrast reproduction in r- and Kselected animals. What are the advantages of each?

SUGGESTED READINGS Blake, R., ed. 1991. Efficiency and Economy in Animal Physiology. Cambridge: Cambridge University Press. (This book considers, in an evolutionary framework, the efficiency and metabolic costs of various types of animal locomotion.) Block, B. A. 1994. Thermogenesis in muscle. Annu. Rev. Physiol. 56535-577. (This comprensive review describes the biochemical and cellular mechanisms behind specialized heater tissues in endothermic animals.) Carrey, C., ed. 1993. Life in the Cold: Ecological, Physiological and Molecular Mechanisms. Boulder: Westview Press. (This book presents a collection of multilevel re-

723 U S I N G ENERGY: M E E T I N G E N V I R O N M E N T A L C H A L L E N G E S ............................................................................... views on hibernation, torpor, and other mechanisms by which animals survive life in the cold.) Chadwick, D. J., and K. Ackrill, eds. 1995. Circadian Clocks and Their Adjustment. New York: Wiley. (Reviews of genetic, molecular, and neural bases of biological clocks controlling circadian rhythms.) Cossins, A. R., and K. Bowler. 1987. Temperature Biology of Animals. London: Chapman and Hall. (This book focuses on the cellular aspects of thermoregulation in animals.) Edmunds, L. N., Jr. 1988. Cellular and Molecular Bases of Biological Clocks. Berlin: Springer-Verlag. (Th'is monograph explores the phenomenon of annual rhythmicity in a wide range of animals and plants.) Heinrich, B. 1993. The Hot-Blooded Insects: Strategies and Mechanisms of Thermoregulation. Cambridge, Mass.: ,Harvard University Press. (This book explores the physiological and biochemical mechanisms of endothermy among the insects.) Jones, J. H., and S. L. Lindstedt. 1993. Limits to maximal performance. Annu. Rev. Physiol. 55:547-569. (The metabolic and physiological factors that limit animal locomotor performance are the focus of this review.) McMahon, T. A., and J. T. Bonner. 1983. On Size and Life. New York: Scientific American Books. (This delightfully illustrated book examines how scaling and allometry pervade the world around us.)

McNeil, R. A. 1992. Exploring Biomechanics: Animals in Motion. New York: Scientific American Library. (McNeil provides a comprehensive treatment of the anatomy and biomechanics of animal locomotion.) Peters, R. H. 1983. The Ecological Implications of Body Size. Cambridge: Cambridge University Press. (This book is valuable not only for its lucid descriptions of allometry, but also for the extensive tabulated data in its numerous appendices.) Ruben, J. 1995. The evolution of endothermy in mammals and birds: from physiology to fossils. Annu. Rev. Physiol. 995:69-95. (In this comprehensive review, Ruben speculates on the evolutionary processes leading to endothermy in vertebrates.) Schmidt-Nielsen, K. 1983. Scaling: Why is Animal Size So Important? New York: Cambridge University Press. (This now-classic book provides a wealth of information on how anatomy and physiology are affected by body size and why physiologists should care.) Trayhurn, P., and D. G. Nicholls, eds. 1986. Brown Adipose Tissue. London: E. Arnold. (Neural control mechanisms, biochemistry, metabolism, physiology, and anatomy of brown fat are all covered in this book.) Woakes, A. J., and W. A. Foster, eds. 1991. The comparative physiology of exercise. J. Exp. Biol. 160. (This entire volume consists of a series of brief review papers describing the physiological implications of, and adapations foy exercise.)

Appendix 1: SI Units Basic SI units Physical quantity

SI multipliers Name of unit

Symbol for unit

Multiplier

Prefix

Symbol

Length

meter

m

10l2

tera

T

Mass

kllogram

kg

1o9

glga

G

Time

second

s

1O6

mega

M

Electric current

ampere

A

1o3

kllo

k

Temperature

kelvin

K

102

hecto

h

Lum~nous~ntensity

candela

cd

10

deka

da

lo-'

decl

d

10-2

cent1

c

IO-~

mllll

m

10-6

micro

P

1o

nano

n

PIC0

P

-~

Derived SI units -

Physical quantity

-

Name of unit

Symbol for unit

meter per second squared

m . s2

Activity

1 per second

s-'

Ekctrlc capacitance

farad

F

A.s.V-'

Electrlc charge

coulomb

C

A.s

Electric field strength

volt per meter

J . s-'

Accelerat~on

Electrical resistance

ohm

Entropy

joule per kelvin

Force

newton

Frequency

hertz

Illumination

lux

Luminance

candela per square meter

Definition of unit

Luminous flux

lumen

Power

watt

W

Pressure

newton per square meter

N . m2

Voltage, potential difference

volt

V

W . A-'

Work, energy, heat

joule

J

N .m

APPENDIXES

725

............................................................................... Appendix 2: Logs and Exponentials Straight line equations:

If a straight line describes a plot of x against y, then b is the value of the intercept of the line on the ordinate and a is the slope of the line. The relationship between x and y is:

IJ

Abscissa

x

Exponential equations:

In biological systems there is often an exponential relationship between values, described by the equation: y

=

b.ax

The logarithmic form of this equation is: logy = logb

+ xloga

Using semi-log graph paper (linear abscissa) log y can be plotted against x, giving a straight line to determine the values of slope a and intercept b.

X

X

(Examplesof the use of straight line and exponential equations may be found throughout the book, especially in chapters 3 and 16.) Use of logarithm terms:

, In the equation y = lox,x is the logarithm of y. That is, x is the power to which 10 must be raised in order to yield y. For example, the logarithm of 10 is 1,and the logarithm of 100 is 2. The equation

can be tranformed into a l o g a r i t h c equation: a logy = log-= b

loga

-

logb

just as multiplication can be transformed into the addition of logarithms. A convenient identity that is useful in calculating equilibrium potentials using the Goldman equation is: log-

a b

=-

log-

b a

a b This identity is true because log - = log a - log b and log - = log b - log a. Notice that log a - log b b a - (log b - log a), which proves the identity.

=

Appendix 3: Conversions, Formulas, Physical and Chemical Constants, Definitions Units and conversion factors To convert from angstroms

atmospheres

to i

multiply by

~nches

3.937 x lo-'

meters

1 X 10-lo

micrometers (pm)

1x

bars dynes per square centlmeter grams per square centlmeter torr (= mmHg; PC) pounds per square ~nch pascals

bars

calories

atmospheres

0.9869

dynes per square centmeter grams per square centmeter

1 x lo6 1019.716

pounds per square inch

14.5038

torr (= mmHg, 0°C)

750.062

pascals

1o5

British thermal units ergs foot-pounds kilocalories horsepower-hours joules watt-hours watt-seconds

ergs

British thermal units calor~es dynes per centimeter foot-pounds gram-centimeters joules watt-seconds

grams

daltons grams ounces (avdp) pounds (avdp)

inches

angstroms centimeters feet meters

joules

calories ergs foot-pounds watt-hours watt-seconds

APPENDIXES

727

............................................................................... Units and conversion factors (Continued) To convert from

multiply by

to

lrters

gallons (US, Irq)

1o3 0 2641794

cubrc centrmeters

lumens

prnts (US, Irq)

2 113436

quarts (US, Irq)

1.056718

candle power

7.9577 x

lumens per square meter meters

angstroms micrometers Cum) centimeters feet inches kilometers miles (statute) millimeters yards dynes

newtons pascals

atmospheres

IO-~ 9 87 x I 0-6

dynes per square centimeter

10

bars

grams per square centrmeter

1.0197 x

torr (= mmHg; P C )

7.52 X

pounds per square inch

1,450 x 1o

Britrsh thermal unrts per second

watts

calories per minute ergs per second foot-pounds per minute horsepower joules per second

Temperature conversions

"C

=

519 ("F

-

32)

"F = 915 ("C) + 32

0 K = -273.15"C 0°C

=

=

-459 67°F + 32

273 15 K = 32°F

..

~

-~

Useful formulas Electrlc potentla1

E = IR = q/C E = electrlc potentla1(voltage) I= current R = resistance q = charge

C = capacitance

Power

cr2

force of attraction

r = dlstance separating ql and q, E

=

d~electr~c constant

Potential energy

E = mgh h = height of mass above surface of Earth

Kinetic energy

E

p = w/t w = work

t Electric power

F=- q l q2

Electrostat~c

=

=

1/2mv2

v = velocity of mass

p = RI2= El

E

=

time

E

Energy of a charge

=

electric potential

1I2qV

q = charge

V = electric potential Work

W = R12t= Elt = Pt Perfect gas law

Pressure

P = pressure

P = force (f)/unit area

V = volume Weight

W = mg m

n = number of moles

mass

=

R = gas constant

g = acceleration of gravity

Force

T = absolute temperature

f = ma

F = kT

Hooke's law of elasticity

m = mass

k = spring constant

F = force

a = acceleration

T = tension Dalton's law of partial pressures

+

PV = V(p, + p2+ p3+ . . . p,) P = pressure of gas mixture

h = Planck's constant

p = pressure of each gas alone

v = frequency

Physical and chemical constants Avogadro's number

E = hv

Energy of a photon

V = volume

Dimensions of plane and solid figures N,

=

6.022 x 1023

Area of a square = l2

Faraday constant

F = 96,487 C . rnol-I

Suface area of a cube

Gas constant

R = 8.314 J . K-'

. mol

Volume of a cube

1.98 cal . K-'

. rnol

Circumferenceof a circle = 27rr

=

.

= 0.082 L atm

Planck's constant Speed of light in a vacuum

h c

. K-'

. mol

=

6.62 x 10-"ergs . s-'

=

1.58

=

2.997 x 101° cm . s-'

X

cal . s-I

= 186,000 mi . s-'

Chemical definitions 1 rnol = the mass in grams of a substance equal to its molecular or atomic weight: this mass contains Avogadrok number (N,) of molecules or atoms Molar volume = the volume occupied by a mole of gas at standard temperature and pressure (25"C, 1 atm) = 22.414 L

1 molal solution = 1 mol per 1000 g of solvent 1 molar solution= 1 mol of solute in 1 L of solution 1 equivalent = 1 mot of 1 unit charge

1 einstein = Irnol of photons

=

= 61'

l3

Area of a circle = 7rr2 Surface area of a sphere = 47rr2 Volume of a sphere = 4/3m3 Surface area of a cylinder

=

27rrh

Volume of a cylinder = 7rr2h

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'

a-adrenergic receptors Receptors on cell surfaces that bind norepinephrine and, less effectively, epinephrine; the binding leads to enzymatically mediated responses by the cells. A band A region of a muscle sarcomere that corresponds to the myosin thick filaments. abomasum The true digestive stomach of the ruminant digastric stomach. absolute temperature Temperature measured from absolute zero, the state of no atomic or molecular thermal agitation. The absolute scale is divided into kelvin units (K), with 1°K having the same size as 1 Celsius degree. Thus, 0°K is equal to - 273.15"C or - 459.67"F. acclimation The persisting change in a specific function due to prolonged exposure to an environmental condition such as high or low temperature. acclimatization The persisting spectrum of changes due to prolonged exposure to environmental conditions such as high or low temperature. accommodation The temporary increase in threshold that develops during the course of a subthreshold stimulus. acetylcholinesterase An enzyme that hydrolyzes ACh and resides on postsynaptic membrane surface. acetylcholine (ACh) An acetic acid ester of choline (CH3-CO-O-CH,-CH,-N(CH3)3-OH), important as a synaptic transmitter in most species and in many different types of neurons. acid Proton donor. acidosis Excessive body acidity. acid tide Acidification of the blood following pancreatic secretion. acini Plural of acinus. See acinus. acinus A small sac or alveolus, sometimes lined with exocrinesecreting cells. acromegaly Hypersecretion of growth hormone in adulthood, causing enlargement of the skeletal extremities and facial structures. actin A ubiquitous protein that participates in muscle contraction and other forms of cellular motility. G-actin is the globular monomer that polymerizes to form F-actin, the backbone of the thin filaments of the sarcomere of muscle. action potential (nerve impulse; spike; AP) Transient all-or-none reversal of a membrane potential produced by regenerative inward current in excitable membranes. action spectrum The degree of response to different wavelengths of incident light.

activating reaction A reaction that changes an inactive enzyme into an active catalyst. activation energy The energy required to bring reactant molecules to velocities sufficiently high to break or make chemical bonds. activation heat Heat produced during excitation and activation of muscle tissue, but independent of shortening. active hyperemia The increase in blood flow that follows increased activity in a tissue, particularly skeletal muscle. active site The catalytic region of an enzyme molecule. active state In muscle, the condition when myosin cross-bridges are attached to actin, causing the muscle fibers to resist a force that would pull them to a greater length. active transport Energy-requiring translocation of a substance across a membrane, usually against its concentration or electrochemical gradient. Primary transport: Transport of a substance directly related to hydrolysis of ATP or other ~hosphagen.Secondary transport: Uphill transport of one substance coupled to and energy derived from the downhill transport of another substance. active zone Local region, within a presynaptic terminal, at which synaptic vesicles dock and are prepared for release by exocytosis. activity Capacity of a substance to react with another; the effective concentration of an ionic species in the free state. activity coefficient A proportionality factor obtained by dividing the effective reactive concentration of an ion (as indicated by its properties in a solution) by its molar concentration. actomyosin A complex of muscle proteins formed when myosin cross-bridges bind to actin in thin filaments. acuity Resolving power. adaptation Evolution through natural selection leading to an organism who's physiology, anatomy and behavior are matched to the demands of its environment. adaptation (sensory) Decrease in sensitivity during sustained presentation of a stimulus. adaptive See adaptation. addiction A physiological state of chemical dependence in which neuronal function shifts so that the individual experiences intense-even life-threatening-discomfort unless there are regular doses of the exogenous chemical. adductor muscle One that br.ings a limb toward the median plane of the body.

adenine A white, crystalline base 6-amino-purine, CSHSNs;purine base constituent of DNA and RNA. adenohypophysis (anterior pituitary gland; anterior lobe) The glandular anterior lobe of the hypophysis, consisting of the pars tuberalis, pars intermedia, and pars distalis. adenosine diphosphate (ADP) A nucleotide formed by hydrolysis of ATP, with the release of one high-energy bond. adenosine triphosphatase See ATPase. adenosine triphosphate (ATP)An energy-rich nucleotide used as a common energy currency by all cells. adenylate cyclase (adenyl cyclase) A membrane-bound enzyme that catalyzes the conversion of ATP to CAMP. adipose Fatty. ADP See adenosine diphosphate. Adrenalin Trade name for epinephrine. adrenal medulla Central portion of the adrenal gland. adrenergic Relating to neurons or synapses that release epinephrine, norepinephrine, or other catecholamines. adrenergic receptors (adrenoreceptors)Receptors on cell surfaces that bind norepinephrine and epinephrine; the binding leads to enzymatically mediated responses by the cells. adrenocorticotropic hormone (ACTH; adrenocorticotropin; corticotropin) A hormone released by cells in the adenohypophysis that acts mainly on the adrenal cortex, stimulating growth and corticosteroid production and secretion in that organ. adrenoreceptors See adrenergic receptors. aequorin A protein extracted from the jellyfish Aequorea; on combining with Ca2+,it emits a photon of blue-green light. aerobic Utilizing molecular oxygen. aerobic metabolic scope The ratio of the maximum sustainable metabolic rate to the BMR (or the SMR). aerobic metabolism Metabolism utilizing molecular oxygen. afferent Transporting or conducting toward a central region; centripetal. afferent fiber (afferent neuron) An axon that relays impulses from a sensory receptor to the central nervous system. affinity sequence (selectivity sequence) The order of preference with which an electrostatic site will bind different species of counterions. agiomerular Lacking glomerulae in the kidney. agonist A substance that can interact with receptor molecules and mimic an endogenous signalling molecule. aldehydes A large class of substances derived from the primary alcohols by oxidation and containing the -CHO group. aldosterone A mineralocorticoid secreted by the adrenal cortex; the most important electrolyte-controlling steroid, acting on the renal tubules to increase the reabsorption of sodium. alimentary canal A hollow, tubular cavity extending through animals open at both ends; for ingestion, digestion and secretion of food materials alkali earth metals A group of grayish-white, malleable metals easily oxidized in air, comprising Be, Mg, Ca, Sr, Ba, and Ra. alkaline tide A period of increased body and urinary alkalinity associated with excessive gastric HCI secretion during digestion. alkaloids A large group of organic nitrogenous bases found in plant tissues, many of which are pharmaceutically active (e.g., codeine, morphine). alkalosis Excessive body alkalinity.

dantoic membrane One of the membranes within a bird eggshell; important in the respiration of the unhatched chick. allantoin Waste product of purine metabolism. allometry Systematic changes in body proportions with increasing species size. all-or-none Describes a condition in which the magnitude of a cell's response is independent of the strength of a stimulus above some threshold value. If an input signal brings the cell to its threshold, the amplitude of the response is maximal; if the stimulus fails to bring the cell to threshold, there is no response. allosteric site Area of an enzyme that binds a substance other than the substrate, changing the conformation of the protein so as to alter the catalytic effectiveness of the active site. a-bungarotoxin neurotoxin in the venom of the krait (a snake) that blocks neuromuscular transmission in vertebrates by binding to nicotinic acetylcholine (ACh)receptors. alpha helix Helical secondary structure of many proteins in which each NH group is hydrogen bonded to a CO group at a distance equivalent to three amino acid residues; the helix makes a complete turn for each 3.6 residues. alpha motor neurons Large spinal neurons that innervate extrafusal skeletal muscle fibers of vertebrates. alveolar ventilation volume The volume of fresh atmospheric air entering the alveoli during each inspiration. alveoli Small cavities, especially those microscopic cavities that are the functional units of the lung. amacrine cells Neurons without axons, found in the inner plexiform layer of the vertebrate retina. ambient Surrounding, prevailing. amide An organic derivative of ammonia in which a hydrogen atom is replaced by an acyl group. amine Derivative of ammonia in which at least one hydrogen atom is replaced by an organic group. amino acids Class of organic compounds containing at least one carboxyl group and one amino group; the alpha-amino acids, RCH(NH,)COOH, make up proteins. amino group -NH2 . ammonia NH, ; toxic, water-soluble, alkaline waste product of deamination of amino acids and uric acid. ammonotelic Pertaining to the excretion of nitrogen in the form of ammonia. ampere (A)MKS unit of electric current; equal to the current produced through a 1ohm (0)resistance by a potential difference of 1 volt (V); the movement of 1coulomb (C) of charge per second. amphipathic Pertaining to molecules bearing groups with different properties, such as hydrophilic or hydrophobic groups. . amphoteric Having opposite characteristics; behaving as either an acid or a base. amylase Carbohydrases that hydrolyze all but the terminal glycosidic bonds within starch and glycogen, producing disaccharides and oligosaccharides. anabolism Synthesis by living cells of complex substances from simple substances. anaerobic Oxygen-free. anaerobic metabolism Metabolism not utilizing molecular oxygen. anastomose To interconnect.

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s

anatomical dead space The nonrespiratory conducting pathways in the lung. androgens Hormones having masculinizing activity. aneurism Localized dilation of an artery wall. angiotensin A protein in the blood, converted from angiotensinogen by the action of renin; it first exists as a decapeptide (angiotensin I) that is acted upon by a peptidase, which cleaves it into an octapeptide (angiotensin II), a potent vasopressor and stimulator of aldosterone secretion. angiotensin II See angiotensin. anion Negatively charged ion; attracted to the anode or positive pole. anode Positive electrode or pole to which negatively charged ions are attracted. anoxemia A lack of oxygen in the blood. anoxia A lack of oxygen. antagonist remuscle A muscle acting in opposition to the movement of another muscle. antagonists Agents that inhibit, block, or counteract an effect; for example, antagonists of synaptic transmitters typically block the postsynaptic receptor molecules that bind the neurotransmitter. antenna1 gland Crustacean osmoregulatory organ. antibody An immunoglobulin, a four-chain protein molecule of a specific amino acid sequence; an antibody will interact only with the antigen that brought about its production or one very similar to it. antidiuretic hormone (ADH,vasopressin) A hormone made in the hypothalamus and liberated from storage in the neurohypophysis; acts on the epithelium of the renal collecting duct by stimulating osmotic reabsorption of water, thereby producing a more concentrated urine; also acts as a vasopressor. antigen A substance capable of bringing about the production of antibodies and of then reacting with them specifically. antimycin An antibiotic that is isolated from a Streptomyces strain; acts to block electron transport from cytochrome 6 to cytochrome c in the electron-transport chain. antiports Membrane transport proteins that transfer two solutes, each in the opposite direction. anus The opening of the alimentary canal through which feces are expelled. aorta The main artery leaving the heart. aortic body A nodule on the aortic arch containing chemoreceptors that sense the chemical composition of the blood. apical Pertaining to the apex; opposite the base. apnea The suspension or absence of breathing. apoenzyrneThe protein portion of an enzyme; the apoenzyme and coenzyme form the functioning holoenzyme. apolysis Release; loosening from. aporepressor A repressor gene product that, in combination with a corepressor, reduces the activity of particular structural genes. aquaporin 28kDa protein, tetramers of which form water channels in membranes. area centralis (fovea)In the mammalian retina, the area with the highest visual resolution due to the small divergence and convergence in the pathway linking photoreceptors to ganglion cells; in primates contains closely packed cone cells. arginine phosphate A pho~phor~lated nitrogenous compound

found primarily in muscle; acts as a storage form of high-energy phosphate for the rapid phosphorylation of ADP to ATP. arteriole A tiny branch of an artery; in particular, one nearest a capillary. arteriosclerosis A class of diseases marked by an increase in thickness and a reduction in elasticity of the arterial walls. association cortex Areas of the cerebral cortex that neither directly receives sensory information nor directly contributes to motor output; instead neurons of association cortex typically receive input from many sensory modalities and are broadly connected to other areas in the cortex and other brain centers. asynchronous muscle A type of flight muscle found in the thorax of some insects; contracts at a frequency that bears no one-toone relation to motor impulses. See also fibrillar muscle. ATP See adenosine triphosphate. ATPase (adenosine triphosphatase) A class of enzymes that catalyze the hydrolysis of ATP. ATPS Ambient temperature and pressure, saturated with water vapor; referring to gas volume measurements. atria A chamber that gives entrance to another structure or organ; usually used alone to refer to an atrium of the heart. atrial natriuretic peptide (ANP) One of a family of peptide hormones, cleaved from a single precursor peptide and produced in the cardiac atria, the physiological effects of which include increased urine output, increased sodium excretion, and a receptor-mediated vasodilation, the net result of whlch is lowered blood pressure. atrioventricular node Specialized conduction tissue in the heart, which, along with Purkinje tissue, forms a bridge for electrical conduction of the impulse from atria to ventricles. auditory cortex Regions of the cerebral cortex that are associated with hearing. auditory ossicle One of the bones of the middle ear (the malleus, the incus, and the stapes), connecting the tympatic membrane and the oval window. autocrine A hormonal pathway characterized by the production of a biologically active substance by a cell; the substance then binds to receptors on that same cell to initiate a cellular response. autoinhibition Self-inhibition. autonomic nervous system The efferent nerves that control involuntary visceral functions; classically subdivided into the sympathetic and parasympathetic sections. autoradiography The process of making a photographic record of the internal structures of a tissue by utilizing the radiation emitted from incorporated radioactive material. autorhythmicity The generation of rhythmic activity without extrinsic control. autotrophic Pertaining to the ability to synthesize food from inorganic substances by utilizing the energy of the sun or of inorganic compounds. Avogadro's law Equal volumes of different gases at the same temperature and pressure contain equal numbers of molecules. One mole (mol) of an ideal gas at 0°C and 1standard atmosphere (atm) occupies 22.414 liters (L). Avogadro's number equals 6.02252 X molecules/mol. axon The elongated cylindrical process of a nerve cell along which action potentials are conducted; a nerve fiber. axonemme Complex of microtubules and associated structures within the flagellar or ciliary shaft.

axon hillock The transitional region between an axon and the nerve cell body. axon terminal The end of each axon, which typically is the site where signals are passed to another neuron or other cell. axoplasm The cytoplasm within an axon. azide Any compound bearing the N,- group.

