Current Topics in Membranes and Transport, Volume 06

Current Topics in Membranes and Transport

Advisory Board

Robert W . Berliner Peter F. Curran (Deceased) I . S. Edelman I . M . Glynn Franpis Morel Aser Rothstein Philip Siekewitz Torsten Teorell Daniel C . Tosteson Hans H. Ussing

Contributors

W . McD. Armstrong Halvor N . Christensen Torben Clausen Mahendra Kurnar J a i n A . A. Lev H . J . Schatzmann

Current Topics in Membranes and Transport

VOLUME 6

Edited by Felix Bronner

Department of Oral Biology University of Connecticut Health Center Farmington, Connecticut and Arnort Kleinzeller

Graduate Division of Medicine University of Pennsylvania Philadelphia, Pennsylvania

1975

Academic Press

New York

San Francisco

London

A subsidiary of Harcourt Brace Jouanovich, Publishers

COPYRIGHT 0 1975, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NWl

LIBRARY OF CONGRESS CATALOG CARDNUMBER:70-1 17091 ISBN 0-12-153306-9 PRINTED IN THE UNITED STATES OF AMERICA

List of Contributors, vii Contents of Previous Volumes, ix Peter Ferguson Curran, 1931-1974, xiii Role of Cholesterol in Biomembranes and Related Systems MAHENDRA KUMAR JAIN

I. Introduction and Scope, 1 11. Distribution of Cholesterol in Biological Systems, 5 111. Solubilisation and Dispersion of Cholesterol, 13 IV. Correlative Relationships of Cholesterol Content, 16 V. State of Cholesterol in Organized Lipid Aggregates, 23 VI. Molecular Aspects of Organization of Cholesterol in Bilayers, 39 References, 41 Ionic Activities in Cells A. A. LEV AND W. McD. ARMSTRONG I. Introduction, 59 11. Definition of “Single Ion Activities,” 63 111. Experiments with Model Polyelectrolyte Systems as Supporting Evidence for the Physical Validity of Single Ion Activity Parameters, 66 IV. Microelectrodes for Measuring Intracellular Ionic Activities, 72 V. Techniques for Measuring Intracellular Ionic Activities, 84 VI. Intracellular Ionic Activities, 90 VII. Conclusion, 113 References, 113

Active Calcium Transport and Ca2+-ActivatedATPase in Human Red Cells H. J. SCHATZMANN

I. Calcium Transport, 126 11. Membrane ATPases Activated by Calcium in Human Red Cells, 142 111. Relationship between Calcium Transport and Calcium Magnesium-Activated ATPase, 149 IV. Relation between Calcium-Transport-ATPase and SodiumPotassium Transport-ATPase, 154 V. Comparison with Other Systems Transporting Calcium, 155

+

V

vi

CONTENTS

VI. Physiological Significance of Calcium Pumps, 157 References] 161

The Effect of Insulin on Glucose Transport in Muscle Cells TORBEN CLAUSEN

I. Introduction, 169 11. Preparations Used for the Study of Insulin Action on Sugar Transport, 170 111. Cellular Structures Involved in Sugar Transport, 172 IV. The Function of the Glucose Transport System, 178 V. Cellular Signals Controlling Glucose Transport, 196 VI. Mechanisms for the Mode of Act.ion of Insulin, 209 References] 211 Recognition Sites for Material Transport and Information Transfer HALVOR N. CHRISTENSEN

I. Introduction. Summary List of Principal Transport Systems for the Amino Acids, 227 11. Description of the Neutral Systems, 229 111. The Cationic Amino Acid Systems, 232 IV. Extension to System ASC of Approaches to Site Description Taught by the Basic Amino Acids, 235 V. Efforts to Develop System-Specific, Nonmetabolizable Substrates for the Neutral Systems, 237 VI. System-Specific Substrates for the Transport System for the Cationic Amino Acids, 241 VII. Stimulation of Release of Pancreatic Hormones by Amino Ac?ds, 243 VIII. Concluding Discussion, 250 References, 255 Subject Index, 259

List of Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin. Department of Physiology, Indiana University School of Medicine, Indianapolis, Indiana (59)

W. McD. Armrfrong,

Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan (227)

Halvor N. Christensen,

Torbon Clauren,

Fysiologisk Institut, Aarhus Universitet, Aarhus, Denmark (169)

Department of Chemistry and Health Sciences, University of Delaware, Newark, Delaware (1)

Mahendra Kumar Join,

Institute of Cytology of the Academy of Sciences of the USSR, Leningrad, USSR (59)

A. A. Lov,

Institute of Veterinary Pharmacology, University of Bern, Bern, Switzerland (125)

H. J. Schalrrnann,

vii

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Contents of Previous Volumes Volume 1

Some Considerations about the Structure of Cellular Membranes MAYNARD M. DEWEYAND LLOYDBARR The Transport of Sugars across Isolated Bacterial Membranes H. R. KABACK Galactoside Permease of Escherichia coli ADAMKEPES Sulfhydryl Groups in Membrane Structure and Function ASERROTHSTEIN Molecular Architecture of the Mitochondrion DAVIDH. MACLENNAN Author Index-Subject Index Volume 2

The Molecular Basis of Simple Diffusion within Biological Membranes W. R. LIEB AND W. D. STEIN The Transport of Water in Erythrocytes ROBERTE. FORSTER Ion-Translocation in Energy-Conserving Membrane Systems B. CHANCEAND M. MONTAL Structure and Biosynthesis of the Membrane Adenosine Triphosphatase of Mitochondria ALEXANDER TZAGOLOFF Mitochondria1 Compartments: A Comparison of Two Models HENRY TEDESCHI Author Index-Subject Index

X

CONTENTS

OF PREVIOUS VOLUMES

Volume 3

The Na+, K+-ATPase Membrane Transport System: Importance in Cellular Function ARNOLDSCHWARTZ, GEORGE E. LINDENMAYER, AND JULIUS C. ALLEN Biochemical and Clinical Aspects of Sarcoplasmic Reticulum Function ANTHONY MARTONOSI The Role of Periaxonal and Perineuronal Spaces in Modifying Ionic Flow across Neural Membranes W. J. ADELMAN, JR.AND Y. PALTI Properties of the Isolated Nerve Endings GEORGINA HODR~GUEZ DE LORESARNAIZAND EDUARDO DE ROBERTIS Transport and Discharge of Exportable Proteins in Pancreatic Exocrine Cells : I n Vitro Studies J. D. JAMIESON The Movement of Water across Vasopressin-Sensitive Epithelia RICHARD M. HAYS Active Transport of Potassium and Other Alkali Metals by the Isolated Midgut of the Silkworm WILLIAMR. HARVEY AND KARLZERAHN Author Index-Subject Index Volume 4

The Genet>icControl of Membrane Transport CAROLYN W. SLAYMAN Enzymic Hydrolysis of Various Components in Biomembranes and Related Systems MAHENDRA KUMAR JAIN Regulation of Sugar Transport in Eukaryotic Cells HOWARD E. MORGAN AND CAROL F. WHITFIELD Secretory Events in Gastric Mucosa RICHARD P. DURBIN Author Index-Subject Index Volume 5

Cation Transport in Bacteria: K+, Na+, and H+ FRANKLIN M. HAROLD AND KARLHEINZ ALTENDORF Pro and Contra Carrier Proteins; Sugar Transport via the Periplasmic Galactose-Binding Protein WINFRIED Boos

CONTENTS OF PREVIOUS VOLUMES

Xi

Coupling and Energy Transfer in Active Amino Acid Transport ERICH HEINZ The Means of Distinguishing between Hydrogen Secretion and Bicarbonate Reabsorption : Theory and Applications to the Reptilian Bladder and Mammalian Kidney AND THEODORE P. SCHILB WILLIAM A. BRODSKY Sodium and Chloride Transport across Isolated Rabbit Ileum STANLEY G. SCHULTZ AND PETER F. CURRAN A Macromolecular Approach to Nerve Excitation ICHIJI TASAKI AND EMILIO CARBONE Subject Index

Peter Ferguson Curran

Peter Ferguson Curran, 1931-1974 Historiographers may argue that a scientist’s contributions should be subjected to the tests of time and appraised by a detached reviewer-not by a still stunned and saddened collaborator barely two weeks after the death of his friend. Yet, it is a testimony to the high caliber of Peter F. Curran’s accomplishments that many of his contributions have already admirably weathered these tests and have been so widely acclaimed by objective critics that unnecessary, but inevitable, praise from friends and admirers may be forgiven. Curran’s first major publication (with A. K. Solomon), “Ion and water fluxes in the ileum of rats” (J. Gen. Physzol. 41, 143, 1957) heralded the caliber and set the theme of his lifelong scientific endeavors. The principal conclusion, that water absorption is a passive process coupled to solute transport, challenged the then-viable notion of “active” water absorption and, within a very few years, became a truism. However, the notion of “active” water transport could not be dismissed until a formal mechanism was developed which could account for both the stoichiometric coupling between water and solute flows and the fact that water can be absorbed against adverse hydrostatic and/or osmotic pressure differences. In 1961, Curran, drawing upon the work of Staverman, laid the foundation of the widely acclaimed “double (series)-membrane model” which was experimentally verified a year later in the now classic paper (with R. MacIntosh), “A model system for biological water transport” [Nature (London) 193, 347, 19621. The almost immediate recognition and acclamation of this model stemmed from its power, elegance, and simplicity-hallmarks of creative genius, and recurring themes of many of Curran’s subsequent contributions. Active transport of solute into a constrained intraepithelial compartment could bring about passive water absorption in the absence of, and against, transepithelial gradients of water activity, providing the barriers bounding this compartment have different passive permeation properties; such asymmetries are sufficient to effect net water transport without having to postulate a direct link to metabolic energy. In short, the major characteristics of transepithelial water transport can be mimicked by a simple system that consists of two different artificial membranes arranged in series. The need to invoke “active” water absorption could be dismissed and the conceptual foundation for the ultrastructural analysis of transepithelial water transport was laid. A milestone in the application of theory to the elucidation of biological processes had been achieved! The “double (series)-membrane model” was born only one year after Ora Kedem and Aharon Katchalsky brilliantly thrust the formalisms of irreversible thermodynamics onto the biological stage. Curran was captivated by the beauty and power of this relatively new discipline and by the sparkle and genius of Katchalsky; Katchalsky, in turn, was immediately attracted to this talented young investigator who had already employed the concepts of irreversible thermodynamics to “tame” a major biological problem. Katchalsky and Curran became lifelong friends and co-authored the book, “Nonequilibrium Thermodynamics in Biophysics” (Harvard University Press, 1965)-a treatise that glows with lucidity, insight, and logic and which is destined to become a classic. [It is sad and ironic that one piece of unfinished business a t the time of Pete’s death xiii

xiv

PETER FUROUSON CURRAN, 1931-1974

was a dedication to Katchalsky (assassinated in 1972) which was to be published in the Biophysical Journal.] Curran was a gifted theoretician and experimentalist. Between 1957 and 1974 he authored or co-authored more than eighty papers dealing with transport across artificial membranes, frog skin, and intestine; the interaction between sodium transport and the transport of sugars and amino acids by small intestine; and, most recently, the molecular characteristics of the amino acid transport mechanisms in small intestine. The common thread running through all of his contributions is the attempt to marry theory with observation successfully-to construct the simplest model that could explain and unify a set of apparently unrelated observations and thereby lay a logical foundation for subsequent studies. This credo is clearly set forth in the paper entitled “Coupling between transport processes in intestine’’ (Physiologist 11, 3, 1968) delivered in 1967, when he was honored by being chosen the American Physiological Society’s Twelfth Bowditch Lecturer. The past quarter of a century has witnessed exciting and significant advances in our understanding of solute and water transport across epithelial tissues, advances to which the name of Peter F. Curran will always be closely linked. Peter F. Curran was born on November 5, 1931 in Waukesha, Wisconsin. He received his B.A. from Harvard College in 1953 and his Ph.D. from Harvard University in 1958. During his professional life he was involved in a host of activities reflecting the high esteem in which he was held by his colleagues. He was Section Editor for Gastrointestinal Physiology of the American Journal of Physiology (1968-1971), Chairman of the Publications Committee of the American Physiological Society (1971-1974), served on the Editorial Boards of the Biophysical Journal, Biochimica et Biophysica Acta, and the Journal of General Physiology, and was on the Advisory Board of This Publication. He was President of the Society of General Physiologists (1972-1973) and a Council Member of the American Physiological Society and the Biophysical Society. He waa a member of the Molecular Biology Advisory Panel of the National Science Foundation and Chairman of Physiology Study Section of the National Institutes of Health. At the time of his death, he was Professor of Physiology and Director of the Division of Biological Sciences at Yale University. Peter F. Curran died a t the age of forty-two. His professional career was rich with achievement and recognition. We will never know the full potential of his talents nor the further heights to which this extremely gifted and energetic scientist might have risen. His untimely death is not only a tragic loss to those who knew him but t o the entire scientific community. STANLEY G. SCHULTZ

Current Topics in Membranes and Transport Volume 6

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Related Systems* MAHENDRA K U M A R J A I N Department of Chemistry and Health Sciences, University of Delaware, Newark, Delaware

1. Introduction and Scope

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

. . . . . . . . Solubilization and Dispersion of Cholesterol. . . . . . . . . . Correlative Relationships of Cholesterol Content . . . . . . . .

11. Distribution of Cholesterol in Biological Systems 111.

IV.

A. With Barrier Properties of Biomembranes . . . . . B. With Permeability of Model Systems . . . . . . C. With Electrical Properties of Bilayer . . . . . . V. State of Cholesterol in Organized Lipid Aggregates . . . A. In Monolayers. . . . . . . . . . . . . B. In Bilayers. . . . . . . . . . . . . . C. I n Biomembranes . . . . . . . . . . . . VI. Molecular Aspects of Organization of Cholesterol in Bilayers . References . . . . . . . . . . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . . .

1 5 13 16 16 18 22 23 23 27 38 39 41

We must be grateful to Lord Russell for the unequaled skill with which he has left the vast darkness of the subject unobscured. Alfred North Whitehead

1. INTRODUCTION AND SCOPE

For quite some time now, considerable attention has been focused on the role of cholesterol in arterial degeneration and on its role as an essential

* Abbreviations used : CTAB, cetyl trimethylammonium bromide; BLM, black lipid membrane; DSC, differential scanning calorimetry; ESR, electron spin resonance; NMR, nuclear magnetic resonance; PC, phosphatidylcholine; PE, phosphatidylethanolamine; RBC, red blood cell; SM, sphingomyelin. 1

2

MAHENDRA KUMAR JAlN

component of many cell organelles and body fluids. The turnover and the organizational and physical state of cholesterol and its esters in tissues, cells, and body fluids have been the subject of intensive study. I n the solid state, cholesterol displays considerable pleomorphism and is not readily dispersed in water or electrolyte solutions. Cholesterol has a maximum solubility in aqueous solutions of 4.7 pM and c.m.c. of 25-40 nM a t 25’ C (Haberland and Reynolds, 1973). Cholesterol can, however, be “solubilized” and carried in ternary systems with lecithin, or in quaternary systems containing bile salts and lecithin (Small et aE., 1966), or in certain proteins and phosophlipids (see below). I n these dispersed systems cholesterol enters the mesophase only in the unsubstituted forms. Plasma lipoproteins, however, can also carry some cholesterol esters (Table I). The fatty acid chain length of the amphipath does influence the order and stability of incorporation of unsubstituted cholesterol in the mesophase. TABLE I

PERCENTAGE COMPORITION OF SOME HUMAN BLOOD SERUMLIPOPROTEINS

Lipoprotein characteristic Average hydrated densit.y (gm/ml) Molecular weight Particle size (diameter, A) Shape Percentage composition (w/w) Protein Phospholipid Cholest,erol Free Ester Trigly ceride Free fatty acid Fraction of plasma lipids Cholesterol Phospholipid Trigly ceride Presence in blood

