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Current Topics n i Membranes and Transport Volume 3

Advisory Board

Robert W . Berliner Britton Chance I . S. Edelman Aharon Katchalsky (deceased) Adam Kepes Richard D. Keynes Philip Siekevitz Torsten Teorell Daniel C. Tosteson Hans H . Ussing

Contributorr

W . J . Adelman, J r . Julius C . Allen Eduardo De Robertis William R. Harvey Richard M . Hays J . D. Jamieson George E. Lindenmayer Anthony Martonosi Y , Palti G'eorgina Rodriguez De Lores Arnaiz Arnold Schwartz Karl Zerahn

Current Topics in Membranes and Transport

VOLUME 3

Edited by Felix Bronner Department of Oral Biology School of Dental Medicine University of Connecticut Storrs, Connecticut and

Arnost Kleinzeller Graduate Division of Medicine University of Pennsylvania Philadelphia, Pennsylvania

1972

Academic Press

New York and London

INC.

COPYRIGHT 8 1972, BY ACADEMIC PRESS, ALL RIGHTS RESERVED. N O PART O F 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 T H E PUBLISHER.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York. New York 10003

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

LIBRARY OF

CONGRESS CATALOG CARD

NUMBER: 70- 117091

PRINTED IN TH E UNITED STATES OF AMERICA

List of Contributors, ix Preface, xi Contents of Pmvious Volumes, xii The Na+, K+-ATPase Membrane Transport System: Importance in Cellular Function ARNOLD SCHWARTZ, GEORGE E. LINDENMAYER, AND JULIUS C. ALLEN I. Introduction and History, 2 11. Cell Membrane, 5 111. A Review of Studies on the Mechanism of the Sodium Pump, 9 IV. Some Physiological Aspects of Na+, K+-ATPase, 43 V. Other Effects of Cardiac Glycosides on Membrane-Linked Functions, 68 VI. Role of Membrane Transport in Biogenic Amine Transport, 69 VII. Effects of Phlorizin on Membranes, 70 References, 73 Biochemical and Clinical Aspects of Sarcoplasmic Reticulum Function ANTHONY MARTONOSI I. Introduction, 84 11. The Mechanism of Ca Transport, 86 111. The Regulation of Sarcoplasmic Reticulum Function, 112 IV. The Regulation of Sarcoplasmic Ca2f Concentration in Cardiac Muscle, 122 V. Sarcoplasmic Reticulum in Red Skeletal Muscles, 136 VI. The Structure and Function of the Transverse Tubular System and the Triad, 141 VII. The Content of Sarcoplasmic Reticulum Tubules, 151 VIII. The Sarcoplasmic Reticulum in Diseases of Skeletal Muscle, 159 References, 175 Note Added in Proof, 195

The Role of Periaxonal and Perineuronal Spaces in Modifying Ionic Flow Across Neural Membranes W. J. ADELMAN, JR., AND Y. PALTI I. Introduction, 199 11. External Potassium Ion Accumulation, 201 111. Significance of Potassium Ion Accumulation for Axon and Neuron Behavior, 220 IV. Significance of Potassium Ion Accumulation, in Brain Behavior, 223 Appendix A: Model for Ion Accumulation in Periaxonal Space, 226 Appendix B: Calculation of [KB]Changes upon Voltage Clamping the Squid Giant Axon, 229 Appendix C: Reconstruction of a Membrane Action Potential, 231 References, 233 V

vi

CONTENTS

Properties of the Isolated Nerve Endings GEORGINA RODRIGUEZ de LORES ARNAIZ AND EDUARDO De ROBERTIS

I. Introduction, 238 11. Isolation of Nerve Endings and Their Limiting Membrane, 239 111. Chemical Composition, 244 IV. Immunological Properties of Isolated Nerve Endings (INE), 250 V. Osmotic Properties of the INE, 251 VI. Synthesis of High-Energy Compounds, 252 VII. Metabolism of Amino Acids, 255 VIII. Metabolism of Phospholipids, 256 IX. Amino Acid Uptake and Protein Synthesis, 258 X. Uptake Mechanisms Related t o the Transmitter Function, 259 XI. Ion Permeability, 262 XII. Concluding Remarks, 266 References, 268 Transport and Discharge of Exportable Proteins in Pancreatic Exocrine Cells: In Vitro Studies

J. D. JAMIESON I. Introduction, 273 11. The Secretory Process in Resting Pancreatic Exocrine Cells, 274 111. Physiological Modulation of the Secretory Process in Pancreatic Exocrine Cells, 315 IV. Interrelationships of Intracellular Membranes during the Secretory Process, 333 References, 336 The Movement of Water Across Vasopressin-Sensitive Epithelia RICHARD M. HAYS

I. Introduction, 339 11. The Pore Enlargement Hypothesis, 340 111. The True Diffusion Rate of Water across the Luminal Membrane, 346 IV. The Activation Energy for Water Diffusion, 357 V. The Solvent Drag Effect, 359 VI. Conclusions, 364 References, 365 Active Transport of Potassium and Other Alkali Metals by the Isolated Midgut of the Silkworm WILLIAM R. HARVEY AND KARL ZERAHN I. Introduction, 368 11. Methods, 375 111. Active K-Transport, 378

vii

CONTENTS

IV. V. VI. VII. VIII.

Influence of [K] on PD and ZaC,379 Coupling of K-Transport to Metabolism, 384 Transport of Other Alkali Metal Ions and Other Substances, 386 Competition between Alkali Metal Ions, 389 Route of Ion Transport, 393 References, 409