b-adrenergic receptors Epinephrine-binding sites; normally coupled to activation of adenylate cyclase. baroreceptor A sensory nerve ending that is stimulated by changes in pressure, as those in the walls of blood vessels. basal body (kinetosome) Microtubular structure from which a cilium or flagellum arises; homologous with centriole. basal cell A cell in a chemoreceptive organ that regularly gives rise to new chemoreceptor cells during adult life. basal metabolic rate (BMR) The rate of energy conversion in a homeotherm while it is resting quietly within the thermal neutral zone without food in the intestine. base Proton acceptor. basilar membrane The delicate ribbon of tissue bearing the auditory hair cells in the cochlea of the vertebrate ear. Bell-Magendie rule The dorsal root of the spinal cord contains only sensory axons, whereas the ventral root contains only nlotor axons. Bernoulli's effect Fluid pressure drops as fluid velocity increases. beta (b)adrenergicreceptors The class of adrenergic membrane receptors that are blocked by the drug propranolol; their activation is less sensitive to norepinephrine than is that of the alpha receptors. beta keratin Insoluble, sulfur-rich scleroprotein;constituent of epidermis, horns, hair, feathers, nails, and tooth enamel. Beta refers to the protein's secondary structure, which is in pleated sheets. beta pleated sheet Form of protein secondary structure in which two or more distinct amino acid chains lay side by side, held together by hydrogen bonds. bile Viscous yellow or greenish alkaline fluid produced by the liver and stored in the gallbladder; containing bile salts, bile pigments, and certain lipids, it is essential for digestion of fats. bile duct Duct carrying bile fluid from liver to duodenum. bide fluid Fluid secreted by liver that emulsifies fats and neutralizes acid intestinal contents. bile pigments Pigments in bile fluid derived from breakdown products of hemoglobin. bile salt Bile acid such as cholic acid conjugated with glycine or .taurine, promoting emulsification and solubilization of intestinal fats. binocular convergence Positioning of the eyes so that the images formed fall on analogous portions of the two retinas, avoiding double vision. biogenic amine Any of a number of signalling molecules that are synthesized in the body from single amino acid molecules. bipolar cell A neuron with two axons emerging from opposite sides of the soma; one class of these neurons is found in the vertebrate retina, where they transmit signals from the photoreceptor cells to the retinal ganglion cells. birefringence Double refraction; the ability to pass preferentially light that is polarized in one plane. bleaching Fading of photopigment color upon absorption of light.

blubber A fatty, insulating tissue typically found under the skin in cetaceans. Bohr effect (Bohr shift) A change in hemoglobin-oxygen affinity due to a change in pH. Bolus A discrete plug or collection of food material moving through the alimentary canal. bombykol Sex attractant pheromone of the female silkworm moth (Bombyx mori). book lungs The respiratory surface in spiders. Bowman's capsule (glomerular capsule) A globular expansion at the beginning of a renal tubule and surrounding the glomerulus. Boyle's law At a given temperature, the product of pressure and volume of a given mass of gas is constant. bradycardia A reduction in heart rate from the normal level. bradykinin A hormone formed from a precursor normally circulating in the blood; a very potent cutaneous vasodilator. brain The major neuronal center within the body; typically located at the anterior of the body. brain hormone (prothoracicotropin; activating hormone) A hormone synthesized by the neurosecretory cells of the pars intercerebralis and released by the corpora cardiaca of insects; activates the prothoracic glands to secrete ecdysone. bronchi Conducting airways in the lung; branches of the tracheae. bronchioles Small conducting airways in the lung; branches of the bronchi. brood spot A prolactin-induced bald area on the ventral surface of some brooding birds that receives a rich supply of blood for the incubation of eggs. brown fat Fat deposits with extensive vascularization, mitochondria and enzyme systems for oxidation. Found in small, specific deposits in a few mammals and used for nonshivering thermogenesis. Brunner's glands Exocrine glands that are located in the intestinal mucosa and secrete an alkaline mucoid fluid. brush border A free epithelial cell surface bearing numerous microvilli. BTPS Body temperature, atmospheric pressure, saturated with water vapor. buccal Pertaining to the mouth cavity. buffer A chemical system that stabilizes the concentration of a substance; acid-base systems serve as pH buffers, preventing large changes in hydrogen ion concentration. bundle of His The conducting tissue within the interventricular septum of the mammalian heart. bungarotoxin (BuTX) A blocking agent composed of a group of neurotoxins isolated from the venom of members of the snake genus Bungarus (the krait) of the cobra family; binds selectively and irreversibly to nicotinic acetylcholine receptors. Bunsen solubility coefficient The quantity of gas at STPD that will dissolve in a given volume of liquid per unit partial pressure of the gas in the gas phase. This coefficient is used only for gases that do not react chemically with the solvent. bursicon A hormone secreted by neurosecretory cells of the insect central nervous system; tans and hardens the cuticle of freshly molted insects. cable properties The passive electrical properties (resistance and capacitance) of a cell; these properties were first worked out for submarine cables.

GLOSSARY G-5 ......................................

calcitonin (thyrocalcitonin) A protein hormone secreted by the mammalian parafollicular cells of the thyroid in response to elevated plasma calcium levels. calcitriol A steroid-like compound produced from vitamin D ingested with some foods and from vitamin D3, or synthesized from cholesterol in the skin. The physiological actions of calcitriol are similar to those of parathyroid hormone. calcium response A graded depolarization due to a weakly regenerative inward calcium current. caldesmon A calcium-binding regulatory protein in smooth muscle, which plays a role in the "latch" mechanism of some smooth muscles. calmodulin A troponinlike calcium-binding regulatory protein found in essentially all tissues. calorie (cal)The quantity of heat required to raise the temperature of 1g of water from 14.5" to 15.S°C; most commonly used as kilocalorie (kcal) = 1000 cal. calorimetry Measurement of heat production in an animal. calsequestrin A calcium-binding protein that contributes to the regulation of contraction in smooth muscles. canines Pointed, dagger-like teeth are used for piercing and tearing food. capacitance The property of storing electric charge by electrostatic means. capacitive current Current entering and leaving a capacitor. capacity The ability of a capacitor or other body to store electric charge. The unit of measure is the farad (F), which describes the proportionality between charge stored and potential for a given voltage, C = q N = coulombs divided by volts. carbohydrases Enzymes that specifically break down carbohydrates. carbohydrate Aldehyde or ketone derivative of alcohol; utilized by animal cells primarily for the storage and supply of chemical energy; most important are the sugars and starches. carbonic anhydrase An enzyme catalyzing the reversible interconversion of carbonic acid to carbon dioxide and water. occurs in such comcarbonyl The organic radical-C=O,which pounds as aldehydes, ketones, carboxylic acids, and esters. carboxyhemoglobin The compound formed when carbon monoxide combines with hemoglobin; carbon monoxide competes successfully with oxygen for combination with hemoglobin, producing tissue anoxia. carboxylates R-COO-, salts or esters of carboxylic acids. carboxyl group The radical -COOH, which occurs in the carboxylic acids. cardiac output The total volume of blood pumped by the heart per " unit of time; cardiac output equals heart rate times stroke volume. carotid body A nodule on the occipital artery just above the carotid sinus, containing chemoreceptors that sense the chemical composition of arterial blood. carotid rete Countercurrent heat exchanger at base of skull that helps prevent overheating of the brain of certain hoofed animals and carnivores. carotid sinus A dllat~onof the Internal carot~dartery wlth many baroreceptor (pressure receptor) endlngs In the wall. carotid sinus baroreceptors Receptors that sense arter~alblood pressure; located in the carot~dsmus, a dllatat~onof the Internal carot~dartery at ~ t orrgln. s carrier-mediated transport Transmembrane transport of solutes

achieved by membrane embedded carrier (e.g., facilitated diffusion). carrier molecules Lipid-soluble molecules that act within biological membranes as carriers for certain molecules that have lower mobility in the membrane. catabolism Disassembly of complex molecules into simpler ones. catalysis An increase in the rate of a chemical reaction promoted by a substance-the catalyst-not consumed by the reaction. catalyst A substance that increases the rate of a reaction without being used up in the reaction. cataract A condition in which proteins of the lens of the eye become cross-linked, causing it to become cloudy and reducing visual acuity. catecholamines A group of related compounds exerting a sympathomimetic action on nervous tissue; examples are epinephrine, norepinephrine, and dopamine. catecholandues A group of related compounds exerting a sympathomimetic action on nervous tissue; examples are epinephrine, norepinephrine, and dopamine. cathode The negative electrode, so called because it is the electrode to which cations are attracted. cation A positively charged ion; attracted to a negatively charged electrode. caudal Pertaining to the tail end. cecum A blind pouch in the alimentary canal. Cellulase An enzyme that digests cellulose and hemicellulose, produced by gut-borne symbiotic micro-organisms. central chemoreceptors Sensory structures in the brain monitoring pH and initiating appropriate changes in breathing. central lacteal Small, blind-ended lymph vessel in center of intestinal villus for the uptake of fats and some vitamins. central nervous system The collection of neurons and parts of neurons that are contained within the brain and spinal cord of vertebrates, or within the brain, ventral nerve cord, and major ganglia of invertebrates. central pattern generator A group of neurons that produces and maintains a rhythmic pattern of action, such as breathing, walking, chewing, or swimming. central pattern-generating network A set of interconnected neurons whose collective activity produces patterned behavior. central sulcus A deep, almost vertical furrow on the cerebrum, dividing the frontal and parietal lobes. cephalic Pertaining to the head. cephalic phase Gastric secretion occurring in response to sight, smell, and/or taste of food, or in response to conditioned reflexes. cephalization The evolutionary tendency for the neurons of higher organisms to be concentrated in a brain located at the anterior end of the animal. cerebellum A part of the hindbrain that contributes to the coordination of motor output. cerebral cortex The thin layer of gray matter that covers the cerebrum of mammals, and probably of birds. cerebral hemispheres The large paired structures of the cerebrum, connected by the corpus callosum. cerebral ventricles A series of confluent fluid-filled cavities within the brain of vertebrates; the fluid in the ventricles is cerebrospinal fluid. cerebrospinal fluid In vertebrates, clear fluid that fills the cavities

G-6

GLOSSARY

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(ventricles)within the brain and the central canal of the spinal cord; it is a complex filtrate of blood plasma and is modified by brain cells before it returns to the venous system. cerebrum The largest part and highest center of the mammalian brain; it evolved from the olfactory centers of lower vertebrates. charge, electric ( q ) Measured in units of coulombs (C). To convert 1 g .equiv weight of a monovalent ion to its elemental form (or vice versa) requires a charge of 96,500 C (1faraday, F). Thus, in loose terms, a coulomb is equivalent to 1196,500 g .equiv of electrons. The charge on one electron C. If this is multiplied by Avogadro's number, is - 1.6 x the total charge is 1 F, or -96,487 C . molk'. chelating agent A chemical that binds calcium, or other ions, and removes it from solution. chemical energy Energy contained in the chemical bonds holding molecules together. chemical synapse A junction between a neuron and another cell in which the signal from the presynaptic neuron is carried across the synaptic cleft by neurotransmitter molecules. chemoreceptor A sensory receptor specifically sensitive to certain molecules. chief cells (zygomatic)Ep~thelialcells of the gastric epithelium that release pepsin. chimera An animal with extra copies of a normal gene or a copy of a mutated gene. chitin A structural polymer of D-glucosamme that serves as the primary constituent of arthropod exoskeletons. chloride cells Epithelial cells of fish gills that engage in active transport of salts. chloride shift The movement of chloride ions across the red blood cell membrane to compensate for the movement of bicarbonate ions. chlorocruorin A green respiratory pigment found in some marine polychaetes; similar to hemoglobin. choanocytes Flagellated cells lining the body cavity of sponges. cholecystokinin (CCK, pancreozymin; CCK-PZ) A hormone liberated by the upper intestinal mucosa that induces gallbladder contraction and release of pancreatic enzymes. cholesterol A natural sterol; precursor to the steroid hormones. cholinergic Relating to acetylcholine or substances with actions similar to ACh. choroidplexus Highly vascularized, furrowed projections into the brain ventricles that secrete cerebrospinal fluid. choroid rete A countercurrent arrangement of arterioles and venules behind the retina in the eyes of teleost fish. chromaffin cells Epinephrine-secreting cells of the adrenal medulla; named for their high affinity for chromium salt stains. chromatography A general technique that exploits the fact that different components in a sample will move at different rates through a substrate such as chromatography paper or other porous solid matrices. chromophore A chemical group that lends a dist~nctcolor to a compound containing it. chronobiology The study of biological rhythms. chronotropic Pertaining to rate or frequency, especially in reference to the heartbeat. chylomicrons Protein-coated tiny droplets of triglycerides, phospholipids and cholesterol formed within vesicles of absorptive

cells from the digestion produces of fats, monoglycerides, fatty acids, and glycerol. chyme The mixture of partially digested food and digestive juices found in the stomach and the intestine. chymotrypsin A proteolytic enzyme that specifically attacks peptide bonds containing the carboxyl groups of tyrosine, phenylalanine, tryptophan, leucine, and methionine. chymotrypsinogen Inactive precursor of chymotrypsin. ciliary body A thick region of the anterior vascular tunic of the eye; joins the choroid and the iris. ciliary muscle A muscle of the ciliary body of the vertebrate eye; influences the shape of the lens in visual accommodation. ciliary reversal A change in the direction of the power stroke of a cilium, causing it to beat in reverse. cilium A motile organelle with a "9 + 2" tubular substructure; when present generally in large numbers, a small flagellum. circadian rhythms Biological rhythms with daily cycles. circalunar Referring to biorhythms related to lunar cycles. circannual Referring to biorhythms related to yearly cycles. circatidal Referring to biorhythms related to tidal cycles. circular smooth muscle Inner layer of smooth muscle that encircles small intestine. cis A configuration with similar atoms or groups on the same side of the molecular backbone. cis-trans isomerization Conversion of a cis isomer into a trans isomer. citric acid cycle (also Krebs cycle, TCA cycle) A series of eight major reactions following glycolysis, in which acetate residues are degraded to CO, and H20. cladogram A form of diagrammatic analysis of taxonomic relationships between organisms that relates animals according to suites of common characters. clathrin The protein surrounding the cytoplasmic surface of a coated vesicle membrane. clefts Lateral intercellular spaces between cells in epithelium. cloaca The terminal area of hindgut of some fishes, amphibians, reptiles, birds and a few mammals; aids in urinary ion and water resorption. clone A population of genetically identical cells all derived from a single original cell. coated pits Receptor-lined membrane depressions that eventually form coated vesicles during the process of receptor-mediated endocytosis. coated vesicle Vesicle with its cytoplasmic surface covered with the protein clathrin, formed in process of receptor-mediated endocytosis. cochlea A portion of the inner ear, a tapered tube would into a spiral like the shell of a snail, containing hair cell receptors for detecting sound. cochlear microphonics Electrical signals recorded from the cochlea, having a frequency identical to that of the sound stimulus. coelenteron A blind-ended tube or cavity in coelenterates that serves as a "batch reactor" site for chemical digestion. coelom The body cavity of higher metazoans, situated between the gut and the body wall and lined with mesodermal epithelium. coenzyme An organic molecule that combines with an apoenzyme to form the functioning holoenzyme.

GLOSSARY G-7 ......................................

coenzyme A (CoA) A derivative of pantothenic acid to which acetate becomes attached to form acetyl CoA. cofactor An atom, ion, or molecule that combines with an enzyme to activate it. coitus Sexual intercourse. colchicine An antimitotic agent that disrupts microtubules by interfering with the polymerization of tubulin monomers. collar cells Flagellated cells h i n g the internal chambers of sponges (Porifera). collateral processes Branches of an axon that terminate in locations other than the major target location. collaterals Side branches of a nerve or blood vessel. collecting duct The portion of the renal tubules in which the final concentration of urine occurs. colligative properties Characteristics of a solution that depend on the number of molecules in a given volume. colloid A system in which fine solid particles are suspended in a liquid. command system A set of neurons that, when stimulated, elicit a set pattern of coordinated movements. competitive inhibition Reversible inhibition of enzyme activity due to competition between a substrate and an inhibitor for the active site of the enzyme. compliance The change in length or volume per unit change in the applied force. compound eye The multifaceted arthropod eye; the functional unit is the ommatidium. concentration gradient Difference in solute concentration across a membrane, or between two different regions of a solution. condensation A reaction between two or more organic molecules leading to the formation of a larger molecule and the elimination of a simple molecule, such as water or alcohol. conditioned reflex Reflexes learned or modified through behavioral repetition. conductance, electrical (G)A measure of the ease with which a conductor carries an electric current; the unit is the siemen (S), re-ciprocal of the ohm (a). conductance, thermal A quantity describing the ease with which heat flows by conduction under a temperature gradient across a substance or an object. conductivity The intrinsic property of a substance to conduct electric current; reciprocal of resistivity. conductor A material that carries electric current. cone A vertebrate visual receptor cell that has a tapered outer segment in which the lamellar photosensitive membranes remain ' continuous with the surface membrane. confocal scanning microscope A microscope using a focused laser beam to rapidly scan different areas of the specimen in a single plane. Light reflected from that plane is assembled by a computer into a composite image of the specimen. conformer An animal in which internal body fluid condition tends to parallel that of the external environment. conjugate acid-base pair Two substances related by gain or loss of an H+ ion (proton). connective A collection of axons that carry information between neuronal centers, such as ganglia, in many invertebrate nervous systems. contracture A more or less sustained contraction in response to an abnormal stimulus.

contralateral Pertaining to the opposite side. conus A chamber invested with cardiac muscle and found in series with and downstream from the ventricle in elasmobranchs. convection The mass transfer of heat due to mass movement of a gas or liquid. convergence A pattern in which inputs from many different neurons impinge upon a single neuron. corepressor A low-molecular-weightmolecule that unites with an aporepressor to form a substance that inhibits the synthesis of an enzyme. cornea The clear surface of the eye through which light passes as it enters the eye. corneal lens The clear structure at the outside surface of an ommatidium; it admits and focuses light entering the ommatidium. corpora allata Nonneuronal insect glands existing as paired organs or groups of cells dorsal and posterior to the corpus cardiaca; the corpora allata secrete juvenile hormone (JH). corpora cardiaca Major insect neurohemal organs existing as paired structures immediately posterior to the brain; they liberate brain hormone. corpus luteum The yellow ovarian glandular body that arises from a mature follicle that has released its ovum; it secretes progesterone. If the ovum released has been fertilized, the corpus luteum grows and secretes during gestation; if not, it atrophies and disappears. cortex External or surface layer of an organ. corticospinal tract A group of axons of neurons whose somata and dendrites are located in the motor cortex and whose axon terminals synapse on motor neurons in the spinal cord. corticotropin A hormone released by cells in the adenohypophysis that acts mainly on the adrenal cortex, stimulating growth and corticosteroid production and secretion in that organ. cortisol A steroid hormone secreted by the adrenal cortex. cotransmitter A second neurotransmitter molecule synthesized in and released from an axon terminal, along with a small transmitter molecule such as acetylcholine or GABA. cotransport Carrier-mediated transport in which two dissimilar molecules bind to two specific sites on the carrier molecule for transport in the same direction. coulomb (C)MKS unit of electric charge; equal to the amount of charge transferred in 1 second (s) by 1 ampere (A) of current. See also charge, electric. counter current heat exchanger A specialized parallel arrangement of incoming arteries and outgoing veins forming a heat exchanger that conserves heat in the body core. countercurrent multiplier A pair of opposed channels containing fluids flowing in opposed directions and having an energetic gradient directed transversely from one of the channels into the other. Since exchange due to the gradient is cumulative with distance, the exchange per unit distance will be multiplied, so to speak, as a function of the total distance over which exchange takes place. counterion An ion associated with, and of opposite charge to, an ion or an ionized group of a molecule. countertransport The uphill membrane transport of one substance driven by the downhill diffusion of another substance. coupled transport Uptake of one substance into a cell that depends on the downhill diffusion exit of another substance into the cell.

G-8

GLOSSARY

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covalent bond A bond formed by electron-pair sharing between two atoms. creatine phosphate (phosphocreatine)A phosphorylated nitrogenous compound found primarily in muscle; acts as a storage form of high-energy phosphate for the rapid phosphorylation of ADP to ATP. creatinine Nitrogenous waste product of muscle creatine. cretinism A chronic condition caused by hypothyroidism in childhood; characterized by arrested physical and mental development. cristae The folds of the inner mitochondria1 membrane. critical fusion frequency The number of light flashes per second at which the light is perceived to be continuous. critical thermal maximum The temperature above which longterm survival is not possible. crop A muscular organ in the upper alimentary canal of birds; receives swallowed food items and small stones, and churns the mixture together to break down the food into more digestible particles. crop milk A nutrient-rich substance fed by regurgitation to pigeon chicks by both parents. cross-bridges Spirally arranged projections from myosin thick filaments that bind to sites on actin thin filaments during muscle contraction. crustecdysone A steroid hormone that promotes molting in crabs. crypt of Lieberkiihn A circular depression around the base of each villus in the intestine. cupula A small upside-down cup or domelike cap; in the lateralline and the vertebrate organs of equilibrium, the cupula covers hair cells in a gelatinous matrix. curare (D-tubocurarine) South American arrow poison; blocks synaptic transmission at the motor endplate by competitive inhibition of nicotinic acetylcholine receptors. current, electric The flow of electric charge. A current of 1 coulomb (C) per second is called an ampere (A). By convention the direction of current flow is the direction that a positive charge moves (i.e., from the anode to the cathode). cuticle The hard outer coat of insects and crustacea secreted by an epidermal layer, the hypodermis. cyanide A compound containing cyanogen and one other element; blocks transfer of electrons from the terminal cytochrome a and a, to oxygen in the respiratory chain. cybernetics The science of information communication and control in animals and machines. cyclic AMP (CAMP)A ubiquitous cyclic nucleotide (adenosine 3'5'-cyclic monophosphate) produced from ATP by the enzymatic action of adenylate cyclase; important cellular regulatory agent that acts as the second messenger for many hormones and transmitters. cyclic GMP (cGMP) A cyclic nucleotide (guanosine 3'5'-cyclic monophosphate) analogous to CAMP but present in cells at a far lower concentration and producing target cell responses that are usually opposite to those of CAMP. cyclostomes A group of jawless vertebrates, including lampreys and hagfishes. cytochalasin A drug that disrupts cytoplasmic microfilaments. cytochromes A group of iron-containing proteins that function in the electron-transport chain in aerobic cells; they accept and pass on electrons.

cytoplasm The semifluid substance within a cell, exclusive of the nucleus but including other organelles. cytosine Oxyamino-pyrimidine, C,H,N,O; base component of nucleic acid. cytosol The unstructured aqueous phase of the cytoplasm between the structured organelles. D600 Methoxyverapamil; an organic drug that blocks calcium influx through cell membranes. D-tubocurarine See curare. Dalton's law The partial pressure of a gas in a mixture is independent of other gases present. The total pressure is the sum of the partial pressures of all gases present. dark current Steady sodium current that leaks into a vertebrate visual receptor cell at the outer segment. The sodium is actively pumped out of the inner segment, completing the circuit. The dark current is reduced by photoexcitation. decerebration Experimental elimination of cerebral activity by section of the brain stem or by interruption of the blood supply to the brain. decussation Crossing over from one side to the other. defecation The process of expelling feces. dehydrogenase An enzyme that "loosens" the hydrogen of a substrate in preparation for passage to a hydrogen receptor. dehydroretinal An aldehyde of dehydroretinol. dehydroretinol (retinol2; vitamin A2) The form of vitamin A occurring in the liver and the retina of freshwater fishes, some invertebrates, and amphibians. delayed outward current (late outward current) Current carried by Kt through channels that open with a time lag after onset of a depolarization; responsible for repolarization of the action potential. denaturation Alteration or destruction of the normal nature of a substance by chemical or physical means. dendrites Fine processes of a neuron, often providing the main receptive area of the cell onto which synaptic contacts are made. densitometer An instrument that measures the amount of exposure of the film emulsion produced by autoradiography. deoxyhemoglobin Hemoglobin in which oxygen is not combined to the Fe,, of the heme moiety. deoxyribonucleic acid See DNA. depolarization The reduction or reversal of the potential difference that exists across the cell membrane at rest. desmosome A type of cell junction serving primarily to aid the structural bonding of neighboring cells. diabetes mellitus A metabolic malady in which there is a partial or complete loss of activity in the pancreatic islets; the concomitant insulin insufficiency leads to inadequate uptake of glucose into cells and loss of blood glucose in the urine. diacylglycerol (DAG) A diglyceride, present as a constituent of cellular phospholipids; when released from these phospholipids by an agonist-activated phospholipase, this molecule serves as the endogenous activator of the calcium- and calmodulin-dependent protein kinase (protein kinase C) and is part of an important intracellular signalling cascade. dialysis The process by which crystalloidsand macromolecules are separated by utilizing the differences in their diffusion rates through a semipermeable membrane. diaphragm The dome-shaped muscle that separates the thoracic

and abdominal cavities and serves as the chief muscle of respiration. diastole The phase in the heartbeat during which the myocardium is relaxed and the chambers are filling with blood. dielectric constant A measure of the degree to which a substance is able to store electric charge under an applied voltage; the dielectric constant of a material depends on charge distribution within molecules. diffusion Dispersion of atoms, molecules, or ions as a result of random thermal motion. diffusion coefficient Coefficient relating rate of diffusional flux to concentration gradient, path length, and area across which diffusion occurs. digastric stomach The multichambered stomach of ruminants. digestive enzymes Enzymes secreted by alimentary canal to aid in chemical digestion. digestive epithelium Epithelium lining the small intestine. dimer A molecule made by the joining of two molecules (i.e., monomers) of the same kind. dinitrophenol (DNP) Any of a group of six isomers, C,H,(OH)(NO,), ,that act as aerobic metabolic inhibitors by virtue of their ability to uncouple oxidation from phosphorylation in mitochondria1 electron transport. dipole A molecule with separate regions of net negative and net positive charge, so that one end acts as a positive pole and the other as a negative pole. dipole moment The electrostatic force required to align a dipolar molecule parallel to the electrostatic field; the force required increases as the separation of the molecular charges decreases. disaccharide sugars A double sugar formed when two monosaccharides (single sugars) are joined together by dehydration synthesis. disinhibition Release of a neuron from inhibitory input when the inhibitory neuron is, itself, synaptically inhibited. dissociation Separation; resolution by thermal agitation or solvation of a substance into simpler constituents. dissociation constant K' = [H][A-]/[HA]. The empirical measure of the degree of dissociation of a conjugate acid-base pair in solution. distal More distant from a point of reference in the centrifugal direction; i.e., away from the center. distal tubule The renal tubules located in the renal cortex leading from (and continuous with) the ascending limb of the loop of Henle to the collecting duct. distance of closest approach Shortest possible span between the centers of two atoms. disulfide linkage Bond between sulfide groups that determines protein tertiary structure by linking together portions of polypeptide chains. diuresis An increase in urine excretion. diuretic An agent that increases urine secretion. divalent Carrying an electric charge of two units; a valence of 2. divergence A pattern in which the axon of a single neuron branches, allowing it to synapse onto more than one synaptic target. DNA (deoxyribonucleic acid) The class of nucleic acids responsible for hereditary transmission and for the coding of amino acid sequences of proteins. Donnan equilibrium Electrochemical equilibrium that develops

when two solutions are separated by a membrane permeable to only some of the ions of the solutions. dopanitine (hydroxytyramine) A product of the decarboxylation of dopa, an intermediate in norepinephrine synthesis; a central nervous system transmitter. dormancy The general term for reduced body activities, including sleep, torpor, hibernation, "winter sleep," and estivation. dorsal horn The dorsal part of the gray matter in the vertebrate spinal cord; contains the cell bodies of neurons that receive, process, and transmit sensory information. dorsal root A nerve trunk that enters the spinal cord near the dorsal surface; contains sensory axons only. dorsal root ganglion (DRG) On the surface of the dorsal root, a collection of the cell bodies of sensory neurons that send processes into the region of the body that is innervated by that spinal segment; each spinal segment contains bilaterally paired DRGs. drag The resistance to movement of an object through a medium, increasing with the viscosity and density of the medium and the surface area and shape of the object. ductus arteriosus The vessel connecting the pulmonary artery and the aorta in fetal mammals. duodenum The initial section of the small intestine, situated between the pylorus of the stomach and the jejunum. dwarfism An abnormally small size in humans; a result of insufficient growth hormone secretion during childhood and adolescence. dynamic range The range of energy over which a sensory system is responsive and can encode information about stimulus intensity. dynein A ciliary protein with magnesium-activated ATPase activity. dynein arms Projections from tubule A of one microtubule doublet toward tubule B of the next, composed of a protein exhibiting ATPase activity. dyspnea Labored, difficult breathing.