Very low density Low density High density ‘(VLDL) (LDL) (HDL) pre-6p-lipoa-lipoChylomicron lipoprotein proteins proteins cholesterol > campesterol > sitosterol (Bruckdorfer et al., 1868b, 1969). It may be pertinent to note that compared with animal sterols some plant and fungal sterols are poorly absorbed in the intestine. This specificity of absorption has been attributed to the ability of sterols to enter membranes and soluble lipoproteins of the intestinal cell (Glover and Green, 1957; Desai and Glover, 1963; Edwards and Green, 1972). Interestingly, cholesterol-deficient cells remove cholesterol from P-lipoproteins only when dimethylsulfoxide (DMSO) is added to the medium (Bruckdorfer et al., 1969). Exchange of cholesterol from a-lipoproteins occurs without DMSO. This suggests different boundary arrangements for cholesterol in a- and P-lipoproteins. With the cautionary note just described, one may examine the proportion of cholesterol in total lipids from a variety of proteolipids (Table I) and membranes (Table 111). The mole fraction of cholesterol seems to vary from 0 to only 0.5. Therefore, in contrast to phospholipids, cholesterol is not a necessary constituent of all membranes and thus must perform a particular, rather than a general, function. Cholesterol is well recognized as a prominent lipid constituent of many biological interfaces. Besides cholesterol, only sterols such as 7-dehydrocholesterol, lathosterol, and cholestanol have been reported in animal cells (Lesser and Clayton, 1966; Werbin et al., 1962; Glover and Green, 1957; Subbiah et al., 1971). Cholestanol (dihydrocholesterol) has been identified in fractions of brain from patients with cerebrotendinous xanthomatosis

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

7

TABLE I11 COMPOSITION OF CERTAINMEMBRANE TYPEW

Source

Cholesterol (mole % of total Total lipid (% dry weight) phospholipid)

References

Myelin-human central nervous system Myelin-beef M y e l i n s p i n a l root (beef) Platelet-human

78.7

40.1

O’Brien (1967)

75.9 76 56

39 37 25

Cow RBC

38

43

Rat RBC

40

42

Sheep RBC

30

40

Dog RBC Rabbit RBC

43 39

48 47

Chicken RBC

55

38

Guinea pig RBC

47

47

Goat RBC Horse RBC Pig RBC Cat RBC Elephant seal RBC Harp seal Human RBC Rat liver plasma membrane

48 39 38 50 36 36 46 40

40 50 47 46 47 44 42 26

Rat myometrium

59

41

Rat liver microsomes

32

8

Guinea pig brain microsomes

30-35

8

Retinal rod outersegment

41

4

Squid retinal axon

45

33

O’Brien (1967) O’Brien (1967) Barber and Jamieson (1970) Cornwell et al. (1968);Nelson (1967a) Cornwell et a2. (1968); Nelson (1967a) Cornwell et al. (1968); Nelson (1967a) Nelson (1967a) Cornwell et al. (1968); Nelson (1967a) Kates and James (1961) Reed and Roberts (1968) Nelson (196713) Nelson (1967b) Nelson (1967b) NeIson (1967b) Nelson (1970) Nelson (1970) O’Brien (1967) Benedetti and Emmelot (1968) Kidwai et al. (1971) Glaumann and Dallner (1968) Fleischer and Rouser (1965) Eichberg and Hess (1967) Fischer et a1 (1970)

(Continued)

8

MAHENDRA KUMAR JAlN

TABLE I11 (Continued)

Source

Cholesterol (mole % of total Total lipid (% dry weight) phospholipid)

Synaptic vesicles (guinea pig brain) Ehrlich ascites carcinoma cells Bovine heart mitochondria

34

Rat liver mitochondria

21

. .5 i

Guinea pig kidney mitochondria Guinea pig brain mitochondria

15

0.5

27-30

1

Pig lymphocyte

42

50

Rat intest,inal microvillus

38

63

HeLa cell

40

52

Bovine olfactory epithelium

30

13

Mycoplasma laidlawii B Azobacter azilis

3.5-37 10

0

Escherichia coli

10

0

Agrobacteriicm tumefaciens

10

0

33 24

5.6 12 1

0

References Eichberg et al. (1964) Wood et al. (1970) Fleischer et al. (1967) Fleischer and Rouser (1965) Rouser et al. (1968) Fleischer and Rouser (1965) Allan and Crumpton (1970) Forstner et al. (1968) Bosman et al. (1968) Koyoma et al. (1971) Razin (1963) Kaneshiro and Marr (1962) Kaneshiro and Marr (1962) Kaneshiro and Marr (1962)

a The calculations used in the preparation of this table have often required making the assumption that total lipid has molecular weight 750. Because of this and because of the variations in experimental results, the reader is advised that the tabulated values should be considered &s estimates only, presented to emphasize the mole proportions and their variation. Neutral lipids of erythrocytes (RBC) contain almost exclusively cholesterol.

(Stahl et al., 1971). The role played by these sterols is unknown. The cholesterol content of biological membranes is highly variable. I n a given cell, the cholesterol content (both relative and absolute) of various organelle membranes seems to vary by almost an order of magnitude. For example, in rat liver the molar ratio of phospholipid to cholesterol is 0.76 for plasma membrane, 0.24 for smooth endoplasmic reticulum, 0.12 for microsomes and outer mitochondria1 membranes, 0.06 for rough endo-

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

9

plasmic reticulum (Colbeau et al., 1971). The highest levels of cholesterol so far reported have been for myelin, followed (in that order) b y plasma membranes from a variety of sources, microsomes, outer and inner mitochondrial and nuclear membranes (Lesser and Clayton, 1966). This is also the order in which the proportion of lipids to total membrane dry weight decreases and the proportion of unsaturated acyI chains in membrane lipids increases. The highest mole proportion of cholesterol to phospholipids is always one or below. This suggests some correlation between lipid-cholesterol interaction and its dependence upon degree of unsaturation of fatty acid chains. A rationale for this correlation may be found in the nature of intermolecular association between cholesterol and phospholipids as described later on. However, this poses serious questions regarding the considerable difference in cholesterol content of plasma membranes as compared to membranes of subcellular organelles. This may be partially accounted for in terms of degree of unsaturation. As the membranes of subcellular organelles contain higher proportions of u6,u6 double bonds, their propensity for interaction with cholesterol would be considerably reduced (see below). In contrast, u6 bonds in plasma phospholipids would not interfere with lipid-cholesterol association. Membrane phospholipids with a higher proportion of unsaturated acyl chains or 1-unsaturated-2-saturated phospholipids (see below) would not only be a less effective barrier, but would also be incapable of incorporating cholesterol. Thus the ability of a phospholipid to interact with cholesterol in a biomembrane would parallel the occurrence of these phospholipids in nature. I n many animal tissues there appears to be a positive correlation between levels of cholesterol, sphingomyelin, and total lipids. For example, these three components tend to be high in myelin and erythrocytes (O’Brien, 1967; Coleman, 1968), in aged human arteries (Buck and Rossiter, 1952), and in plasma membrane (Ray et al., 1969). On the other hand, all three of these are relatively low in mitochondria (Fleischer et al., 1967; Parsons and Yano, 1967). Plant tissues contain neither cholesterol nor sphingomyelin. Patton (1970) has found a positive correlstion between the cholesterol/sphingomyelin content of mitochondria, nucleus, endoplasmic reticulum, Golgi apparatus, and plasma membrane of rat hepatocytes. It has been suggested that membrane lipid class composition is determined a t least in part by type and number of protein binding sites (e.g., see Kramer et al., 1972). However, cholesterol and polar lipids do not substitute for each other. Thus the presence of cholesterol in membranes must be explained by binding to polar lipids rather than directly to protein. Since the mole fraction of cholesterol in membrane lipids is always less than 0.5, it appears that cholesterol does not bind to all polar lipids. e9,

w699J2

10

MAHENDRA KUMAR JAlN

Based on lipid composition data of erythrocytes from various mammalian species (Nelson, 1967a, b), it has been suggested that two molecules of cholesterol bind to one molecule of either PE, sulfatide, or acidic phospholipids. These authors also observed that the molar sum of PC, SN, cerebrosides, and acidic phospholipids (i.e., all polar phospholipids except P E and sulfatides) of human brain was almost exactly equal to the number of moles of cholesterol. This correlation is not applicable to subcellular organelles which do not have the same high level of cholesterol as would be expected from their polar lipid composition. It may, however, be noted that the nature and degree of unsaturation are an important factor in determining sterol-phospholipid interaction. A higher degree of unsaturation as observed in acyl chains from phospholipids of subcellular organelles would retard interaction of cholesterol with these polar lipids. Bacteria do not require sterols as membrane constituents. Some strains of Mycoplasma laidlawii such as T strain (Rottem et al., 1971) and strain 07 (Smith, 196413) require sterols. M . laidlawii strain B is not dependent on sterols but can incorporate sterols from the growth medium (Razin et al., 1966; Deliruyff et al., 1972). Thus the strains of Mycoplasma may have between 10 and 30% sterols in their membranes (Smith, 1967, 1968, 1969). Although sterol esters are also incorporated by these strains (23%), there is no evidence that the free sterol molecule is chemically altered by these organisms. The ratio of membrane lipid classes in Myccplasma laidlawii is not significantly affected by variations (induced by adding lipids to growth medium) in fatty acid composition or sterol content (RlcElhaney et al., 1970). However, when a sterol-requiring strain is made to grow in the absence of cholesterol, its polar lipids become more saturated (Rottem et al., 1973a). Also the total lipid from the adapted strain shows phase-transition (Rottem et al., 1973b). Other organisms such as Saccharomyces cerevisiae (Starr and Parks, 1962) and Pythium spp. (Haskins, 1965; Schlosser and Gottlieb, 1966; Sietsma and Haskins, 1967) can maintain viability a t high temperatures (45-50°C) if sterols are added to the growth medium. It thus appears that sterol incorporation into the membrane may protect the membranes against high temperature damage. Similar protection may be provided by certain pigments such as neurosporene and carotenoids, along with sterols. A detailed description of the nature, physiological properties, and requirements for sterols and pigments may be found elsewhere (Smith, 1969). Sterol esters cannot replace free sterols in growth media. I n fact, to support Mycoplasma growth (a) the sterol must be planar, i.e., the A and B rings must be trans fused; (b) it must have a n equatorial 3-p-hydroxyl group; and (c) there must be a hydrocarbon chain. Thus cholesterol, cholestanol, lanosterol, p-sitosterol, stigmasterol, and ergosterol can

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

11

support growth, whereas coprostanol, 7-dehydrocholesterol, epicholesterol, and cholestan-3-one cannot (Smith, 1969). However, there appears to be considerable difference in various strains of mycoplasma. T strain can incorporate cholesterol and p-sitosterol and, to a lesser extent 7-dehydrocholesterol, stigmasterol, ergosterol, and cholestanol (Rottem et al., 1971). For strain 07 it is observed that only cholesterol and cholestanol support growth (Smith, 1964a). Strain B cell can incorporate epicholesterol besides cholesterol (DeKruyff et al., 1972). Proof for membrane localization of these sterols in these organisms is obtained from the ability of digitonin to lyse sterol containing Mycoplasma spp. only (Smith and Rothblatt, 1960), whereas other nonspecific surfactants, such as soaps, alcohols, cationic and anionic detergents, are able to disrupt several Mycoplasma strains (Smith, 1964a). Tetrahymanol, a sterollike triterpenoid, has been identified as a constituent of various membranes of Tetrahymena pyriformis (Thompson et al., 1972). The tetrahymano1:phospholipid ratio is constant in growing and early stationary phase, but increases in various membranes from starved or senescent cells. Tetrahymanol is resistant to catalytic attack. Surviving cells of senescent cultures ingest fragments of dead cells, degrading the phospholipid, but accumulating the triterpenoid. This is consistent with the observation that ergosterol (Conner et al., 1971) or cholesterol (Conner et al., 1968), added to the cultures of Tetrahymena, blocks tetrahymenol synthesis and eventually replaces it in the growing cells. Interestingly, the addition of tetrahymanol itself does not inhibit tetrahymanol biosynthesis (Landrey et al., 1971). Variation in cholesterol content has also been observed in plasma membranes from cells of higher animals. Cholesterol content and lipid composition of red blood cells can be changed by altering growth conditions or diet or by incubation in plasma from cholesterol-fed guinea pigs. Earlier studies had demonstrated that aqueous dispersions of egg lecithin can remove cholesterol from erythrocyte membranes and that cholesterol can exchange between lecithin-cholesterol dispersions and erythrocyte ghosts (Bruckdorfer et al., 1968a). Also large proportions of membrane sterol can be replaced by other lecithin-solubilized sterols (Eruckdorfer et al., 196813). Thus a choIestero1 increase in the diet of hamsters is associated with the appearance of prominent, abnormal spicules on the surface of the red blood cell. Alterations in erythrocyte cholesterol and lecithin levels result in increased osmotic fragility and premature destruction, even when the cells are essentially normal in other respects (e.g., in ATPase level and glucose metabolism). This results in eventual decrease in the viability of the erythrocytes, presumably due to an increase in the interal viscosity of the bilayer. Similarly, a greater osmotic fragility of hamster erythrocytes has

12

MAHENDRA KUMAR JAlN

been observed following prolonged exposure of the animals to a n elevated ambient temperature. Among other effects, a decrease in cholesterol: phospholipid ratio from 1.07 to 0.92 has been observed (Kuiper et al., 1971). This lowering of cholesterol content may be responsible for a variety of effects besides the mechanical stability of the cells. For example, penetration of cells by psychomimetic drugs (Demel and Van Deenen, 1966), plant growth substances (Kennedy and Harvcy, 1972), carcinogens (Belmonte and Swarbrick, 1973), catecholamines (Salt and Iversen, 1972), vitamin A (Bangham et al., 1964), an azo-dye-orangc IV (Horton et ul., 1973), and polyene antibiotics (Norman 6t al., 1972a,b; Hsuchen and Feingold, 1973; Drabikowski et al., 1973; Kinsky et aE., 1968) may all be influenced by the presence of cholesterol in membranes. A decrease in “fluidity” and increased cholesterol content of unilamellar vesicles has been shown to decrease the fusion of mammalian cells by these vesicles (Papahadjopoulos et ul., 1973a). Similar processes may regulate the influence of cholesterol on blood coagulation (Sterzing and Barton, 1973). Also autoxidation of cholesterol has been shown to be influenced by the presencr of lecithin (Weiner et al., 1973). Saponins and polyene antibiotics sekctively lyse membranes containing cholesterol, and their lytic activity is antagonized by free cholesterol in the medium. The polyene antibiotics behave very much like saponins, although there are significant differences in the electron micrographs of negatively strained liposonies containing lecithin cholesterol (7 :1) treated with polyenes and saponins (Rinsky et al., 1966, 1967). The pits and rings observed in these two sets of electron micrographs are similar in form, but not in dimension. It is particularly important to note that erythrocyte membranes treated with saponin do not show the same substructuring as the liposomes containing equimolar lecithin and cholesterol. This may indicate that the mode of packing of cholesterol in these two membranes is different. Furthermore, the interaction of saponins and polyene antibotics with membrane-bound cholesterol is consistent with the fact that cholesterol molecules have a considerable degree of freedom of rotation and reorientation, a condition that would be necessary if these lytic agents were to form channels by aggregation. Similar forces may be involved in the interaction of tyrocidine B with BLM; the presence of cholesterol (1 :1 molar ratio) makes the kinetics bimolecular (Goodall, 1970, 1973). Although these effects may be somewhat specific, a n explanation requires a n understanding of membrane packing, permeability, and partition behavior on a molecular basis (see below). An increase in the levels of cholesterol has been shown to render membranes more lipophilic, less permeable, and more susceptible to mechanical abuse. The modifications of

+

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

13

macroscopic phase behavior of bilayers are reflected in such diverse, membrane-related phenomena as functioning of secretory vesicles (Keenan and Moore, 1970), myelin formation (Vandenheuvel, 1963; O’Brien, 1967), morphology and viability of the red cells (Ostwald and Shannon, 1964; Murphy, 1965; Sardet et al., 1972), photohemolysis (Lamola et al., 1973), ouabain-sensitive sodium transport (Kroes and Ostwald, 1971; Kroes et al., 1972), milk-fat globule secretion (Bargmann and Knoop, 1959; Patton and Fowkers, 1967) accumulation of lipids into atheromatous plaques (Eisenberg et al., 1969; Portman and Alexander, 1970; Forte et al., 1971; Hamilton et al., 1971), and membrane-bound enzymic activity (Cobon and Haslam, 1973). Among rather specific effects of cholesterol may be mentioned activation of Na K - ATPase (Noguchi and Freed, 1971; Jarnefelt, 1972), inhibition of catecholamine uptake by rat heart (Salt and Iversen, 1972), inhibition of 6-glucuronidase and glutamate dehydrogenase (Tappel and Dillard, 1967), and possibly tetrodotoxin binding in nerves (Villegas et al., 1970). This last possibility appears unlikely since squid axon membranes generally have about 108 cholesterol molecules/pm2, whereas only < 100 T T X binding sites/pm2 are present on various nerve membranes.