Author Index, 41 1 Subject Index, 432

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List of Contributors Adelman, Jr., Laboratory of Biophysics, National Institute of Neurological Diseases and Stroke, National Institutes of Health, United States Public Health Service, Bethesda, Maryland Julius C. Allen, Division of Myocardial Biology, Baylor College of Medicine and the Fondren Brown Cardiovascular Research and Training Center, Methodist Hospital, Houston, Texas Eduardo De Robertis, Instituto de Anatomia General y Embriologfa, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina William R. Harvey, Department of Biology, Temple University, Philadelphia, Pennsylvania Richard M. Hays, Department of Medicine, Albert Einstein College of Medicine, New York, New York J. D. Jamieson, The Rockefeller University, New York, New York George E. Lindenmayer,* Division of Myocardial Biology, Baylor College of Medicine and the Fondren Brown Cardiovascular Research and Training Center, Methodist Hospital, Houston, Texas Anthony Martonosi, Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri Y. Palti, Department of Physiology and Biophysics, The Aba Khoushy School of Medicine, Israel Institute of Technology, Haifa, Israel Georgina Rodriguez de Lores Arnaiz, Instituto de Anatomia General y Embriologia, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina Arnold Schwartz, Division of Myocardial Biology, Baylor College of Medicine and the Fondren Brown Cardiovascular Research and Training Center, Methodist Hospital, Houston, Texas Karl Zerahn, Institute of Biological Chemistry A, University of Copenhagen, Denmark

W. J.

* Present address: Cardiology Branch, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland. ix

This Page Intentionally Left Blank

I n presenting the third volume of Current Topics in Membranes and Transport the cditors, as in previous volumes, have endeavored to encourage contributions that deal with some fundamental aspects of biological transport. Thus this volume includes a detailed analysis of the characteristics and functions of Na+,K+-ATPase, one of the major enzyme systems thought to be involved in sodium transport, a description of the sarcoplasmic reticulum and the central regulatory role the calcium ion plays in muscular contraction and relaxation, and a review of the role played by ionic concentration changes in spaces and layers adjacent to excitable membranes. This is followed by a broad discussion of the properties of isolated nerve endings and the sequence of reactions involved in precursor entry and transmittef synthesis and release, leading to nerve impulse. A review of the factors that determine the orderly flow of cellular products from their site of synthesis to ultimate discharge from storage granules is followed by a critical analysis of the movement of water across cell membranes, with the final chapter a discussion of the movement of potassium across a layer of epithelium, as distinguished from movement across individual cells. Here, as in earlier volumes, we believe the authors have maintained the high standards of critical evaluation and analysis for which we have striven from the inception of the series. If precise coverage or choice of an individual topic does not always reflect the editors’ views, this is due to the burdens and occasional frustrations experienced by editors and authors alike in transforming a topic into the final review. Yet we feel sure the wide range of topics presented here will be of interest to all biologists. Since the second volume of this series went to press, the editors, the advisory board, and the publishers of Current Topics in Membranes and Transport were shocked by the untimely death of Aharon Katzir-Katchalsky, whose senseless murder at the Lod Airport has deprived us of his wise counsel and advice. His contribution to the understanding of membrane and transport phenomena was literally unique. We shall continue our work inspired by his analytical viewpoint, as well as by his indomitable enthusiasm, and we dedicate this volume to his memory. F E L I X BRONNER ARNOST KLEINZELLER xi

Contents of Previous Volumes Volume 1

Some Considerations about the Structure of Cellular Membranes MAYNARD M. DEWEYAND LLOYD BARR 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. LIEBAND W. D. STEIN The Transport of Water in Erythrocytes ROBERT E. FORSTER Ion-Translocation in Energy-Conserving Membrane Systems B. CHANCE AND M. MONTAL Structure and Biosynthesis of the Membrane Adenosine Triphosphatase of Mitochondria ALEXANDERTZAGOLOFF Mitochondria1 Compartments: A Comparison of Two Models HENRYTEDESCHI Author Index-Subject Index

xii

The Na', K+-ATPase Membrane Transport System: Importance in Cellular Function* ARNOLD SCHWARTZ,I GEORGE E . LINDENMAYER,$ and JULIUS C . ALLEN Division of Myocardial Biology, Baylor College of Medicine and the Fondren Brown Cardiovascular Research and Training Center, Methodist Hospital, Houston, Texas

I. Introduction and History . . . . . . . . . . . . . . 11. Cell Membrane . . . . , . . . . . . . . . . . . 111. A Review of Studies on the Mechanism of the Sodium Pump . . . . A. Monovalent Cation Activation and Transport . . . . . . . B. Mechanism of Energy Transduction . . . . . . . . . C. K+-Phosphatase , . . , . . . . . . . . . . . D. Cardiac Glycoside Inhibition . . . . . . . . . . . . IV. Some Physiological Aspects of Na+,K+-ATPase . . . . . . . . A. The Possible Role of the Na+,K+-ATPase Enzyme System in Amino Acid Transport . . . . . . . . . . . . . . . . B. The Possible Relationship Between Na+,K+-ATPase and Sugar . . . . . , . . . . . . . . . . . Transport C. Some Complications of Sugar Transport in Relation to the Na+,K+ATPase . . . . . . . . . . . . . . . . . . V. Other Effects of Cardiac Glyeosides on Membrane-Linked Functions . . Antilipolytic Effects of Cardiac Glycosides . . . . . . . . . VI. Role of Membrane Transport in Biogenic Amine Transport . . . . VII. Effects of Phloriein on Membranes . . . . . . . . . . . References . . . . . . . , . . . . . . . . . . .