early inward current Depolarizing current of excitable tissues, carried by Na+ or Ca2+;responsible for the upstroke of the action potential. early receptor potential (ERP)An almost instantaneous potential change recorded from the retina in response to a short flash of light that probably corresponds to a movement of charge that occurs as the photopigment undergoes conformational change. eccentric cell In Limulus, the afferent neuron of each ommatidium; it is surrounded by and receives information from photoreceptive retinular cells. ecdysis Shedding of the outer shell; molting in an arthropod. ecdysone A steroid hormone secreted by the thoracic gland of arthropods that induces molting. echolocation The ability of some species to recognize and locate objects in their environment by emitting sounds and receiving the sound that is reflected back to them by the objects. eclosion hormone Insect hormone that induces the emergence of the adult from the puparium. ectopic pacemaker A pacemaker situated outside the area where it is normally found. ectoplasm The gel-state cytoplasm surrounding the endoplasm.

G-10 GLOSSARY ......................................... ectotherms Refers to animals whose body temperature is dependent on heat in the environment. edema Retention of interstitial fluid in organs or tissues. EDTA Ethylenediaminetetraacetic acid; a calcium- and magnesium-chelating agent. effector A cell, tissue, or organ that acts to change the condition of an organism (e.g., by contracting muscles or secreting a hormone) in response to neuronal or hormonal signals. efferent (nerve or neuron) Centrifugal; a neuron that carries information from higher brain centers toward structures in the periphery. efflux Movement of solute or solvent out of a cell across the cell membrane. EGTA Ethyleneglycol-bis(p-aminoethylether)-N,N'-tetraacetic acid; a calcium chelating agent. elastic Capable of being distorted, stretched, or compressed with subsequent spontaneous return to original shape; resilient. electrical potential Electrostatic force, analogous to water pressure; a potential difference (i.e., voltage) is required for the flow of electrical current across a resistance. electrical synapse A junction between two cells at which a signal is carried from one cell to the other by the passage of charged particles through gap junctions. electrocardiogram (ECG) The record of electrical events associated with contractions of the heart; obtained with electrodes placed on other portions of the body. electrochemical equilibrium The state at which the concentration gradient of an ion across a membrane is precisely balanced by the electric potential across the membrane. electrochemical gradient The sum of the combined forces of concentration gradient and electrical gradient acting on an ion. electrochemical potential The electrical potential developed across a membrane due to a chemical concentration gradient of an ion that can diffuse across the membrane. electrode An electrical circuit element used to make contact with a solution, a tissue, or a cell interior; used either to measure potential or to carry current. electrogenic Giving rise to an electric current or voltage. electrolyte A compound that dissociates into ions when dissolved in water. electromotive force (emf) The potential difference across the terminals of a battery or any other source of electric energy. electronegativity Affinity for electrons. electroneutrality rule For a net potential of zero, the positive and negative charges must add up to zero; a solution must contain essentially as many anionic as cationic charges. electron shells Energy levels of electrons surrounding the nucleus. electron-transport chain (respiratory chain) A series of enzymes that transfer electrons from substrate molecules to molecular oxygen. electro-olfactogram An extracellular electrical recording of the summed activity in many olfactory receptors. electro-osmosisMovement of water through a membrane of fixed charge in response to a potential gradient. electrophoresis A technique for separating proteins using the net positive or negative charge on their surface amino acids. electroplax organ An organ in electric fishes such as Torpedo that builds up and delivers a powerful electrical charge sufficient to stun or even to kill prey. The electroplax organ is derived

from embryonic muscle and uses synapses similar to neuromuscular junctions to build up the charge. electroreceptors Sensory receptors that detect electrical fields. electroretinogram (ERG) An extracellular electrical recording, made at the surface of the eye, of activity in many visual receptors and other retinal neurons. electrotonic potential A graded potential generated locally by currents flowing across the membrane; not actively propagated and not all-or-none. Embden-Meyerhof pathway See glycolysis. endergonic Characterized by a concomitant absorption of energy. endocardium The internal lining of the heart chamber. endocrine A hormonal pathway characterized by the production of a biologically active substance by a ductless gland; the substance then is carried through the bloodstream to initiate a cellular response in a distal target cell or tissue. endocrine glands Ductless organs or tissues that secrete a hormone into the circulation. endocytosis Bulk uptake of material into a cell by membrane inpocketing to form an internal vesicle. endogenous Arising within the body. endogenous opioids Neurotransmitter or neuromodulator molecules (e.g., endorphins and enkephalins) whose receptors bind the opioid drugs, such as opium and heroin; in some parts of the nervous system, when these transmitters bind to their receptor molecules, the net result is reduction in the perception of pain. endolymph Aqueous liquid with a high K+ concentration and a low Na+ concentration found in the vertebrate organs of equilibrium and in the scala medza of the cochlea. endometrium An epithelium that lines the uterus. endopeptidases Proteolytic enzymes that break up large peptide chains into shorter polypeptide segments. endoplasm The sol-state cytoplasm that streams within the cell interior. endorphins Endogenous neuropeptides that exhibit morphinelike actions, found in the central nervous system of vertebrates; different types consist of 16,17, or 31 amino residues. endothelium Single cell layer forming the internal lining of blood vessels. endotherms animals whose body temperature is dependent on heat production by the body. endplate A traditional name of the vertebrate neuromuscular synapse, where the motor axon forms many fine terminal branches that end over a specialized system of folds in the postsynaptic membrane of the muscle cell. endplate potential (epp)A postsynaptic potential in the muscle at the neuromuscular junction (or motor endplate). end-product inhibition (feedback inhibition) Inhibition of a biosynthetic pathway by the end product of the pathway. energy Capacity to perform work. energy metabolism The complex collection of biochemical reactions within cells that generate ATP and other high-energy compounds, which serve as the immediate source of energy for all biological events. enkephalins Endogenous nuropeptides exhibiting morphinelike actions, found in the central nervous system of vertebrates; these peptides consist of five amino acid residues.

GLOSSARY G-11 ........................................

enterogastric reflex A reflex that inhibits gastric secretion, triggered when duodenum is stretched by chyme pumped from the stomach. enterogastrone A hormone that is secreted from the duodenal mucosa in response to fat ingestion and that suppresses a gastric motility and secretion. enterokinase Intestinal proteolytic enzyme. enthalpy The heat produced or taken up by a chemical reaction. entropy Measure of that portion of energy not available for work in a closed system; measure of molecular randomness. Entropy increases with time in all irreversible processes. enzyme A protein with catalytic properties. enzyme activity A measure of the catalytic potency of an enzyme: the number of substrate molecules that react per minute per enzyme molecule. enzyme induction Enzyme production stimulated by the specific substrate (inducer) of that enzyme or by a molecule structurally similar to the substrate. epicardium The external covering of the heart wall. epididymis A long, stringlike duct along the dorsal edge of the testis; its function is to store sperm. epinephrine Generic name for the catecholamine released from the adrenal cortex; also known by the trade name Adrenalin. equilibrium The state in which a system is in balance as a result of equal action by opposing forces arising from within the system. equilibrium potential The voltage difference across a membrane at which an ionic species that can diffuse across the membrane is in electrochemical equilibrium; it is dependent on the concentration gradient of the ions and is described by the Nernst equation. equimolar Having the same molarity. equivalent pore size Cell membrane pore diameter that accounts for the rate of diffusion of polar substances across the membrane. eserine (physostigmine)An alkaloid (C,,H,,N,O,) of plant origin that blocks the enzyme cholinesterase. esophagus The region of the alimentary canal that conducts food from the headgut to the digestive areas. essential amino acids Amino acids that cannot be synthesized by an animal, but are required for synthesis of essential proteins. esterases Enzymes that hydrolyze esters. estivation Dormancy in response to high ambient temperatures and/or danger of dehydration. estradiol-17P The most active natural estrogen. estrogens A family of female sex steroids responsible for producing estrus and the female secondary sex characteristics; also prepares the reproductive system for fertilization and implantation of the ovum; synthesized primarily in the ovary, although some is made in the adrenal cortex and male testis. estrous cycle Periodic episodes of "heat," or estrus, marked by sexual receptivity in mature females of most mammalian species. ethers A class of compounds in which two organic groups are joined by an oxygen atom, R1-0-R,. eupnea Normal breathing. euryhaline Able to tolerate wide variations in salinity. exchange diffusion A process by which the movement of one molecule across a membrane enhances the movement of another molecule in the opposite direction; most likely involves a common carrier molecule.

excitability The property of altered membrane conductance (and often membrane potential) in response to stimulation. excitation-contraction coupling In muscle fibers, the process by which electrical excitation of the surface membrane leads to activation of the contractile process. excitation-secretion coupling Sequence of molecular events responsible for secretion of stored chemicals from an activated secretory cell. excitatory In neurophysiology, pertaining to the enhanced probability of producing an action potential. excitatory postsynaptic potential (epsp) A change in the membrane potential of a postsynaptic cell that increases the probability of an action potential in the cell. exergonic Characterized by a concomitant release of heat energy. exocrine Of or relating to organs or structures that secrete substances via a duct. exocrine gland A gland that secretes a fluid via a duct. exocytosis Fusion of the vesicle membrane to the surface membrane and subsequent expulsion of the vesicle contents to the cell exterior. exopeptidases Enzymes that attack only peptide bonds near the end of a pept~dechain, providing free amino acids, plus dipeptides and tripeptides. exothermic See exergonic. expiratory neurons Neurons in the medulla of the brain controlling actlvity in motor neurons innervating muscles involved in expiration (breathing out). explants Small pieces of tissue removed from a donor animal kept alive and grown in a flask filled with the appropriate mixture nutrients. extensor A muscle that extends or straightens a limb or other extremity. exteroceptors Sense organs that detect stimuli arriving at the surface of the body from a distance. extracellular digestion Digestion occurring outside of the cell in an alimentary system. extrafusal fiber Contractile muscle fibers that make up the bulk of skeletal muscle. See also intrafusal fibers. extravasation Forcing fluid out of blood vessels, usually blood, serum, or lymph. eye An organ of visual reception that includes optical processing of light as well as photoreceptive neurons. facilitated transport (carrier-mediated transport) Downhill transmembrane diffusion aided by a carrier molecule that enhances the mobility of the diffusing substance in the membrane. facilitation An increase in the efficacy of a synapse as the result of a preceding activation of that synapse. factorial scope for locomotion A ratio between the basal metabolic rate and the maximum metabolic rate that can be achieved with intense exercise. Fahraeus Lindqvist effect The reduction in apparent viscosity of blood as it flows into small arterioles. farad (F)The unit of electrical capacitance. faraday Measure of electric charge, -96,487 C .mol-'. Faraday's constant (F)The equivalent charge of a mole of electrons, equal to 9.649 X lo4 coulombs (C) per mole of electrons. fast chemical synaptic transmission Synaptic transmission at a chemical synapse that is mediated by neurotransmitters that

G-12

GLOSSARY

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bind on the postsynaptic membrane to receptor protein complexes, each of which includes an ion channel. Binding of the transmitter to the receptor complex is sufficient to open (or to close) the ion channel. feces Undigested material and bacteria eliminated from the hindgut. feedback The return of output to the input part of a system. In negative feedback, the sign of the output is inverted before being fed back to the input so as to stabilize the output. In positive feedback, the output is unstable because it is returned to the input without a sign inversion, and thus becomes selfreinforcing, or regenerative. fermentation Enzymatic decomposition;anaerobic transformation of nutrients without net oxidation or electron transfer. ferritin A large protein molecule opaque to electrons, used as a marker in electron microscopy; normally present in the spleen as a storage protein for iron. fever Disease-induced increase in body temperature above normal levels. fibrillar muscle Oscillatory insect flight muscle; also termed asynchronous muscle because contractions are not individually controlled by motor impulses. fibroblast A connective-tissuecell that can differentiate into chondroblasts, collagenoblasts, and osteoblasts. Fick diffusion equation An equation defining the rate of solute diffusion through a solvent. field metabolic rate (FMR) The average rate of energy utilization as the animal goes about its normal activities, which may range from complete inactivity during resting to maximum exertion. filter feeding See suspension feeding. h a 1 common pathway The concept that the sum total of neuronal integrative activity expressed in motor output is channeled through the motor neurons to the muscles. firing level Potential threshold for the generation of an action potential. first law of thermodynamics Net energy is conserved in any process. first-order enzyme kinetics Describes enzymatic reactions, the rates of which are directly proportional to one reactant's concentration (either substrate or product). fixed action pattern A behavior that is performed in a stereotyped fashion in response to specific stimuli. flagellum A motile, whiplike organelle similar in organization to a cilium, but longer and generally present on a cell only in small numbers. flame cells Flagellated cells at the ending of the excretory collecting tubules of flatworms and nemerteans. flavin adenine dinucleotide (FAD)A coenzyme formed by the condensation of riboflavin phosphate and adenylic acid; it performs an important function in electron transport and is a prosthetic group for some enzymes. flavoproteins Proteins combined with flavin prosthetic groups that are important as intermediate carriers of electrons between the dehydrogenases and cytochromes in the respiratory chain. flexer A muscle that flexes or bends an extremity. fluid mosaic model The accepted model for the cell membrane, in which globular proteins are integrated with the lipid bilayer. fluorescence The property of emitting light upon molecular excitation by an incident light; the emitted light is always less

energetic (has a longer wavelength) than the light producing the excitation. flux The rate of flow of matter or energy across a unit area. folds of Kerckring See plica circularis. follicle-stimulating hormone (FSH) An anterior pituitary gonadotropin that stimulates the development of ovarian follicles in the female and testicular spermatogenesis in the male. follicular phase That part of the estrous and menstrual cycles that is characterized by formation of and secretion by the Graafian follicles. food vacuole Enzyme-filled vesicle in Protozoa. foramen An orifice or opening. foregut The upper region of the alimentary canal involved in food conduction, storage and digestion. Fourier's law The rate of flow of heat in a conducting body is proportional to its thermal conductance and to the temperature gradient. fovea (area centralis) In the mammalian retina, the area with the highest visual resolution due to the small divergence and convergence in the pathway linking photoreceptors to ganglion cells; in primates, contains closely packed cone cells. Frank-Starling mechanism The increase in mechanical work from the ventricle caused by an increase in end-diastolic volume (or venous filling pressure). free energy The energy available to do work at a given temperature and pressure. frequency modulated The property of a signal in which information is encoded by varying the frequency at which the strength of the signal changes. fructose A ketohexose, C,H,,O,, found in honey and many fruits. fusimotor system The gamma motor neurons and the intrafusal fibers that they innervate. gain The increase in signal produced by amplification. gallbladder The organ associated with liver that concentrates and stores bile for eventual discharge into the intestine. gamma aminobutyric acid (GABA) Inhibitory transmitter identified in crustacean motor synapses and in the vertebrate central nervous system. gamma efferents The motor axons innervating the intrafusal muscle fibers of spindle organs. gamma motor neurons Nerve cells of the ventral spinal cord that innervate the intrafusal muscle fibers. gamma rays Electromagnetic radiation of very short wavelength (lo-'' cm) and very high energy. ganglia Collections of neuronal cell bodies. ganglion An anatomically distinct collection of neuron cell bodies. ganglion cells A nonspecific term applied to some nerve cell bodies, especially those located in ganglia of invertebrates or outside the vertebrate central nervous system proper. ganglion cells (retinal) The afferent neurons that carry visual information from the vertebrate retina to higher centers of the brain. gap junctions Specializationsfor electrical coupling between cells, where cell membranes are only about 2 nm apart and tubular assemblies of proteins connect the apposed membranes. gastric Pertaining to the stomach. gastric cecum The outpouching of insect alimentary canal,

GLOSSARY G-13 ...................................... ........................................

lined with enzyme-secretingand phagocytic cells and serving as a stomach. gastric inhibitory peptide (GIP) A gastrointestinal hormone released into the bloodstream from the duodenal mucosa, inhibiting gastric secretion and motility. gastric juice Fluid secreted by the cells of the gastric epithelium. gastric phase Secretion phase of digestion stimulated by presence of food in stomach. gastrin A protein hormone that is liberated by the gastrin cells of the pyloric gland and induces gastric secretion and motility. gastrointestinal peptide hormones Hormones that regulate the basic electric rhythm of smooth muscle in the alimentary canal. Gay-Lussac's law Either the pressure or the volume of a gas is directly proportional to absolute temperature if the other is held constant. Geiger counter An instrument that detects the presence of ionizing radiation. gel The stiff, high-viscosity state of cytoplasm. gene Regions of coded information carried within the subunits of DNA forming chromosomes. generator potential A change in transmembrane potential within the receptive part of a sensory neuron; its amplitude is graded with stimulus intensity, and the potential is conducted eletrotonically in the neuron. If the potential is sufficiently large at the spike-initiating zone of the axon, action potentials will be generated. geniculate body A thalamic nucleus that relays incoming sensory (auditory and visual) information to the cortex; named for its kneelike shape in cross section. gestation Pertaining to pregnancy. gigantism Excessive growth due to hypersecretion of pituitary growth hormone from birth. gizzard See crop. gland An aggregation of specialized cells that secrete or excrete substances, such as the pituitary gland, which produces hormones, and the spleen, which takes part in blood production. glial cells (neuroglia)Inexcitable supportive cells associated with neurons in nervous tissue. globin Protein in hemoglobin made of two equal parts each consisting of two polypeptide chains. glomerular filtrate rate (GFR) The amount of total glomerular filtrate produced per minute by all nephrons of both kidneys; equal to the clearance of a freely filtered and nonreabsorbed substance such as insulin. glomerulus A coiled mass of capillaries. glucagon A protein hormone released by the alpha cells of the pancreatic islets; its secretion is induced by low blood sugar or by growth hormone; it stimulates glycogenolysis in the liver. glucocorticoids Steroids synthesized in the adrenal cortex with wide-ranging metabolic activity; included are cortisone, cortisol, corticosterone, and 11-deoxycorticosterone. gluconeogenesis Synthesis of carbohydrates from noncarbohydrate sources, such as fatty acids or amino acids. glucose 6-carbon sugar comprising the cell's primary metabolic fuel; blood sugar. glutamate A putative excitatory synaptic transmitter in the vertebrate central nervous system and in arthropod neuromuscular junctions.

glutamine Amino acid used, because it is less toxic, to transport ammonia between the liver and kidneys in mammals. glycocalyx A meshwork of acid mucopolysaccharide and glycoprotein filaments that arise from the membrane covering the microvilli of the intestinal brush border. glycogen A highly branched D-glucose polymer found in animals. glycogenesis The synthesis of glycogen. glycogenolysis The breakdown of glycogen to glucose 6phosphate. glycogen synthetase An enzyme that catalyzes the polymerization of glucose to glycogen. glycolipid A lipid containing carbohydrate groups, in most cases galactose. glycolysis (Embden-Meyerhof pathway) The metabolic pathway by which hexose and triose sugars are broken down to simpler substances, especially pyruvate or lactate. glycosidases Carbohydrases that break down disaccharides (sucrose, fructose, maltose, lactose) by hydrolyzing alpha-1,6 and alpha-1,4 glucosidic bonds into their constituent monosaccharides for absorption. glycosuria (glucosuria)The excretion of excessive amounts of glucose in the urine. goblet cells See mucous cells. goiter An abnormal increase in size of the thyroid gland, usually due to a dietary lack of iodine. Goldman equation The equation describing the equilibrium potential for a system in which more than one species of diffusible ions are separated by a semi-permeable membrane; if only one species can diffuse across the membrane the equation reduces to the Nernst equation. Golgi tendon organs Tension-sensing nerve endings of the Ib afferent fibers found in muscle tendons. gonadotropic hormones (gonadotrophic hormones; gonadotropins) Hormones that influence the activity of the gonads; in particular, those secreted by the anterior pituitary. gonadotropins Hormones that act on the gonads. Graafian follicle A mature ovarian follicle in which fluid is accumulating. graded response One that increases as a function of the energy applied; a membrane response that is not all-or-none. Graham's law The rate of diffusion of a gas is proportional to the square root of the density of that gas. gray matter Tissue of the vertebrate central nervous system consisting of cell bodies, unmyelinated fibers, and glial cells. growth hormone (GH, somatotropin) A protein hormone that is secreted by the anterior pituitary and stimulates growth; directly influences protein, fat, and carbohydrate metabolism and regulates growth rate. guanine 2-amino-6-oxypurine (CjH,N,jO);a white, crystalline base; a breakdown product of nucleic acids. guano White, pasty waste product of birds and reptiles; high in uric acid content. guanosine triphosphate (GTP) A high-energy molecule similar to ATP that participates in several energy-requiring processes, such as peptide bond formation. guanylatecydase An enzyme that converts GTP to cyclic GMP. gustation The sense of taste; chemoreception of ions and molecules in solution by- specialized epithelial sensory receptors.