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111. SOLUBlLlZATlON AND DISPERSION OF CHOLESTEROL

I n the solid state, cholesterol is not readily dispersed in water or electrolyte solutions which could occur naturally. Yet, since cholesterol is integrated into all cells and most body fluids in nonparticulate form, some very efficient methods of microdispersion and phase change must operate biologically (e.g., see Rohmer et al., 1972; Rampone, 1973). Solubilization of cholesterol can be achieved in ternary systems with lecithin as the amphiphile, in which case a complex, stable mesophase develops at a certain critical concentration. Also complete solution has been achieved in quaternary aqueous systems containing bile salt and lecithin (Small et al., 1966). In these amphiphilic systems, cholesterol enters the mesophase only in the unsubstituted state; cholesterol esters, irrespective of the chain length of the fatty acid tail, do not form ordered phases. On the other hand, the fatty acid chain length of the ester used as amphiphile does influence the order and stability of cholesterol in mesophase. This suggests that the packing of cholesterol molecules may induce a steric pattern entirely different from that of phospholipids in bilayers. Cholesterol exhibits polymorphism (Spier and Van Senden, 1965; Bernal and Crowfoot, 1933; Bulkin and Krishnan, 1971; Scanu and Tardieu, 1971), and

14

MAHENDRA KUMAR JAlN

forms liquid crystals when mixed with a variety of simple compounds (Zull and Sciotto, 1969; Zull et al., 1968; Rowel1 et al., 1965; Gibson and Pochan, 1973; Snart, 1967a, b). The principal structural requirement of the second component is that it be a n amphipath with a chain-length greater than twelve methylene residues. There is an upper limit to the proportion of sterol that can be introduced into liposomes (Table IV, see also Horwitz et al., 1971). A closer examination of data presented in this table reveals that the orientation of hydroxyl group a t position 3, ring junction A/B, the position of double bonds, unsaturation and length of side chain, all seem to determine the extent of incorporation of these sterols into bilayers. The pattern of incorporation TABLE IV

THESOLUBILIZATION OF STEROLS BY SONICATION WITH EGGLECITHIN" Sterol

Cholesterolb Cholestanolb Lat hosterolb 7-Dehydroch~lesterol~ Ergosterol Stigmasterolb Androstan-3p-olb p-Norcholesterolb Caprostanol* Epicholesterolb Androstan-3-cr-01~ Cholestan-3-one Cholest-4-en-3-oneb Cholest-5-en-3-oneb Choleste-4, 6-dien-3-oneb Testosterone crotonoatec Testosterone benzoatec Testosterone 2-octenoatec Testosterone 3-octenoatec Testosterone undecylenatec Testosterone 2-methylpropionate" Testosterone 4-methyl pentanoateC

Sterol :phospholipid (molar ratio) 1.04:l 1.11:l 1.07:l 0.55:l 0.3931 0.57:l 0.72:l 1.16:l 1.04:l 0.30:l 0.58:l 0.43:l 0.59:l 0.57:l 0.55:1 0.14:l 0.04:l 0.56:l 0.55:l 0.56:l 1.11:l

0.2.5 :1

* See also Bourges et al. (1967a, b). Demel et al. (1972a). Stevens and Green (1972) ;see also Kellaway and Sanders (1967).

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

15

does not seem to correlate with the lipophilic character of the sterols (Stevens and Green, 1972). These observations are best rationalized in terms of conformational adaptation of sterols into polymethylene chains. Within this limitation, optimum incorporation (maximum solubilization) would be obtained with the molecule giving van der Waals interactions, and so side chain structural requirements may be somewhat specific. Solubilization of sterols by lysolecithin (Gale and Saunders, 1971) and incorporation of sterols into phospholipid monolayers (see below) may not necessarily depend upon these considerations since the geometry of the aggregates would introduce serious restrictions for intermolecular association and organization. Bile salts also have the solubilizing capacity for cholesterol (Admirand and Small, 1968). However, the addition of lecithin decreases the dissolution rate even though lecithin increases the equilibrium solubility of cholesterol in these solutions. The reduction in rates caused by lecithin has been attributed to a large crystal (cholesterol)solution interfacial barrier (Higuchi et al., 1972); cholesterol is also “solubilized” by lysolecithin (Neiderhiser and Roth, 1972). Similarly a change in the type of bile salts does not markedly affect the quantity of cholesterol solubilized by added lecithin (Neiderhiser and Roth, 1968). Admirand and Small (1968, 1972; see also Mufson et al., 1972a) have shown that the solubility of cholesterol in human bile is determined by the relative concentration of bile salts and lecithin in the quaternary aqueous system. I n some pathological states they found that the concentration of cholesterol was increased beyond the point of solubilization. This led to the deposition of cholesterol and the formation of gallstones. Admirand and Smith’s findings amount to an attractively simple biophysical explanation of the occurrence of some forms of gallbladder disease especially in conditions of cholestasis, or when the excess of cholesterol is attributable to metabolic cholesteroisis. Of particular interest in relation to the aging process in humans is the partitioning of cholesterol in plasma lipoproteins (Stewart, 1959, 1961a). The total concentration tends to increase in middle age, but it is the distribution rather than the total amount that shows the most striking change. I n youth, cholesterol and its esters are carried in the high-density lipoprotein (cf. Table I) fraction. I n men from the mid-twenties onward and in women after menopause there is sharp increase in low-density lipoproteins which carry a relatively higher concentration of all forms of lipid. Normally, excess lipid is split hydrolytically, but this process is less effective against low-density lipoproteins. Thus the lipolysis fails when it is most needed and cholesterol remains in the blood stream or deposits in various tissues (cf. atherosclerosis). The relative immunity of women to atherosclerosis and its consequences during their reproductive years

16

MAHENDRA KUMAR JAlN

may find a qualitative biophysical explanation in the fact that the excess cholesterol is directed to or held in ovarian cells (corpora lutea), where it is a precursor in hormonal synthesis (Stewart, 1961b). Consistent with this hypothesis is the finding that, in diseases associated with signs of premature aging, like untreated diabetes, hypothyrodism, and chronic nephritis, the low--density lipoprotein is markedly increased and deposition of cholesterol in arteries and elsewhere is very conspicuous.

IV. CORRELATIVE RELATIONSHIPS OF CHOLESTEROL CONTENT A. With Barrier Properties of Biomembranes

Permeability of biomembrane bilayer for small solutes, particularly water, has been measured in a variety of systems (Table V). I n most cases permeability has been measured by osmotic swelling on the underlying assumption that the osmotic response (fragility and permeability) of whole cells, vesicles, and liposomes may reflect the lipid composition and organization. Replacement of cholesterol in erythrocyte membranes by lanosterol, 7-dehydrocholesterol, 0-norcholesterol, or by exchange with lecithinsterol dispersions shows that these sterols have a n effect on membrane permeability comparable to that of cholesterol (Bruckdorfer et al., 1969). Replacement of cholesterol by cholestan-3-one1 cholestene-4 ,6-dien-3-one in erythrocyte membranes results in an increased permeability. Permeability of lecithin liposomes is not affected, however, when a variety of other ketosteroids is incorporated. Similarly, cholesterol-enriched mitochondria are more resistant to swelling than control mitochondria (Graham and Green, 1970). Pythium mycelium gown in the presence of cholesterol has a decreased rate of release of metabolites (Child et al., 1969). I n comparison to other membrane systems, however, no change in nonelectrolyte permeability has been observed in pig erythrocyte depleted of 35% of cholesterol (Deuticke and Zollner, 1972). The permeability of the cholesterol-loaded erythrocytes for nonelectrolytes has the same activation energy as for the untreated cells (Kroes and Ostwald, 1971). A similar observation has been made for Mycoplasma (DeGier et al., 1969). This suggests that the added choIesterol lowered the permeability without affecting the threshold energy for the process. Since cholesterol in the bilayer is unlikely to influence the rate of interfacial solute transfer, it may affect the rate of solute permeation through the hydrocarbon region of the bilayer.

17

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

TABLE V P.4SSIVE

PERME.1BILITY OF BIOMEMBRANES AS A FUNCTION OF CHOLESTEROL CONTENT Source

Permean t

*

Effect

Mycoplaarna laidlawii B

Glycerol, erythritol

Cholesterol-containing membranes have lower permeability; epicholesterol does not affect permeability (DeKruyff et al., 1972, 1973a; McElhaney et al., 1973). No change in activation energy for transport

Pig and rabbit RBC

Glycerol, erythritol

Cholesterol-depleted cells show higher permeability (Bruckdorfer et al., 1969)

Cholesterol-fed guinea pig ItBC

Erythritol, thiourea, monoacetin, Na

Permeability decreases following incorporation of cholesterol (Kroes and Ostwald, 1971). Act.ivation energy for hemolysis remain unchanged. Ouabain-sensitive and unsensitive permeability of Na is also lowered

Heart froin cholesterol-fed rats

Catecholamine

Permeability is inhibited (Salt and Iversen, 1972)

Both the active (ouabain-sensitive) and passive (ouabain-insensitive) components of sodium efflux are decreased in cholesterol-loaded erythrocytes (Kroes and Ostwald, 1971). However, the binding of ouabain to normal and loaded cells was approximately the same. This implies that the decrease in sodium permeability of the cholesterol-loaded cells was not due to a decrease in the number of sodium pump sites. These results could arise from a decreased leakage of cholesterol-loaded cells as active efflux will also decrease in such a situation. It may be pertinent to note that in delipidated Naf I 73 MHz. The same effect occurs a t much lower cholesterol: lecithin ratios. The ordering effect has been analyzed in terms of the spin labels being oriented about a cone, the axis of which is perpendicular to the plane of the film. Increasing the cholesterol content decreases the solid angle contained by the cone. The highest degree of orientation was observed a t 25 mole percent cholesterol. This corresponds to a deviation of the long axis of the spin label from the perpendicular by 10 =t3". Between 25 and 50 mole percent the degree of order is constant (Lapper et al., 1972; Schreier-Muccillo et al., 1973;

+

36

MAHENDRA KUMAR JAlN

Long et al., 1971). The structural features of the steroid that can effect order in spin-labeled multibilayers are: a planar nucleus, a 3p-OH group, and a tail a t C-17 (Butler et al., 1970b). Sterols in the bilayer also affect the mobility gradient along the hydrocarbon chain. When spin-labeled fatty acids or lecithins are incorporated into egg lecithin (EL) bilayers, the decrease in order parameters S ( = 0 for an isotropic liquid and =1 for a perfect crystalline solid) is greater than logarithmic as the number of methylene groups separating the label from the polar head group increases. The data can be reconciled by assuming that a net tilt of 30" is present in the head group region. In the presence of cholesterol (EL:cholesterol, 2:1), it has been found that the first eight carbon atoms from the bilayer surface can be thought of as a rigid rod, with the remaining carbons greatly increasing their motion toward the center of the bilayer (Hubbell and XcConnell, 1971; Oldfield and Chapman, 1971; Seeling, 1971). I n dry egg lecithin film the cholesterol label undergoes isotropic rotational diffusion characterized by a correlation time of 10-8 second. Thus the immediate environment of the label appears to be liquidlike. I n hydrated lecithin film, the steroid label is oriented with its long axis approximately perpendicular (24" tilt) to the plane of the film, with the label undergoing rapid rotational reorientation about the long steroid axis at a rate of about lo8 sec-' (Hsia et al., 1970a). I n dry lecithin cholesterol films there is a considerable degree of spectral anisotropy. The long axis of the steroid label is perpendicular to the plane of the dried film, with no motion about the long axis of the label. In hydrated lecithin cholesterol films the steroid label has rotational diffusion about the long axis a t a rate of about lo8 sec-'. There appears to be almost perfect orientation of the label: the long axis is perpendicular to the plane of the multilayer film (Lapper et al., 1972). Thus cholesterol has a significant effect on the orientation of chains in the bilayer. Large amounts of cholesterol cause a stiffening of the hydrocarbon chains. Hydration of mixed lipid film induces a high degree of order, with a spacing between the hydrocarbon chains sufficiently large to allow rotation of the steroid label about its long axis. Similar conclusions have also been arrived a t from the study of brain and erythrocyte phospholipids (Butler et al., 1970b). Using fatty acid spin labels, Hsia et al. (1970a,b, 1971) found that cholesterol not only restricts probe motion, but moves the probe to a new and more symmetrical polar environment, with the tilt angle of acyl-chains decreased from 28' to 10'. Also, addition of cholesterol reduces the rotational freedom for the probes (Barrett et al., 1969; Hubbell and McConnell, 1968). These studies also indicate that the polarity of the hydrocarbon

+

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ROLE OF CHOLESTEROL IN BIOMEMBRANESAND RELATED SYSTEMS

37

region dccrcascs as onc moves the label toward trrminal methyl group (Hubbell and McConnell, 1971). I n addition, as one goes from the outside to the ccntrr of the bilaycr, freedom of motion increases markedly. Low-angle X-ray diffraction data suggest that for a given hydration level the domains in the egg lecithin-cholesterol hydrocarbon region are better oriented than those in the hydrocarbon region (Levine and Wilkins, 1971). At 21% water content, the terminal methyl groups in egg lecithin lipsomes are distributed over a wide region. I n the presence of cholesterol, however, the terminal methyl groups appear to be well localized. It is particularly significant that the peak-to-peak distance across the hydrocarbon region for egg lecithin bilayers decreases from 39.6 A (at 14% water content) to 3 6 . 8 i a t 21% water content. I n contrast, for egg lecithin cholesterol bilayers, the peak-to-peak distance remains almost constant a t 42 A. These observations indicate that addition of cholesterol reduces the molecular motion in the bilayer and extends the chains so that the membrane thickness increases. This conclusion is consistent with the studies on thermal coefficient of expansion (Rand and Pangborn, 1973) and sedimentation coefficient (Johnson, 1973) of liposomes measured as a function of cholesterol mole fraction in lecithin. The 1,2-dipalmitoyllecithin-cholesterol-water systems show integral orders of a principal long spacing in the low-angle region (Ladbrooke et al., 196813). With increasing cholesterol content, the long spacing increases initially and reaches a maximum a t 7.5 mole percent cholesterol (Fig. 1). A parallel increase has been observed in the water permeability of liposomes that contain low (-10 mole percent) concentrations of cholesterol. On addition of further cholesterol the long spacing gradually doecreases to 64 a t mole percent. The sharp high-angle spacing of 4.2 A increases to 4.45 A and become diffuse. These results are consistent with the view that cholesterol causes a reduction in the cohesive forces between the adjacent hydrocarbon chains of lecithin. Nevertheless the passage of various permeants is reduced through cholesterol-containing liposomes. The results presented in this section show that the chains in bilayers exhibit considerable orientation and that their free ends are near the center. With increasing hydration the chains become disoriented and their ends localized or less mobile. Addition of cholesterol reduces the molecular motion in the bilayer and extends the chains so that they are well oriented with their terminal methyl groups well localized. From the analysis of ESR data, it has been suggested that below the phase transition temperature the spin-labeled steroid molecules are present as clusters in the bilayer (Trauble and Sackman, 1972). The density of clusters appears to be independent of the molar ratio of steroid :lipid, but the size of clusters increases with increasing steroid concentration. Above the phase

+

dO

38

MAHENDRA KUMAR JAlN

transition temperature the clusters dissolve and a homogeneous mixture is formed. C. In Biomembranes

Although cholesterol is a major component of plasma membranes of eukaryotic cells, very Iittle is known about the state of cholesterol in these membranes. It is certain that cholesterol in biomembranes is localized anisotropically with its long axis perpendicular to the plane of the membrane. The conclusions derived from studies on model bilayers may be applicable to biomembranes. The natural abundance 13C-NP\IR spectra of various biomembranes show very broad signals. This suggests that the correlation times for molecular motion are a t least an order of magnitude slower than those observed by the use of ESR and fluorescent probes in biomembranes and model membranes. Rothman and Engelman (1972) have shown that in liposomal membranes that contain cholesterol lipids, the lower region of the phospholipid chains will be in a “liquidlike” state and the upper region will be more ordered. This is because the cross-sectional area of the cholesterol side chain is only about half that of the ring system. X-ray diffraction studies suggest that biomembranes from a variety of sources contain extended regions of lipid bilayer (Wilkins et al., 1971). Membranes that contain high concentrations of cholesterol, such as in erythrocyte and myelin, have their chains extended and fairly perpendicular to the membrane plane. These membranes show little disorder (Chapman et al., 1968, 1969a; Williams et al., 1973). However, the broad band a t 4.6 seen in the X-ray diffraction pattern suggests that no long range order is present in the plane of the bilayer in these membranes. I n membranes containing little or no cholesterol, such as in mycoplasma and retinal rod mcmbranes, disorder of the chains is relatively significant. The electron density curves obtained from rabbit optic and sciatic membrane profiles are similar, but the profile from frog sciatic nerve membrane has a narrower central minimum and thus presumably shorter hydrocarbon chains (Casper and Kirschner, 1971). Furthermore there appears to be a slight asymmetry in the bilayer, which these authors interpret as being caused by an uneven distribution of cholesterol. The data indicate a n approximate equimolar ratio of cholesterol and polar lipid on the outer side of the hydrocarbon layer, and a ratio of about 3 :7 on the inner side. This interpretation accounts for the measured cholesterol content (40% mole percent, cf. Table 111). These X-ray measurements indicate that the hydrocarbon chains are predominantly close packed in the “steroid step regions” (it is up to ninth carbon atom, which is the most frequent position

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ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

39

for the double bond). They also indicate that the ends of the chains near the bilayer center are pliant and disordered. The asymmetric cholesterol distribution presumably results from (or in) specific interactions with protein. However, the protein does not seem to modify significantly the local lipid packing arrangement. These and other (Oldfield and Chapman, 1972b; Marsh and Smith, 1973) observations suggest that cholesterol may have a dual role of preventing formation of crystalline gel areas in some membranes, while also inhibiting the motion of hydrocarbon chains in more fluid, liquid crystalline, regions.