.

2 5 9 9 20 33 35 43 43 59 62 68 68 69 70 73

* The original studies cited were supported by U.S. Public Health Service grants, HL 07906, HL 05435-p8, NIH-71-2493, HL 13870, HL 05925 and by the American Heart Association, Houston Chapter, Texas Affiliate. t Recipient of a Career Research and Development Award (Ka-HL 11,875). $ Present address : Cardiology Branch, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland. 1

2

ARNOLD SCHWARTZ, GEORGE E. LINDENMAYER, AND JULIUS C. ALLEN

1. INTRODUCTION AND HISTORY

It is almost axiomatic that the more primitive, in terms of evolution, a structure or function is, the more difficult it is to understand. Life processes tend to remain tantalizingly elusive. The truly creative scientist pulls and tugs the layers of secrets away so that he can relieve the frustrations of ignorance. As Chargaff discussed quite recently (Chargaff, 1971), man cannot live without mysteries. Even though a definitive discipline “molecular biology” is taught in every college and medical school, basically we are “still very far from an actual grammar of the living cell. . . the processes of cell differentiation, morphogenesis and cellular organization still are entirely obscure” (Chargaff, 1971). We have practically no concept of the inside of a living cell; we have very little knowledge of the outside of the cell. Yet, who would deny that during the past one hundred years, advances in science have been of such magnitude that several diseases have been conquered and life has been prolonged and made more productive. When one considers the first living organism, it seems almost naive to suggest that anything but some type of membrane must have been the first structure. Oparin, Miller, and many others have suggested that, even before the advent of life, organic substances must have been formed on the earth by abiogenic means, that is, the reactions of the synthesis of specific molecular compounds must have served as a type of nutrient ,broth, and the final emergence of living matter probably initially involved a simple separation from the aqueous milieu, by a type of membranous barrier. Development of this barrier might not have been particularly difficult since it has been clearly shown that even under natural conditions small enclosed bladders of a lipoprotein composition can develop from infoldings induced by wind in surface films on bodies of water (Bresnick and Schwartz, 1968). In fact, Bungenberg de Jong (1949) has demonstrated that, in the presence of lipids, the surface of coacervate droplets can assume a protein-lipid membrane “sandwich” structure. It is well recognized that almost all living cells are rich in potassium (in fact, potassium represents the primary cation) and poor in sodium, while the reverse situation exists with respect to extracellular fluids. It is equally well known that sea water, which undoubtedly formed the first broth, contains an abundance of sodium. Why is it that the evolved cells should contain more potassium and less sodium than the extracellular fluid? Reasoning backward, it is well established that, in all tissue in general, the excitatory event depends upon the differences in concentrations and activities of sodium as well as potassium on both sides of the cell membrane. Figure 1 depicts an idealized action potential in nerve. The resting membrane potential depends, in part, upon the diffusion gradient for potassium.

THE No’, Kt-ATPase MEMBRANE TRANSPORT SYSTEM

-> A

3

O--

WE E

+ I

Resting membrane potential ( K f )

FIG 1. Idealized action potential in nerve.

The rising phase of the action potential represents a “sodium current.” The permeability to sodium suddenly increases. The cessation of sodium permeability is reflected by the “overshoot” and the beginning of repolarization; the latter is presumably dependent upon the diffusion of minute amounts of potassium ‘Ldownhill,” and the recovery phase involves a movement of the two monovalent cations against the concentration and electrical gradients, with presumably the expenditure of energy derived from cellular metabolism. This is active transport or “pumping.” Excitability is a primitive feature of all living cells, including liver and red blood cells/ and it may be that the difference in cation composition of intracellular and extracellular fluid evolved as part of some kind of excitability mechanism. On the other hand, the difference in salt concentration may have come first and been made use of in the evolution of an excitability mechanism later. In living cells, there are several enzymes which are activated by potassium ions and inhibited by sodium ions; here again, it seems likely that the enzymes have evolved to suit the internal medium rather than the other way around. “One rather plausible hypothesis to account for the difference in composition of the two ions, is that the expulsion of sodium was developed as a way of overcoming the osmotic entry of water which must have presented a problem as soon as cells began to accumulate large non-penetrating molecules inside the cells.. . .” (Chargaff, 1971). One way of accomplishing this is to simply “pump anything out of the cell so that its excess concentration outside balances the osmotic pressure of the cell proteins and phosphates. And sodium would be, after all, the most abundant solute in the cell” (Glynn, 1966). SO we really