G-14

GLOSSARY

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H zone The light zone in the center of the resting muscle sarcomere, where myosin filaments do not overlap with actin filaments; the region between actin filaments. habituation The progressive loss of behavioral response probability with repetition of a stimulus. hair cell A mechanosensory epithelial cell bearing stereocilia and in some cases a kinocilium. Haldane effect Reduction in total C02content of the blood at constant PCOz when hemoglobin is oxygenated. half-width The length of time during which a transient physiological variable is half of its maximum value or greater. halide A binary compound of a halogen and another element. halogens A family of related elements that form similar saltlike compounds with most metals; they are fluorine, chlorine, bromine, and iodine. headgut The anterior (cranial)region of the alimentary canal providing an external opening for food entry. heat Energy in the form of molecular or atomic vibration that is transferred by conduction, convection, and radiation down a thermal gradient. heat capacity Amount of heat required to raise 1g of substance 1°C. heater tissues Tissues specialized for heat production, e.g. modified eye muscles in billfishes. heat of shortening Thermal energy associated with muscle contraction; it is proportional to the distance the muscle has shortened. heat of vaporization Heat necessary per mass unit of a given liquid to convert all of the liquid to gas at its boiling point. heavy meromyosin (HMM; H-meromyosin) The "head" and "neck" of the myosin molecule, the part of the myosin molecule that has ATPase activity. helicotrema The opening that connects the scala tympani and the scala uestibuli at the cochlear apex. hematocrit The percentage of total blood volume occupied by red blood cells; in humans, the hematocrit is normally 40 to 50%. heme C,,H,,04N4FeOH; an iron protoporphyrin portion of many respiratory pigments. hemerythrin An invertebrate respiratory pigment that is a protein but does not contain heme. hemimetabolous Refers to insects, like bugs, that show incomplete metamorphosis during. their life cycle. See also holometabolous. hemocoel Space between ectoderm and endoderm in many invertebrates containing blood (hemolymph). hemocyanin An invertebrate respiratory pigment that is a protein, contains copper, and is found in mollusks and crustaceans. hemoglobin The oxygen-carrying pigment of the erythrocytes, formed by the developing erythrocyte in bone marrow. It is a complex protein composed of four heme groups and four globin polypeptide chains. They are designated a(alpha),P(beta), y(gamma), and S(delta)in an adult, and each is composed of several hundred amino acids. hemolymph The blood of invertebrates with open circulatory systems. hemopoiesis (hematopoiesis) The processes leading to the production of blood cells. hemopoietic factor See intrinsic factor. Henderson-Hasselbalch equation pH = pK + log ([H+accep-

tor]/[H+ donor]). The formula for calculation of the pH of a buffer solution. Henry's law The quantity of gas that dissolves in a liquld is nearly proportional to the partial pressure of that gas in the gas phase. hepatocyte A liver cell. herbivores Animals that feed solely on plant material. Hering-Breuer reflex A reflex in which lung inflation activates pulmonary stretch receptors that inhibit further inspiration during that cycle; activity from stretch receptors is carried in the vagus nerve. hertz (Hz)Cycles per second. Hess's law The total energy released in the breakdown of a fuel to a given set of end products is always the same, irrespective of the intermediate chemical steps or pathways used. heterodimers Dimers consisting of two nonidentical subunits. heterosynaptic facilitation Increased effectiveness of synaptic transmission between two neurons that occurs as a result of activity in a third neuron. heterosynaptic modulation A change in synaptic efficacy at one synapse that occurs due to activity at another, separate synapse. In heterosynaptic facilitation synaptic efficacy increases. In heterosynaptic depression, the synaptic efficacy decreases. heterotherm An animal that derives essentially all of its body heat from the environment. heterotrophic Depending on energy-yieldingcarbon compounds derived from the ingestion of other plants or animals. hexose A six-carbon monosaccharide. hibernation A period of deep torpor, or winter dormancy, in animals in cold climates, lasting weeks or months. hindgut The terminal region of the alimentary canal, responsible for storing and eventually eliminating the remnants of digested food. hindgut fermentation Fermentative digestion occurring in distal portions of the alimentary canal. histamine The base formed from histidine by decarboxylation; responsible for dilation of blood vessels. histaminergic Referring to nerves that release histamine. histone A simple, repeating, basic protein that combines with DNA. Hodgkin cycle The regenerative, or positive-feedback, loop responsible for the upstroke of the action potential; depolarization causes an increase in the sodium permeability, permitting an increased influx of Na+, which further depolarizes the membrane. holometabolous Refers to insects, like flies, that show complete metamorphosis during their life cycle. See also hemimetabolous. homeostasis The condition of relative internal stability maintained by physiological control systems. homeotherm An animal (mammal, bird) that regulates its own internal temperature within a narrow range, regardless of the ambient temperature, by controlling heat production and heat loss. homeoviscous adaptation Molecular level adaptations, especially of cell membranes, that help minimize temperature-induced differences in viscosity. homonymous Pertaining to the same origin. homosynaptic modulation A change in synaptic efficacy that

GLOSSARY G-15 ...............................................................................

occurs subsequent to activity at a synapse. In synaptic facilitation, efficacy increases. In synaptic depression, efficacy decreases. horizontal cell A nerve cell whose fibers extend horizontally in the outer plexiform layer of the vertebrate retina; interconnects adjacent photoreceptors. hormone A chemical compound synthesized and secreted by an endocrine tissue into the bloodstream; influences the activity of a target tissue. horseradish peroxidase A large protein molecule that is opaque in the electron microscope, and is used to trace neuronal pathways in the central nervous system. hybridomas Hybrid cells that are formed by the fusion of two different cell types, used in the production of monoclonal antibodies and other cellular products. hydration Combination with water. hydraulic permeability Refers to the sieve-like properties of the Bowman's capsule in the kidney. hydride Any compound consisting of an element or a radical combined with hydrogen. hydrofuge Pertaining to structures with nonwetting surfaces. hydrogen bond A weak electrostatic attraction between a hydrogen atom bound to a highly electronegative element in a molecule and another highly electronegative atom in the same or a different molecule. hydrolase transport The mechanism by which monosaccharides are taken up into absorptive cells, using a membrane-bound glycosidase to break down and transport the parent disaccharide across the absorptive cell's membrane. hydrolysis Fragmentation or splitting of a compound by the addition of water, whereupon the hydroxyl group joins one fragment and the hydrogen atom the other. hydronium ion (H,O+)A hydrogen ion (H+)combined with a water molecule; H 2 0 + . hydrophilic Having an affinity for water. hydrophobic Lacking an affinity for water. hydrostatic pressure Force exerted over an area due to pressure in a fluid. hydroxyapatite Ca,o(PO,),(OH),, a crystalline material lending hardness and rigidity to the bones of vertebrates and shells of mollusks. hydroxyl group(radica1)The -OH- group. hvdroxvl , , ion OH-. hypercalcemia Excessive plasma calcium levels. hypercapnia Increased levels of carbon dioxide. hyperemia Increased blood flow to a tissue or an organ. hyperglycemia Excessive blood glucose levels. hyperosmotic Containing a greater concentration of osmotically active constituents than the solution of reference. hyperpnea Increased lung ventilation; hyperventilation. hyperpolarization An increase in potential difference across a membrane, making the cell interior more negative than it is at rest. hyperthermia A state of abnormally high body temperature. hypertonic Having a higher tonicity or osmotic pressure than reference solution. hypertrophy Excessive growth or development of an organ or a tissue.

hyperventilation See hyperpnea. hypoglycemia Low blood glucose levels. hypoosmotic Containing a lower concentration of osmotically active constituents than the solution of reference. hypophysis The pituitary gland. hypopnea Hypoventilation; decreased lung ventilation. hypothalamic releasing hormones Hormones from the hypothalamus that cause the release of other hormones from the pituitary. hypothalamus The part of the diencephalon that forms the floor of the median ventricle of the brain; includes the optic chiasma, mammillary bodies, tuber cinereum, and infundibulum; many subregions contribute to the regulation of the autonomic nervous system and of endocrine function. hypothalmo-hypophyseal relay system Portal veins linking the capillaries of the hypothalamic median eminence with those of the adenohypophysis; these transport hypothalamic neurosecretions directly to the adenohypophysis. hypothermia A state of abnormally low body temperature. hypothesis A specific prediction that can then be tested by performing further experiments. hypothyroidism Reduced thyroid activity. hypotonic Having a lower tonicity or osmotic pressure than reference solution. hypoventilation Reduced lung ventilation. hypoxia Reduced oxygen levels. hysteresis A nonlinear change in the physical state of a system, such that the state depends in part on the previous history of the system.

I band The region between the A band and Z disk of a resting muscle sarcomere; it appears light when viewed microscopically and contains part of the actin thin filaments that does not overlap with myosin filaments. ileum Posterior section of the small intestine. impedance The dynamic resistance to flow met by fluids moving in a pulsatile manner. impulse-initiatingregion (spike-initiatingzone) The proximal portion of the axon, which has a lower threshold for action potential generation than either the soma or the dendrites. incus The middle bone of the three bones in the mammalian inner ear; it connects the malleus and the stapes. influx Movement of solute or solvent into a cell across the cell membrane. incisors Chisel-like teeth used for gnawing. inertia The tendency of a mass to resist acceleration. infradian rhythms Biological rhythms with a periodicity of less than a day in length. infrared Thermal radiation; electromagnetic radiation of wavelengths greater than 7.7 x 10-'cm and less than 12 x 10-4cm; the region between red light and radio waves. inhibitory In neurophysiology, pertaining to a reduction in probability of generating an action potential. inhibitory postsynaptic potential (ipsp) A change in the transmembrane potential of a postsynaptic cell that reduces the probability of an action potential in the cell. initial segment The portion of axon and axon hillock proximal to the first myelinated segment; the spike initiating zone of many neurons. inner plexiform layer In the vertebrate retina, the layer of

connecting processes that lies between the bipolar cells and the ganglion cells. inner segment The portion of a vertebrate photoreceptor cell that contains the cell organelles and synaptic contacts. inositol trisphosphate (InsP,) The intracellular second messenger produced by the action of phospholipase C on membrane phosphatidylinositolphosphate in response to stimulation of cell-surface receptors by growth factors, hormones, or neurotransmitters. inotropic Pertaining to the strength of contraction of the heart. inspiratory neurons Neurons in the medulla of the brain controlling motor neurons of muscle associated with breathing in. instars The stages between molts in insect development. instinct A species-specific set of unlearned behaviors and responses. insulin A protein hormone synthesized and secreted by the beta cells of the pancreatic islets; controls cellular uptake of carbohydrate and influences lipid and amino acid metabolism. integral proteins Proteins spanning the cell membrane that form selective filters and active transport devices that get nutrients into and cellular products and waste out of the cell. integration, neuronal Synthesis of an output based on the sum of inputs to a neuron or neuronal network. intercalated disk The junctional region between two connected cardiac muscle cells. interferon anti-viral and anti-tumor agent produced by animal cells interneuron A nerve cell that is entirely contained within the central nervous system; interneurons typically connect two or more other neurons. internode The space along a myelinated axon that is covered by the myelinating cell (i.e., it is covered by the Schwann cell or the oligodendrocyte). interoceptive receptors Internal sensory receptors responding to changes inside the body. interstitial Between cells or tissues. interstitial cell-stimulating hormone (ICSH)Identical to luteinizing hormone but in the male. interstitium The tissue space between cells. intestinal chyme Semifluid mass of partially digested food. intestinal gastrin Hormone stimulating gastric glands to increase secretion rate. intestinal juice See succus entericus. intestinal phase Phase of digestion control by gastrin and other hormones. intracellular digestion Nutrient breakdown occurring within cells. intracellular milieu The general physio-chemical characteristics within the cell. intrafusal fibers The muscle fibers within a muscle spindle organ. intrinsic factor (or hemopoietic factor) A mucoprotein produced by the H+-secreting parietal cells of the stomach; involved in vitamin BI2 absorption. inulin An indigestible vegetable starch; used in studies of kidney function because it is freely filtered and not actively transported. in vitro "In a glass"; in an artificial environment outside the body. in vivo Wlthm the llv~ngorganlsm or tlssue.

iodoacetic acid An agent that poisons glycolysis by inhibiting glyceraldehyde phosphate dehydrogenase. ion An atom bearlng a net charge due to loss or gain of electrons. ion battery The electromotive force capable of driving an ionic current across a membrane; results from unequal concentrations of an ion species in the two compartments separated by the membrane. ion bonding sites Partially ionized regions of protein and other molecules that electrostatically interact with ions in the surrounding solution. ion-exchanger site (ion-binding site) An electrostatically charged site that attracts ions of the opposite charge. ionic bond Electrostatic bond. ionization The dissociation into ions of a compound in solution. ipsilateral Relating to the same side. iris The pigmented circular diaphragm located behind the cornea of the vertebrate eye. ischemia The absence of blood flow (to an organ or a tissue). islets of Langerhans Microscopic endocrine structures dispersed throughout the pancreas. They consist of three cell types: the alpha cells, which secrete glucagon; the beta cells, which secrete insulin; and the delta cells, which secrete gastrin. isoelectric point The pH of a solution at which an amphoteric molecule has a net charge of zero. isomer A compound having the same chemical formula as another, but with a different arrangement of its atoms. isometric contraction Contraction during which a muscle does not shorten significantly. isometry Proportionality of shape regardless of size. isosmotic Having the same osmotic pressure. isoteric interaction Chemical interaction involving molecules with the same number of valence electrons in the same configuration, but made up of different types and numbers of atoms. isotonic Having an equivalent tonicity or osmotic pressure than reference solution. isotonic contraction Contraction in which the force generated remains constant while the muscle shortens. isotope Any of two or more forms of an element with the same number of protons (atomic number), but a different number of neutrons (atomic weight). isovolumic Having the same volume. isozymes Multiple forms of an enzyme found in the same animal species or even in the same cell. Jacobs-Stewart cycle The cycling of CO, and H C 0 3 between intracellular and extracellular compartments, which functions to transfer H+ ions between the cell interior and the extracellular fluid. jejunum The portion of small intestine between the duodenum and the ileum. joule (J) SI unit of work equivalent to 0.239 calories (cal). junctional fold A fold in the cell membrane of a postsynaptic cell that lies under the axon terminals of the presynaptic cell. Junctional folds are typical of skeletal muscle fibers at neuromuscular junctions. juvenile hormone UH) h class of insect hormones that are secreted by the corpora allata and that promote retention of juvenile characteristics. juxtaglomerular apparatus Apparatus comprised of specialized secretory cells situated in the afferent glomerular arterioles; act

as receptors that respond to low blood pressure by secreting renin, which converts angiotensinogen to angiotensin, resulting in vasoconstriction and aldosterone secretion. juxtaglomerular cells Specialized secretory cells situated in the afferent glomerular arterioles; act as receptors that respond to low blood pressure by secreting renin, which converts angiotensinogen to angiotensin, resulting in the stimulation of aldosterone secretion. juxtapulmonary capillary receptors Sensory receptors found in the lung that, when stimulated, elicit the sensation of breathlessness. k selection Pattern of energetic investment in reproduction in which small numbers of large offspring are produced, each with a high chance of survival due to extensive parental care. kelvin (K) See absolute temperature. keratin Structural protein found in skin, feathers, nails, and hoofs. ketone Any compound having a carbonyl group (CO) attached (by the carbon) to hydrocarbon groups. ketone bodies Acetone, acetoacetic acid, and P-hydroxybutyric acid; products of fat and pyruvate metabolism formed from acetyl CoA in the liver; oxidized in muscle and by the central nervous system during starvation. key stimulus (releasing stimulus) The stimulus that is effective in producing a fixed action pattern. kinematic viscosity Viscosity divided by density; gases of equal kinematic viscosity will become turbulent at equal flow rates in identical airways. kinetic energy Energy inherent in the motion of a mass. kininogen Precursor of bradykinin. kinocilium A true "9 + 2" or "9 + 0" cilium present in sensory hair cells. Kirchhoff's laws First law:The sum of the currents entering a junction in a circuit equals the sum of the currents leaving the junction. Second law: The sum of the ~otentialchanges encountered in any closed loop in a circuit is equal to zero. Kleiber's law 0.75 exponent relates metabolic rate to body mass. knock-outs Animals that lack the ability to express the function originally coded for by the gene. Krebs cycle See tricarboxylic acid (TCA) cycle and citric acid cycle. labeled line coding A pattern of information processing in the nervous system in which each neuron encodes only one particular type of information (e.g., sour stimuli in the taste system) and all of the axons that carry that type of information project to the same location or locations. labeled-lines concept The idea that sensory modalities are determined by the stimulus sensitivity of peripheral sense organs and the anatomical specificities of their central connections. lactation The production of milk by the mammary glands (breasts). lactogen Hormone that prepares the breasts for milk production. lagena A structure associated with hair cells in the vertebrate organs of equilibrium. lamella A thin sheet or leaf. laminar flow Turbulence-free flow of fluid in a vessel or past a moving object; a gradient of relative velocity exists in which the fluid layers closest to the wall or body have the lowest relative velocity. Laplace's law The transmural pressure in a thin-walled tube is

proportional to the wall tension divided by the inner radius of the tube. larva The immature, active feeding stage characteristic of many invertebrates. latent heat of vaporization The amount of energy required to change a liquid to its gaseous form (evaporate) at the same temperature. latent period The interval between an action potential in a muscle fiber and the initiation of contraction. lateral geniculate A region of the brain in birds and mammals that processes visual information coming from the retina. lateral geniculate nuclei The major relay nuclei between the retina and the visual cortex in the mammalian visual system; they are included in the thalamic nuclei. lateral inhibition Reciprocal suppression of excitation by neighboring neurons in a sensory network; it produces enhanced contrast at boundaries and an increase in dynamic range. lateral-linesystem Series of hair cells (seeneuromast) in canals running the length of the head and body of fishes and many amphibians; these channels have openings to the outside, and the system is sensitive to water movement. lateral plexus In the compound eye of Limulus, the collection of neurons that interconnect eccentric cells of the ommatidia, producing lateral inhibition. lecithin Any of a group of phospholipids found in animal and plant tissues; composed of choline, phosphoric acid, fatty acids, and glycerol. length constant (1) The distance along a cell over which a potential change decays in amplitude by (1- l/e), or 63%. lens The major light-focusing structure in the vertebrate eye. leukocytes White blood cells. Leydig cells (interstitial cells) Cells of the testes that are stimulated by luteinizing hormone to secrete testosterone. ligand-gated ion channel An ion channel through the cell membrane that opens when a molecule, or molecules, binds to the extracellular domain of the protein. light Electromagnetic radiation with wavelengths between those of x-rays and those of heat (infrared radiation). light meromyosin (LMM) The rodlike fragment of the myosin molecule that constitutes most of the molecule's backbone. limited heterothermy A survival mechanism used by camels and other normally homeothermic animals, in which body temperature is allowed to rise and fall somewhat as ambient temperature ranges through extremes. Lineweaver-Burk equation Straight line transformation of the Michaelis-Menton equation. lipases Enzymes that specifically break down lipids. lipid Any of the fatty acids, neutral fats, waxes, steroids, and phosphatides; lipids are hydrophobic and feel greasy. lipid bilayer Continuous double layer of lipid molecules forming the basic structure of the cell membrane. lipogenesis The formation of fat from nonlipid sources. lipophilic Having an affinity for lipids. lipoprotein Protein-lipid complex in the plasma membrane. local circuit current The current that spreads electrotonicallyfrom the excited portion of an axon during conduction of the nerve impulse, flowing longitudinally along the axon, across the membrane, and back to the excited portion.

G-18

GLOSSARY

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longitudinal smooth muscle Outer layer of smooth muscle running along the long axis of small intestine. long-term potentiation An increase in synaptic efficacy that occurs due to sustained synaptic input and that lasts for a relatively long time-even days, weeks, or months. loop of Henle A U-shaped bend in the portion of a renal tubule that lies in the renal medulla. lower critical temperature (LCT) ambient temperature below which the BMR becomes insufficient to balance heat loss, resulting in falling body temperature. lumen The interior of a cavity or duct. luminosity Brightness; relative quantity of light reflected or emitted. luteal phase The part of the estrous or menstrual cycle characterized by formation of and secretion by the corpus luteum. luteinizing hormone (LH) A gonadotropin that is secreted by the aden~h~pophysis and that acts with follicle-stimulating hormone (FSH)to induce ovulation of the ripe ovum and liberation of estrogen from the ovary; also influences formation of the corpus luteum and stimulates growth in and secretion from the male testicular Leydig cells. lymph Plasmalike fluid collected from interstitial fluid and returned to the bloodstream via the thoracic duct; contains white, but not red, blood cells. lymphatic system A collection of blind-ending tubes which drain filtered extracellular fluid from tissues and return it to the blood circulation. lymph heart A muscular pump found in fish and amphibia causing movement of lymph. lymph nodes Aggregations of lymphoid tissue in the lymphatic system that produce lymphocytes and filter the lymphatic fluid. lymphocytesWhite blood cells, produced in lymphoid tissue, lacking cytoplasmic granules but having a large, round nucleus. lysolecithin A lecithin without the terminal acid group. lysosomes Minute electron-opaque organelles that occur in many cell types, contain hydrolytic enzymes, and are normally involved in localized intracellular digestion. M line In a muscle sarcomere, the darkly staining structure in the middle of the H band. maculae (Greek for "spots.") Organs of equilibrium in the vertebrate inner ear. magnetite A magnetic mineral composed of Fe,O, and found in some animals; believed to play a role in geomagnetic orientation. malleus The outermost bone of the three bones in the mammalian inner ear; it connects the tympanic membrane with the incus. Malpighian tubules Insect excretory osmoregulatory organs responsible for the active secretion of waste products and the formation of urine. mass action law The velocity of a chemical reaction is proportional to the active masses of the reactants. mass-specific metabolic rate The metabolic rate of a unit mass of tissue. mastication The chewing or grinding of food with the teeth. mastoid bone The posterior process of the temporal bone, situated behind the ear and in front of the occipital bone. maximum aerobic velocity (MAV)Locomotor speed at which the maximum rate of aerobic respiration is reached.

mechanism A theory that proposes that life is based purely on the action of physical and chemical laws. mechanoreceptor A sensory receptor tuned to respond to mechanical distortion or pressure. median eminence A structure at the base of the hypothalamus that is continuous with the hypophyseal stalk; contains the primary capillary plexus of the hypothalamo-h~pophyseal portal system. medulla oblongata In vertebrates, a cone-shaped neuronal mass that lies between the pons and the spinal cord. medullary cardiovascular center A group of neurons in the medulla involved in the integration of information used in the control and regulation of circulation. medullary respiratory centers Groups of neurons in the medulla of the brain controlling the activity in motor neurons associated with breathing. melanocyte-stimulating hormone (melanophore-stimulatinghormone) A peptide hormone released by the adenohypophysis that effects melanin distribution in mammals and creates skincolor changes in fishes, amphibians, and reptiles. melting point The lowest temperature at which a solid will begin to liquefy. membrane potential The electric potential measured from within the cell relative to the potential of the extracellular fluid, which is by convention at zero potential; the potential difference between opposite sides of the membrane. membrane pumps Membrane-based cellular mechanisms that actively transport substances against a gradient. membrane recycling Recovery and reformation into new secretory vesicles of membrane lost from the cell membrane due to exocytosis. membrane transport proteins Integral proteins that transport particular classes of molecules across membranes; see active transport. menarche The onset of menstruation during puberty. menopause The cessation of the menstrual cycle in the mature female human. menses Shedding of the uterine lining during a menstrual cycle. menstrual cycle Recurring physiological changes that include menstruation. menstruation The shedding of the endometrium, an event that usually occurs in the absence of conception throughout the fertile period of the female of certain primate species, including humans. mesencephalicus lateralis dorsalis (MLD)The nucleus to which auditory information projects in the owl, contributing to the bird's ability to locate objects in its environment based entirely on audition. messenger molecules Hormones, synaptic transmitters, and other chemicals that regulate biological processes. messenger RNA (mRNA)A fraction of RNA that is responsible for transmission of the informational base sequence of the DNA to the ribosomes. metabolic acidosis A decrease in blood pH at constant PCO, usually as a result of metabolism or kidney function. metabolic alkalosis An increase in blood pH at constant PCO, usually as a result of metabolism or kidney function. metabolic intensity The metabolic rate of a unit mass of tissue. See also mass-specific metabolic rate.