VI. MOLECULAR ASPECTS

OF ORGANIZATION OF CHOLESTEROL IN BILAYERS

The biomembranes are probably as variable in composition as the number of different cells and organelles which they delimit; perhaps even more so. Nevertheless, the close packing of polymethylene chains (C16-C22)in two parallel arrays form the essential structural feature of biomembranes (Jain, 1972). Since cholesterol is an integral component of plasma membranes and is present in the same concentrations as phospholipids, the mode of packing of the polymethylene chains in a bilayer is definitely affected by the penetration of cholesterol. This is shown by monolayer studies, passive permeability measurements, phase transition characteristics, and various physicochemical studies. The experimental data suggest that, in general, the presence of cholesterol tends to condense a liquidcrystalline film in the sense that the combined molecules take up less space than that predicted for them from simple additivity of molecular cross sectional areas. The observed departure from additivity could be due to either formation of some stoichiometric phospholipid/cholesterol complex or may arise from the incorporation of cholesterol in the cavities (or void volume) between phospholipid molecules. The experimental data available so far does not eliminate either of these possibilities. Since most plasma membranes have both phospholipids and cholesterol as their main constituents, a study of the nature of intermolecular interactions in the mixed lipid bilayers is of interest. The effect of cholesterol on orientation, clustering, rotation, and lateral mobility of lipid molecules may be biologically significant. Incorporation and localization of cholesterol into phospholipid bilayer appear to be a consequence of the extended hydrophobic overlap between hydrocarbon chains of phospholipid and the planar ring system of cholesterol. If it is assumed that the cholesterol packs closely alongside or even forms a complex with the fatty acid chains or

40

MAHENDRA KUMAR JAIN

polar groups of phospholipids, such an interaction would be expected to hinder any rotational movements which the pure lecithin molecules may have. It would also allow a closer packing of the molecules. Various characteristics of bilayer show changes as a function of mole proportion of cholesterol present in the bilayer. Most of these changes are observed until the cholesterol mole proportion reaches a maximum of 0.5. At higher mole proportions, cholesterol crystallizes in the bilayer. However, not all bilayer properties show a maximum or minimum a t 50 mole percent cholesterol. I n fact, studies on phase transition characteristics of dipalmitolylecithin with varying mole proportions of cholesterol have yielded conflicting results. Ladbrooke et al. (1968b) reported that the enthalpy of transition vanished at 50 mole percent cholesterol. I n contrast Hinz and Sturtevant (1972) have reported that the transition enthalpy vanished a t 33 mole percent cholesterol. A model consisting of cholesterol surrounded by seven lecithin molecules in the membrane plane has been suggested (Engelman and Rothman, 1972) to account for the loss of transition enthalpy a t 33 mole percent cholesterol. This model is based on the assumptions that the two hydrocarbon chains of lecithin are freely mobile and that the phospholipid/cholesterol pair does not show any specificity of interaction or orientation. These assumptions are contrary to studies discussed earlier in this chapter. There is ample evidence for the involvement or modification of surface group region following incorporation of cholesterol in a bilayer (Abramson and Katzman, 1968). Other models consistent with a maximum incorporation of 50 mole percent cholesterol also suffer from several limitations. It appears that successively higher concentrations of cholesterol bring about changes in the packing and/or orientation of phospholipid chains in at least two (maybe three) different regions. These different orientation and packing characteristics of hydrocarbon chains or polar groups may be present a t about 10, 35, and 50 mole percent (for a hypothetical model see Jain and Cordes, 1974). All these changes, of course, may not be reflected by the use of a single technique. Thc molecular states of lipids corresponding to these three cholesterol concentrations may be distinct. As a result different regions of the bilayer may be affected as thc cholesterol molr proportion in the bilayer increases. Also a t certain mole proportion (lower range) cholesterol may be present as a distinct stoichiomrtric aggregate with lipids or floating as a n island in the bilayer of excess lipid. At higher cholesterol concentrations (30 mole percent or more), however, it is almost certain that cholesterol is randomly distributed among lipid molecules in the plane of the bilayer and thus has a disrupting effect on the “cooperative islands” of lipids. Also in biomembranes containing various phospholipid species one would expect that certain lipids (saturated species for example) would tend to accumulate

ROLE OF CHOLESTEROL IN BIOMEMBRANESAND RELATED SYSTEMS

41

in the vicinity of cholesterol. Highly unsaturated phospholipids would, however, tend to segregate cholesterol. Thus the effect of cholesterol on molecular orientation and organization of phospholipid would be sensitive to structural and compositional variation in hydrocarbon chains. A physiologically important question regarding the biological role of cholesterol pertains to a possible relationship between membrane-bound and plasma solubilized cholesterol. Questions relating to the mode of exchange of cholesterol between membranes and surrounding plasma, its physiological and biochemical regulation, possible presence of specific cholestero1 binding and/or exchange proteins, and the role and significance of various lipoproteins are yet to be answered. The relationship of this exchange process on the structural and organizational states of lipid molecules (fluidity, rotational, translational, and lateral mobility) may yield relevant information. Finally, it may be pertinent to note that the cholesterol-induced variation in membrane fluidity on the function of membrane-bound proteins (Fourcans and Jain, 1974) may have a farreaching regulatory role. ACKNOWLEDGMENT

I wish to thank Mmes Davis and Bergner for help in preparing this manuscript. REFERENCES Abramson, M., and Katzman, R. (1965). Ionic interactions of sulfatide with choline lipids. Science 161, 576-577. Adam, N., and Jessop, W. G. (1925). Structure of thin films. Part X I I : Cholesterol and its effect in admixture with other substances. Proc. Roy. Soc., Ser. A 120, 473-482. Admirand, W., and Small, D. M. (1968). The physicochemical basis of cholesterol gallstone formation in man. J . Clin. Invest. 47, 1043-1052. Admirand, W., and Small, D. M. (1972). The effect of modifications of lecithin and cholesterol on the micellar solubilization of cholesterol. Biochim. Biophys. Acta 270, 407413. Allan, D., and Crumpton, M. J. (1970). Preparation and characterization of the plasma membrane of pig lymphocyte. Biochem. J. 120, 133-143. Back, P., Hamprecht, B., and Lynen, F. (1969). Regulation of cholesterol biosynthesis in rat liver: Diurnal changes of activity and influence of bile acids. Arch. Biochem. Biophys. 133, 11-21. Badley, R. A., Schneider, H., and Martin, W. G. (1971). Organization and motion of a fluorescent probe in model membranes. Bzochem. Biophys, Res. Commun. 45, 174-183. Bangham, A. D. (1972). Lipid bilayers and biomembranes. Annu. Rev. Biochem. 41, 753-776. Bangham, A. C., Dingle, J. T., and Lucy, J. A. (1964). Studies on the mode of action of excess of vitamin A. Penetration of lipid monolayers by compounds in the vitamin A series. Biochem. J . 90, 133-140. Bangham, A. D., Standish, M. M., and Weissman, G. (1965). The action of steroids and

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Stewart, G. T. (1969). Change of phase and change of state in biological systems. Mol. Cryst. Liquid Cryst. 7, 75-102. Subbiah, M. T. R., Kottke, B. A., and Carlo, I. A. (1971). 5a-Cholestan-3p-01: High concentration in testis of white carneau pigeon. Lipids 6, 517-519. Sunshine, G. H., Williams, D. J., and Rabin, B. R. (1971). Role of steroid hormones in the interaction of ribosomes with the endoplasmic membranes of rat liver. Nature (London),New Biol. 230, 133-136. Tappel, A. L., and Dillard, C. J. (1967). Inhibition of P-glucuronidase by cholesterol and retinol. J . Biol. Chem. 242, 2463-2469. Thompson, G. A., Bambery, R. J., and Nozawa, Y. (1972). Environmentally produced alterations of the tetrahymano1:phospholipid ratio in Tetrahymena pyriformis membranes. Biochim. Biophys. Acta 260, 630-638. Tinoco, J., and McIntosh, D. J. (1970). Interactions between cholesterol and lecithins in monolayers a t the air-water interface. Chem. Phys. Lipids 4 , 72-84. Tinoco, J., Ghosh, D., and Keith, A. D. (1972). Interactions of spin-labeled lipid molecules with natural lipids in monolayers a t the air-water interface. Biochim. Biophys. Acta 274,279-285. Tinsley, J. J., Hague, R., and Schmedding, D. (1971). Binding of DDT to lecithin. Science 174, 145-147. Torsvik, H., Berg, K., Magnani, H. N., McConathy, W. J., Alaupovic, P., and Gjone, E. (1972). Identification of the abnormal cholestatic lipoprotein (LP-X) in familial lecithin :cholesterol acyltransferase deficiency. FEBS (Fed. Eur. Biochem. SOC.), Lett. 24, 165-168. Trauble, H. (1971a). Phasenumwandlungen in Lipiden Mogliche Schaltprozesse in biologischen Membranen. Naturwissenschaften 58, 277-284, Trauble, H. (1971b). The movement of molecules across lipid membranes: A molecular theory. J . Membrane Biol. 4, 193-208. Trauble, H., and Haynes, D. H. (1971). The volume change in lipid bilayer lamellae a t the crystalline-liquid crystalline phase transition. Chem. Phys. Lipids 7, 324-335. Trauble, H., and Sackman, E. (1972). Studies of the crystalline-liquid crystalline phase transition of lipid model membranes. 111. Structure of a steroid-lecithin system below and above the lipid-phase transition. J . Amer. Chem. SOC. 94,44994510. Van Deenen, L. L. M. (1966). Some structural and dynamic aspects of lipids in biological membranes. Ann. N.Y. Acad. Sci. 137. 717-730. Van Deenen, L. L. M. (1972). Permeability and topography of membranes. Chem. Phys. Lipids 8, 366-373. Vandenheuvel, F. A. (1963). Study of biological structure a t the molecular level with stereo model projections. I. The lipids in the myelin sheath of nerve. J . Amer. Oil Chem. Soc. 40,455471. Vandenheuvel, F. A. (1965a). Lipid-protein interactions and cohesional forces in the lipoproteins system of membranes. J. Amer. 0 2 1 Chem. SOC.43, 258-270. Vandenheuvel, F. A. (1965b). Structural studies of biological membranes: The structure of myelin. Ann. N . Y . Amd. Sci. 122, 57-76. Vanderkooi, J. M., and Martonosi, A. (1971). Sarcoplasmic reticulum. XVI. The permeability of phosphatidyl choline vesicles for calcium. Arch. Bwchem. Biophys. 147, 632-646. Van Putte, K., Skoda, W., and Petroni, M. (1968). Phase transition and CH8-rotation in solid cholesterol. Chem. Phys. Lipids 2, 361-371. Verkleij, A. J., Ververgaert, P. H. J., Van Deenen, L. L. M., and Elbers, P. F. (1972).

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

57

Phase transitions of phospholipid bilayers and membranes of Acholeplasma laidlawii B visualized by freeze fracturing electron microscopy. Biochim. Biophys. Acta 288,326-332. Verma, S. P., and Wallach, D. F. H. (1973). Effects of cholesterol on the infrared dichroism of phosphatide multibilayers. Biochim. Biophys. Acta 330, 122-131. Villegas, R., Barnola, F. V., and Camejo, G. (1970). Ionic channels and nerve membrane lipids. Cholesterol-tetrodotoxin interaction. J. Gen. Physiol. 55, 548-561. Weiner, N. D., and Felmeister, A. (1970). Comparison of physical models used to explain condensation effects in lecithin-cholesterol mixed films. J. Lipid Res. 11, 220-222. Weiner, N. D., Bruning, W. C., and Felmeister, A. (1973). Influence of lecithins on auto-oxidation of cholesterol from aqueous dispersions a t 85”. J. Pharm. Sci. 62, 1202-1206. Weissmann, G., Sesoa, G., and Weissmann, S. (1965). Phospholipid/cholesterol structures: Effect of steroids. Nature (London) 208, 649-651. Werbin, H., Chaikoff, J. L., and Imada, M. R. (1962). 5a-Cholestan-3&01: Its distribution in tissues and its synthesis from cholesterol in the guinea pig. J. Biol. Chem. 237, 2072-2077. White, S.H. (1970). A study of lipid bilayer membrane stability using precise measurements of specific capacitance. Biophys. J. 10, 1127-1148. Wilkins, M. H. F., Blaurock, A. E., and Engelman, D. M. (1971). Bilayer structure in membranes. Nature (London), New Biol. 230, 71-76. Williams, E., Hamilton, J. A., Jain, M. K., Allerhand, A., Cordes, E. H., and Ochs, S. (1973). Natural abundance carbon-13 nuclear magnetic resonance spectra of the canine sciatic nerve. Science 181, 869-871. Willmer, E. N. (1961). Steroids and cell surfaces. Biol. Reu. Cambridge Phil. SOC.36, 368-398. Wood, R., Anderson, N. G., and Swartzendruber, D. C. (1970). Tumor lipids: characterization of the lipids isolated from membranous material. Arch. Biochem. Biophys. 141, 190-197. Yamamoto, A., Susumu, A., Ishikawa, K., Yokomura, T., Kitani, T., Nasu, T., Imato, T., and Nishikawa, M. (1971). Studies on drug-induced lipidosis. 111. Lipid composition of the liver and some other tissues in clinical cases of “Niemann-Pick-like syndrome” induced by 4,4‘-diethylaminoethoxyhexestrol.J. Biochem. (Tokyo) 70, 775-784. Zull, J. E., and Sciotto, C. G. (1969). Hydrogen bonding of vitamin D and egg lecithin in the solid state. A possible molecular role for vitamin D in calcium binding by membranes. Biochim. Biophys. Acta 173, 409-418. Zull, J. E., Greenoff, S., and Adam, H. K. (1968). Interaction of egg lecithin with cholesterol in the solid state. Biochemistry 7, 4172-4176.

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A . A . LEV and W. McD . ARMSTRONG Institute of Cytology of the Academy of Sciences of the USSR. Leningrad. USSR. and Department of Physiology. Indiana Unicersity School of Medicine. Indianapolis. Indiana

I . Introduction . . . . . . . . . . . . . . . . . 11. Definitjion of “Single Ion Activities” . . . . . . . . . . . 111. Experiments with Model Polyelectrolyte Syst.ems as Supporting Evidence for the Physical Validity of Single Ion Activity Parameters . . . . IV . Microelect.rodes for Measuring Intracellular Ionic Activities . . . A . Glass Membrane Microelectrodes . . . . . . . . . . B. Liquid Ion Exchanger Microelectrodes . . . . . . . . . C . Metal Microelectrodes . . . . . . . . . . . . . D . Other Microelectrodes . . . . . . . . . . . . . V . Techniques for Measuring Intracellular Ionic Activities . . . . . A . Calibrat~ionof Ion Selective Microelectrodes . . . . . . . B. Measurement of the Intracellular Activity of a Single Ion . . . C . Simultaneous Measurement of t.he Intracellular Activities of Two Ions, e.g., Kf and Na+ . . . . . . . . . . . . . VI . Intracellular Ionic Activities . . . . . . . . . . . . . A. Intracellular H+ Activity-Intracellular pH . . . . . . . B. Intracellular Na+, K+, and C1- Activities and Their Relationship to Cellular Function . . . . . . . . . . . . . . VII . Conclusion . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

. .