4


do not know why most cells did develop a mechanism (quite early in terms of evolution) for maintaining high concentrations of potassium inside the cell and high concentrations of sodium outside the cell. We do know, however, that this feature is characteristic of most animal cells and has a variety of functions consistent with the maintenance of life. Certainly the ionic gradients provide energy for the propagation of impulses in nerve and muscle. Salt transport in the proximal kidney tubule and the gallbladder is responsible for the concentration of urine and of bile, respectively; in the loop of Henle and the distal convoluted tubule the transport of sodium is apparently concerned with the formation of a hyper- or hypotonic urine, also in the maintenance of the balance of total body sodium and potassium and possibly also in controlling hydrogen ion movements (Kunau, 1970). Sodium and potassium transport play very important roles in fish. For example, in Electrophorus electricus (electric organ-containing fish) thousands of excitable membranes are arranged in series so that the electrical event actually adds up to several hundred volts. The avian salt gland is responsible for the removal of huge amounts of sodium chloride. It is well known that several species of fish can adapt to salt water living from a fresh water environment. The adaptation phenomenon is always accompanied by specific membrane changes. For example, the Coho salmon can be removed from fresh water and adapted to salt water living. A membranous fraction can be isolated from the gills of such animals. This fraction exhibits a specific adenosine triphosphatase activity that is stimulated by sodium and potassium in the presence of magnesium and inhibited by the cardiac glycoside ouabain. As salt water adaptation proceeds, a specific and significant increase occurs in sodium-stimulated activity of this enzyme. It has been suggested that a magnesium-dependent, ouabain-insensitive enzyme site associated with this system is converted to a form requiring sodium and potassium during the adaptation phenomenon of both the Coho and Chinook salmon (Zaugg and McLain, 1971). It is of interest that the specific activity of the sodium-potassium membrane transporting ATPase is high in the gills of salt water teleosts and low in gills of elasmobranchs and fresh water teleosts. When fresh water eels (Anguilla rostrata) are adapted to sea water for 2-3 weeks, a specific increase of Na+,K+-ATPase occurs (Jampol and Epstein, 1970). It is of importance that, when adaptation phenomena occur, specific alterations in the cell membrane accompany the changes in enzyme activity. For example, in response to osmotic stress the secretory epithelium of the avian salt gland develops surface specialization; the lateral and basal surfaces of the cells become deeply folded, forming complex intra- and extracellular compartments. This leads to a tremendous increase in absorptive surface area which, interestingly enough, is paralleled by an increase in the membrane transport ATPase activity

THE Na+, K+-ATPase MEMBRANE TRANSPORT SYSTEM

5

(Ernst and Ellis, 1969). During the proccss of metamorphosis, either natural or thyroid-induced, a dramatic increase in membrane transport also can occur (Taylor et al., 1967). In fact, during growth, differentiation, and development in general, all living organisms seem characteristically to alter their membrane propcrtirs accompanying an increase in sodium and potassium transport. It is of further interest that membrane transport in general appears to be specifically involved in the movements of a large variety of compounds required for t h r maintenance of life, such as glucose, amino acids, iodide, and possibly calcium. So, whilc we cannot be certain of the evolutionary reasons for the aphorism, “high internal potassium, low internal sodium,” wc do know that this characteristic is a function of the cell membrane and indeed must have been one of the first functional developments. It is pertinent, therefore, prior to a specific discussion of sodium and potassium transport, to take a “modern” look at the cell membrane. We would like to emphasize at the outset that this is not to be a comprehensive review of the Na+, Kf-ATPase field. Since the first publication on this complicated enzyme system about 10 years ago, there have been approximately 1500 published scientific papers. We intend, instead, to emphasize the characteristics of the enzyme system and, in particular, conjecture about physiological function. 11. CELL MEMBRANE

It is of interest that the importance of the membrane in distinguishing between sodium, calcium, and potassium, thereby maintaining cellular viability, was recognized as early as 1883 by Sidney Ringer in his classic studies on frog heart. Ringer found, for example, that ventricular contraction could be maintained for several hours when aupplied with a neutral circulating fluid composed of sodium chloride to which chloride and potassium chloride had been added. Dr. Ringer stated that “in the blood therefore, sodium. . . m u s t exert a very small influence, if any direct influence on the cardiac contraction, and this is regulated by the antagonizing action of calcium and potassium salts.” As so often happens in science, Ringer made his initial discoveries by accident. He was attempting to substitute saline solutions for blood in maintaining cardiac contractility but, instead of using distilled water, employed “pipe” water supplied by a local distributor. He found that the pipe water was much more effective than pure saline solution and proceeded t o analyze the impure water, discovering the presence of minute traces of various inorganic substancesamong them, calcium and potassium.