GLOSSARY

G-19

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .' . metabolic pathway A sequence of enzymatic reactions involved in the alteration of one substance into another. metabolic water Water evolved from cellular oxidation. metabolism The totality of physical and chemical processes involved in anabolism, catabolism, and cell energetics. metachronal waves Waves of activity that spread over a population of beating cilia. metachronism The progression of in-phase activity in a wavelike manner over a population of organelles, such as cilia. metamorphic climax The last stage of amphibian metamorphosis, in which the adult form is attained. metamorphosis A change in morphology-in particulal; from one stage of development to another, such as juvenile to adult. metarhodopsin Product of the absorption of light by rhodopsin; decomposes to opsin and trans-retinal. metarteriole An arterial capillary. metazoa Multicellular organisms. methemoglobin Hemoglobin in which the Fe3+of heme has been oxidized to Fez+. micelle A microscopic particle made from an aggregation of amphipathic molecules in solution. Michaelis-Menton equation The rate equation for a single enzymecatalyzed substrate reaction. miaoclimate A small refugium (e.g., burrow, crack in bark) that provides protection from general climatological conditions. microelectrodes Tiny glass "needles" inserted into tissues or even individual cells for recording physiological data. microfilaments Actin filaments within the cytoplasmic substance; diameter of less than 10 nm. micromanipulator A mechanical device that holds and moves microelectrodes incrementally in three different planes. miaotome A device used in microscopy to cut ultrathin sections from small blocks of tissues. microtubules Cylindrical cytoplasmic structures made of polymerized tubulin and found in many cells, especially motile cells, as constituents of the mitotic spindle, cilia, and flagella. microvilli Tiny cylindricalprojections on a cell surface that greatly increase surface area; frequently found on absorptive epithelia, but also in photoreceptors. micturition Urination. midgut Major alimentary canal site for the chemical digestion of protein, fat, and carbohydrates. mineralocorticoids Steroid hormones that are synthesized and secreted by the adrenal cortex and that influence plasma electrolyte balance-in particular, by sodium and chloride reabsorption in the kidney tubules. See also aldosterone. miniature endplate potentials (mepps) Tiny depolarizations (generally 1 mV or less) of the postsynaptic membrane at a motor endplate; produced by presynaptic release of single packets of transmitter. miniature postsynaptic potentials (mpps)Potentials produced in a postsynaptic neuron by presynaptic release of single vesicles of transmitter substance. mitochondria Membrane-enclosed organelle where ATP is produced during aerobic metabolism. mixed nerve A nerve that contains axons of both sensory and motor neurons. mobility, electrical A quantlty proportional to the migration rate of an ion in an electric field.

mobility, mechanical A quantity proportional to the rate at which a molecule will diffuse in a liquid phase. modulatory agent One that either increases or decreases the response of a tissue to a physical or chemical signal. molality The number of moles of solute in a kilogram of a pure solvent. molarity The number of moles of solute in a liter of solution. molars Teeth used in a side-by-side grinding motion to break down food. mole Avogadro's number (6.023 x loz3)of molecules of an element or a compound; equal to the molecular weight in grams. molecular phylogeny A system of phylogenetic relations inferred from similarities and differences in the nucleic acid sequences coding for identified proteins. monoclonal antibody A homogeneous antibody that is produced by a clone of antibody-forming cells and that binds with a single type of antigen. monocytes White blood cells lacking cytoplasmic granules, but having an indented or horseshoe-shaped nucleus. monogastric stomach A stomach consisting of a single strong muscular tube or sac. monomer A compound capable of combining in repeating units to form a dimer, trimer, or polymer. monopole An object or a particle bearing a single unneutralized electric charge, as, for example, an ion. monosaccharide sugar An unhydrolyzable carbohydrate, a simple sugar. Such sugars are sweet-tasting, colorless crystalline compounds with the formula C,(H20), . See also saccharide. monosynaptic Requiring or transmitted through only one synapse; for example, the stretch reflex of vertebrate limbs. monovalent Having a valence of one. monozygotic Arising from one ovum or zygote. motility The ability of the alimentary tract to contract and transport ingested material along its length. motor cortex The part of the cerebral cortex that controls motor function; situated anterior to the central sulcus, which separates the frontal and parietal lobes. motor neuron (motoneuron) A nerve cell that innervates muscle fibers. motor program An endogenous coordinated motor output of central neuronal origin and independent of sensory feedback. motor unit The unit of motor activity consisting of a motor neuron and the muscle fibers it innervates. mRNA See messenger RNA. mucin The mucopolysaccharide forming the chief lubricant of mucus. mucosa Mucous membrane facing a cavity or the exterior of the body. mucosal See mucosa. mucous cells Mucus-secreting cells of the intestine. mucus A viscous, protein-containing mixture of mucopolysaccharides secreted from specialized mucous membranes; often plays an important role in filter feeding (invertebrates)or in lubricating or protecting internal or external surfaces. Mullerian ducts Paired embryonic ducts originating from the peritoneum that connect with the urogenital sinus to develop into the uterus and fallopian tubes. multineuronal innervation Innervation of a muscle fiber by

G-20 GLOSSARY ......................................... several motor neurons, as in many invertebrates, especially arthropods. multiterminal innervation Numerous synapses made by a single motor neuron along the length of a muscle fiber. multi-unit muscle A smooth muscle in which individual muscle fibers contract only when they receive excitatory input from neurons; contraction of these muscles is neurogenic. muscarinic Pertaining to muscarine, a toxin derived from mushrooms; refers to acetylcholine receptors that respond to muscarine, but not to nicotine. muscle fiber A skeletal muscle cell. muscle spindle (stretch receptor) A length-sensitive receptor organ located between and in parallel with extrafusal muscle fibers; gives rise to the myotatic, or stretch, reflex of vertebrates. mutagens Compounds that produce mutations in the germ cell line. mutation A transmissible alteration in genetic material. myelination Forming a myelin sheath. myelin sheath A sheath formed by many layers of the membrane of Schwann cells or oligodendrocyte glial cells that are wrapped tightly around segments of axon in vertebrate nerve; serves as electrical insulation in saltatory conduction. myoblast Embryonic precursor for skeletal muscle fibers. myocardium Heart muscle. myofibril A longitudinal unit of muscle fiber made up of sarcomeres and surrounded by sarcoplasmic reticulum. myogenic Capable of producing an intrinsic cycle of electrical activity. myogenic pacemaker A pacemaker that is a specialized muscle cell. myoglobin An iron-containing protoporphyrin-globin complex found in muscle; serves as a reservoir for oxygen and gives some muscles their red or pink color. myoplasm The cytosol in a muscle cell. myosin The protein that makes up the thick filaments and cross bridges in muscle fibers; it is also found in many other cell types and is associated with cellular motility. myotatic reflex (stretch reflex) Reflex contraction of a muscle in response to stretch of the muscle. myotube A developing muscle fiber. Na+-K+ pump See sodium-potassium pump. Naloxone An analog of morphine that acts as an opioid antagonist. nares Nostrils. nematocysts Stinging cells of hydras, jellyfish, and anemones. nephron The morphological and functional unit of the vertebrate kidney; composed of the glomerulus and Bowman's capsule, the proximal and distal tubules, the loop of Henle (birds, mammals), and the collecting duct. Nernst equation Equation for calculating the electrical potential difference across a membrane that will just balance the concentration gradient of an ion. nerve As a noun: A bundle of axons held together as a unit by connective tissue. As an adjecttve: Neuronal. nerve net A collection of interconnected neurons that are distributed through the body, rather than concentrated in a central location; these neuronal systems are most typical of lower organisms, such as coelenterates. nerve-specific energy The term used by Johannes Muller in his hy-

pothesis that the sensory modality of a stimulus is encoded in the projection pattern of sensory neurons and not on particular features of the cellular response in the stimulated neurons. nervous system The collection of all neurons in an animal's body. net flux Sum of influx and efflux through a membrane or other material. neurilemma Connective tissue sheath covering a bundle of axons. neurites Cell processes extending from the soma of neurons. neurogenic pacemaker A pacemaker that is a specializednerve cell. neuroglia (glia) Inexcitable supporting tissue of the nervous system. neurohemal organ Organ for storage and discharge into the blood of the products of neurosecretion. neurohormone A substance that exists within the neurons of the nervous system and exert hormonal effects outside the nervous system. neurohumor Synaptic transmitters and neurosecretory hormones. neurohypophysis (pars nervosa) A neuronally derived reservoir for hormones with antidiuretic and oxytocic action; consists of the neural lobe, which makes up its bulk, and the neural stalk, which is connected to and passes neurosecretions from the hypothalamus. neuromast A collection of hair cells embedded in a cupula in lateral-line mechanoreceptor of the lower vertebrates. neuromodulation A change in neuronal function caused by chemical messengers (neuromodulators) that are released from axon terminals, but that diffuse more widely than do typical neurotransmitters; neuromodulatory effects can be relatively long-lasting. neuromuscular junction (NMJ)The synapse that connects a motor neuron with a skeletal muscle fiber. neuron Nerve cell. neuronal circuit A set of interconnected neurons. neuronal integration Ongoing summing of all synaptic input onto a postsynaptic cell, which determines whether or not the postsynaptic cell will produce an action potential. neuronal network (neuronal circuit) A system of interacting nerve cells. neuronal plasticity Modification of activity in a neuronal circuit based on experience and changes in input. neuropeptide A peptide molecule identified as a neurotransmitter substance. neuropeptide Y A 36 amino acid peptide, co-localized with norepinephrine in sympathetic ganglia and adrenergic nerves as well as localized in some nonadrenergic fibers, the physiological effects of which include amelioration of actions of catecholamines on the mammalian heart and potentiation of actions of catecholamines in fish hearts. neurophysins Proteins associated with neurohypophyseal hormones stored in granules in the neurosecretory terminals; cleaved from the hormones before secretion. neuropil A dense mass of closely interwoven and synapsing nerve cell processes (axon collaterals and dendrites) and glial cells. neurosecretory cells Nerve cells that liberate neurohormones. neurotoxin A substance that interferes with the proper firing of nerve impulses. neurotransmitter A chemical mediator released by a presynaptic nerve ending that interacts with receptor molecules in the

postsynaptic membrane. This process generally induces a permeability increase to an ion or ions and thereby influences the electrical activity of the postsynaptic cell. nicotinamide adenine dinucleotide (NAD)A coenzyme widely distributed in living organisms, participating in many enzymatic reactions; made up of adenine, nicotinamide, and two molecules each of d-ribose and phosphoric acid. nicotinic Pertaining to nicotine, an alkaloid derived from tobacco; refers to acetylcholine receptors that respond to nicotine, but not to muscarine. node of Ranvier Regularly spaced interruption (about every millimeter) of the myelin sheath along an axon. noncompetitive inhibition Enzyme inhibition due to alteration or destruction of the active site. nonsaturation kinetics Kinetics occurring when the rate of influx increases in proportion to the concentration of the solute in the extracellular fluid. nonshivering thermogenesis A thermogenic process in which enzyme systems for fat metabolism are activated, breaking down and oxidizing conventional fats to produce heat. nonspiking neuron A neuron that receives and transmits information without action potentials; synaptic transmitters are released by these neurons in proportion to their membrane potential, a process called nonspiking release. nonspiking release The release of neurotransmitter from a presynaptic neuron that occurs independent of action potentials; typically graded changes in membrane potential modulate the activation of voltage-gated Ca2+channels, and changes in the intracellular concentration of free Ca2+modulate the release of transmitter. norepinephrine (noradrenaline) A neurohumor secreted by the peripheral sympathetic nerve terminals, some cells of the central nervous system, and the adrenal medulla. nucleases Enzymes that hydrolyze nucleic acids and their residues. nucleic acids Nucleotide polymers of high molecular weight. See also DNA; RNA. nucleosidases Enzymes that hydrolyze nucleic acids and their residues. nucleotide A product of enzymatic (nuclease) splitting of nucleic acids; made up of a purine or pyrimidine base, a ribose or deoxyribose sugar, and a phosphate group. nucleus Of an atom: The central, positively charged mass surrounded by a cloud of electrons. Of a cell: The membranebound body within eukaryotic cells that houses the genetic material of the cell. Of newe cells: A related group of neurons in the central nervous system. nymph A juvenile developmental stage in some arthropods; morphology resembles the adult. nystatin A rod-shaped, antibiotic molecule that creates channels through membranes that allow the passage of molecules of a diameter less than 0.4 nm. obligatory osmotic exchange An exchange between an animal and its environment that is determined by physical factors beyond the animal's control. occipital lobe The most posterior region of the cerebral hemsphere. occular dominance column A set of neurons arranged vertically through the mammalian visual cortex, all of which receive input from one of the two eyes. ohm (a) MKS unit of electrical resistance, equivalent to the resis-

tance of a column of mercury 1 mm2 in cross-sectional and 106 cm long. Ohm's law I = V I R. The strength of an electric current, I, varies directly as the voltage, V, and inversely as the resistance, R. olfaction The sense of smell; chemoreception of molecules suspended in air. oligodendrocytes A class of glial cells with few processes. These cells wrap axons in the central nervous system, forming myelin sheaths. oligopeptides Polypeptide residues of two or three amino acids. oligosaccharides Carbohydrates made up of a small number of monosaccharide residues. omasum The part of the ruminant stomach lying between the rumen and the abomasum. ommatidium The functional unit of the invertebrate compound eye, consisting of an elongated structure with a lens, a focusing cone, and photoreceptor cells. oncotic pressure Osmotic pressure plus hydrostatic pressure caused by distribution of ions according to the Donnan equilibrium. l a afferent fiber An axon with a peripheral sensory ending innervating a muscle spindle organ and responding to stretch of the organ; its central terminals synapse directly onto alpha motor neurons of the homonymous muscle. l b afferent fiber An axon whose sensory terminals innervate the tendons of skeletal muscle and respond to tension. 1,25-dihydrozycholecalciferolA substance that is converted from vitamin D in the liver and increases Ca2+absorption by the kidney. onophores Molecules or molecular aggregates that promote the permeation of ions across membranes; these may be carrier molecules or ion-permeable membrane channels. oocyte A developing ovum. operator gene A gene that regulates the synthetic activity of closely linked structural genes via its association with a regulator gene. operon A segment of DNA consisting of an operator gene and its associated structural genes. opiates Opium-derived narcotic substances. opioids Substances that exert opiatelike effects; some are synthesized endogenously by neurons within the vertebrate central nervous system. opsin Protein moiety of visual pigments; it combines with 11-cisretinal to become a visual pigment. optic axis An imaginary straight line passing through the center of curvature of a simple lens. optic chiasm (optic chiasma) A swelling under the hypothalamus of the vertebrate brain where the two optic nerves meet; depending on the species, some axons cross the midline here and project to the contralateral side of the brain. optic tectum Region of brain in fish and amphibia involved in processing visual information coming from the retina. organ of Corti The tissue in the cochlea of the inner car that contains the hair cells. organs of equilibrium Regions of the inner ear that sense the position of the body relative to gravity or changes of the body's position with respect to gravity. ornithine-urea cycle A cyclic succession of reactions that eliminate ammonia and produce urea in the liver of ureotelic organisms.

G-22

GLOSSARY

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osmoconformer An organism that exhibits little or no osmoregulation, so that the osmolarity of its body fluids follows changes in the osmolarity of the environment. osmolarity The effective osmotic pressure. osmole The standard unit of osmotic pressure. osmolyte A substance that serves the special purpose of raising the osmotic pressure or lowering the freezing point of a body fluid. osmometer An instrument for the measurement of the osmotic pressure of a solution. osmoregulation Maintenance of internal osmolarity with respect to the environment. osmoregulator An organism that controls its internal osmolarity in the face of changes in environmental osmolarity. osmosis The movement of pure solvent from a solution of an area of low solvent concentration to an area of high solvent concentration through a semipermeable membrane separating the two solutions. osmotic flow The solvent flux due to osmotic pressure. osmotic pressure Pressure that can potentially be created by osmosis between two solutions separated by a semipermeable membrane; the amount of pressure necessary to prevent osmotic flow between the two solutions. ossicles Little bones. Auditory ossicles are the tiny bones (malleus, incus, stapes) of the middle eal; which transmit sound vibrations from the tympanic membrane to the oval window. ostia The small, mouth-like openings in the body wall of sponges. otolith A calcareous particle that lies on hair cells in the organs of equilibrium. ouabain Cardiac glycoside, a drug capable of blocking some sodium pumps. outer plexiform layer In the vertebrate retina, the layer of connecting processes that lies between the photoreceptor cells and the bipolar cells. outer segment The part of a vertebrate photoreceptor that contains the pigmented receptor membranes; it is attached to the inner segment by a thin bridge that has microfilaments arranged as in a cilium. oval window The connection between the middle ear and the cochlea; it is covered by the base of the stapes. overshoot The reversal of membrane potential during an action potential; the voltage above zero to the peak of the action potential. ovulation The release of an ovum from the ovarian follicle. ovum An egg cell; the reproductive cell (gamete) of the female. oxidant An electron acceptor in a reaction involving oxidation and reduction. oxidation Loss of electrons or increase in net positivity of an atom or a molecule. Biological oxidations are usually achieved by removal of a pair of hydrogen atoms from a molecule. oxidative phosphorylation Respiratory chain phosphorylation; the formation of high-energy phosphate bonds via phosphorylation of ADP to ATP, accompanied by the transport of electrons to oxygen from the substrate. oxyconformer Animal that allows oxygen consumption to fall as ambient oxygen falls. oxygen debt The extra oxygen necessary to oxidize the products of anaerobic metabolism that accumulate in the muscle tissues during intense physical activity. oxygen dissociation curves Curves that describe the relationship

between the extent of combination of oxygen with the respiratory pigment and the partial pressure of oxygen in the gas phase. oxyhemoglobin Hemoglobin with oxygen combined to the Fe atom of the heme group. oxyntic cells (parietal cells) HCl-secreting cells of the stomach lining. oxyregulator Animal that maintains oxygen consumption as ambient oxygen falls. oxytocin An octapeptide hormone secreted by the neurohypophysis; stimulates contractions of the uterus in childb~rthand the release of milk from mammary glands. P-wave That portion of the electrocardiogram associated with depolarization of the atria. pacemaker An excitable cell or tissue that fires spontaneously and rhythmically. pacemaker potentials Spontaneous and rhythmical depolarizations produced by pacemaker tissue. Pacinian corpuscles Pressure receptors found in skin, muscle, joints, and connective tissue of vertebrates; they consist of a nerve ending surrounded by a laminated capsule of connective tissue. pancreas An organ that produces exocrine secretions, such as digestive enzymes, as well as endocrine secretions, including the hormones insulin and glucagon. pancreatic duct Duct carrying secretions from the pancreas to the small intestine. pancreatic juice A secretion of the pancreas containing proteases, li~ases,and carboh~drasesessential for intestinal digestion. pancreozymin See cholecystokinin. parabiosis The experimental connection of two individuals to allow mixing of their body fluids. parabronchi Air-conduction pathways in the bird lung. paracellular pathways Solvent and solute pathway through epithelium passing between rather than through cells. paracrine A hormonal pathway characterized by the production of a biologically active substance that passes by diffusion within the extracellular space to a nearby cell where it initiates a response. parafollicular cells (C cells) Cells in the mammalian thyroid that secrete calcitonin. parallel processing A pattern of information processing in the nervous system in which multiple pathways simultaneouslycarry information about a particular input or output; the information carried in multiple channels is synthesized where the pathways converge. parasympathetic nervous system The craniosacral part of the autonomic nervous system; in general, increased activity of these neurons supports vegetative functions such as digestion. parathyroid glands Small tissue masses (usuallytwo pairs) close to the thyroid gland that secrete parathormone (parathyroid hormone). parathyroid hormone (PTH; parathormone) A polypeptide hormone of the parathyroid glands secreted in response to a low plasma calcium level; stimulates calcium release from bone and calcium absorption by the intestines while reducing calcium excretion by the kidneys. paraventricular nucleus A group of neurosecretory neurons in the supraoptic hypothalamus that send their axons into the neurohypophysis.

GLOSSARY

G-23

...................................... parietal cells Cells of the stomach lining that secrete hydrochloric acid. See also oxyntic cells. pars intercerebralis The dorsal part of the insect brain; contains the cell bodies of neurosecretory cells that secrete brain hormones from axon terminals in the corpora cardiaca. partition coefficient Ratio of the distribution of a substance between two different liquid phases (e.g., oil and water). parturition The process of giving birth. pawalbumin Calcium-binding protein found in vertebrate muscle. patch-clamping A recording technique in which a glass pipette electrode is brought into contact with the outside of a cell membrane and sealed tightly against it. Current is then passed to hold the potential difference across the membrane at a constant value, and the magnitude of the current required is recorded. The method can be used to measure ionic currents through single ion channels or across the membrane of an entire cell. patch clamp recording A method of investigating epithelial ion current transfer on very localized regions of cells. patella The bone of the knee-cap. pentose A five-carbon monosaccharide sugar. pepsin A proteolytic enzyme secreted by the stomach lining. pepsinogen Proenzyme of pepsin. peptide A molecule consisting of a linear array of amino residues. Protein molecules are made of one or more peptides. Short chains are oligopeptides; long chains are polypeptides. peptide bond The center bond of the -CO-NHgroup, created by the condensation of amino acids into peptides. peptide hormones Hormones that regulate alimentary canal basic electric rhythms. perfusion The passage of fluid over or through an organ, a tissue, or a cell. pericardium The connective-tissue sac that encloses the heart. perilymph The aqueous solution, similar to other body fluids, that is contained within the scala tympani and scala uestibuli of the cochlea. peripheral nervous system The set of neurons and parts of neurons that lie outside of the central nervous system. peripheral resistance units (PRUs) The drop in pressure (in millimeters of mercury, mmHg) along a vascular bed divided by mean flow in milliliters per second. peristalsis A traveling wave of constriction in tubular tissue produced by contraction of circular muscle. peritoneum The membrane that lines the abdominal and pelvic cavities. permeability The ease with which substances can pass through a membrane. pH scale Negative log scale (base 10) of hydrogen ion concentration of a solution. pH = - log[H+]. phagocyte A cell that engulfs other cells, microorganisms, or foreign particulate matter. phagocytosis The ingestion of particles, cells, or microorganisms by a cell into its cytoplasmic vacuoles. phase contrast microscopy A microscopic technique using differential light refraction by different components of the specimen to enhance viewed images. phasic Transient. pheromone A species-specificsubstance released into the environ-

ment for the purpose of signaling between individuals of the same species. phlorizin A glycoside that inhibits active transport of glucose. phonon A quantum of sound energy. phosphagens High-energy phosphate compounds (e.g., phosphoarginine and phosphocreatine) that serve as phosphate-group donors for rapid rephosphorylation of ADP to ATP. phosphoarginine A compound that has phosphagen properties similar to those of phosphocreatine and that occurs in the muscles of some invertebrates. phosphocreatine (aeatine phosphate) A phosphorylated nitrogenous compound found primarily in muscle; contains a highenergy phosphate bond, which can be rapidly transferred to ADP, regenerating ATP. phosphodiesterase An hydrolytic cytoplasmic enzyme that degrades CAMPto AMP. phosphodiester bonds Bonds that link individual nucleotides in nucleic acids. phosphodiester group -0 -P-0 -. phosphoglycerides Glycerine-based lipids of cell membrane. phospholipid A phosphorus-containing lipid that hydrolyzes to fatty acids, glycerin, and a nitrogenous compound. phosphorylase a Activated (phosphorylated)form of phosphorylase that catalyzes the cleavage of glycogen to glucose 1phosphate. phosphorylase kinase Enzyme that, when phosphorylated by a protein kinase, converts phosphorylase b to the more active phosphorylase a. phosphorylation The incorporation of a PO3- group into an organic molecule. photon A quantum of light energy (the smallest amount of light that can exist at each wavelength). photopigments Pigment molecules that change their energy state when they absorb one or more photons of light. photoreceptor A sensory cell that is tuned to receive light energy. physiological dead space That portion of inhaled air not involved in gas transfer in the lung. pilomotor Pertaining to the autonomic control of smooth muscle for the erection of body hair. pinna The outer structure of the mammalian ear, which can be more or less elaborate and which captures and funnels sound into the ear. pinnate Resembling a feather, with similar parts arranged on opposite sides of the axis. pinocytosis Fluid intake by cells via surface invaginations that seal off to become vacuoles filled with liquid. pituitary gland (hypophysis) A complex endocrine organ situated at the base of the brain and connected to the hypothalamus by a stalk. It is of dual origin: the anterior lobe (adenohypophysis) is derived from embryonic buccal epithelium, whereas the posterior lobe is derived from the diencephalon. pK' The negative log (base 10) of an ionization constant, K' .pk' = -log,,K1. placebo A physiologically neutral substance that elicits curative or analgesic effects, apparently through psychological means. placental lactogen A hormone from the placenta that prepares the breasts for milk production. plane-polarized light Light vibrating in only one plane. plasma kinins Peptide hormones formed in the blood after injury-for example, bradykinin.