59 63

.

66 72 72 79 81 83

. . .

. . . .

84

.

85 87

. . .

88 90 90

. 94 . 113

.

113

.

1 INTRODUCTION

The role of inorganic ions in a variety of cellular functions. including such bioelectric phenomena as membrane. resting. and action potentials. has usually been interpreted in terms of conceptual models of the cell 59

60

A. A.

LEV AND W. McD. ARMSTRONG

that are based on certain assumptions concerning intracellular ionic concentrations. These assumptions are that all the apparent intracellular water* acts as a solvent for intracellular solutes and that small intracellular ions are uniformly distributed throughout this water volume. Such models require the further assumption that the intracellular activity coefficients of small ions are essentially the same as their activity coefficients in the bathing medium. I n recent years the theoretical and experimental limitations of these models have become increasingly clear. The cell interior is a highly organized system that is extremely heterogeneous, both structurally and chemically. Biopolymers (e.g., proteins, polysaccharides, nucleic acids), mainly of a polyelectrolyte nature, are of prime importance in determining the structure and function of cytoplasm and cytoplasmic organelles. Lipids and phospholipids are not polyelectrolytes in the strict sense, but, when they form lamellar and/or micellar aggregates, they may in many respects be regarded as polyelectrolyte structures (Luzzati et al., 1969). As discussed below, the theoretical description and experimental determination of the thermodynamic state of small ions, even in homogeneous polyelectrolyte solutions, is not a simple problem. In heterogeneous multicompartment polyelectrolyte systems the problem becomes much more complex. Despite these complications, it has long been apparent that the influence of the polyelectrolyte components of cytoplasm on the physical state of intracellular ions may be highly significant and should be taken into consideration. On the basis of electrostatic interactions alone one would expect the thermodynamic state of mobile inorganic ions to be altered in the presence of polyelectrolyte molecules or phospholipid aggregates with a high surface or volume charge density. The extent of such interactions may be expected to depend on such factors as the nature and spacing of the fixed charges on the polyelectrolyte molecules and the distance of closest approach to them of the mobile ions. Variations in degree of electrostatic interaction which arise from such causes may be interpreted as differences in the “binding” of inorganic ions by polyelectrolyte systems. The above concept has been extensively developed in a number of hypotheses concerning solute distribution between the cell interior and the external medium. Notable among these are the “phase” or “sorption” hypothesis (Nasonov and Alexandrov, 1940, 1943; Troschin, 1956, 1961) and the association-induction hypothesis (Ling, 1952, 1960, 1962). I n these hypotheses specific association between inorganic ions and fixed

* This is normally computed as total tissue water minus that fraction (the “extracellular volume”) which equilibrates with an external nonpenetrating solute such as inulin, mannitol, polyethylene glycol (Conway, 1957).

IONIC ACTIVITIES IN CELLS

61

charge groups in cytoplasmic macromolecules is considered to be the sole reason for the marked discrepancies in ionic distribution between extracellular and intracellular fluid. This now appears unlikely in view of the vast and growing body of evidence for the existence in living cells of transmembrane active transport processes, in particular the ubiquitous “sodium pump.” Nevertheless the problem of inorganic ion interaction with cytoplasmic macromolecules or specific ion binding in cytoplasm continues to be an important one in relation to ion transport and electroselectivity in cells. For example, the difference in cytoplasmic binding between a divalent cation such as calcium and a monovalent ion such as potassium seems beyond reasonable doubt. Also the Donnan effect in the distribution of inorganic ions between the cell interior and the external medium may be regarded in a general sense as a “phase” property of the cytoplasm. Thus, a knowledge of the physical state of intracellular cations, particularly in the vicinity of the plasma membrane, is of obvious importance in relation t o bioelectric phenomena. The existence of discrete intracellular organelles creates further possibilities for inhomogeneity in the distribution of intracellular ions. Kleinzeller et al. (1969) introduced the terms structural and chemical compartmentation to describe different types of intracellular organelles which may differ in their ionic content. Organelles surrounded by membranelike diffusion barriers (e.g., endoplasmic reticulum and its derivatives, mitochondria, chloroplasts, vacuoles, lysosomes) and in which the internal ionic content is largely determined by active ion transport processes were designated structural compartments. Subcellular organelles not separated from the main intracellular space (hyaloplasm or ground cytoplasm) * by membrane structures were defined as chemical compartments. Such compartments include myofibrils, neurofibrils, tonofibrils, fibrils of flagella and cilia, ribosomes, lipid granules, and perhaps membrane structures themselves. In contrast to structural compartments, the ionic composition of chemical compartments should be a function of the ionic composition of the ground cytoplasm, a t least within the limits of the constancy of the polyelectrolyte content of the corresponding subcellular organelles, and the distribution of ions between ground cytoplasm and a given chemical compartment should depend primarily on the relative charge density of their polyelectrolyte components. If the ability to discriminate between different ions with the same sign of charge is not significant in either compartment, the distribution of mobile ions between them should obey the Donnan principle. *The existence within the cell of such a phase as “ground cytoplasm” is open to argument., but there is no doubt that, as an assumption, i t is extremely useful at present in constructing electrochemical models to explain bioelectric phenomena.

62

A. A. LEV AND W. McD. ARMSTRONG

If the mobile ions have significantly different affinities for the fixed charge groups in different compartments, their equilibrium distribution between these compartments is still subject to the restriction that their electrochemical potentials must be the same in all chemical compartments and in the ground cytoplasm, i.e., Pi,g.o.

=

Pi.1

= pio = pio

=

pi.2

=

+ RT In

* . a

-

Pi,n

+ RT In + z,Fpl + RT In a i , 2+ ziFpz = ... = pio + RT In a i , , + ziFpn ai,g.c.

= pio

where p i , g . c . , P ~ J p, i . 2 - . * p i , n are the electrochemical potentials of the i t h ion in the ground cytoplasm and chemical compartments, a i , g . c . , ai,2 a i , na r e its corresponding activities, pio its chemical potential in the standard state, z i its valence, and p1p2 * pn are the phase boundary potentials between the ground cytoplasm and chemical compartments 1,2. It follows from Eq. (1) that, a t equilibrium, the concentrations, and indeed the activities, of an ion may be different in different chemical compartments. Thus it is clear that intracellular ionic concentrations calculated from chemical or isotopic determinations of tissue water and ion contents are only a first approximation to real intracellular ionic concentrations. Methods in which ground cytoplasm is separated from whole cells and analyzed for its ionic content (see, e.g., Bruce and Marshall, 1965) inevit,ably involve tissue or cell homogenization, during which profound changes may occur in the ionic composition of many if not all cytoplasmic fractions. At present the only direct method available for determining the ionic composition of the ground cytoplasm of cells is the measurement of intracellular ionic activities with ion-sensitive microelectrodes. The position, with respect to cellular ultrastructures, of the tip of the ion selective electrode following impalement of a cell is usually not exactly known, and one cannot prove beyond doubt that the medium surrounding the electrode tip is in fact ground cytoplasm. However, it is clear that the position of these microelectrodes is exactly the same as that of ordinary capillary microelectrodes used for transmembrane potential difference measurements. Therefore ion activities found by the method of ion-sensitive microelectrodes are plausible for the description of electrochemical plasma membrane properties and transmembrane ionic fluxes.

... a n .

-

IONIC ACTIVITIES IN CELLS

63

II. DEFINITION OF "SINGLE ION ACTIVITIES"

The concept of "single ion activity" is somewhat uncertain from a strict thermodynamic viewpoint. This uncertainty arises from the negation in classical thermodynamics of the physical reality of ions of one sign of charge as independent components of macroscopic systems. Actually, ions of one sign of charge may not be added in macroscopic quantities to a system without simultaneous addition of an equivalent amount of oppositely charged ions or removal of an equivalent amount of another ion of the same charge. Thus the principle of macroscopic electroneutrality forces one to use such parameters as single ion activities and single ion activity coefficients only in combinations capable of being transformed into other parameters which are characteristic of electrolytes as a whole (Guggenheim, 1929, 1933). This situation is paradoxical in that real physical components of electrolyte systems (ions with one sign of charge) are under a forma.1restriction in the rigorous thermodynamic approach to the electrochemistry of solutions. In fact some investigators consider determinations of single ion parameters (including single ion activities and activity coefficients) to be in certain respects invalid. Hence it is not surprising to find attempts aimed at finding new theoretical and experimental approaches to the definition of single ion activities and activity coefficients and the provision of a physical meaning for these parameters (Rabinovich, 1964; Rabinovich et al., 1960, 1967; Manning, 1969; Manning and Zimm, 1966; Frank, 1967; Nikerov and Rabinovich, 1968; Lev et al., 1971). If the physical validity of single ion activities as parameters of thermodynamic systcms may be assumed, then there are two main experimental restrictions to their exact determination. The first concerns the question of standardization. This problem is the same as that which arises in pH measurements (Bates, 1973), since a pH measurement is a particular case of a single ionic activity determination. The second restriction originates from the difficulty of estimating precisely the liquid junction potential which necessarily arises when galvanic cells with transference are used in electrochemical measurements. We shall discuss briefly the importance of these two restrictions in the determination of single ionic activities. The mean activity of a salt in solution is a thermodynamically rigorous parameter which may be defined by several independent methods, including potentiometric measurements with the aid of galvanic cells without transference (Robinson and Stokes, 1965). For such measurements it is sufficient to have a pair of electrodes, one of which is reversible for the cation and the other for the anion of the salt under investigation. A commonly used method for standardizing the mean activity of the salt is based on the

64

A. A. LEV AND W. McD. ARMSTRONG

assumption of equality between salt activity and salt concentration in very M ) solutions. dilute (in practice, say less than To determine the activity of a single ion, one requires a galvanic cell consisting of an electrode which is reversible for the cation or anion in question and a reference electrode connected to the solution by a salt bridge. For the moment we shall postpone discussion of the liquid junction potential between the solution in the salt bridge and the solution under investigation and regard this potential as known, or a t least as constant. I n this case the problem of single ion activity standardization becomes simply a question of common agreement on a primary standard. This may be a solution of any salt or acid provided reversible electrodes are available for both its cations and anions. For example, one may choose a KC1 solution since highly selective electrodes reversible to K+ ions are now available (e.g., Orion model 92-19 liquid ion exchange potassium electrodes). An Ag/AgCl or calomel electrode may be used as the C1- reversible electrode. On the basis of the Debye-Huckel limiting law, one may assume that in a dilute KC1 solution the K+ ion activity ( U K ) is equal to the C1activity ( u c ~ and ) that both are equal to the mean activity ( u K c ~ ) of KC1 in the reference solution. If it is assumed that the liquid junction potential remains constant for all solutions used, one may then extend this method to provide secondary standards for other ions; e.g., a galvanic cell with transference which includes a -C1- reversible electrode can be used with a dilute NaCl solution to obtain a reference standard for Naf activity. If the cell is first calibrated for C1- activity in the primary KCl standard solution it can be used to determine the C1- activity in a dilute NaCl solution. If the mean NaCl activity of this solution is known, its Na+ activity is readily calculated from the relationship 2

UNs = uNaCl/uCl

(2)

Data thus obtained from a series of NaCl solutions can then be used to calibrate a reversible Na+ electrode. In like manner, secondary standards can be obtained for other cations and anions.* As already mentioned, the above standardization procedure is correct under the assumption of constant liquid junction potential for all solutions used (the actual magnitude of this potential is relatively unimportant). Unfortunately this assumption is usually not justified. Further, estimation of the probable size of the liquid junction potential and of the amount by

* pH standards conforming to an accepted convention (Bates and Alfenaar, 1969) map be used as primary standards from which secondary standards for the activities of other ions may be derived.

65

IONIC ACTIVITIES IN CELLS

which it may change under the different experimental conditions employed is not easy. Liquid junction potentials may be regarded as a particular case of phase boundary potentials, and it is well known that the potential difference between two phases is not theoretically determinable. Our inability in principle to determine the liquid junction potential for a given set of conditions implies a similar inability to determine its constancy or lack of constancy under varying conditions. I n the most general approach, the liquid junction of diffusion potential between two solutions can be represented by the following equation :

where t i and t, are the transference numbers of the ith ionic species and the solvent respectively, ai‘ and a:’ are the ith ion activities in the two solutions in contact with each other, and a,’ and a:’ are the corresponding solvent activities. Equation (3) can be integrated only under certain assumptions. The problem has been considered by several authors (Planck, 1890a,b; Behn, 1894; Henderson, 1907, 1908) who obtained satisfactory results subject to certain specific limitations. These limiting conditions depend on the model chosen for the boundary between the mixing solutions. The most popular integrated form of Eq. (3) is the diffusion equation of Henderson (1907, 1908).

RT

c ui(c:’-

Ci’)

c

UiZiCi’

n

(4) n

n

This equation was obtained quasithermodynamically with the diffusion zone regarded as a continuous series of mixtures of solutions (’) and (”). Ionic mobilities were supposed to be independent of concentration, and single ion activity coefficients of unity were assumed. A more rigorous determination of the diffusion potential difference requires a knowledge of the actual profile of single ion activities in the zone of solution mixtures. However, as already pointed out, determination of these parameters depends on the possibility of determining the liquid junction potential. Thus, the range of unsolved questions is closed. Fortunately, this state of affairs does not mean that approximate estimations of diffusion potentials, from Eq. (4) for example, are useless, especially in the case of contacts between simple salt solutions containing cations and anions of nearly equal mobilities (e.g., such pairs as K+ and C1- or NH4+and NOa-) a t low concentrations. Under these conditions, diffusion potentials estimated from the

66

A. A. LEV A N D W. McD. ARMSTRONG

Henderson Eq. (4) range from a few tenths of a millivolt to 2-3 mV. These limits of the estimated diffusion potentials introducc an uncertainty of approximately 5-150/, in the determination of single ion activities in M to lo-' ill salt solutions when the method of standardizing described above is used. Because of the unccrtainties in the assumptions underlying Eq. (4), this estimate of the error inherent in single ion activity determinations is not quite certain, even for simple salt solutions. The situation with respect to liquid junction potentials in polyelectrolyte solutions is much less sure. Hence the reliability of estimates of single ion activities in such solutions is considerably reduced, and a similar situation holds for corresponding estimates in cytoplasm. Obviously, additional criteria are required to establish the validity of ionic activity measurements in polyelectrolyte solutions and, by inference, in cytoplasm. Such criteria can be found in the following ways: (a) comparison of experimental data on single ion activities in polyelectrolyte solutions with theoretically predicted values : (b) comparison of single ionic activities found in polyelectrolyte solutions with other physical parameters (found by independent methods) in these solutions. Some results of such comparisons are given in the next section.