6


Basic understanding of membrane structure began with the observation by Overton that certain anesthetic substances that were lipoidal in nature probably acted by dissolving in or interacting with the membrane. This observation defined the lipid naturc of the membrane (Overton, 1895). Gorter and Grendrl (1925) extracted lipids from red blood cell membranes, measured the surface area of monolayers of the lipid material, and then calculated the surface area of the red blood cell membrane. They reported that the lipid of the erythrocytes provided just enough surface area to cover the cell twice and hence suggested that the membrane existed as a lipid bilayer. Korn (1968a) reconsidered the data of Gortcr and Grendel and found that the lipid content of the red blood cell ghosts was underestimated and was, in fact, sufficient to cover the cell surface only 1.3 times. Furthermore, Korn stated that X-ray diffraction studies have demonstrated that phospholipid-water systems can assume stable structures other than bimolecular leaflets (Korn, 1968b). Therefore, Korn maintains that alternative structures for phospholipid-cholesterol complexes should be considered since natural membranes seem to exhibit a low surface tension compared to the rather high surface tension observed with neutral lipids. Because of the presence of large amounts of protein in the area of cell membranes, Gorter and Grendel and others assumed that the lipid bilayer must be coated with protein. However, as Korn pointed out, some synthetic bimolecular leaflets exhibit a surface tension as low as that of the natural cell surface (Korn, 1968b). Recently, however, the work of Gorter and Grendel has been reexamined, and it was found that the original investigators extracted only 70% to 80% of the total lipids, and ironically enough they also underestimated the cell surface by a comparable amount (Thompson, 1964; Westerman et al., 1961). Therefore, as Hendler pointed out (1971), the ratio of lipid to surface membrane area is still 2: 1. Bar and his co-workers (1966) used modern methods for complete lipid extraction and more accurate values for the area of the cell surface. They showed that pressure begins to be exerted by the lipid film a t a film to cell area ratio of 2-2.2: 1 and that pressure mounts with further compression to the ratio 1.2-1.4:l. Further compression caused a collapse of the film so that it was impossible to achieve a monolayer coating with a ratio of 1.O:l. This experiment shows that, depending on the state of compression of lipids to the cell membrane, they can cover the cell surface 1.3-2.2 times. The values around 2.0 are obtained when the lipids are close enough together to exert an influence on each other. Engelman (1969) calculated hydrophobic volumes per cell occupied by phospholipids and neutral lipids from data on the lipid content per cell and the volumes of various groups contained in acyl groups. He assumed that an average fatty acid contained 17.5 carbons and

7

THE No+, K+-ATPare MEMBRANE TRANSPORT SYSTEM

1.26 double bonds. The hydrophobic volume for cholesterol per cell was also calculated. If the assumption is made that the lipids are evenly distributed over the cell surface, the area available per molecule of phospholipid would be the relative volume occupied by two fatty acid residues times the cell surface area. If a bilayer exists around the cell, the phosphoof surface area available. lipid cholesterol combinations would have 117 Measured a t high compression, monolayers made from 1 : l mixtures of human red cell lecithin and cholesterol gave areas reported to be 90-104 AZ and other 1 :1 mixtures of cholesterol and various phospholipid areas of 100-100 b. The thickness and area per phospholipid plus cholesterol corresponds closely to values predicted from a bilayer configuration of a membrane having a liquid hydrocarbon interior. Engelman found that his for the cholesterol-phospholipid complex was calculated value of 117 somewhat larger than expected from published data and proposed that perhaps 10% to 20% of the surface area allowed for the lipids might actually be occupied by some nonlipid elements. Hendler noted that Bar and his colleagues had calculated from their studies that the area of the phospholipid-cholesterol complex, corresponding to a lipid to cell area ratio of 2.0, was obtained a t a compression of 9 dynes per centimeter and was equal t o 125.5 k. The value derived by Engelman was just slightly less than the value of Bar and his colleagues. Hendler discussed in detail the use of newer techniques to demonstrate that, in fact, a lipid bilayer basis for membrane structure is still entirely possible. These new methods include techniques of X-ray diffraction on dispersions of isolated membranes; electron spin resonance of paramagnetic substances to study the orientation of lipids; reversible thermotropic gel-liquid crystal phase transition experiments; freeze-cleavage techniques for preparing specimens for electron microscopic examination; newer extractive techniques. Korn argues, on the other hand, that the typical trilaminar image of the cell membrane revealed by fixations in osmium remains unchanged, even when all the lipid material was removed prior to fixation. This argument represents a serious flaw in the unit-membrane hypothesis, which we will discuss below. Korn pointed out that the original assumption that osmium specifically labels the polar groups of phospholipids cannot be correct since it now has been clearly shown that osmium reacts with proteins, especially the amino and sulfydryl groups and “it would be surprising if the tertiary and secondary structures of protein were unaffected by a fixing reagent, such as osmium.” Osmium, therefore, might be only a marker for the aqueous interface of the membrane, not a specific indicator of the presence of polar groups of phospholipids a t the interface. The experiments of Gorter and Grendel were followed in the early

Az

A2

8


EXTERIOR OF CELL

1

LIPOID AREA

INTERIOR OF CELL

FIG.2. Danielli and Davson model of cell membrane as modified by Robertson (1957).

1930’sby those of Danielli and Harvey (1935) and of Danielli and Davson (1935). These investigators proposed that the biomolecular lipid leaflet represented the basis for all cell membranes and that this was arranged with hydrophobic “tails” opposite to each other on the inside with their polar, presumably phospholipid, heads on the outside. The outside layers of the lipids were supposedly covered with proteins in globular form; this became known as the Danielli-Davson model. In 1957, Robertson made a series of new observations, particularly on myelin, and slightly modified the original Danielli-Davson model, suggesting that the proteins existing on the inner and outer surface of the membranes were in p-conformation. He suggested that this model (see Fig. 2) represented a “universal structure for all biological membranes’’ (Robertson, 1957). Robertson assumed an asymmetric arrangement of the two outer surfaces so that one could conceivably be richer in carbohydrates and might provide a more hydrophilie area. It appears, therefore, that the membrane consists of some orderly array of lipids. The placement of proteins is as variable as there are investigators in the field. Consequently the number of models available is too numerous for in-depth discussion and is of little value for understanding mechanisms of active transport. See also the first chapter of Volume 1 of this series.