G-24

GLOSSARY

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plasmalemma Cell membrane; surface membrane. plasma membrane Cell membrane; surface membrane. plasma skimming The separation of plasma from blood w i h the circulation. plasticity Compliance to external influence. plastron The ventral shell of a tortoise or turtle; also a gas film held in place under water by hydrofuge hairs, creating a large airwater interface. pleura The membranes that line the pleural cavity. pleural cavity The cavity between the lungs and the wall of the thorax. plicae circularis The extensive fold of intestinal mucosa bearing villi. pneumotaxic center A group of neurons in the pons, thought to be involved in the maintenance of rhythmic breathing in mammals. pneumothorax Collapse of the lung due to a puncture into the pleural cavity of the chest wall or the lung. poikilotherm An animal whose body temperature tends to fluctuate more or less with the ambient temperature. Poiseuille's law In laminar flow, the flow is directly proportional to the driving pressure, and resistance is independent of flow. Poisson distribution A theoretical description of the probability that random, independent events based on a unitary event of a particular size will occur. polyclonal Derived from different cell lines or clones. polyestrous The state of having many estrous cycles throughout the year. polymer A compound composed of a linear sequence of simple molecules or residues. polypeptide chain A linear arrangement of more than two amino acid residues. polypnea Increased breathing rate. polysaccharides Carbohydrases that hydrolyze the glycosidic bonds of long-chain carbohydrates (cellulose,glycogen, and starch). polysynaptic Referring to transmission through multiple synapses in series. polytene Having many duplicate chromatin strands. pons The region of the vertebrate brain that lies just rostra1 to the medulla oblongata. pores of Kohn Small holes between adjacent regions of the lung, permitting collateral air flow. porphyrins A group of cyclic tetrapyrrole derivatives. porphyropsin A purple photopigment, based on ll-cis dehydroretinal, that is present in the retinal rods of some freshwater fishes. portal vessels Blood vessels that carry blood directly from one capillary bed to another. positive phototaxis Referring to the movement of an animal toward light. postsynaptic Located on the receiving side of a synaptic connection. posttetanic depression Reduced postsynaptic response following prolonged presynaptic stimulation at a high frequency; believed to be due to presynaptic depletion of transmitter. posttetanic potentiation (PTP)Increased efficacy of synaptic transmission following presynaptic stimulation at a high frequency; often follows posttetanic depression.

potassium activation An increase in the conductance of a membrane to potassium in response to depolarization. potential The voltage above zero to the peak of the action potential. potential energy Stored energy that can be released to do work. premetamorphosis The developmental stage just preceding amphibian metamorphosis, during which iodine binding and hormone synthesis occur in the thyroid gland. presbyopia The tendency for human eyes to become less able to focus on close objects ("far-sighted") with age; occurs as the lens becomes less compliant. pressure pulse The difference between the systolic and diastolic pressures. presynaptic Located on the sending side of a synaptic connection. presynaptic inhibition Neuronal inhibition resulting from the action of a terminal that ends on the presynaptic terminal of an excitatory synapse, reducing the amount of transmitter released. primary follicle An immature ovarian follicle. primary projection cortex A region of cerebral cortex that directly receives sensory signals from lower centers; the first cortical cells to receive sensory information projected to the brain. primary sensory neurons Neurons that directly receive sensory stimulation. primary structure The sequence of amino acid residues of a polypeptide chain. proboscis An elongated, protrudmg mouth part, typically in sucking insects. procaine 2-diethylaminoethyl-p-aminobenzoate; a local anesthetic that interferes with some of the ion conductances of excitable membranes. proenzyme (zymogen) The inactive form of an enzyme before it is activated by removal of a terminal segment of peptide. progesterone A hormone of the corpus luteum, adrenal cortex, and the placenta that promotes growth of a suitable uterine lining for implantation and development of the fertilized ovum. prolactin An adenohypophyseal hormone that stimulates milk production and lactation after parturition in mammals. prometamorphosis The first stage of amphibian metamorphosis, during which there is increased development and activity in the thyroid gland and median eminence. propeptide A large peptide that contains the amino acid sequences of several smaller peptides, which are released when the large peptide is enzymatically cleaved. proprioceptors Sensory receptors situated primarily in muscles and tendons that relay information about the position and motion of the body. prostaglandins A family of natural fatty acids that arise in a variety of tissues and are able to induce contraction in uterine and other smooth muscle, lower blood pressure, and modify the actions of some hormones. prostate gland A gland located around the neck of the bladder and urethra in males that contributes to the seminal fluid. prosthetic group An organic compound essential to the function of an enzyme. Prosthetic groups differ from coenzymes in that they are more firmly attached to the enzyme protein. protagonistic muscles Muscles whose contractions cooperate to produce a movement.

GLOSSARY G-2.5 ......................................

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protease Enzymes that break down peptide bonds of proteins and polypeptides. protein kinase Any enzyme that catalyzes the transfer of a phosphate group from ATP to a protein, creating a phosphoprotein. proteins Large molecules composed of one or more chains of alpha amino acid residues (i.e., polypeptide chains). proteolysis The splitting of proteins by hydrolysis of peptide bonds. proteolytic Protein-hydrolyzing. prothoracic glands Ecdysone-secreting tissues situated in the anterior thorax of insects. prothoracicotropic hormone (MTH)A neurohormone produced by neurosecretory cells in the pars intercerebralis of the brain. PTTH activates the prothoracic gland to synthesize and secrete molting hormones. proximal tubules Coiled portions of the renal tubules located in the renal cortex, beginning at the glomerulus and leading to (and continuous with) the descending limb of the loop of Henle. pseudopodium Literally, false foot; a temporary projection of an amoeboid cell for engulfment of food or for locomotion. pseudopregnancy A false pregnancy. psychophysics The branch of psychology concerned with relationships between physical stimuli and perception. pulmonary Pertaining to or affecting the lungs. pupa A developmental stage of some insect groups; between the larva and the adult. pupil The opening at the center of the iris through which light passes into the eye. purinergic Referring to nerve endings that release purines or their derivatives as transmitter substances. purines A class of nitrogenous heterocyclic compounds, C5H4N4, derivatives of which (purine bases) are found in nucleotides; they are colorless and crystalline. pyloric Pertaining to the caudal portion of the vertebrate stomach where it joins the small intestine. pyloric sphincter Sphincter guarding the opening of the stomach into the small intestine. pylorus The distal stomach opening, ringed by a sphincter, that releases the stomach contents into the duodenum. pyramidal tract A bundle of nerve fibers originating in the motor cortex and descending down the brain stem to the medulla oblongata and to the spinal cord; responsible for mediating control of voluntary muscle movements. pyrimidine A class of nitrogenous heterocyclic compounds, C4H4N,, derivatives of which (pyrimidine bases) are found in nucleotides. pyrogen Substance that leads to a resetting of a homeotherm's body thermostat to a higher set point, thereby producing fever. Q,, The ratio of the rate of a reaction at a given temperature to its rate at a temperature 10°C lower. QRS-wave That portion of the electrocardiogram related to depolarization of the ventricle. quality A property that distinguishes sensory stimuli within a sensory modality; e.g., color is a quality of visual stimuli. quantal content The number of neurotransmitter molecules in one synaptic vesicle.

quantal release The release of neurotransmitter in discrete packets which correspond to vesicles containing transmitter molecules. quantal synaptic transmission The concept that neurotransmitter is released in multiples of discrete "packets." It is now apparent that the quantal packets represent individual presynaptic vesicles. quaternary structure The characteristicways in which the subunits of a protein containing more than one polypeptide chain are combined. r selection A pattern of energetic investment in reproduction in which large numbers of very small offspring are produced, each with a low chance of survival due to lack of parental care. radial finks Extensions from peripheral doublets to the central sheath in cilia and flagella. radiation, thermal The transfer of heat by electromagnetic radiation without direct contact between objects. radioimmunoassays (RIAs)An immunological technique for the measurement of minute quantities of antigen or antibody, hormones, certain drugs, and other substances with the use of radioactively labeled reagents. radioisotope A radioactive isotope. radula A rasp-like structure in the mouth of many gastropods. range fractionation The pattern in which receptors within one sensory modality are tuned to receive information within relatively narrow, but not identical, intensity ranges, so the entire dynamic range of the modality is divided among different classes of receptors. For example, in the human eye rods respond to dim light but are saturated in bright light; cones are less sensitive to dim light but remain responsive in bright light. rate constant (specificreaction rate) The proportionality factor by which the concentration of a reactant in an enzymatic reaction is related to the reaction rate. reactive hyperernia Higher than normal blood flow that occurs following a brief period of ischemia. receptive field That area of an organism's body (e.g., on the skin or in the retina) that when stimulated influences the activity of a given neuron is the receptive field of that neuron. receptor Molecules that are situated on a membrane and that interact specifically with messenger molecules, such as hormones or transmitters. receptor cell A neuronal cell that is specialized to respond to some particular sensory stimulation. receptor current A stimulus-induced change in the movement of ions across a receptor cell membrane. receptor-mediated endocytosis Specialized process of endocytosis that requires solutes being transported to temporarily bind to receptor molecules embedded in the cell membrane prior to transport across the cell membrane. receptor molecules Molecules that are situated on the outer surface of the cell membrane and that interact specifically with messenger molecules, such as hormones or neurotransmitters. receptor potential A change in membrane potential elicited in sensory receptor cells by sensory stimulation, which changes the flow of ionic current across the cell membrane. receptor tyrosine kinases (RTKs) Receptors with intrinsic tyrosine kinase activity, which are known to bind insulin and a number of growth factors. When activated by external signal binding, RTKs transfer the phosphate group from

G-26

GLOSSARY

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ATP to the hydroxyl group on a tyrosine residue of selected proteins in the cytosol. RTKs also phosphorylate themselves when activated; this autophosphorylation enhances the activity of the kinase. reciprocal inhibition Inhibition of the motor neurons innervating one set of muscles during the reflex excitation of their antagonists. recombinant DNA An engineered DNA molecule resulting from the two different organisms. recruitment The pattern in which neurons with increasingly high thresholds become active as intensity increases. This pattern can occur in sensory neurons (range fractionation) or in motor neurons. rectal gland Organ near the rectum of elasmobranchs that excretes a highly concentrated NaCl solution. redox pair Two compounds, molecules, or atoms involved in mutual reduction and oxidation. reductant Donor of electrons in a redox reaction. reduction The addition of electrons to a substance. reduction potential A measurement of the tendency of a reducer to yield electrons in a redox reaction, expressed in volts. reflex An action that is generated without the participation of the highest neuronal centers and is thus not voluntary; an involuntary motor response mediated by a neuronal arc in response to sensory input. reflex arc A neuronal pathway that connects sensory input and motor output; consists of afferent nerve input to a nerve center that produces activity in efferent nerves to an effector organ. refraction The bending of light rays as they pass from a medium of one density into a medium of another density. refractive index The refractive power of a medium compared with that of air, designated 1. refractory period The period of increased membrane threshold immediately following an action potential. Absolute refractory period: The initial phase of the refractory period when no AP can be generated. Relative refractory period: The later phase of the refractory period when the threshold is elevated, but an AP can be generated with sufficiently intense stimulation. regenerative Self-reinforcing; utilizing positive feedback; autocatalytic. regulator Animals that use biochemical, physiological, behavioral, and other mechanisms to maintain internal homeostasis. regulator gene Genes that code for repressor proteins, which suppress the action of structural genes. regurgitation Reverse movement of intestinal luminal contents produced by reverse peristalsis. Reissner's membrane A membrane within the mammalian cochlea. release-inhibitinghormone (RM;release-inhibitingfactor, RIF) A hypothalamic neurosecretion carried by portal vessels to the adenohypophysis, where it restrains the release of a specific hormone. releasing hormone (RH, releasing factor) A hypothalamic neurosecretion that stimulates the liberation of a specific hormone from the adenohypophysis. releasing stimulus (key stimulus) The stimulus that is effective in producing a fixed action pattern. renal clearance That volume of plasma containing the quantity of a freely filtered substance that appears in the glomerular

filtrate per unit time. Total renal clearance is the amount of ultrafiltrate produced by the kidney per unit time. renin A proteolytic enzyme produced by specialized cells in renal arterioles; converts angiotensinogen to angiotensin. rennin An endopeptidase enzyme that coagulates milk by promoting the formation of calcium caseinate from the milk protein casein; found especially in the gastric juice of young mammals. Renshaw cells Small inhibitory interneurons in the ventral horn that are excited by branches of a-motor neuron axons that feed back on the motor neuron pool. repolarization The return to resting polarity of a cell membrane that has been depolarized. repression proteins Proteins that can bind to a short region of DNA preceding the structural geneis), thus preventing transcription. repressor gene (regulator gene) A gene that produces a substance (repressor) that shuts off the structural-gene activity of an operon by an interaction with its operator gene. reserpine A botanically derived tranquilizing agent that interferes with the uptake of catecholamine from the cytosol by secretory vesicles; its effect is to deplete the catecholamine content of adrenergic cells. residual volume The volume of air left in the lungs after maximal expiratory effort. resistance (R)The property that hinders the flow of current. The unit is the ohm (a), defined as the resistance that allows 1ampere (A)of current to flow when a potential drop of 1volt (V) exists across the resistance. It is equivalent to the resistance of a column of mercury 1 mm2in cross-sectional area and 106.3 cm long. R = p x length + cross-sectional area. resistivity (p) The resistance of a conductor 1 cm in length and 1 cm2 in cross-sectional area. respiratory acidosis A decrease in blood pH associated with a fall in blood PCO, as a result of lung hypoventilation. respiratory alkalosis An increase in blood pH associated with a rise in blood PCOz as a result of lung hyperventilation. respiratory chain See electron-transport chain. respiratory pigment A substance that combines reversably with oxygen-for example, hemoglobin. respiratory quotient (RQ)The ratio of CO, production to 0, consumption; depends on type of food oxidized by the animal. respirometry Measurement of an animal's respiratory exchange. resting potential The normal, unstimulated membrane potential of a cell at rest. rete mirabile An extensive countercurrent arrangement of arterial and venous capillaries. reticulum A small network. retina The photosensitive inner surface of the vertebrate eye. retinal The aldehyde of retinol obtained from the enzymatic oxidative cleavage of carotene; in the 11-cis form it unites with opsins in the retina to form the visual pigments. retinal streak (visualstreak) A retinal structure-found in the eyes of some species that inhabit plains-in which photoreceptors are packed into a horizontal streak across the retina, providing high resolution along the visual horizon. This pattern is similar to the fovea of primates, but it has a different shape. retinol Vitamin A (C,,H,,O), an alcohol of 20 carbons; converted reversibly to retinal by enzymatic dehydrogenation. retinular cell A photoreceptor cell of the arthropod compound eye.

GLOSSARY

G-27

...................................... reversal potential The membrane potential at which no current flows through membrane ion channels, even though the channels are open; it is equal to the equilibrium potential for the ion or ions that are conducted through the open channels. Reynolds number (Re) A unitless number; the tendency of a flowing gas or liquid to become turbulent is proportional to its velocity and density and inversely proportional to its viscosity. Calculated from these parameters, the Reynolds number indicates whether flow will be turbulent or laminar under a particular set of conditions. rhabdome The aggregate structure consisting of a longitudinal rosette of rhabdomeres located axially in the ommatidium. rhabdomere The light-absorbing part of a retinular cell that faces the central axis of an ommatidium; the photopigment-bearing surface membrane is expanded into closely packed microvilli, increasing the amount of photosensitive membrane. rheogenic Producing electric current. rhodopsin (visual purple) A purplish red, light-sensitive chromoprotein with 11-cisretinal as its prosthetic group; found in the rods and cones of the retina; bleaches to "visual yellow" (alltrans-retinal) when it absorbs incident light. ribonucleic acid See RNA. ribose A pentose monosaccharide with the chemical formula HOCH,(CHOH),CHO; a constituent of RNA. ribosome Ribonucleoprotein particles found within the cytoplasm; the sites of intersection of mRNA, tRNA, and the amino acids during the synthesis of polypeptide chains. rigor mortis The rigidity that develops in dying muscle as ATP becomes depleted and cross bridges remain attached. Ringer solution Physiological saline solution. RNA (ribonucleic acid) A nucleic acid made up of adenine, guanine, cytosine, uracil, ribose, and phosphoric acid; responsible for the transcription of DNA and the translation into protein. rods One class of vertebrate visual receptor cells, the cones being the other; very sensitive to light, based on cellular physiology and on a high degree of convergence onto second-order cells. In most species, there is only one class of rods in the retina, so rods cannot convey information about color. Root effect (Root shift) A change in blood oxygen capacity as a result of a pH change. round window A membrane-covered opening, separating the rniddle ear and the cochlea, through which pressure waves leave after traveling through the cochlea. rumen The storage and fermentation chamber in the digastric stomach of ruminants. rumination The chewing of partially digested food brought up by reverse peristalsis from the rumen in ungulate animals and in other ruminants. saccharide A family of carbohydrates that includes the sugars; they are grouped as to the number of saccharide (C,H,,O,-I) groups comprising them: the mono-, di-, tri-, and polysaccharides. sacculus One of the vertebrate organs of equilibrium. safety factor A factor relating input and output in a system, describing how likely transmission through the system will fail; the higher the safety factor, the less likely it is that transmission will fail. saliva A water-like fluid secreted in the upper alimentary canal (headgut);aids in mechanical and chemical digestion.

salivary glands Glands that secrete saliva into the headgut. saltatory (conduction)Jumping; discontinuous. salt glands Osmoregulatory organs of many birds and reptiles that live in desert or marine environments. A hypertonic aqueous exudate is formed by active salt secretion into the small tubules situated above the eyes and is excreted via the nostrils. "salting out" A decrease in Bunsen solubility coefficient as a result of increased ionic strength of the solvent. sarcolemma The surface membrane of muscle fibers. sarcomere The contractile unit of a myofibril, it is bounded by two Z disks. sarcoplasm Cytosol of a muscle cell. sarcoplasmic reticulum (SR) A smooth, membrane-limited network surrounding each myofibril. Calcium is stored in the SR and released as free Ca2+during muscle excitation-contraction coupling. sarcotubular system The sarcoplasmic reticulum plus the transverse tubules. saturated In reference to fatty acid molecules, indicates that the carbon-carbon bonds are single, with each carbon atom bearing two hydrogens. Without free valence electrons. scala media The cochlear duct, a membranous labyrinth containing the organ of Corti and the tectorial membrane; it is filled with endolymph. scala tympani A cochlear chamber connected with the scala vestibuli through the helicotrema; it is filled with perilymph. scala vestibuli A cochlear chamber beginning in the vestibule, connecting with the scala tympani through the helicotrema; it is filled with perilymph. scaling The study of how both anatomical and physiological characteristics change with body mass. Schwann cell A neuroglial cell outside the central nervous system that wraps its membrane around axons during development to produce the insulating myelin sheath that envelops peripheral axons in the regions between nodes of Ranvier. scintillation counter An instrument that detects and counts tiny flashes of light produced in scintillation fluid produced by particles emitted from radioisotopes. SDA (specificdynamic action) The increment in metabolic energy cost that can be ascribed to the digestion and assimilation of food; it is highest for proteins. secondary structure Refers to the straight or helical configuration of polypeptide chains. secondary vacuole Vacuole formed when food containing vacuole merges with enzyme-containing lysosomes. second law of thermodynamics All natural or spontaneous processes are accompanied by an increase in entropy. second messenger A term applied to CAMP,cGMP, Ca2+,or any other intracellular regulatory agent that is itself under the control of an extracellular first messenger, such as a hormone. second-order enzyme kinetics Describes enzymatic reactions whose rates are determined by the concentrations of two reactants multiplied together or of one reactant squared. second-order neuron A neuron that receives input from primary sensory neurons. secretagogue A substance that stimulates or promotes secretion. secretin A polypeptide hormone secreted by the duodenal and jejunal mucosa In response to the presence of acid chyme in the intestine; induces pancreatic secretion into the intestine and is chemically identical to enterogastrone.

G-28

GLOSSARY

.........................................

secretory granules (secretory vesicles) Membrane-bound cytoplasmic granules containing secretory products of a cell. segmentation Rhythmic contractions of the circular muscle layer of the intestine that mixes the intestinal contents. selectivity sequence See affinity sequence. self-repair capability Ability of cell organelles and membranes to reseal themselves when chemically or mechanically disturbed. semicircular canals Three of the vertebrate organs of equilibrium, which sense acceleration of the body with respect to the gravitational field. seminal vesicles Paired sacs attached to the posterior urinary bladder that have tubes joining the vas deferens in the male. semipermeable membrane A membrane that allows certain molecules but not others to pass through it. sensation The perception of a sensory stimulus (as opposed to the reception of stimulus energy in a primary sensory receptor). sensilla (plural; sensillum, singular) Collections of sensory receptors in the periphery of an organism, usually an invertebrate; sensilla are typically very simple in structure, lacking accessory structures. sensillum A chitinous, hollow, hairlike projection of the arthropod exoskeleton that serves as an auxiliary structure for sensory neurons. sensor Mechanical, electrical, or biological device that detects changes in its immediate environment. sensory adaptation The property of sensory systems to become less sensitive to stimuli during prolonged or repeated stimulation. sensory fiber An axon that carries sensory information to the central nervous system. sensory filter network Neuronal circuits that selectively transmit some features of a sensory input and ignore other features. sensory modality A set of sensory structures that are tuned to receive a particular class of energy; e.g., vision, hearing, and the sense of smell are three sensory modalities. sensory reception The absorption of stimulus energy by a neuron that produces a receptor potential in the neuron and that may produce action potentials that travel to the central nervous system. series elastic components (SEC)Elastic structures ( such as tendons and other connective tissues) that are arranged in series with the contractile elements in muscle. serosa Outermost layer of alimentary tract. serosal Pertaining to the side of an epithelial tissue facing the blood, as opposed to the mucosal side, which faces the exterior or luminal space. serotonin 5-Hydroxytryptamine, 5-HT; a neurotransmitter, CIOH12N2O. serum The clear component of blood plasma. servo mechanism A control system that utilizes negative feedback to correct deviations from a selected level, the set point. set point In a negative-feedback system, the state to which feedback tends to bring the system. siemen (S) The unit of electrical conductance; reciprocal of the ohm. sign stimulus The most basic essential pattern of sensory input required to release an instinctual pattern of behavior.

signal-to-noise ratio The relation between a signal and the random background activity that arises as the result of kinetic energy or other irrelevent events; information theory has thoroughly considered this relation and generated rules that describe efficient and effective information transfer. single-unit muscle A smooth muscle in which individual fibers are coupled through gap junctions, allowing excitation to spread through the muscle independent of neuronal activity; contraction in these muscles is often myogenic, driven by internal pacemaker cells. sinoamal node A mass of specialized cardiac tissue that lies at the junction of the superior vena cava with the right atrium; it acts as the pacemaker of the heart in initiating each cardiac contraction. sinus A cavity or sac; a dilated part of a blood vessel. sinus node (sinoatrial node, SA node) The junction between the right atrium and the vena cava, the location of the pacemaker. sinus venosus The membrane chamber attached to the heart that receives Venus blood in fish, amphibians, and reptiles, and transmits it to the atrium. skeletal muscle The striated muscle whose contraction is responsible for moving the bodies of animals. Contraction of these fibers is neurogenic; that is, contraction occurs only when the fibers are excited by synaptic input from motor neurons. sliding-filament theory The theory that muscle sarcomeres shorten when actin thin filaments are actively pulled toward the middle of myosin thick filaments by the action of myosin crossbridges. sliding-tubule hypothesis Bending movements of cilia and flagella are produced by active longitudinal sliding of the axonemal microtubules past one another. slow chemical synaptic transmission Synaptic transmission at a chemical synapse mediated by neurotransmitters that bind on the postsynaptic membrane to receptor molecules that affect intracellular second messenger systems, typically through G proteins. Binding of the transmitter to the receptor complex activates the second messengers, which in turn modify the state of ion channel proteins. smooth muscle Muscle without sarcomeres and hence without striations. Myofilaments are nonuniformly distributed within small, mononucleated, spindle-shaped cells. sodium activation An increased conductance of excitable membranes to sodium ions in response to membrane depolarization; believed to result from an opening of sodium gates associated with membrane channels. sodium hypothesis The upstroke of an action potential is due to an inward movement of Na+ down its electrochemical gradient as a result of a transient increase in sodium permeability. sodium inactivation Loss of responsiveness of sodium gates to depolarization; develops with time during a depolarization and persists for a short period after repolarization of the membrane. sodium-potassium ATPase (sodium pump) Membrane protein responsible for maintaining the asymmetrical concentrations of Na+ and K+ions across the cell membrane; it actively extrudes Na+ from the cell and takes up K+ from extracellular fluids at the expense of metabolic energy. In some sodium pumps, there is a 3 :2 exchange of intracellular Na+ for extracellular K+. sol The low-viscosity state of cytoplasm.