111. EXPERIMENTS WITH MODEL POLYELECTROLYTE SYSTEMS AS SUPPORTING EVIDENCE FOR THE PHYSICAL VALIDITY OF SINGLE ION ACTIVITY PARAMETERS

Some experimental evidence that the physical state of inorganic ions in solutions containing biological polyelectrolytes differs from their state in aqueous solutions of simple salts was found many years ago (Hammersten, 1924; Teunissen van Zijp and Bungenberg de Jong, 1938; Creeth and Jordan, 1949; Shack et al., 1952). Convincing quantitative data bearing on this problem, however, were obtained only after the development of ion-sensitive electrodes (Scatchard et al., 1957). In a series of studies on the state of Na+ ions in DNA solutions (Botre et al., 1958; Ascoli et al., 1959, 1961; Vorobjev et al., 1971), a marked decrease in the activity coefficient of Na+ was noted in solutions containing native DNA. The converse of this, an increase in Na+ activity (i.e., a decrease in counterion binding) was observed during thermal or dilution denaturation of DNA. The state of small inorganic ions (counter- and co-ions) in solutions containing polyelectrolytes of known structure was considered theoretically by Manning and Zimm (1966) and by Manning (1969). The equations obtained by these authors can be applied to biological systems because they

67

IONIC ACTIVITIES IN CELLS

were derived for solutions containing concentrations of polyelectrolyte and simple salt which are comparable to those found in cytoplasm. Those obtained earlier by Katchalsky et al. (1966) are less applicable since they are based on a consideration of salt-free polyelectrolyte solutions or polyelectrolyte solutions containing a large excess of salt. I n deriving cquations for counter- and co-ion activity in polyelectrolyte solutions, Manning (1969) made certain assumptions. Among these is a model of polyelectrolyte molecules as linear chains with uniformly distributed surfacc charges. Interactions between charged groups on the polyelectrolyte molecule were ignored, and the dielectric constant of the polyelectrolyte solution was considered to be the same as that for a simple salt solution. Also, the Debye-Hiickel approximation was applied to all mobile ions except those in the region of ion condensation. Manning's (1969) equations for singly charged counter- and co-ions (in the case of DNA and polyacrylate solutions these will be cations and anions, respectively) are yi = y(f-lx

+ 1)(x + l)-l exp[YA

Y ~ A=

y2(E-'X

= y exp[- &'x/(E-'

+ 1) ( X + l)-'

&lx/(t-l

+ 2)l

+ 2)1

exp[ - €-'X/(t-'

(5) (6)

+ 211

(7)

In these equations y i is the activity coefficient of the counterion, Y A that of the co-ion and Y ~ Ais the mean activity coefficient of the (univalent) salt; X = n,/n, where n, is an equivalent concentration of the charged group of the polyelectrolyte (e.g., the concentration of phosphate groups in the case of DNA) ; n, is the concentration of uni/univalent salt added to the polyelectrolyte solution and E = e2DkTb where e is the unit charge, D is the dielectric constant (80 for water), k is Boltzmann's constant, T is the absolute temperature, and b is the distance between charges on the polyelectrolyte molecule (projected onto its axis). The factor y (Manning and Zimm, 1966) was introduced to take account of interactions between small inorganic ions in the solution and is given by the equation

where X = 4re2/DkT = (8.76 x lo-' cm for aqueous solutions) and C, is the total concentration of inorganic ions in the solution. The above equations permit one t o calculate activity coefficients for polyelectrolyte solutions containing a known concentration of polyelectrolyte and different concentrations of NaC1. These can then be compared with the experimentally observed values of these parameters under the same conditions. We have made such a comparison for DNA and poly-

68

A. A. LEV AND W. McD. ARMSTRONG

acrylate solutions containing NaC1. DNA and polyacrylate were chosen for this study because their charge distribution is well known. Galvanic cells with transference were used to measure Na+ and C1activities. These cells consisted of a sodium-sensitive glass electrode and an Ag/AgCl electrode. The mean NaCl activity was either determined directly with a galvanic cell without transference or taken as the geometric mean of u N a and ucl. In both cases the results should be the same. Since the concentration of NaCl added to the polyelectrolyte solution was known, experimental values for the activity coefficients of Naf (yNa), C1- (ycl), and the mean activity coefficient of NaCl (yaScL)were readily obtained from the activity data. These were then compared with the theoretical values for these parameters calculated from Eqs. ( 5 ) , (6), and (7). The results obtained for native sodium-DNA and sodium-polyacrylate solutions are shown in Figs. 1 and 2. The quantitative agreement between calculated and observed activity coefficients for DNA (Fig. 1) is not very good although the mode of change of the experimental values and their general position on the graph are in accordance with theoretical predictions. Agreement between theoretical and observed values is better for the polyacrylate/NaCl solutions (Fig. 2) although there is some scatter in the experimental points. The deviation from theory of the y N a points for DNA

0.6

*

0.4

.

0.2

.

0

Y

{O-’

40-3

(0-2

AO-4

CNaCL

FIG.1. Dependence of YNa, yc1, and Y N ~ C Ion NaCl concentration in solutions of native DNA. The curves are theoretical values calculatedofrom Eqs. ( 5 ) , (6), and (7), the parameter b in these equations being taken as 7 A. The points are experimentally determined activity coefficients. The DNA concentration ranged from 0.159 to 0.171 gm/100 ml.

69

IONIC ACTIVITIES IN CELLS

0.8 Y

0.6 0.4 0.2

t

i

!O-'

10-2

10-4 CNoCL

FIG.2. Data as in Fig. 1, but for sodium polyacrylate solutions (0.05 g m / l O O ml). The degTee of neutralization of the polyacrylic acid was 95%. Parameter b was taken as 2.55 A.

solutions (Fig. 1) may be due, in part a t least, to some denaturation with time of the polymer a t room temperature resulting in changes in the mean intercharge spacing, 6. An important feature of the data shown in Figs. 1and 2 is that the differences between theoretical and experimental values for mean activity coefficients appear to be very similar to the differences between predicted and observed values for single ion activity coefficients. This similarity is particularly apparent in Fig. 1. The mean activity coefficient of a salt (yfNaa in Figs. 1 and 2) is a rigorously defined thermodynamic parameter. As such it is free from the theoretical objections which are sometimes raised against the concept of single ion activities (page 63). Furthermore, mean activity coefficients can be determined without the complications connected with liquid junction potentials. The fact that mean activity coefficients measured in this way proved to be in no better agreement with theory than experimentally determined single ion activity coefficients can be interpreted as an indication that the liquid junction potential did not vary significantly over the whole range of salt concentrations employed in these experiments (in the experiments with DNA this was to lo-' M ) . This conclusion is important supporting evidence for the validity of single ion activities measurements and of standardization procedures (page 64). Further support for the validity of single ion activity determinations was obtained during an investigation of the conditions necessary for the so-called isoionic dilution of polyelectrolytes. It is known that in the

70

A. A. LEV AND W. McD. ARMSTRONG

measurement of some hydrodynamic and optical parameters of polymer solutions (e.g., characteristic viscosity determination and measurement of rotatory diffusion by flow birefringence) extrapolation to zero polymer concentration must be used. This extrapolation can be done most precisely under conditions where the measured parameter (reduced viscosity when characteristic viscosity is determined, or the x angle when rotatory diffusion is studied) shows a linear dependence on polymer concentration. To obtain such linear dependence in polyelectrolyte solutions it is necessary to dilute the initial solution with the simple salt solution at a Concentration which does not alter the screening of polyelectrolyte charged groups by counterions. This is the condition of isoionic dilution. Its importance is that the dimensions (and hence the intercharge spacing) of the polyelectrolyte molecules are held constant during dilution. Where the thermodynamic state of the counterions is not influenced by the polymer (i.e., in solutions of uncharged polymers), isoionic dilution is quite simply obtained by adding simple salt a t the same concentration as that in the initial sample of polymer solution. If, as in the case of polyelectrolyte solutions, the activity of counterions may be significantly changed by “ion-binding,” then, to ensure that dilution is isoionic, the activities rather than the concentrations of counterions in the polyelectrolyte solution must be known. Dranitskaya et al. (1967, 1974) and Lev et al. (1971) approached thc problem of isoionic dilution of a number of biopolymers and synthetic polyelectrolytes in solutions containing NaCl as follows: Reduced viscosity and counterion activity were measured in the initial sample of polymer solution. As expected, counterion activities were frequently much smaller than their corresponding concentration. For example, in solutions of native DNA, Y N was ~ found to be as low as 0.25, and in sodium polyacrylate solutions Y N appeared ~ to be between 0.3 and 0.4 when a minute amount or no NaCl was added. Polymer solutions with measured counterion activities were diluted successively with increasing amounts of salt solutions having the same counterion activity, this activity being measured independently in the salt solution. Reduced viscosity was determined a t each dilution state and plotted as a function of polymer concentration. A linear relationship between reduced viscosity and polymer concentration was taken as an indication that dilution was in fact isoionic. In the majority of cases studied, it was found that when the measured counterion activity of the diluent solution was adjusted to equal its measured activity in the initial polymer sample, the reduced viscosity was in fact a linear function of polymer concentration. This is illustrated in Fig. 3 which shows the results of an experiment with solutions of sodium polyacrylate containing NaCl a t initial mean activities ranging from 3.4 t o 7.2 X M. I n all

71

IONIC ACTIVITIES IN CELLS

30

10

I

1

0.01

0.02

0.03 CPA

0.04

0.05

(Oh)

FIG.3. Dependence of reduced viscosity, (7 - l ) / C N a p A on the concentration of sodium polyacrylate ( C ~ A(MW ) 5.1 x 106) in samples with different initial concentrations of NaCl. Isoionic dilutions (judged by a linear dependence of reduced viscosity on polymer concentration) were obtained when the NaCl solutions used for dilution (the “solvent”) had the same UN* as that in the initial samples of polyeleetrolyte. The broken line shows the dependence when a N s in the “solvent” was the same as a N s c l in the I for the initial polyelectrolyte solution. The numbers near the lines are the U N ~ C values initial polyelectrolyte solutions.

cases where the diluting solution (“solvent”) had the same aNaas the initial polymer sample a linear dependence of reduced viscosity on polymer concentration was obtained. It is highly significant that in the one instance where dilution was performed by adding NaCl at the same mean activity (aNaci) as that of the initial sample (dashed line in Fig. 3) the plot of reduced viscosity versus polymer concentration was not a straight line. Also, a differenceof 10-15% between aNa in the “solvent” and the initial sample =as sufficient to cause a significant deviation from linearity. Essentially similar results have been obtained for natural and reconstituted nucleohistone complexes (Frisman et al., 1970a,b), ribosomal RNA solutions (Schagina et al., 1969), and solutions of polymethacrylic acid (Dranitskaya et al., 1974). The significance of these results in the present context may be summa.rized as follows: Single ion activity measurements performed with the aid of galvanic cells with transference permit one to define the conditions

72

A. A. LEV A N D W. McD. ARMSTRONG

for constant screening of polyelectrolyte charged groups in solution. Constancy of screening under these conditions can be checked by an absolutely independent hydrodynamic method. If the screening of polyelectrolyte charged groups in solution is a physically real phenomenon, then single ionic activities in solution have the same physical reality. Furthermore, these experiments strongly support the concept that ionic activity determinations in solutions containing polymers have the same validity as corresponding measurements in simple salt solutions.

IV. MICROELECTRODES FOR

MEASURING ACTIVITIES

INTRACELLULAR IONIC

The use of ion selective electrodes for the determination of ionic activities on a macroscale is well established as a routine analytical method. A large and rapidly growing variety of these electrodes is now commercially available and the list of their application to biological and nonbiological systems is almost bewildering (see, for instance, Beljustin and Lev, 1965; Eisenman, 1967a; Feder, 1968; Durst, 1969). To date, the number of types of electrodes used and the extent of their use in intracellular studies, although rapidly increasing, is much more modest, so that it is still possible to review this subject within a reasonable space. For convenience, we have classified the different kinds of microelectrodes so far used for measuring intracellular ionic activities under four general headings. These are, glass membrane electrodes, liquid ion exchanger microelectrodes, metallic microelectrodes, and other microelectrodes. A. Glass Membrane Microelectrodes

1. SIMPLIFIED THEORY OF THE GLASSELECTRODE

A rigorous discussion of the theory of glass membrane electrodes (commonly called glass electrodes) is outside the scope of this review. An excellent account of their properties is that of Eisenman (1965), on which much of the brief outline presented below is based (see also Nicolsky et al., 1967). A glass electrode consists of a thin glass membrane enclosing a solution of known ionic composition. When it is inserted in an external solution of different composition a potential difference develops across the membrane. This potential difference can be measured by immersing reversible reference electrodes (e.g., Ag/AgCl or calomel half-cells) in the inner and outer solutions, respectively. Depending on the composition of the glass mem-

73

IONIC ACTIVITIES IN CELLS

brane, glass electrodes show various selectivities; that is, they respond preferentially to a specific ion. This is invariably a cation because glass is a fused mixture of the oxides of elements with a valence of 1+ or 2+ and oxides of elements with a valence of 3f or more and univalent cations are the only readily mobile ions in the fused mixture. Bivalent cations have much smaller mobilities and the mobility of anions is essentially zero (Anderson and Stuart, 1954). Also, because of the high concentration of fixed negative charges in the glass network, its concentration of small free anions is very low. Thus, glass membranes function essentially as univalent cation exchangers (Eisenman, 1965). The detailed relationship between the composition of glasses and their ionic selectivity is discussed by Eisenman (1965, 196713, 1969), HBbert (1969), and Bates (1973). Depending on its composition, the selectivity of a glass membrane may be so great as to be virtually absolute in practice. In other words, cations other than those to which the glass is primarily responsive will, under normal conditions of use, have only negligible effects on the total electrode potential. Perhaps the best-known example of this behavior is the H+ glass electrode routinely used in pH measurements. Alternatively, one or more ions, in addition to the preferred ion, may contribute significantly to the potential recorded by the electrode in solutions containing mixtures of cations. Examples are electrodes made from K+ or Na+ selective glasses (e.g., NAS 25-4 K+ selective glass or NAS 11-18 Na+ selective glass*). Both of these are sensitive to H+ ions and, in addition, +(I selective glasses show significant sensitivity to Na+. The latter fact, as discussed below, has some interesting consequences in the determination of intracellular Na+ and K+ activities. If a glass membrane separates two solutions containing an ion i to which it is sensitive, the potential across it is given by the Nernst equation

E

=

(RT/F) ln(ail/a:’)

(9)

where air and a:’ are the activities of ion i on the two sides of the membrane. If one of these activities, e.g., a:’ is held constant, then

E

=

const.

+ (RTIF) lna,’

(10)

and, following appropriate calibration, this equation may be used to determine the activity of i in solutions in which it is the only cation. Further,

* These and similar codes for the composition of glasses are interpreted as follows (Eisenman et al., 1957) : The letters NAS indicate that the glass is made from a mixture of NazO, AlzO,, and SiOz. The numbers following the letters are the moles percent of NazO and Alzo, respectively; e.g., NAS 27-4 contains 27 moles percent of Na202, 4 moles percent of AlzOaand, hence, 69 moles percent of SiOz.

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A. A. LEV A N D W. McD. ARMSTRONG

if the electrode type and the conditions are such that i is the only ion which contributes significantly to the overall potential (e.g., the pH glass electrode under ordinary conditions), or, if interfering cations are present in minute amounts compared to i, Eq. (10) can also be applied. Nicolsky (1937) has shown theoretically that if a glass electrode is immersed in a solution containing two univalent cations to which it is sensitive in different degrees, the total electrode potential is given by the equation E = E, (RT/F) U a , k,,aJ (11)

+

+

where i is the preferred cation.* E, corresponds to the constant term in Eq. (10);and i t is the potential registered by the electrode in a mixture of i and j, for which the term in parentheses is unity. k , , is a selectivity coefficient which expresses the selectivity of the electrode for i compared to j. It is readily determined from the potentials recorded by the electrode in solutions of i a n d j , respectively, when a, = a,. Under these conditions Ink,, = F(E,

- E,)/RT

(12)

where Ej and E , are the potentials observed in the solution containing j only and in that containing i only. Note that the value of k , , is inversely proportional to the selectivity of the glass for i relative to j. A k , , of 0.1 means that the glass is 10 times more sensitive to i than to j.

When an electrode is sensitive, in varying degrees, t o a number of ions, Eq. (11) takes the general form

E

=

E,

+ (RT/F)ln(a, + kt,a, +

k,,a,)

(13)

if the electrode is immersed in a solution containing all these ions and on the assumption that the solution approximates ideal behavior. The physical meaning of the selectivity coefficient k , , in Eq. (11) deserves comment. Nicolsky’s (1937) original derivation of this equation was for the response of glass electrodes to Na+ and H+ and had its origin

* The experimental validity of

Nicolsky’s equation has been confirmed by Eisenman al. (1957), who also found that an even wider range of phenomena is adequately described by the empirical equation et

E

=

Ea

+ ( n R T / F )In

+ (kij

~ j ) ” ~ ]

(114

where n is a constant for a specific glass and a given pair of cations. Obviously, when n = 1, Eq. ( l l a ) reduces to Eq. (11). This usually turns out to be the case for Na+ or K + selective glasses when the conditions are such that Naf and K + are the only ions contributing significantly to the overall electrode potential. Hence, Eq. (11) has proved adequate for the determination of intracellular Na+ and K+ activities with K + selective glass electrodes (see, e.g., Lev, 1964; Armstrong and Lee, 1971).