THE No+, K+-ATPase MEMBRANE TRANSPORT SYSTEM

9

111. A REVIEW OF STUDIES O N THE MECHANISM OF THE SODIUM PUMP

The term “sodium pump” is hereafter used to designate the system responsible for the energy-requiring efflux of sodium usually, but not always, coupled to the influx of potassium across the plasma membranes of most mammalian cells. The following discussion is based on three assumptions: (1) the sodium pump is contained within, or is part of, the membrane; (2) the energy source of the pump is ATP; and (3) the Naf, K+ATPase enzyme system is synonymous with the sodium pump. Considerable evidence has been accumulated to validate these assumptions (Albers, 1967; Glynn, 1964; Hokin and Hokin, 1963b; Judah and Ahmed, 1964; Post and Sen, 1965; Skou, 1965), but opposing theories have not been convincingly eliminated (Hoffman, 1962b; Conway, 1960; Ling, 1962, 1969a,b). The molecular mechanism by which the sodium pump carries out its transport function is unknown, as are the mechanisms of most, if not all, complex particulate enzymes (Koshland and Neets, 1968). Inability to purify most particulate enzyme systems represents a major obstacle in the quest for mechanisms. It is possible, however, that certain “membrane enzymes” are not single entities, require structural integrity of multiple sites and hence really cannot be purified in the classical enzymological sense. The problem of the Na+, I> k4), is suggested by the fact that glycosides raise the amount of potassium required to half maximally activate Na+,K+ATPase (Dunham and Glynn, 1961; Ahmed et al., 1966; Auditore and

-

40


Murray, 1962; Post and Albright, 1959; Allen et al., 1971b). This implies that potassium can overcome part of the early glycoside effect. It is assumed that all intermediate forms in the turnover cycle are in equilibrium with the free system. Thus, by stabilizing one form, the glycoside could induce a buildup of an “unstable” enzyme state, for instance, enzyme-phosphate, using inorganic phosphate as the precursor. Alternatively, exchange or phosphorylysis reactions (Siege1 et al. , 1969) could explain the formation of E-I’-32P from Pi-32P. It is equally possible that different forms of the receptor explain the data. Allen et al. (1971a), for example, showed that the rates of glycoside-receptor dissociation depend on the ionic environment used to induce binding. Specifically, the complex induced by [ATP magnesium sodium] “dissociated” at faster rates, a t 37” and 45”C, than the complex formed in the presence of [magnesium inorganic phosphate]. It should be emphasized that the dissociation reaction occurs only when the prelabeled enzyme is placed into an entirely fresh and different medium (Allen et al., 1971a,b) the reaction is temperature sensitive. Both forms cause inhibition of enzyme activity; dissociation of inhibitor; from enzyme results in complete recovery of enzyme activity. This difference, of course, may simply reflect the differences in affinities of the nonglycoside ligands for the system. It should be pointed out that although potassium retards binding of the glycoside, it also inhibits dissociation of the “stable” drug-receptor complex (Allen and Schwartz, 1970a,b; Akera et al., 1970). Thus, the consequence of “stable” ouabain-receptor interaction, further stabilized by potassium, is to ultimately fix a11 “reactive pump molecules” in an unreactive form. This hypothesis is consistent with the observed effects of cardiac glycosides on the phosphorylation, i.e., the sodium-dependent step of the ATP hydrolysis sequence. Sen et al. (1969) found that E-P-32P,formed from ATP-32P in the presence of magnesium and sodium (viz., a condition favorable for glycoside binding), is susceptible to “chase” of the label by the addition of unlabeled ATP and potassium. Ouabain prevented the chase and the (E-P-32P)-ouabain complex was slowly hydrolyzed leaving a dephospho-(enzyme)-ouabain complex. The latter form dissociated very slowly; furthermore, this form was unable to catalyze ATP hydrolysis, nor could i t be rephosphorylated with ATP-32P.Thus, it appears that glycosides inhibit the sodium-dependent step in a somewhat indirect manner. Another approach has provided insight into the sodium-potassium effectson glycoside-3H interaction with the system. Membranes containing Na+,K+-ATPase activity which are exposed to [ATP magnesium 4sodium], or t o [magnesium inorganic phosphate] bind ~ u a b a i n - ~ H at relatively slow rates in the presence of potassium (Lindenmayer, 1970; Lindenmayer and Schwartz, 1970b). Thus, under the appropriate experi-