GLOSSARY

G-29

...................................... solvation The process of dissolving a solute in a solvent; hydration, or clustering, of water molecules around individual ions and polar molecules. solvent drag Process in which smaller solute molecules are carried passively along with the water as it flows down its osmotic gradient through hydrated channels. soma The nerve cell body, or perikaryon; in general, the body. somatic Referring to the body tissues as distinct from the germ cells. somatic system The part of the nervous system that receives input from the body. somatosensory cortex The region of the cerebral cortex that receives sensory input from the body surface. somatostatin Growth-hormone-inhibiting hormone, which inhibits growth-hormone release from the pituitary. spatial summation Integration by a postsynaptic neuron of simultaneous synaptic currents that arise from the terminals of different presynaptic neurons. specific dynamic action (SDA)A marked increase in metabolism accompanying the digestion and assimilation of food. specific resistance (R,) Resistance per unit area of a membrane in ohms per square centimeter. spectrophotometer A device that passes a beam of visible or W light through a fluid-filled vial and measures the emerging wavelengths. spectrum Specific, charted bands of electromagnetic radiation wavelengths produced by refraction or diffraction. sphincter A ring-shaped band of muscle fibers capable of constricting an opening or a passageway. sphingolipid A lipid formed by a fatty acid attached to the nitrogen atom of sphingosine, a long-chain, oily amino alcohol (C18H,,0,N). The sphingolipids occur primarily in the membranes of brain and nerve cells. spike-initiating zone Region of the nerve axon where an action potential is initiated. In many-but not all-neurons, the axon hillock. spinal canal The fluid-filled cavity that runs longitudinallythrough the vertebrate spinal cord; it is confluent with the cerebral ventricles. spinal cord The portion of the vertebrate central nervous system that is encased in the vertebral column, extending from the caudal end of the medulla oblongata to the upper lumbar region; constructed of a core of gray matter and an outer layer of white matter. spinal root A large bundle of axons that enters or leaves the spinal cord at each spinal segment. spindle organ A stretch receptor of vertebrate skeletal muscle. spiracle Surface opening of the tracheal system in insects. spiral ganglion The ganglion lying near the cochlea that contains the somata of neurons whose axons carry auditory information from the hair cells of the organ of Corti to the auditory centers of the brain. spirometry Measurement of an animal's 0, and CO, turnover. standard metabolic rate (SMR)Similar to basal metabolic rate, but utilized for metabolic rate of a heterotherm maintained at a selected body temperature. standard temperature and pressure (STP, dry, STPD) 2S°C, 1atmosphere (atm). standing-gradient hypothesis Hypothesis describing process of solute-coupled water transport, involving the active transport

of salt across the portions of the epithelial cell membranes facing intercellular clefts. standing wave A resonating wave with fixed nodes. stapes (stirrup) The innermost auditory ossicle, which articulates at its apex with the incus and whose base is connected to the oval window. starch A polysaccharide of plant origin, formula (C,H,,O,), . Starling curves Curves that describe the relationship between heart work and filling pressure. statocyst Gravity-sensing sensory organ made up of mechanoreceptive hair cells and associated particles called statoliths. statolith A small, dense, solid granule found in statocysts. steady state Dynamic equilibrium. stenohaline Able to tolerate only a narrow range of salinities. stereocilia Nonmotile filament-filled projections of hair cells; these "hairs" lack the internal structure of motile "9 + 2" cilia. steric Pertaining to the spatial arrangement of atoms. steroid hormones Cyclic hydrocarbon derivatives synthesized from cholesterol. sterols A group of solid, primarily unsaturated polycyclic alcohols. stimulus A substance, action, or other influence that when applied with sufficient intensity to a tissue causes a response. stomach The major digestive region of the alimentary canal. stomatogastric ganglion A ganglion in Crustacea that contains the central pattern generator neurons that control the rhythmic activity of the stomach and associated organs. STP See standard temperature and pressure. stretch receptor Sensory receptors that respond to stretch, typically associated with lungs or muscle tissue. stria vascularis Vascular tissue layer over the external wall of the scala media; secretes the endolymph. striated muscle Characterized by sarcomeres aligned in register. Skeletal and cardiac muscle are striated. stroke volume The volume of blood pumped by one ventricle during a single heartbeat. structural gene A gene coding for the sequence of amino acids that make up a polypeptide chain. strychnine A poisonous alkaloid (C,,H,,N,O,) that blocks inhibitory synaptic transmission in the vertebrate central nervous system. submucosa Second-most inner layer of alimentary canal, underlying inner mucosa. submucosal plexus Neural plexus that acts to stimulate gut motility and secretion. substrate A substance that is acted on by an enzyme. substrate-level phosphorylation Mechanism by which chemical energy of oxidation is stored in the form of ATP. succus entericus Digestive juice secreted by the glands of Lieberkiihn in the small intestine. sulfhydryl group The radical -SH. supercooling Cooling of a fluid below its freezing temperature without actual freezing because of the failure of ice crystals fail formation. supraoptic nucleus A distinct group of neurons in the hypothalamus, just above the optic chiasma; their neurosecretory endings terminate in the neurohypophysis. surface charge Electric charge at the membrane surface, arising from fixed charged groups associated with the membrane surface.

G-30 GLOSSARY ...................................... surface hypothesis Hypothesis of Rubner that metabolic rate of birds and mammals should be proportional to body surface area. surface tension The elasticity of the surface of a substance (particularly a fluid), which tends to reduce the surface area at each interface. surfactant A surface-active substance that tends to reduce surface tension, for example, in the lung. swim bladder A gas-filled bladder used for flotation; found in many teleost fishes. sympathetic nervous system Thoracolumbar part of the autonomic nervous system; increased activity in sympathetic neurons typ~cally provides metabolic support for vigorous physical activity, so this system has been called "the fight or flight system." symports Coupled membrane transporters that transfer two solutes in the same direction across the cell membrane. synapse A specialized area that connects two directly interacting nerve cells. At a synapse, activity in the presynaptic (transmitting) cell influences the activity of the postsynaptic (receiving)cell. synaptic cleft The space separating the cells at a synapse. synaptic current The ionic current that flows across a postsynaptic membrane when ion channels open after neurotransmitter molecules bind to membrane receptors; the flow of ions causes the membrane potential to change toward the equilibrium potential of the ion or ions that flow through the synaptic channels. synaptic delay The time separating the arrival of an impulse at a presynaptic nerve terminal and a change in the membrane potential of the postsynaptic cell. synaptic efficacy Effectiveness of a presynaptic impulse in producing a postsynaptic potential change. synaptic facilitation Increase in synaptic efficacy. synaptic inhibition A change in a postsynaptic cell that reduces the probability of its generating an action potential; produced by a transmitter substance that elicits a postsynaptic current having a reversal potential more negative than the threshold for the action potential. synaptic noise Irregular changes in the transmembrane potential of a postsynaptic cell, produced by random subthreshold synaptic input. synaptic transmission Transfer of a signal between two cells, usually between a neuron and another cell, which could be a neuron, a muscle, or some other effector cell such as a gland. synaptic vesicles Membrane-bound vesicles containing neurotransmitter molecules; located within axon terminals. syncitium A network of cells that are connected by lowresistance intercellular pathways. syneresis Contraction of a gelled mixture so that a liquid is squeezed out from molecular interstices. systemicPertaining to or affecting the body; for example, systemic circulation. systole The portion of the cardiac sequence when the heart muscle is contracting; it takes place between the first and second heart sounds as the blood flows through the aorta and pulmonary artery. T-wave That portion of the electrocardiogram associated with repolarization (and usually relaxation) of the ventricle.

...................................... tachycardia An increase in heart rate above the normal level. target cells Cells that preferentially bind and respond to specific hormones. taxis Locomotion that is oriented with respect to a stimulus direction or gradient. tectorial membrane A fine gelatinous sheet lying on the organ of Corti in contact with the cilia of cochlear hair cells. tectum The highest center for processing visual information in fishes and amphibians. teleost Bony fish, of the infraclass Teleostei. temporal Referring to the lateral areas of the head above the zygomatic arch. Also, relating to time; time limited. temporal lobe A lobe of the cerebral hemisphere, situated in the lower lateral area, at the temples. temporal summation Summation of postsynaptic membrane potentials that occur close to one another in time, but not simultaneously. tendon A band of tough fibrous connective tissue that anchors a skeletal muscle to the skeleton, allowing contraction of the muscle to move the body of an animal. terminal cisternae The closed spaces that make up part of the sarcoplasmic reticulum on both sides of the Z line, making close contact with the T tubules. tertiary structure Refers to the way a polypeptide chain is folded or bent to produce the overall conformation of the molecule. testosterone A steroid androgen synthesized by the testicular interstitial cells of the male; responsible for the production and maintenance of male secondary sex characteristics. tetanus An uninterrupted muscular contraction caused by high frequency motor impulses. Also the name of a neurotoxin that is retrogradely (toward the cell body) transported in axons and that causes prolonged excitation of muscle fibers, causing tetanic contraction. tetraethylammonium (TEA) A quarternary ammonium agent, (C,H,),N, that can be used to block some potassium channels in membrane. tetrodotoxin (TTX) The pufferfish poison, which selectively blocks voltage-gated sodium ion channels in the membranes of excitable cells. thalamus A major center in the midbrain of birds and mammals that receives and transmits both sensory and motor information. theca interna The internal vascular layer encasing an ovarian follicle; responsible for the biosynthesis and secretion of estrogen. theophylline A crystalline alkaloid (C,H,N,H,O) found in tea; inhibits the enzyme phosphodiesterase, thereby increasing the level of CAMP;also releases Ca2+from calcium-sequestering organelles. thermal conductance Ability of a material to conduct heat, which is poor in insulating materials such as fur or feathers. thermal neutral zone (thermoneutral zone) That range of ambient temperatures within which a homeotherm can control its temperature by passive measures and without elevating its metabolic rate to maintain thermal homeostasis. thermogenesis The production of body heat by metabolic means such as brown-fat metabolism or muscle contraction during shivering. thermophilic behavior Heat-seeking behavior. thermophobic behavior Heat-avoiding behavior.

c

thermoreceptor Sensory nerve ending specifically responsive to temperature changes. thick filament A myofilament made of myosin. thin filament A myofilament that contains actin and regulatory proteins. thoracic cage The chest compartment formed by the ribs and diaphragm containing the lungs and heart. thoracic duct Duct draining the lymphatic system into the anterior cardural vein. 3-dehydroretinal A derivative of vitamin A that is found in the visual pigments of freshwater fishes and amphibia. 3,5,3-triiodothyronine An iodine-bearing tyrosine derivative synthesized in and secreted by the thyroid gland; raises cellular metabolic rate, as does thyroxine. threshold potential The potential at which a response (e.g., an action potential or a muscle twitch) is produced. threshold stimulus The minimum strength of stimulation necessary to produce a detectable response or an all-or-none response. thymine A pyrimidine base, 5-methyluracil (C,H6N,0), a constituent of DNA. thyroid-stimulating hormone (TSH)An adenohypophyseal hormone that stimulates the secretory activity of the thyroid gland. thyroxine An iodine-bearing, tyrosine-derived hormone that is synthesized and secreted by the thyroid gland; raises cellular metabolic rate. tidal volume The volume of air moved in or out of the lungs with each breath. tight junction An area of membrane fusion between adjoining cells; prevents passage of extracellular material between adjoining cells; prevents passage of extracellular material between the cells. time constant (t) A measure of the rate of accumulation or decay in an exponential process; the time required for an exponential process to reach 63% completion. In electricity, it is proportional to the product of resistance and capacitance. tonic Steady; slowly adapting. tonicity (hyper-, hypo-, iso-) The relative osmotic pressure of a solution under given conditions (e.g., its osmotic effect on a cell relative to the osmotic effect of plasma on the cell). tonotopic map A pattern of auditory projection to the brain in which neurons are arranged and make synapses based on the frequency of the sound to which they respond. tonus Sustained resting contraction of muscle, produced by basal neuromotor activity. torpor A state of inactivity, often with lowered body temperature and reduced metabolism, that some homeotherms enter into SO as to conserve energy stores. trachea The large respiratory passageway that connects the pharynx and bronchioles in the vertebrate lung. tracheal system Consists of air-filled tubules that carry respiratory gases between the tissues and the exterior in insects. tracheoles Minute subdivisions of the tracheal system of insects. tragus The tab that extends from the ventral (anterior) edge of the outer ear and partially covers the opening of the ear. train of impulses A rapid succession of action potentials propagated down a nerve fiber. trans A configuration with particular atoms or groups on opposite sides.

transcellular pathways Routes through or around a cell by which substances are actively transported across an epithelium. transcription The formation of an RNA chain of a complementary base sequence from the informational base sequence of DNA. transducer A mechanism that translates energy or signals of one form into a different kind of energy or signals. transducer molecule Intermediate molecule (G protein) within the cell membrane that transmits a hormone-initiated signal from the externally facing hormone receptor to the internally facing enzyme. transducin The G protein that links the capture of light by rhodopsin molecules with a change in the current flowing across the membrane of photoreceptors. transduction General term for the modulation of one kind of energy by another kind of energy. Thus, sense organs transduce sensory stimuli into nerve impulses. transfer RNA (TRNA)A small RNA molecule that is responsible for the transfer of amino acids from their activating enzymes to the ribosomes; there are 20 tRNAs, one for each amino acid. transgenic animal An animal in which its genetic constitution has been experimentally altered by the addition or substitution of genes from other animals of that species or other species. translation Utilization of the DNA base sequence for linear organization of amino acid residues on a polypeptide; carried out by mRNA. transmitter substance A chemical mediator liberated from a presynaptic ending, producing a conductance change or other response in the membrane of the postsynaptic cell. transmural pressure The difference in pressure across the wall of a structure, for example, a blood vessel. transphosphorylation The transfer of phosphate groups between organic molecules, bypassing the inorganic phosphate stage. transverse tubules ( T tubules) Branching membrane-limited, intercommunicating tubules that are continuous with the surface membrane and are closely apposed to the terminal cisternae of the sarcoplasmic reticulum. traveling wave A wave that moves through the propagating medium, as opposed to a standing wave, which remains stationary. tricarboxylic acid cycle (TCA cycle; Krebs cycle; citric acid cycle) The metabolic cycle responsible for the complete oxidation of the acetyl portion of the acetyl coenzyme A molecule. trichromacy theory The theory that three kinds of photoreceptor cone cells exist in the human retina, each with a characteristic maximal sensitivity to a different part of the color spectrum. triglyceride A neutral molecule composed of three fatty acid residues esterified to glycerol; formed in animals from carbohydrates. triglycerol A neutral molecule composed of three fatty acid residues esterified to glycerol; formed in animals from carbohydrates. trirner A compound made up of three simpler identical molecules. trimethylamine oxide A nitrogenous waste product, probably from choline decomposition. tritium A radioactive isotope of hydrogen with an atomic mass of three (H3). Triton X-100 A nonionic detergent used in cell biology to solubilize lipids and certain cell proteins.

G-32 GLOSSARY ...................................... trituration Grinding; mastication. tRNA See transfer RNA. trophic level Individual level within a food chain. trophic substances Chemical substances believed to be released from neuron terminals and to influence the chemical and functional properties of the postsynaptic cell. tropomyosin A long protein molecule located in the grooves of actin filaments of muscle; inhibits muscle contraction by blocking the interaction of myosin cross bridges with actin filaments. troponin A complex of globular calcium-binding proteins associated with actin and tropomyosin in the thin filaments of muscle. When troponin binds Ca2+, it undergoes a conformational change, allowing tropomyosin to reveal myosinbinding sites on the actin filament. trypsin An enzyme specifically attacking peptide bonds in which the carboxyl group is provided by arginine or lysine. trypsinogen A proenzyme of trypsin. tubulin An actinlike, 4 nm globular protein molecule that is the building block of microtubules. tunica adventitia The fibrous outer layer of arterial blood vessel walls. tunica intima The inner lining of arterial blood vessel walls. tunica media The middle layer of arterial blood vessel walls consisting of smooth muscle and elastic tissue. turbinates Chambers of the nasal passages with olfactory receptors in the surface epithelium. turbulent flow Flow pattern in which exists sharp gradients and inconsistenciesin velocity and direction of flow fluid. turgor Distension; swollenness. twitch muscle (fast muscle) The most common striated vertebrate skeletal muscle type, usually pale in color because of its low myoglobin content. It has few mitochondria, and its fibers are constructed of many clearly defined fibrils. These contract rapidly and derive most of their energy from anaerobic metabolism. 2-deoxyribose 5-carbon sugar that forms a major structural component of DNA. tympanic membrane The eardrum. tympanum The middle ear cavity; houses the auditory ossicles. type J receptors Abbreviation for juxtapulmonary capillary receptors found in the lung that, when stimulated, elicit the sensation of breathlessness. ultradian rhythms Biological rhythms with a periodicity of greater than a day in length. ultrafiltrate The product of ultrafiltration. ultrafiltration The process of separating colloidal or molecular particles by filtration, using suction or pressure, by means of a colloidal filter or semipermeable membrane. ultraviolet light Light of wavelengths between 180 and 390 nm. uniports Carrier proteins which transport a single solute from one side of the membrane to the other. unitary currents Electrical currents due to the sudden opening of individual channels in the plasma membrane. unit membrane The sandwich-like profile of biological membrane seen in electron micrographs and believed to represent the bimolecular leaflet with a hydrophobic center region between hydrophilic surfaces. unsaturated In reference to fatty acid molecules, indicates that

some of the carbon-carbon bonds are double. Having free valence electrons. upper critical temperature (UCT)Ambient temperature beyond which normal heat loss mechanisms cannot prevent increase in body temperature. uracil A pyrimidine (C,H,O,N,) constituent of RNA. urea (NH,),CO, the primary nitrogenous waste product in the urine of mammals. ureotelic Pertaining to the excretion of nitrogen in the form of urea. ureter A muscular tube passing urine to the bladder from the kidney. urethra The channel passing urine from the bladder out of the body. uric acid A crystalline waste product of nitrogen metabolism found in the feces and urine of birds and reptiles; poorly soluble in water. uricolytic See uricotelic. uricolyticpathway The pathway through which uric acid or urates are cleavaged. uricotelic Pertaining to the excretion of nitrogen in the form of uric acid. Ussing chamber A chamber used to suspend tissue such as frog skin and measure its epithelial transport properties. utriculus One of the vertebrate organs of equilibrium. vacuole A membrane-limited cavity in the cytoplasm of a cell. vagus nerve (tenth cranial nerve) A major cranial nerve that sends sensory fibers to the tongue, pharynx, larynx, and car; motor fibers to the esophagus, larynx, and pharynx; and parasympathetic and afferent fibers to the viscera of the thoracic and abdominal regions. valence The number of missing or extra electrons of an atom or a molecule. van der Waals forces The close-ranging, relatively weak attraction exhibited between atoms and molecules with hydrophobic properties. van't Hoff equation An equation used to calculate the Q,, of a biological function. varicosities Swellings along the length of a vessel or fiber. vasa recta The capillary network that surrounds the loop of Henle in the tubules of the mammalian kidney. vasa vasorum The tiny arteries and veins that supply nutrients and remove waste products from the tissues in the walls of larger blood vessels. vas deferens A testicular duct that joins the excretory duct of the seminal vesicle to form the ejaculatory duct. vasoactive intestinal peptide Peptide hormone regulating intestinal phase of gastric secretion. vasoconstriction Contraction of circular muscle of arterioles, decreasing their volume and increasing the vascular resistance. vasodilation A widening of the lumen or interior space of the blood vessels, increasing blood flow. vasomotor Pertaining to the autonomic control of arteriolar constriction or dilation by contraction or relaxation of circular muscle. vasopressin See antidiuretic hormone. vasopressor A substance that induces arterial and capillary smooth-muscle contraction. vector A carrier; an animal transferring an infection from host to

host. Also, a mathematical term for a quantity with direction, magnitude, and sign. venous shunt A direct connection between arterioles and venules, bypassing the capillary network. ventilation In respiratory physiology, the process of exchange of air between the lungs and the ambient air. ventral Toward the belly surface. ventral horn The ventral part of gray matter in the vertebrate spinal cord in which motor neuron cell bodies are situated. ventral root A nerve trunk leaving the spinal cord near its ventral surface; contains only motor axons. ventricle A small cavity. Also, a chamber of the vertebrate heart. ventricular zone The region of the brain that surrounds the cerebral ventricles; in embryonic vertebrates, cells of the ventricular zone remain mitotic and generate the neurons of the brain and spinal cord. venule A small vessel that connects a capillary bed with a vein. vestibular apparatus The collection of vertebrate organs of equilibrium in the inner ear. villi Small, fingerlike projections of the intestinal epithelium. viscosity A physical property of fluids that determines the ease with which layers of a fluid move past each other. visible light Light of wavelengths between 390 and 740 nm. visual cortex The cerebral cortex in the occipital region of the cerebrum; devoted to processing visual information. visual streak (retinal streak) A retinal structure-found in the eyes of some species that inhabit plains-in which photoreceptors are packed into a horizontal streak across the retina, providing high resolution along the visual horizon. This pattern is similar to the fovea of primates, but it has a different shape. vital capacity The maximum volume of air that can be inhaled into or exhaled from the lungs. vitalism The theory that postulates that biological processes cannot adequately be explained by physical and chemical processes and laws. volt (V) MKS unit of electromotive force; the force required to induce a 1ampere (A) current to flow through a 1 ohm (R) resistance. voltage (E or V) The electromotive force, or electric potential, expressed in volts. When the work required to move 1coulomb (C)of charge from one point to a point of higher potential is 1 joule (J),or 114.184 calories (cal), the potential difference between these points is said to be 1 volt (V). voltage clamping An electronic method of imposing a selected

membrane potential across a membrane by means of feedback control. voltage-gated ion channels Protein channels through the cell membrane that allow ions to cross the membrane when they are open. The conductance of these channels depends upon the transmembrane electrical potential difference. watt (W)A unit of electrical power; the work performed at 1joule (J)per second. Weber-Fechner law Sensation increases arithmetically as a stimulus increases geometrically; the least perceptible change in stimulus intensity above any background bears a constant proportion to the intensity of the background. white matter Tissue of the central nervous system that consists mainly of myelinated nerve fibers. Wolffian ducts The embryonic ducts that are associated with the primordial kidney and that become the excretory and reproductive ducts in the mate. work Force exerted upon an object over a distance; force times distance. X-ray diffraction The method of examining crystalline structure using the pattern of scattered X rays. Xylocaine The trade name for lidocaine, a local anesthetic related to procaine.

Z disk (Z line, Z band) A narrow zone at either end of a muscle sarcomere, consisting of a latticework into which the actin thin filaments are anchored. zeitgeber Environmental factors that entrain biological rhythms. zero-order kinetics Kinetics in which the rate of the reaction is independent of the concentration of any of its reactants. This would occur if the enzyme concentration were the limiting factor. zonula Zone. zonula adherens Form of desmosome in epithelial cells that form a belt of cell-to-cell adhesion under tight junctions. zonula occludens Tight junctions between epithelial cells, usually having a ring-shaped configuration and serving to occlude transepithelial extracellular passages. zwitterion A molecule carrying both negatively and positively ionized or ionizable sites. zygomatic cells See chief cells. zygote A fertilized ovum before first cleavage. zymogen See proenzyme.