75

IONIC ACTIVITIES IN CELLS

in earlier studies that sought to explain the well known “alkaline error” of pH glass electrodes (Hughes, 1922).* Nicolsky assumed a reversible exchange of Na+ and H+ between glass and solution with the same population of sites available to both ions. It was further assumed that the activities of Na+ and H+ in the glass phase m r e proportional to their mole fractions therein and that the total electrode potential was the sum of the phase boundary potentials a t the two glass/solution interfaces. No account was taken of possible contributions from diffusion potentials within the glass membrane. More recently it has been shown (Karreman and Eisenman, 1962; Conti and Eisenman, 1965a,b, 1966) that the glass electiode potential, like the potentials generated across other fixed site ion-exchange membranes (Helfferich, 1956, 1959, 1962; Mackay and Meares, 1960), is a composite of diffusion potentials within the glass membrane together with equilibrium phase boundary potentials a t the glass/solution interfaces so that one may write kt,

=

kt(u,/u,) = P,/Pa

(14)

where ual u,,P a , P , are the mobilities and permeability coefficients of i and j in the glass membrane and k,“,is the ion exchange equilibrium constant. The fact that k,,, as determined, for example, from electrode potential measurements using Eq. (12), is a composite of exchange equilibrium and nonequilibrium kinetic components can impose important practical restrictions on the selectivity of glass membrane electrodes. For instance, the K+/Na+ selectivity of NAS 27-4 glasses as determined by electrode potential measurements is about 8-10-fold. Eisenman (1965) has shown that the equilibrium selectivity of such glasses is in the general range of 50-80. The much smaller overall selectivities are due to the fact that U N * in these glasses is 5-10 times greater than U K . When an electrode is responding, under conditions of zero current flow, in ideal fashion to a given ion, i.e., in a pure solution of that ion or where the effects of other ions on the electrode potential are negligible (cf. the linear portions of the curves in Fig. 5), changes in total electrode potential are determined by changes in the phase boundary potential alone. Under these conditions the diffusion potential is constant, maximal, and is given by RT log uK/uNain the case under discussion (Eisenman, 1965). This explains why Eq. ( l l ) , which is based on simple ion-exchange theory, can successfully predict the behavior of K+ selective glass electrodes in many situations. The condition of zero current flow is effectively that under which intracellular ionic activities are normally measured. * For a review of early work on the reiationship between the composition of glass and its relative sensitivity to H + and Na+ ions, see Eisenman (1965).

76

A. A. LEV AND W. McD. ARMSTRONG

2. DESIGNOF GLASSMEMBRANE MICROELECTRODES FOR MINATION OF IONIC ACTIVITIES

THE

DETER-

Glass membrane microelectrodes have been used successfully for the measurement of intracellular H+, Na+, and I 10,000) across the membrane exceeds the Na2 ratio (10x10) (Portzehl et al., 1964; Baker, 1972), this heteroexchange will not account for the internal Ca2+ concentration a t physiological external Ca concentrations. The possibility exists that the mechanism operates on a cellular compartment underneath the plasma membrane in which the Ca2+concentration is raised by another type of Ca pump to a level above that in the bulk cytoplasm (Reuter, 1973; see below). For the giant axon of the squid, Baker and Glitsch (1973) leave the possibility open that in addition to the exchange mechanism an ATP-driven pump may exist.

VI. PHYSIOLOGICAL SIGNIFICANCE OF CALCIUM PUMPS A. Muscle

Calcium-sequestering systems within the cell (SR, mitochondria) have undoubtedly the task of rapidly restoring low intracellular Ca2+ concentration in the cytosol when it. has been raised by the events during excitation. In skeletal muscle the SR is the ‘Lrelaxingfactor” (see Ebashi and Endo, 1968) which in 20-100 msec removes Ca released by an action potential from the space where the actomyosin is located, regardless of the source from which Ca entered this space during the action potential. I n skeletal muscle this source may be the SR itself whereas in cardiac muscle the main source may be the external medium. At all events these seques-

158

H. J. SCHATZMANN

tering systems do not account for the steadily maintained high Ca gradient between external medium and cytosol unless they can discharge their content to the external medium. For mitochondria any such possibility seems remote and in SR it is a moot point whether a communication between the lumen of the SR and the transverse tubules exists. An alternative is an efficient Ca pump located in the plasma membrane. For quantitative reasons the Na-Ca heteroexchange cannot possibly take this role alone, but it might be one component in a two-step transport. This possibility seems not too far-fetched in cardiac muscle where an anatomical substrate seems to exist for a Ca-rich space underneath the membrane and for which electrophysiological data suggest the existencc of such a compartment (Reuter, 1973).

B.

Red Cells

1. INFLUENCE ON ALKALICATION PERUEABILITY Whereas in muscle relaxation affords a good reason for mechanisms keeping intracellular [Ca] low, these contrivances seem a mere luxury in noncontractile cells. But apart from the fact that contractile processes seem to play a role in many cells, maintenance of low Ca concentrations is necessary for the proper functioning of the cell membrane. I n this respect, red cells of certain species (including man) (Jenkins and Lew, 1973) display a behavior which might be present and important in more highly organized cells, too. Gardos (1958, 1959) demonstrated that metabolic depletion of human red cells increases the K permeability of the membrane provided that Ca is present in the medium. Since its discovery this effect has been confirmed by Hoffman (1962), Whittam (1968), Kregenow (1962), Kregenow and Hoffman (1972), Romero and Whittam (1971), Lew (1970, 1971), Blum and Hoffman (1971), and Dunn (1974), and there seems to be full agreement that the cause for the very dramatic rise in K permeability is a slight rise in intracellular Ca2+ concentration when ATP is missing. Lew (1970) has indicated that an increase in cellular [Caz+]by lop6 M is sufficient to raise the K permeability very markedly. Blum and Hoffman (1971) were able to show that the Ca-induced K outflow exhibits saturation kinetics, and they seem to be inclined to think that internal Ca modifies the Na-K pump into a shuttling carrier for K. In a recent report Jenkins and Lew (1973) showed that the K effect is present in human, rat, guinea pig, and the coypu red cells, but not in sheep, cattle, and goat red cells, although the ruminant red cells also take up Ca when starved. As mentioned before, it is not clear whether the Ca entry is due only to the arrest of the Ca pump or whether the energy-depleted

ACTIVE CALCIUM TRANSPORT AND CaZf-ACTIVATED ATPase IN HUMAN RED CELLS

159

state causes structural changes in the membrane that favor the inward leakage of Ca. 2. INFLUENCE ON MECHANICAL BEHAVIOR AND SHAPE

Entry of Ca into human red cells leads to conspicuous alterations in the mechanical properties and morphological behavior of these cells (Dunn, 1974). Weed (1968), Weed et al. (1969), and Weed and Chailley (1973) have shown that starved red cells with extremely low ATP content become more rigid and that this alteration is strictly paralleled by an increase in cellular Ca content (rigidity is quantitated by measuring filterability or viscosity of cell suspensions or by observing the deformability of a single cell sucked partially into a microcapillary). The effect is reversible if the cells are allowed to pump Ca out again and can be prevented by Ca chelators. I n the depleted state the cells undergo disk-sphere transformation by passing through a phase of crenated disks-crenated spheres (echinocytes). Nakao et al. (1960), Wins and Schoffeniels (1966a), and Palek et al. (1971a, b) have pointed out that Ca2+ can induce shrinking of red cell ghosts. This effect can be attributed only to a Ca action on the internal side of the membrane. There is, however, no agreement as to the role of ATP in this shrinking. Wins and Schoffeniels are inclined to ascribe the effect t o ATP-consuming contractile proteins of the actomyosin type. The existence of a contractile protein was claimed by Ohnishi (1962), and it might be identical or associated with the protein spectrin, which seems to be arranged on the internal membrane surface and was solubilized by Marchesi and Steers (1968). The latter authors showed that their protein forms filaments visible in the electron microscope if ATP and Ca or Mg are present in concentrations between 0.1 and 1 mM. This in turn might be the morphological substrate of Ca-stimulated ATPase activity isolated from ghosts by Rosenthal et al. (1970). Palek et al. (1971b) obtainedmarked shrinking of ghosts in the presence of Ca when isotonicity was restored in the absence of ATP. Weed and Chailley (1973) have considered the possibility that the crenation is due to local activation of contractile processes in membrane areas where Ca pump sites are scarce. The cells become spherical and turn rigid under conditions allowing entry of Ca. Both alterations are unfavorable from a functional point of view. It follows that Ca cntry must be prevented during the lifespan of a red cell, and the Ca pump seems to be an appropriatc mechanism to this end. On the other hand, it seems quite plausible that these processes are initiated when the energy-librrating processes decline in aged cells, thus precipitating their mechanical destruction in the body (LaCelle et al., 1973).

160

H. J. SCHATZMANN

It is quite possible that in certain hemolytic anemias a disturbance in the Ca transport is a t the basis of the increased vulnerability of cells. I n a recent report, Eaton et al. (1973) demonstrated quite convincingly that in sickle cell disease red cell Ca permeability is profoundly disturbed. The Ca content of red cells was 7.6 times that found in cells from healthy subjects, sickled individual cells had higher Ca content than the patient’s cells exhibiting normal morphology, and the 45Cainflux into cells of patients was 9.3 times that of cells of normal subjects in the oxygenated state and 48.5 times larger than in normal cells in the 02-deprived state. It seems that the defect consists mainly in an increased passive permeability for Ca. The function of the Ca transport system and of Ca-ATPase was not tested. As one would expect, the sickle cells also exhibit excessive permeability to alkali cations. Horton et al. (1970) found that in cystic fibrosis patients Ca-ATPase activity of red cells is reduced. However, no functional impairment seems to ensue in this case. It is tempting to speculate that the normal disk shape of red cells is somehow caused by the contractile system mentioned, and that intracellular Ca2+ concentration is kept low by the pump in order to ensure partial rather than maximal activation of the contractile system. However, it is extremely difficult even to come to a coherent view that might be put to a reasonable test. The first difficulty is that the ATPase in the isolated protein has a K d i s s for Ca of lov3M , which is orders of magnitude away from the intracellular Ca concentration. Further, there is no morphological evidence for protein strands running across the intracellular space. Finally, Bull (1973) has shown that a red cell firmly fixed to a glass surface can be rolled by hydraulic force like a caterpillar track so that the convexities and concavities change place on the cell surface, which seems incompatible with any structural elements running across the cell space. However, it is conceivable that activation of a very small fraction of the contractile protein is sufficient to maintain the biconcave shape, and that the width of the protein strands is below the resolution of the electron microscope. If the connection between loose ends of protein chains extending into the lumen of the cell from opposite parts were due to ATP-activated bonding, the mechanism could catch whenever two opposite cell surfaces are brought sufficiently near each other by external forces, such as in Bull’s experiment. A mechanism of this type is compatible with the experiment of Palek et al. (1971b) showing that transient osmotic shrinking with NaCl solutions of ghosts that were probably leaky for alkali cations led to a permanent reduction of volume. Here again the osmotic shrinking might have brought loose ends into contact. But the possible role of a contractile protein in the preservation of the bioconcave disk shape of red cells is highly debatable and requires further experimentation. Bull (1973) has proposed

ACTIVE CALCIUM TRANSPORT AND Ca2+-ACTIVATED ATPase IN HUMAN RED CELLS

16 1

an ingenious model of the membrane which predicts the disk shape without resorting to any contractile mechanism. C. Epithelia

From the work of Loewenstein (1967; Olivera-Castro and Loewenstein, 1971), it is known that high permeability for alkali cations, but also for organic molecules at junctional membranes of epithelia, is abolished if the Ca2+ concentration is allowed to rise inside the cells. It seems, therefore, that maintenance of low intracellular Ca2+ concentration is necessary for the cellular communication in epithelia, and perhaps also a t the nexus of smooth muscle and at intercalated disks in cardiac muscle. D. Secretory Cells

Discharge or exocytosis of storage vesicles in presynaptic nerve endings (Harvey and MacIntosh, 1940; Katz and Miledi, 1967, 1969; Blaustein, 1971), mast cells (Foreman et at., 1973), certain endocrine and possibly exocrine glands is a consequence of Ca entry into the cytosol, whence it follows that low intracellular Ca2+ concentration ensures the stability of these vesicles a t rest and is a prerequisite for a regulatory function of Ca in the release process (for review see Rubin, 1970). REFERENCES Albers, R. W., Koval, G. S., and Siege], G. J. (1968). Studies on the interaction of ouabain and other cardioactive steroids with sodium-potassium-activated adenosinetriphosphatase. Mot. Pharmacol. 4, 324-336. Bader, H. (1971). Two (Ca++)-activated ATPases in human erythrocyte ghosts. Fed. Proc., Fed. Amer. Soc. Exp. Biol. 30, 545. Baker, P. F. (1972). Transport and metabolism of calcium ions in nerve. Progr. Biophys. M o ~Biol. . 24, 177-223. Baker, P. F., and Glitsch, H. G. (1973). Does metabolic energy participate directly in the Natdependent extrusion of Ca2+ ions from squid giant axons? J . Physiol. (London) 233,44P. Baker, P. F., Blaustein, M. P., Hodgkin, A. L., and Steinhardt, R. A. (1967). The effect of sodium concentration on calcium movements in giant axons of Loligo forbesi. J . Physiol. (London) 192,43P. Baker, P. F., Blaustein, M. P., Hodgkin, A. L., and Steinhardt, R. A. (1969) The influence of calcium on sodium efflux in squid axon. J . Physiol. (London) 200, 431458. Balzer, H., Makinose, M., and Hasselbach, W. (1968). The inhibition of the sarcoplasmic calcium pump by prenylamine, reserpine, chlorpromazine and imipramine. N u u n y n Schmiedebergs Arch. Pharmakol. Exp. Pathol. 260, 444-455.

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Blaustein, M. P. (1971). Preganglionic stimulation increases calcium uptake by sympathetic ganglia. Science 172, 391-393. Blum, R. M., and Hoffman, J. F. (1971). The membrane locus of Ca-stimulated K transport in energy depleted human red blood cells. J . Membrane Biol. 6, 31Fi-328. Bodemann, H., and Passow, H. (1972). Factors controlling the resealing of the membrane of human erythrocyte ghosts after hypotonic hemolysis. J . Membrane Biol. 8, 1-26. Bond, G. H. (1972). Ligand induced conformational changes in the (Mgz+ Caz+) dependent ATPase of red cell membranes. Biochim. Biophys. Acta 288,423-433. Bond, G. H., and Clough, D. L. (1973). A soluble protein activator of (MgZ+ Caz+)dependent ATPase in human red cells. Biochim. Biophys. Acta 323, 592-599. Bond, G. H., and Green, J. W. (1971). Effects of monovalent cations on the (Mg2+ C P + ) dependent ATPase of the red cell membrane. Biochim. Biophys. Acta 241, 393-398. Borle, A. B. (1969). Kinetic analysis of calcium movements in HeLa cell culture. J. Gen. Physiol. 53, 57-69. Bramley, T. A., and Coleman, R. (1972). Effects of inclusion of Caz+, Mgz+, EDTA or EGTA during the preparation of erythrocyte ghosts by hypotonic hemolysis. Biochim. Biophys. Acta 290, 219-228. Bull, B. (1973). Red cell biconcavity and deformability. I n “Red Cell Shape” (M. Bessis, R. I. Weed, and P. F. Leblond, eds.), pp. 115-124. Springer-Verlag, Berlin and New York. Carafoli, E. (1967). I n vivo effect of uncoupling agents on the incorporation of calcium and strontium into mitochondria and other subcellular fractions of rat liver. J . Gen. Physiol. 50, 1849-1853. Casteels, R., Goffin, J., Raeymaekers, L., and Wuytack, F. (1973). Calcium pumping in smooth muscle cells of the taenia coli. J. Physiol. (London) 231, 19P. Cha, Y. N., Shin, B. C., and Lee, K. S. (1971a). Further studies on Ca++ stimulated Mg-ATPase of red blood cell membrane fragments. Fed. Proc., Fed. Amer. SOC. Exp. Biol. 30, 199. Cha, Y. N., Shin, B. C., and Lee, K. S. (1971b). Active uptake of Ca2+and Ca*+-activated Mgz+ATPase in red cell membrane fragments. J . Gen. Physiol. 57,202-215. Chance, B. (1965). The energy linked reaction of calcium with mitochondria. J . Biol. Chem. 240, 2729-2738. Christinaz, P., and Schatzmann, H. J. (1972). High potassium and low potassium erythrocytes in cattle. J . Physiol. (London) 224, 391-406. Cittadini, A,, Scarpa, A., and Chance, B. (1973). Calcium transport in intact Ehrlich ascites tumor cells. Biochim. Biophys. Acta 291, 246-259. Comar, L. C., and Bronner, F., ed. (1969). “Mineral Metabolism, Vol. 111: Calcium Physiology.” Academic Press, New York. Cotton, F. A., and Wilkinson, G. W. (1966). “Advanced Inorganic Chemistry,” 2nd Ed. Wiley, New York. Davis, P. W., and Vincenzi, F. F. (1971). Ca-ATPase activation and Na-K-ATPase inhibition as a function of calcium concentration in human red cell membranes. Life Sci. 10, 401-406. De Pont, J. J. H. H. M., van Prooijen-van Eeden, A., and Bonting, S. L. (1973). Studies on (Na+-K+)-activated ATPase. XXXIV. Phosphatidylserine not essential for (Na+-K+)-ATPase activity. Biochim. Biophys. Actu 323, 487494. Duffy, M. J., and Schwarz, V. (1973). Calcium binding to the erythrocyte membrane. Biochim. Biophvs. Acta 330, 294-301.