+

+

+

+

+

41

THE N a + , K+-ATPare MEMBRANE TRANSPORT SYSTEM

mental conditions, constant binding rates can be measured (Barnett, 1970; Lindenmayer, 1970; Lindenmayer and Schwartz, 1970b). These experiments measured the amount of stabEe ouabain-receptor complex formed because the technique of reaction termination used was the rapid addition of an excess of unlabeled ouabain. The amount of potassium required to produce a 50% decrease in this rate was equal to the amount required to half maximally activate the enzyme, approximately 0.2 mM (Lindenmayer, 1970; Lindenmayer and Schwartz, 1970b,c). Conversely, sodium stimulated ~ u a b a i n - ~ H binding rates (i.e., in the presence of ATP magnesium). The amount of sodium required to cause 50% of its maximum effect, however, was five to ten times higher than that required to halfmaximally activate the enzyme for catalysis of ATI’. Potassium increased the amount of sodium required to stimulate both enzyme activity and gly~oside-~H binding rates. The potassium effect on the second function, however, was much greater than on the first. Prom these experiments, i t was suggested that sodium stimulates gly~oside-~H interaction rates by binding to the potassium-activation site on the enzyme (Lindenmayer and Schwartz, 1970~).If correct, this suggests that the rate at which glycosides interact with intact systems is partially dependent upon sodium: potassium ratio in the extracellular space, a prediction made by Matsui and Schwartz from kinetic data (1966a). This prediction seems consistent with the sensitivity of glycoside binding or the effect of rates on active transport of cations by intact cells as the mchdium is manipulated with respect to ion content (Baker and Rlanil, 1968; Baker and Willis, 1970). The above discussion of the glycoside-inhibitory mechanism reflects the case for glycoside-sensitive species only (Akera et al., 1969; Allen and Schwartz, 1969). There is evidence that the enzyme isolated from some rat organs (e.g., heart, kidney, but not brain) may demonstrate a partial dissociation of the receptor and enzyme states and perhaps also a different type of receptor. R a t heart Naf ,K+-ATPase preparations, for example, bind high amounts of glycoside-RH but the binding results in only partial enzyme inhibition (Allen and Schwartz, 1969; Warren and Schwartz, 1969), and these preparations do not manifest time-dependent, or temperature-dependent, inhibition. I n addition, the apparent stability of the drug-receptor is much less than that found in sensitive preparations. It is of interest that rat heart does not exhibit a positive staircase phenomenon nor does it react to paired stimulation with an increased force of contraction. Furthermore, rat ventricular action potentials do not exhibit a significant phase 2 plateau region, an area that may represent an important inward calcium current. These interesting phenomena may be due to intrinsic differences in the membrane sodium-potassium pump system, as Langer has suggested (Langer, 1970).

+

42


It is predictable that comparison of sensitive to insensitive preparations will continue t o provide a tool for future insight into the nature of the interaction as increasingly sensitive techniques are employed. It is difficult t o test the hypothesis that glycosides inhibit via stabilization of a specific conformation from both technical and philosophical points of view, Membrane-bound Naf ,Kf-ATPase preparations are impure (i.e., anywhere from ti to 10 to 45% pure) and form turbid suspensions in water. On the other hand, all protein functions are probably conformationally sensitive and many may require structural changes, whether large or minute, in carrying out their functions (Koshland and Neets, 1968). It is difficult, therefore, to prove that the allosteric hypothesis is incorrect. Although we recognized the validity of these arguments, the next logical step in the delineation of the inhibitory mechanism was to demonstrate that glycosides do or do not stabilize a conformer. Without specific knowledge of structure, however, it is impossible to describe the speciJic type of conformational change that may occur. Nevertheless, some information was obtained by the use of four techniques: (1) Fluorescence spectra of Na+,K f - A TPase preparations. Both membrane-bound and Lubroltreated preparations were examined. Neither monovalent cations, magnesium and inorganic phosphate, nor cardiac glycosides reproducibly altered the spectra. (2) Fluorescence of chromophore probes bound to the preparations. Nagai et‘ al. (1970) examined the spectra of 5-anilinonaphthalenesulfonic acid (ANS) exposed to Na+ ,I (Plummer and Hirs, 1964) and DNase (Salnikow et al., 1970) are glycoproteins and since the elements of the Gold complex in other cell types producing exportable glycoproteins are responsible for the synthesis of the polysaccharide moiety (Fleischer et al., 1969; Haddad et aZ., 1971; Zagury et al., 1970). Finally, as will be discussed below, i t is possible that the elements of the Golgi complex, especially its condensing vacuoles, may be responsible for an alteration of the content resulting in the formation of macromolecular aggregates with low osmotic activity. The final step in the transport pathway consists of discharge of the content of the zymogen granule into the acinar lumen. This step involves movement of the granule to the cell apex, where its limiting membrane fuses with that of the apical plasmalemma resulting in release of the granule content into the duct lumen by exocytosis (see Fig. 14). The details of this operation are discussed later in another section. The results to date lead to the general conclusion that secretory proteins, following initial segregation in the cisternal spaces of the RER, remain

288

J. D. JAMIESON

FIG.9. Exocrine cells after 80 minutes of chase incubation in uitro. Label is now highly concentrated in zymogen granules (2) at the cell apex (- 62%). Condensing vacuoles (C), in this micrograph, are already free of label. The arrow indicates filamentous material in the content of a condensing vacuole. Similar filaments are sometimes associated with the content of the acinar lumen (L). From Jamieson and Palade, 1971a. Courtesy of the Journal of Cell Biology. X 13,600.