Page numbers in italics indicate illustrations. A bands, 352,356,357,371 A-type afferent fibers, 508 A-type cell, 601-602 abdominal ganglion, 457 abomasum, 640 absolute refractory period, 146,147, 156 absorption, 657-661 midgut, 636,640-643 nutrients, 636 surface, 627 water and electrolytes, 660 absorption spectra, 266 absorptive cells, 642 absorptive epithelium, 657 acceptor molecules, 66 acclimation, 5 thermal, 678,679 acclimatization, 5,678,682 accommodation of excitable membranes, 146,147 eye lens, 258 acetate, 195 acetate residue, 87 acetyl coenzyme A (acetyl CoA), 87-88,195 acetylcholine ( ACh), 175,177, 179-180,186,189-190, 193-196,198-200,202-205, 281,289,290,293,368,395, 421,423 blood flow, 542 heart, 472 in pacemaker cells, 474-475 salt secretion, 612 acetylcholine-activated channels, 184, 185,199-202 acetylcholine receptor channels, 185, 187,199-201

acetylcholine receptors ( AChRs), 195,199-200,294 acetylcholinesterase (AChE), 180, 182,195 acid-base balance, 15,517-568 acid loading, 534 acidophils, 306 acidosis, 524,531,534,602,603 acids, 4 6 4 7 dissociation of, 48 and pH buffers, 49 production and excretion, 534-535 removal, 534 weak, and buffering action, 53 1 acinar cells, 649 acinar lumen, 292 acinus (acini),292-294,537,538, 649 acoustic stimuli, 269 acoustic trauma, 247 acoustical impedance mismatch, 243 acoustical orientation, see echolocation acromegaly, 336 actin, 5 , 7,56,352,357, 359-361 muscle contraction, 401,402 see also thin (actin)filaments actin-myosin interaction, 367,378, 642 action potential (AP), 128-132,136, 145-158,163-164,173,186, 187,190,192, 193, 198,212, 213,219,221,225,249,433 all-or-none, 163, 180,181,369, 380 arthropods, 396 basic electric rhythm, 646 cardiac, 4 7 M 7 4

cardiac muscle, 398 chemical synapses, 174 chemoreceptors, 232-233 conduction velocity of, 170 contraction-relaxation cycle, 374 and excitatory inputs, 209-210 fast chemical synaptic transmission, 177 impulse conduction, 172 information transmission, 165 ionic basis of, 147-158 muscle contraction, 377 muscle fibers, 371,375 neuromuscular junction, 368 neuronal integration, 205-207 phasic receptors, 227 photoreceptors, 256,261 presynaptic, 207,212 propagation of, 167-169, 180-181 propagation velocity, 172 Renshaw cells, 435 response to light, 440 retinular cells, 255 and secretions, 28 1,282 and sensory pathways, 223,224 skeletal muscle, 472,473 smooth muscle, 401 sound-producing muscles, 425 species propagation, 170 spectrum, 266 stimulus intensities, 225-226 stomach, 656 and stretch receptors, 222 synaptic facilitation, 211 T tubule, 371 and taste receptors, 234 tetanus, 376 thermoreceptors, 251

1-2

INDEX

...........................

and tonic receptors, 227 transmission of, 175,176 twitches, 376,395 activation, 152 ion channels, 153 kinetics of, 387-389 potassium ion channels, 156 state of, and muscle, 382-383 activation energy, 69 activation particle, 155 active hyperemia, 512,513 active sites, 48, 70-75, 77 active state (of muscle), 375,377 active tensin, 356 active transport, 109-114,143-145, 657,658 epithelial, 119 salt, 120-122 secondary, 111 active zones, 178, 193 activity of electrolytes, 45 and metabolic conditions, 512 and thermoregulation, 703-704 actomyosln, 359,367 acupuncture, 198 acyl-phosphate bond, 85 adaption, 225 of action potentials, 146,147 to environment, 4-7 enzymatic, 678 evolutionary, 4 homeoviscous membrane, 679-680 mechanisms of, 227-229 of muscle for power, 3 81-3 84 physiological, in migrating fish, 615-61 6 sensory receptors and stimuli, 220-222 for speed in sound production, 389-392 addiction, 198 adenine, 60-61,66 cells, 308 aden~hypoph~seal adenohypophyseal hormones, 305-307,308 adenohyp~ph~sis, 304-305,329 adenosine, 316,510,511 cardiac activity, 475 adenosine diphosphate (ADP),67, 68,89,110,112,113,359, 694 and ATP, 79,8 1,83-85 muscles, 378 ribosyltransferase, 214

adenosine 5'-phosphate (AMP), 316-317 adenosine receptors, 3 16 adenosine triphosphate (ATP), 66-68,110-114,194,289, 313,314,316-317,325,366, 367,511,524 actomyosin dissociation, 359 cardiac metabolism, 475 chromaffin cells, 289,292 contraction-relaxation cycle, 374 hydrolysis, 144,361,362, 377-378,380-381 metabolic production of, 78-89 and muscles, 378-379 nonshivering thermogenesis, 694 regeneration during muscle activity, 378-379 secretory granules, 287,288 adenylate cyclase, 237,289, 313-317,319,326 adenyl cyclase pathway, 294 adenyl cyclase second-messenger system, 399 adipose cells, 32,54 adrenal cortex, 275-288,290,305, 329,336,338 adrenal glands, 284,286-289 adrenal medulla, 17, 117,281, 286-292,328,329,421 adrenaline, see epinephrine adrenergic neurons, 195,196,510 adrenergic receptors, see adrenoreceptors adrenocortical hormones, 305 adrenocorticosteroids, 612 adrenocorticotropic hormone (ACTH),56,305,306,329, 336,612 adrenoreceptors, 289-292,316, 423 aequorin, 192,323,372 aerobic enzymes, 519 aerobic glycolysis, 89 aerobic metabolic scope, 667 aerobic metabolism, 79, 80-82, 87 insects, 469 aerobic respiration, 79, 89 aerodynamical valving, 544 afferent arteriole, 593-594,598 afferent axons, 454 afferent fibers, 129,224 A- and B-type, 508 C-type, 508-509 skeletal muscle, 509 afferent nerve endings, 508,509

afferent neurons, 129, 130,217,224, 239,412,454-455 afferent pathways, 408,414415 affinity chromatography, 28 affinity sequence, 51 after-hyperpolarization, 145, 155 aggressive encounters, 425 aglomerular kidneys, 608 agonlsts, 194,201,326,421-423 Ahlquist, R. P., 289 alr-breathing animals body temperature, 681-682 diving and submers~on,513-515, 563-564 osmoregulation, 584-587 vertebrates, 561 alr-breathing fishes heart and circulation, 481-482 air bubbles and breathing, 550-552 air sacs birds, 544 insect tracheal system, 549 alanine, 58,61, 113,234 albumin, 592 concentration, 592 alcohol, 654 Aldini, Giovanni, 134 aldosterone, 284,336,594,598-601 alga, 717 alimentary canal, 292, 635,636 motility of, 644-649 alimentary systems, 635-644 alkaline tide, 654 alkalosis, 524,531, 602 allantoic acld, 623 allantoicase, 623-624 allantom, 618,623 allantoinase, 623-624 alligators reproductive energy costs, 719 allometry, 674,676, 718 allosteric regulation, 77,78 alpha (a)-actinin, 352,400 alpha (a)-adrenoreceptors,289-292, 294,316,317,325,327,399 alpha-amino acids, 55,57, 58 alpha (a)-Bungarotoxin (a-BuTX), 182,199,634 alpha-carbon (C,) atom, 48, 55-56 alpha cells, 329,334 alpha (a)chams, 520,525 alpha (a)-ecdysone,344 alpha (a)-factor,276 alpha (a)hehx, 57-59,220

INDEX 1-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .

alpha-keratins, 56,59,296 alpha (a)-ketoglutaricacid, 89 alpha (a)-motorneurons, 206 alpha-nitrogen atom, 58 alpha (a)protein, 157-158 altitude and oxygen levels, 562 alveolar collapse, 546-547 alveolar ducts, 535-538 alveolar gas, 538-539,540 alveolar pressure, 541-544 alveolar sacs, 535-538 alveolar ventilation volume, 538-541 alveoli, 535,537,538,546 amacrine cells, 438,439 American Journal of Physiology, 11 amide groups, 56,57,59 amiloride, 121,234 aminergic neurons, 648 amines, 302,303 amino acid chains spider silk thread, 296 amino acid residues calcium ion binding sites, 323,324 peptides, 308,310 proteases, 652 amino acids, 78,79 absorption, 657,658 amphoteric properties, 46 cell metabolism, 62-63 digestion, 652 DNA, 61 essential, 661 as fast neurotransmitters, 195 as nutrient molecules, 661-662 opsins, 267 as organic buffers, 49 pH, 48 and proteins, 55-59 reabsorption, 596 resynthesis or excretion of, 620 side groups, 96 and taste receptors, 234 transport of, 113,658-659 uptake of, 332 zwitterion, 48 see also amines; biogenic amines; enzymes; neuropeptides amino acid sequences hemoglobin, 522,524 amino groups, 48 aminopeptidases, 657 ammonia, 46,587,620-623 cell permeability to, 621 and cell pH, 532 Earth's atmosphere, 37

elevated levels, 620 fish, 608 production of, 602-603 toxic effects, 622 ammonia-excreting (ammonotelic) animals, 621-623 ammonium chloride (NH,Cl) and cell pH, 532,533 ammonium Ion (NH,'), 64, 618 buoyancy, 623 cell permeability, 621,622 and cell pH, 532 tubular filtrate, 602 ammonotelic animals, 62 1-623 amoebas, 302 amoeboid movement, 351 amperes, 50, 137 amphetamines, 196 Amphibia, 572 amphibians breathing, 559 cardiac muscle, 399 carotld bodies, 559 circadian rhythms, 717 digestive system, 638 egg-laying, 719 embryo nourishment, 719 excretion, 624 heart and c~rculation,480, 483-484 hemoglobin, 525 h~poxia,563 Jacobson's organ, 639 lungs, 535,546 lymph heart, 503 muscle fibers, 379,380 nervous system, 423 optic tectum, 438 osmoregulation, 574 pulmonary air temperature, 547 pulmonary circulation, 542 retinas, 268 skin permeabdlty, 575 tectum, 416 teeth, 631 urine, 578,608 vibrational stimuli, 428 water loss, 577 amphipathic compounds, 4 3 4 4 , 5 4 , 95 Amphzuma, 540 amphoteric molecules, 46,48,55-59 amplification by enzyme cascades, 322 sensory pathway, 223 of sensory receptor events, 225

sensory signals, 221 sensory transduction, 219-221 amplifiers, 12,314 amplitude receptor potential, 225-226 amygdala nucleus, 506 amylases, 284,293,294,649,652, 654 anabol~sm,665-666 anadromous fishes retinas, 268 anaerobes, 80 anaerobic glucose metabolism, 89 anaerobic glycolysis, 89 anaerobic metabolism, 79, 80,87, 378,379,712 oxygen debt, 89 anaerobic respiration, 89 anatomical dead space volume, 538, 539 androgen-binding protein (ABP),338 androgens, 338,339,341 androstenedione, 338 anemia, 661 anesthetics and body temperature, 702 aneurism, 493 angiotensin I, 598 angiotensln 11,74,285,336,512, 594,598,613 angiotensinogen, 512 angiotensinogen-converting enzyme (ACE),598 animal behavior, 4 2 3 4 3 1 measuring and observing, 32-33 animal electricity, 134 animal energetics and temperature, 677-685 animal physiology defined, 1-2 animal size and cost of locomotion, 708 animal spirits, 351 animals and gradients between environment, 575 muscles and movement, 351-403 primitive, 405 surface-to-volume ratio, 575-576 temperature classification of, 682-685 transgenic, 20-21 anion-exchange mechanisms, 533, 534 anion-exchange rates, 529 anionic sites, 104

1-4

INDEX

........................

anions, 43,45,49-52,102-104, 121,138,153,573 annelids, 9-10 ciliary motility, 645 extracellular fluid, 572 venom, 633 ventral nerve cord, 409 Annual Reuzew of Neurosaence, 11 Annual Review of Physzology, 11 anode, 50 anoxia, 563,564 antagonists, 195,458 antenna1 gland, 616-61 7 antibodies, 56,504-505 monoclonal, 17-1 9 polyclonal, 17,18 ant~bodystaining, 17,18 antidluretic hormone (ADH), 197, 308,309, 336,337,508, 597-599,619 see also vasopressin antifreeze substances, 685 antigens, 17,275,504,505 antimycin, 83 antiporters, 109,114 ants, 684 anurans, 576,685 anus, 643 aorta, 493495,497 aortic arch baroreceptors, 560 aortic blood pressures, 496 aortic bodies, 559-560 aortlc pressure, 476 aperture vertebrate eye, 252,253 apical (mucosal)membrane, 122, 279-281,587,609,610 digestive absorption, 657 kidney, 589 apnea, 514,537 apocrine glands, 293 apocrine secretion, 28 1 apoenzyme, 72 apolysis, 346 aporepressor, 77 aporepressor-corepressor complex, 77 appendages reduct~onof heat loss from, 695 aquaporln, 575 aquatic animals breathing, 561 buoyancy, 565 eyes, 253 filter feeding, 629

......................... flow of blood and water, 552 food absorption, 628 hypoxia, 562 aquatic insects breathing, 550-552 aquatic invertebrates excretion of wastes, 621 flow of blood and water, 552 osmoregulation, 580 aqueous environments osmoregulation, 58 1-584 aqueous phase, 106 flow of blood and water, 552 arachnids osmoregulation, 587 Arapaima gigas, 524 arctic fox, 1 area centralis, see fovea Area 17,443-444,448 arginine, 58,70,234,658 arginine vasopressin, 308, 309, 336 arginine vasotocin (AVT),309, 336,607,613 see also vasotocin Aristotle, 4,217 arterial baroreceptors, 506-508 arterial blood carbon dioxide, 568 pH, 525 arterial blood flow velocity of, 497 arterial blood pressure, 492493, 508,509 and body position and gravity, 496 control of, 505 diving and submersion, 514 and exercise, 512 flow to brain and heart, 510 lung, 540-542 sympathetic stimulation, 510 arterial chemoreceptors, 508 arterial system, 467,469,492497 as pressure reservoir, 491492 arteries, 97,467,492497 laminar flow, 488,489 see also arterial system; peripheral circulation arterioles, 421,499-500,560, 593-594 afferent and efferent, 589, 590 and blood flow, 496 nervous control of, 510 smooth muscle, 400 vasoconstriction, 5 10

Arthropoda alimentary canal, 645 blood c~rculation,467 body temperature, 717 command systems, 458 compound eyes, 253-257 excretion of nitrogen, 624 flu~dfeeding, 630 motor control, 396-397 nervous system, 413 osmoregulation, 584-587,617 venom, 633 ventral nerve cord, 409 water loss, 577 artificial lipid bilayers, 98, 108-109 asc~d~ans osmoregulation, 58 1 ascorbic acid, 662,663 asparagine, 58 aspartate, 194,658 aspartic acid, 58 aspirin, 343 association cortex, 417 Asterzas, 9 astrocytes, 18 Astrup, Paul, 518 asynchronous flight muscles, 392-394 atmosphere primitive Earth, 37 atomic structure, 38 atoms, 3 8 4 0 ATP, see adenosine triphosphate ATPase activity, 110-112, 114 basolateral membrane, 280 contraction-relaxation cycle, 374 dark current, 261 myosin, 367 atrial cells, 202,203,473 atrial contraction, 475,477,481 atrial mechanoreceptors, 508-509 atrial wall elasticity, 493494 atrioventricular block, 475 atrioventricular node, 470,471,474, 475 atrium, 470,479 atropine (belladonna),182,423 attachment plaques, 400 auditory canal, 244 auditory cortex, 419 auditory map owl brain, 4 4 9 4 5 3 aud~torymechanisms, 428 auditory neurons, 436 auditory ossdes, 243-244

INDEX 1-5 .........................

auditory sensory reception, 269 auditory systems, 243,248 thermal noise, 269 August Krogh Principle, 15-1 6 Auk, 11 Australian Journal of Zoology, 10 autocrine secretions, 273,275,302 autoimmune disease, 504 autoinhibition, 231,289 autonomic control of blood flow, 682 autonomic nervous system, 412, 420423,457,647,648,682 autophosphorylation, 325 autoradiography, 1 7 autoregulation glomerular filtration rate, 593-594 autotrophic plants, 627,628 avian aorta, 495 Avogadro's law, 520 Avogadro's number, 45,50,153 avoiding response, 407 AW, see arginine vasotocin axon terminal, 128,129, 164,179 axon hillock, 128, 129,206,207, 210 axonal membrane, 129 axonal transport systems, 304 axons, 127-130,131,148 action potentials, 145,167-169, 224 afferent fiber, 129 auditory neurons, 247 cable properties, 167,169 connectives, 409 current distribution, 166 diameter, 172 fast excitor, 397 invertebrate nervous systems, 408, 409 ionic concentrations, 158 postganglionic neurons, 421 rapid, saltatory conduction in, 170-1 74 e signal transmission, 151 slow excitor, 397 spinal cord, 414 azimuth and sound source, 449,451,452 B cells, see B lymphocytes B lymphocytes, 17-18,504-505 B-type afferent fibers, 508 B-type cells, 602 background noise, 269 , bacteria

and digestion, 641 orientation, 431 pyrogenic, 702 bacterial flora control of by saliva, 293 balanced nutritional state, 661 baleen plates, 630 baleen whales, 630 band I11 protein, 527,529,532 Barbour, Henry G., 699 barn owls auditory system, 449-453 barnacle eye, 218 baroreceptors, 506-508,515,560 barrel fields, 419 basal cells taste receptors, 233 basal clefts, 589 basal membrane fish, 249 kidney, 589 base pairs, 60 basement membrane, 500,501 bases, 4 6 4 7 and pH buffers, 49 basic electric rhythm (BER),646,648 basilar membrane, 244,245 basolateral (serosal)membrane, 280, 589,609,610 digest~on,657 kidney, 601-602 basophils, 306 batch reactors, 635 wing venules, 498 bats, 436 brain, 428,453 circadian rhythms, 716 echolocation, 428 insect capture, 428 sensory signals, 406 vampire, 631 Bayliss, William, 302 Baylor, Denis, 265 beaks prey capture, 633 bears winter sleep, 705 beaver breathing, 551 bedbugs fluid feeding, 630 bees, 392 navigation, 429,430 beetles, 392,549 behavior, 423-431

central command systems, 457461 fish muscle, 3 88 genetically programmed, 426 initiation, patterns, and control, 405461 and lack of nervous system, 407 measuring and observmg, 32-33 and sensory networks, 434-453 behavioral concepts, 423426 behavioral condition feeding and digestion, 654 behavioral control heat exchange, 682 behavioral plasticity, 210 behavioral psychology, 408 behavioral research methods, 32-33 behavioral thermoregulation, 683-684,686,701 BCkCsy, Georg von, 247 Bell-Magendie rule, 414 bends, 564 Bernard, Claude, 7-8,301 Bernheim, H. A., 702 Bernoulli's effect, 629 Berthold, A. A., 283 beryllium, 40 beta (P)-adrenoreceptors,289-292, 294,316-319,325,327,510 cardiac cells, 399 beta (P)-Bungarotoxin,56, 182 beta cells, 329,332 beta (p)chains, 520 and hemoglobin, 525 beta (P)-ecdysone,344,345,346 beta-keratins, 59 beta (p)particles, 16 beta pleated sheet, 58-59,60 beta (p)proteins, 158 bicarbonate in tubular filtrate, 602 bicarbonate ions (HCO,), 525-527, 530,532-534,573 blood, 528 and carbon dioxide (Jacobs-Stewart cycle), 533-534 bicarbonates as organic buffers, 49 bicuculline, 182 bilateral symmetry, 411 bile, 650-651 bile acids, 660 bile duct, 641 bile fluid, 641 bile pigments, 650

1-6

INDEX

.........................

bile salts, 641,650-651,653 billfin fishes, 685 blllfishes, 694 blndlng energies, 269 blndlng proteins, 280 binocular convergency, 258 blochemical amplification, 322 b~ochem~cal analysis, 28-3 1 biochemical diversity, 60 blochemlcal molecules orlgin of, 37-38 b~ochemicalprocesses, 38 biogenic amines, 177,195-1 96 blolog~calclocks, 677, 717 blological molecules, 53-62 blological rhythms, 713-717 temperature regulation and metabohsm, 715-717 biosynthetic reaction sequence enzyme synthesis, 77 blotin, 662 bipedal animals locomotion, 708 blpolar cells, 438,439,440,441, 442 on and off, 441 bird eggs gas transfer in, 547-548 shell, 488 bird embryos heart, 480,488,503 wastes, 621 b~rds audltory mechanisms, 428 beaks and feedmg, 633 body temperature, 682,692 bram, 415,416 breathmg, 559 cardiac cells, 473 carotld body, 559 circadian rhythms, 715-716 core temperature, 716 crop, 639 digestive system, 638 divlng, 563 evaporative coollng, 697 excretion, 623,624 extracellular fluid, 572 feeding offspring, 719 filter feedlng, 630 flying, 710-71 1 ground-nestlng, 32 heart, 438,442,480,486487 heat exchangers, 499 hemoglobin, 525 kldneys, 577-578,608,613

......................... lungs, 544-546,561 muscle fibers, 379,380 nasal salt gland, 284,609-614 navigation, 4 2 9 4 3 0 oocytes and ova, 340-341 orientation, 426,430,431 pulmonary circulation, 540 raptorial feeding, 633 reproductive patterns, 718 skin permeability, 575 submersion, 5 14 temperature receptors, 701 urine, 578,603 vision, 406 visual pathway, 438,442 water loss, 577,578 birefringence, 255 birth control pills, 342 bitter taste, 220,233,234,293 black widow spider, 295 venom, 633-634 Black, Joseph, 518 blackfly, 631 bladder, 638 bladder wall release of urine, 588 Block, B. A., 694 blood carbon dioxide transport in, 525-526 circulatory systems, 467-515 density, 490 high-oxygen affinity, 7 hyposomotic, 580 ionic concentration and oxygen release, 567 leukocytes, 505 nitrogen in, 564 oxygen and carbon dioxide in, 5 19-529 oxygen transport in, 521-525 pooling of, 4 9 6 4 9 8 transfer of gases to and from, 518, 526-529 viscosity and blood flow, 491 blood distribution venous system, 498 blood donors, 497 blood flow, 3,488492,509 arterial system, 492-494,497 autonomic control of, 682 capillaries, 501-502 countercurrent, 499 diving and submersion, 514 effect of gravity and body position, 496497

exercise, 5 12-513 fish gills, 553 and heat loss, 695 kidney, 588 maximum veloclty of, 490 pulmonary circulation, 540-542 relationship with blood pressure, 489492 renal, 606 resistance to, 489,490491 sheet flow, 540,552,554 velocity, 488489 venous, 4 9 7 4 9 8 see also circulation; circulatory systems blood glucose, 335 blood osmolarity, 581,606,612,613 blood perfuslon, 475 kldneys, 592-593 respiration eplrhel~um,555-558 blood pH, 49,273,568 acidosis and alkalosis, 524 Bohr effect, 523,524 changes from acid movement, 531 mammalian arterial blood, 525 and oxygen capacity, 524 transfer of carbon dloxide, 527 vertebrates, 530 blood pressure, 3,478480 arterial system, 492,495496, 506,508 and baroreceptors, 506-508 blood ADH level, 607 C-type afferents, 508,509 capillaries, 50 1-502 closed circulation, 469 effect of gravlty and body position, 496497 and extracellular fluid, 598 and glomerular filtration rate, 593 and hemorrhage, 5 15 hlgh altitudes, 562-563 mammalian heart, 470 measuring, 22 relationship with blood flow, 489492 salt-gland secretion, 612 ultrafiltration, 469-470 and urine formation, 616 blood reservoirs, 497 blood supply, 505 blood volume, 488 capillaries, 500 closed clrculatlon, 469470 venous system, 497 see also cardiac output

INDEX 1-7 ....................................................

blotting, 29-30 blow hole, 586 blubber, 683, 695 blue-absorbing pigment, 267 body heat dissipation of, 696-697 and temperature, 680-682 body mass and energy expenditure, 665 and power, 709 body orientation and rate of heat absorbance, 697 body pH, 530,534-535 regulation of, 529-535 body position and blood pressure and flow, 406-407 body rhythms, 713-717 body size and metabolic rate, 672-676 body surface body temperature, 675 food absorption, 627,628 oxygen and carbon dioxide transfer, 517 body temperature, 7 anesthetics and opiates, 702 basal metabolic rate, 666 and biological rhythms, 715-716 and body surface area, 675 control of, 5 endotherm regulation mechanisms, 692-698 and environment temperature, 681-682 and enzymes, 3 hibernation, 705 homeotherms, 682 maintaining, 3 and metabolic activity, 665 nonmammalian thermoregulatory centers, 701 receptors, 250 regulation of, 298 ' reptiles, 547,717 standard metabolic rate, 666 thermostatic regulation of, 698-701 torpor, 704,705 variations, 713 water and control of, 698 body-temperature independence circadian rhythms, 714 Bohr effect (Bohr shift), 523,524, 529 boiling point

elevation of, 45 of water, 42 Boll, R., 263 bolus, 635,639,645,654 bomb calor~meter,669 bombykol, 232,274 bone cell strains, 27 hormonal effects on, 337,339 resorption, 276 Bonner, J. T., 676 boron, 40 Bossert, W. H., 122 Bowman's capsule, 588-594,608 Boyle, Robert, 518,520 Boyle's Law, 3 brachiopods, 629 bradycardia, 484,513,514 diving, 563 bradykinin, 509,512 brain, 130,131,415420 blood perfus~on,510 capillaries, 501 hormones, 3 10 nerve cells and oxygen deficiency, 80 neurons 409,511 and pH changes, 535 preventing overheating, 703 retina, 447 and sensory ~nformation,217 temperature, 685 vertebrate, 416 visual cortex, 442 visual pathway, 438 Braln, Behavzor and Evolutzon, 11 brain function parallel processing, 443 bram-gut hormones, 3 10 brain maps, 4 5 1 4 5 3 bats, 453 owl auditory system, 449453 brancial filter plates, 630 breathing, 535-537,543-544 apnea, 514 bubble breathing, 550-551 chemoreceptors, 508,561 episodic, 559 gas-transfer system, 518 movements, 538 neural regulation of, 558-562 panting, 697 pH increase, 531 rhythmic, 559 and venous system, 498 breath~ngdepth, 559-562

breathing rate, 539-540,559-562 breathlessness, 562 breaths intervals between, 558 Brett, J. R., 716 Breuer, Josef, 558 Briggs, George E., 73 bright field microscopy, 25 bronchi, 535 bronchial circulation, 540 bronchioconstriction, 561 bronchioles, 535-536 brown fat (brown adipose tissue, BAT), 693-694,705 Brownian motion, 269 Brunner's glands, 656 Bruns, Dieter, 193 brush border, 596,642,643,657, 658 bubble breathing, 550-551 buccal cavity, 545,554 buffer systems, 49-50,531 buffers of pancreatic enzymes, 656 and pH, 531,535 bulbis cordis, 482 bulbogastrone, 652 bulbus, 494 bullfrog breathing, 559 Bungarotoxin, see alpha (a)Bungarotoxin; beta @)Bungarotoxin Bunsen solubility coefficient, 519, 520,531 buoyancy, 565,623,709 bursicon, 344-346 Buxton, Dudley W., 366

C (clear) cells, 276 C fibers, 158 14Cisotope, 16 C-type afferent fibers, 508-509 4SCaisotope, 16 Ca2+,see calcium ion cable properties of axon, 167,169 of cells, 165-166 caffeine, 317, 654 calciferol, 662 calcitonin, 275,276,285,336-338 calcitriol, 284, 336,337,660 calcium and muscle contraction, 366368 reabsorption, 596

calcium channels cardiac muscle, 399 calcium-chelating agents, 354,356, 366 calcium chloride, 45 calcium conductance, 289,473 calcium ion-activated kinases, 326-327 calcium ion binding proteins, 323-324 calcium ion/calmodulin complex, 401,402 calcium ion/calmodulin kinase, 324-326 calcium ion channels, 153, 158-159, 185,187,189-192,203 calcium ion-dependent membrane fusion, 193 calcium ion-dependent voltage-gated potassium ion channels, 159 calcium ion equilibrium potential (Eta), 191 calcium ion-induced

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