+ +

+

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The Effect of Insulin on Glucose Transport in Muscle Cells TORBEN CLAUSEN Fysiologisk Institut Aarhus Universitet. Aarhus. Denmark

I . Introduction . . . . . . . . . . . . . . . . . I1. Preparations Used for the Study of Insulin Action on Sugar Transport A Skeletal Muscle . . . . . . . . . . . . . . B . Heart . . . . . . . . . . . . . . . . . C. Smooth Muscle . . . . . . . . . . . . . . I11. Cellular Structures Involved in Sugar Transport . . . . . . A The Localization of the Glucose Transport System . . . . B. The Role of Membrane Lipids . . . . . . . . . . C. The Role of Membrane Proteins . . . . . . . . . IV. The Function of the Glucose Transport System . . . . . . A. The Basal State . . . . . . . . . . . . . . B . The Effect of Insulin . . . . . . . . . . . . . C . The Effects of Other Activators . . . . . . . . . . D The Effect of Inhibitors . . . . . . . . . . . . V Cellular Signals Controlling Glucose Transport . . . . . . . A Enzymic Processes and Metabolites . . . . . . . . . B The Binding, Transport, and Distribution of Electrolytes . . VI Mechanisms for the Mode of Action of Insulin . . . . . . References . . . . . . . . . . . . . . . . .

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1 INTRODUCTION

The fact that the present review concerning the effect of one hormone on one parameter in one cell type comprises 310 references without being complete is paradigmatic for the degree of specialization reached in the biological sciences. The action of insulin on glucose transport is the classical example of metabolism being controlled by the permeability of membranes. and the many investigations have been rewarding for promoting understanding of basic patterns in structures. functions. and signals related to 169

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transport and hormone action. This may justify that the following text concentrates on such common denominators and leaves out a number of specific details. Several recent reviews may be helpful for those who want the complete picture of this corner of nature (Krahl, 1961; Park et al., 1968; Morgan and Neely, 1972; Morgan and Whitfield, 1974; Clausen, 1974). II. PREPARATIONS USED FOR THE STUDY OF INSULIN ACTION ON SUGAR TRANSPORT A. Skeletal Muscle

The mere size of this major target for the action of insulin would indicate that it plays an obvious role in blood glucose homeostasis, and numerous preparations have been proposed for the study of how insulin controls glucose transport in skeletal muscle cells. An early approach to the problem was made with the demonstration of insulin effects in eviscerated cats (Best et al., 1926) and followed up by several studies of the distribution of nonmetabolized sugars in eviscerated dogs (Levine et al., 1949), rabbits (Wick and Drury, 1953), and nephrectomized rats (Helmreich and Cori, 1957). More recently, the hindquarter (Mahler et al., 1968; Ruderman et al., 1971) and the hemicorpus of the rat (Jefferson et al., 1972) have been shown to provide sensitive tools for the evaluation of insulin effects in major intact and composite structures of skeletal muscle. The effect of insulin on glucose consumption in human peripheral skeletal muscle has also been assessed by measurements of the arteriovenous concentration difference in the forearm (Andres et al., 1962). This method has yielded clear-cut evidence that glucose uptake is a function of the insulin concentration in the physiological range (Christensen and grskov, 1968), but since it is complicated, it has not been used in many laboratories. Although perfused tissues have several advantages with respect to integrity and insulin sensitivity, they are often heterogeneous and technically difficult to work with. A series of isolated muscle preparations have been developed in order to obtain more versatile tools for the study of details in the mechanisms of glucose transport and insulin action. Since the work of Gemmill (1940), the isolated rat diaphragm muscle [which was originally proposed by Meyerhof and Himwich (1924)l has become the classical choice for such investigations, being easy to prepare and sufficiently thin to allow rapid equilibration of oxygen and substrates between the muscle cells and the incubation medium. With the purpose of

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preserving the integrity of the muscle fibers, various modifications have been devised in which the muscle is incubated with its attachments to the rib cage (Kipnis and Cori, 1957; Kono and Colowick, 1961; Creese and Northover, 1961 ; Creese, 1968). Whereas such “intact” diaphragm muscles have decisive advantages in studies of sugar accumulation, the adhering central tendon, cartilage, and cut intercostal fibers represent a major difficulty when such parameters as glucose uptake, the production of metabolites, or the efflux of various solutes are to be evaluated. Because of these limitations together with the fact that the muscle is rhythmically contracting up to the moment of its isolation, it is often desirable to consider alternative preparations, which are perhaps more representative 01 peripheral skeletal muscle. The levator ani muscle of small rats is easier to prepare and has been well characterized (Arvill, 1967), but, owing to the presence of adhering tissues, it suffers from part of the disadvantages of the intact diaphragm. The sartorius muscle of frogs has been shown to be suitable for sugar transport studies (Narahara et al., 1960), but seasonal variations and some discrepancies from mammalian muscle present yet another set of limitations to the conclusions that may be drawn from experiments with this preparation. When using baby rats, the extensor digitorum longus (Pain and ManChester, 1970; Rogus and Zierler, 1973) and the soleus muscle (Chaudry and Gould, 1969; Kohn and Clausen, 1971) can easily be prepared with intact fibers and are quite convenient for most in vitro studies of glucose transport and metabolism as well as the measurement of ionic fluxes (Clausen et al., 1973). It should be noted, however, that diffusion through the interstitial space represents a rate-limiting factor for the exchange of solutes in these preparations, and that spontaneous contractures may occur if the muscles are not thoroughly oxygenated. Attempts to prepare intact isolated muscle cells by mechanical separation of fibers from large mammalian muscles have to some extent been successful, but not without appreciable loss of insulin responsiveness (Beatty et al., 1960). Similar problems may explain why isolated human skeletal muscle fibers do not respond at all to insulin in witro (Holm and Schersten, 1972). Quite a long time ago, it was demonstrated that quarter diaphragms have a smaller response to insulin than do hemidiaphragms (Groen et al., 1952; Liebecq, 1956). B. Heart

Some of the earliest studies of insulin actions i n witro were done with the isolated perfused heart of rabbits (Hepburn and Latchford, 1922).

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Since the work of Bleehen and Fisher (1954) and Morgan et al. (1959), the isolated rat heart has become one of the standard preparations for the investigation of glucose transport and insulin action in muscle cells. It has the advantage of being able to maintain a considerable degree of functional integrity and insulin sensitivity, of being easy to prepare for larger series of experiments, and of allowing an evaluation of the role of contractile activity for basal and insulin-stimulated sugar transport. Information about glucose metabolism and insulin action in the human heart has been obtained in measurements of arteriovenous concentration differences (Rudolph et al., 1969). Whole nonperfused hearts of fetal rats (Clark, 1971) and chickens (Guidotti et al., 1961) have turned out to be convenient for the study of the appearance of insulin sensitivity during fetal life. Digestion of fetal hearts with trypsin and hyaluronidase may yield viable muscle cells, which can be cultured, but these cells show a variable and modest response to insulin (Clark, 1971; Dunand et al., 1972). C. Smooth Muscle

The small and variable effect of insulin on glucose uptake in smooth muscle cells may have been discouraging for the study of this otherwise important tissue element. In the isolated aorta of rabbits (Mulcahy and Winegrad, 1962) or rats (Wertheimer and Ben-Tor, 1962) insulin was found to produce no or only a very modest stimulation of glucose uptake. A somewhat better response was found in taenia coli, which contains a larger proportion of smooth muscle cells (Grossmann and Manchester, 1966); and also in bladder wall (Bower and Grodsky, 1963) and the detrusor muscle of rats (Bihler et al., 1971), insulin could be shown to stimulate glucose uptake and the transport of nonmetabolized sugars. However, only recently a more systematic study has revealed that in the aorta of rabbits and rats, in rabbit colon, and in bovine mesenteric arteries insulin has a clear-cut, although delayed, stimulating effect on the transport of both glucose and nonmetabolized sugars (Arnquist, 1973).

111. CELLULAR STRUCTURES INVOLVED IN SUGAR TRANSPORT A. The Localization of the Glucose Transport System

It is generally assumed that the plasma membrane constitutes the major and rate-limiting barrier for the access of glucose to the cytoplasm. Several

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studies have demonstrated that the system of transverse tubules of the sarcoplasmic reticulum is an extension of the outer sarcolemma and thus adds a potential surface available for transport, which is 5-7 times larger than the sarcolemma (Peachey, 1965). Although this electron microscopic evidence has been available for more than a decade, almost no transport studies have taken into consideration the complexity and heterogeneity of the membranes which may be involved in the uptake of glucose. Electron microscopy of tissues which had been exposed to hyperosmolarity or hypoosmolarity has shown that the volume of the sarcoplasmic reticulum is increased or, respectively, decreased under these conditions (Huxley et al., 1963; Girardier et al., 1963; Freygang et al., 1964; Sperelakis and Schneider, 1968; Rapoport et al., 1969; Birks and Davey, 1969). This indicates that the membranes lining the sarcoplasmic reticulum are participating in the exchange of water, and that the lumen of both the transverse and the longitudinal elements is available to sucrose. This was already suggested by Harris (1963), and the observation that, in rat diaphragm muscle, inulin occupies a smaller space than sucrose or mannitol (Kipnis and Parrish, 1965; Hider et aE., 1971) is compatible with the existence of a compartment which is available to compounds of low molecular weight, but from which inulin and larger structures are excluded. This has been suggested to be identical with the lumina of the longitudinal sarcoplasmic tubules, but direct evidence is not available (Rogus and Zierler, 1973). In frog sartorius muscle, the difference between the spaces available to fructose and inulin was found to vary with osmolarity in accordance with the above mentioned changes seen in electron micrographs (Vinogradova, 1968). Several reports indicate that at least the wall of the transverse tubules is permeable to K+ ions (Adrian and Freygang, 1962; Harris, 1963; Hodgkin and Nakajima, 1972; Almers, 1972), and it was recently proposed that they are also of significance for the exchange of amino acids (Hider et al., 1971) and Na+ ions (Rogus and Zierler, 1973). Hyperosmolarity stimulates the transport of glucose and 3-O-methylglucose in skeletal muscle (Kuzuya et al., 1965; Clausen, 1968a; Clausen et al., 1970; Nikolsky et al., 1971), and hypotonicity suppresses or abolishes the stimulating effect of insulin, work, 2, 4-dinitrophenol, and trypsin on sugar transport (Kohn and Clausen, 1972). On this basis, it was suggested that the sarcoplasmic tubules participate in the exchange of sugars, and that the rate of transport is in part a function of the accessibility (diameter) of the transverse tubules (Clausen and Kohn, 1972). (For alternative interpretations, see Section IV, c, 5.) Too little is known about the relative participation of various membrane elements in the transport of glucose and insulin action, but the information

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available about structural heterogeneity suggests that kinetic analysis can be meaningful only if most of these processes take place a t the sarcolemma. Nothing is known about the effect of insulin on the configuration of the sarcoplasmic reticulum, but cells in which this system is poorly developed (fetal and smooth muscle cells) are relatively insensitive to insulin (Clark, 1971 ; Arnquist, 1973). The observations that significant proportions of the enzymes controlling glycolysis (Fahimi and Karnovsky, 1966; Karpatkin and Braun, 1971) and glycogen metabolism (Andersson-Cedergren and Muscatello, 1963; Meyer et al., 1970) are associated with the membranes of the sarcoplasmic reticulum [around which the density of glycogen granules is particularly high (Wanson and Drochmans, 1968)] indicate that glucose entering via the sarcoplasmic tubules a t least has ready access to appropriate metabolic machinery. Compartmentalization of glucose metabolism in muscle has been demonstrated several times and was recently reinvestigated and discussed by Kalant and Beitner (1971).

B. The Role of Membrane lipids Owing to the hydrophilic nature of the glucose molecule, the lipid matrix of the plasma membrane constitutes an important barrier, and in membranes prepared from extracts of the lipids in red cell ghosts, the permeability to glucose was found to be four orders of magnitude lower than in intact cells (Jung, 1971). The same study showed that the permeability of these artificial membranes to various sugars in no way paralleled that found in red cells. Thus, 3-O-methylglucose, which is more lipophilic than glucose, had a 13-fold higher permeability. LeFevre et al. (1968) suggested that membrane phospholipids are not directly involved in the binding and transport of glucose, but could be of importance for the mobility of the glucose carrier. With phospholipase C it is possible to split off phosphorylcholine, which is the hydrophilic component of the phospholipids available from the outside of the plasma membrane. Rodbell (1966) demonstrated that this enzyme stimulates glucose metabolism in fat cells, and in frog sartorius muscle it produced an increase in the Vmax of 3-O-methylglucose transport, thus mimicking the action of insulin, which was proposed to induce an increase in overall mobility of the glucose carrier (Weis and Narahara, 1969). The fluid mosaic model proposed by Singer (1971) would predict that the mobility of membrane proteins (and therefore presumably also the glucose carrier) is determined by the fluidity of the lipid matrix, which is again influenced by temperature, ionic milieu, chemical composition and a number of drugs. In cultured muscle fibers, membrane proteins labeled

THE EFFECT OF INSULIN ON GLUCOSE TRANSPORT IN MUSCLE CELLS

175

with a fluorescent probe showed a temperature-dependent lateral movement in the plane of the plasma membrane (Edidin and Fambrough, 1973). Further, although indirect, evidence for the involvement of membrane lipids in the transport of glucose comes from studies with membrane stabilizrrs. This general term was suggested for a wide variety of compounds that reduce the excitability, cation permeability, and osmotic fragility of the plasma membrane (Shanes, 1958). Some of the most potent membrane stabilizers, the local anesthetics, were shown to increase the lateral pressure in monomolecular films of lipids (Skou, 1961). This effect was correlated to their anesthetic potency, and it was proposed that these compounds occupy the interspaces in the ordered array of lipid molecules leading to lateral expansion and increased overall rigidity of the membrane. Several different membrane stabilizers (local anesthetics, barbiturates, psychotropic and anticonvulsant drugs) have been shown to inhibit the transport of glucose, galactose, 3-O-methylglucose, and sorbose in rat diaphragm muscle (Rafaelsen, 1961; Bihler and Sawh, 1971c), soleus muscle, adipocytes, and erythrocytes of the rat (Clausen et al., 1973) as well as in human erythrocytes (Baker and Rogers, 1973). It should be noted that the inhibitory effects of these compounds are seen only within a certain concentration range, beyond which a nonspecific leakage of the plasma membrane is produced with ensuing loss of potassium and an apparent stimulation of sugar transport (Bihler and Sawh, 1971c; Clausen et al., 1973). Conversely, veratrine or exposure to a calcium-free environment, which has been shown to labilize the plasma membrane (Shanes, 1958; Sperelakis and Pappano, 1969), were found to accentuate the effect of insulin on the transport of 3-O-methylglucose in rat soleus muscle (Table I). The observation that ions with a high charge density (La3+,Ni2+,Zn2+, Mn2+, and Co2+) inhibit the insulin-induced rise in the accumulation of 3-O-methylglucose in rat diaphragm muscle suggests that electrostatic forces between the negative charges of the surface of the plasma membrane can be of significance for the activation and the mobility of the glucose transport system (Bihler, 1972). The same study showed that calcium could overcome this inhibitory effect and that exposure to a calcium-free environment led to a (progressive) decrease in insulin responsiveness. In rat soleus muscle, lanthanum was found to suppress the stimulating effect of insulin on the efflux of 3-O-methylglucose (Table I). Further studies of the time course of these phenomena are required before the data can be encompassed into a meaningful whole, and direct effects of these ions on membrane proteins will have to be identified. However, the collective evidence suggests that the configuration and composition of polar heads and hydrophobic elements of the plasma

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TABLE I

EFFECT OF TETRACAINE, THIOPENTAL, VERATRINE, INSULIN

cAzi,

AND LAS+ ON

RESPONSIVENESS OF RAT SOLEUS MUSCLE^

Additions

Percent of rnethylglu~ose-~~C released per minute

Pb

Control Tetracaine (2 mM) Insulin (1 mU/ml) tetracaine (0.5 mM) tetracaine (2.0 mM) thiopental (1.0 mM)

0.21 0.19 1.62 0.73 0.20 0.82

f 0.03 f 0.04 f 0.16 f 0.08 f 0.02 f 0.03

(4) (4) (4) (3) (3) (4)

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