PROTEINS IN PANCREATIC EXOCRINE CELLS

289

within and are transported through the cell in association with its membrane-limited compartments until they are finally released from the cell to the extracellular space. At no point in the transport pathway was evidence obtained that secretory proteins move free through the cell sap, which is an alternative pathway proposed by others in the past (Redman and Hokin, 1959; Morris and Dickman, 1960). This latter pathway was suggested to explain the large amounts of secretory proteins recovered in the postmicrosomal supernatant after certain homogenizing procedures and was based in part on the observation that cells depleted of their content of granules are able to maintain an output of secretory proteins over long times (Lin and Grossman, 1956). In the scheme proposed above, the secretory proteins need penetrate a membrane only once-at the time of synthesis on attached polysomes. In any other scheme in which a phase of transport in the cell sap is envisaged, the product must enter the cell sap from the RER cisternae (assuming that step 1 in the process is obligatory as appears to be the case so far) and cross at least one other membranethe plasmalemma-during discharge. In any alternative scheme a t least three (and possibly five) membrane crossings must be postulated. Considering that each membrane crossing must be specific, possibly involving special carriers for the exportable product and in view of the apparent irreversibility of step 1 (Jamieson and Palade, 196813) such an alternative pathway seems improbable. As will be noted below, histochemical studies directly support this contention. To what extent does the pathway described above pertain to other cell types? In the main, it most likely pertains to all cells which temporarily store their secretory products in storage granules prior to discharge. For instance, radioautographic studies on p cells of the endocrine pancreas (Howell et al., 1969b), neutrophilic leukocytes (Fedorko and Hirsch, 1966), and rabbit parotid exocrine cells (Castle el al., 1972) have provided evidence for the existence of a pathway similar to that of the exocrine pancreas. In addition, histochemical studies by Bainton and Farquhar (1970) on eosinophilic leukocytes and by Herzog and Miller (1970) on rat parotid exocrine cells have shown the presence of peroxidase in the expected intracellular membrane-enclosed compartments. These studies also dearly demonstrate the absence of reaction product free in the cell sap and thus provide important direct evidence that transport of exportable products through the cell sap is, within the limits of sensitivity of the techniques, unimportant. In the case of cells which do not form morphologically distinctive storage granules, such as hepatocytes (Peters, 1962; Ashley and Peters, 1969), plasma cells (Zagury et al., 1970), and thyroid follicular cells (Nadler et al., 1964), a t least the first part of the pathway up to the level of the Golgi complex has been demonstrated by cell fractionation and/or radioautographic procedures.

290

J. D. JAMIESON

C. Requirements for Protein Synthesis and Metabolic Energy in the Secretory Pathway (Steps 1-5)

From the above considerations, it is clear that intracellular transport involves a number of different types of membrane-mediated transport operations. Consequently, it was of interest to examine the metabolic requirements of the various steps in the pathway, and to this end two main questions were posed. First, is intracellular transport obligatorily coupled to continued protein synthesis, or does it require the ongoing production of either exportable proteins or other specific, nonexportable proteins, such as couplers and carriers; and second, what are the energy requirements, if any, for intracellular transport? For examination of these problems, a number of simple radioassays, based on the radioautographic studies shown, were devised. Operationally, these can be divided into assays which cover, respectively, steps 1-3; step 4, and step 6 of Fig. 1.

STEPS1-3 The assay covering steps 1-3, i.e., transport from the R E R to condensing vacuoles is based on our earlier findings that following a 3-minute pulse labeling with l e ~ c i n e - ~ H up, to 49% of the labeled proteins migrates during a 37-minute chase period to condensing vacuoles with only a small proportion (-11%) reaching zymogen granules a t this time (Jamieson and Palade, 1967b). Upon cell fractionation of the slices, the labeled condensing vacuoles are recovered in the common zymogen granule pellet where they are detected by their characteristic morphological appearance and by their content of labeled secretory proteins. The end point of the assay then simply consists of determining the amount of labeled proteins accumulating in the zymogen granule fraction during a fixed 37-minute chase period. I n order to study the first question mentioned above, we have used this assay to examine the effects of cycloheximide, a potent inhibitor of protein synthesis, on the efficiency of transport of labeled pr,oteins to condensing vacuoles (Jamieson and Palade, 1968a). For this purpose, the antibiotic was added to the assay immediately post-pulse and was present throughout the 37-minute chase period. The results of this experiments (Fig. 10) show that a t concentrations of cycloheximide which block protein synthesis by >95%, transport proceeds with an efficiency of 7 5 4 0 % of that in the control slices. The 15-20% inhibition of transport is probably related to the parallel depression of O2 uptake by the slices. The data can be taken to indicate that movement of secretory proteins through the R E R cisternae and transport to condensing vacuoles does not depend on the maintenance


29 1

Conc. cycloheximide

FIQ.10. Effect of various doses of cycloheximide on intracellular transport of secretory proteins to condensing vacuoles during a 37-minute chase period post pulse. The effect of the drug on incorporation of leucine-3H into total slice protein is also shown (curve c). Curve a, transport to ZG fraction; curve b, oxygen consumption. From Jarnieson and Palade, 1968a. Courtesy of the Journal of Cell Biology.

of a simple concentration gradient which, by mass action, results in the propulsion of the content to the succeeding compartment. With cycloheximide, delivery from attached polysomes stops abruptly (in < 2 minutes), yet despite this, the pool of labeled proteins continues to drain. Evidently the synthesis of other nonsecretory proteins, such as couplers, carriers, and membrane proteins, is not required, at least during the 37minute period examined, although they may be present in a pool sufficiently large that their requirement is not manifest in this time. Because intracellular transport can be uncoupled from ongoing protein synthesis it was possible to examine in the uncoupled state the energy requirements of steps 1-3 by the use of familiar metabolic inhibitors (Jamieson and Palade, 196813).Previously this would not have been possible because the potential effect of any metabolic inhibitor would have been also to inhibit protein synthesis by virtue of limiting the energy production of the cell. For this series of studies, the assay for transport from the RER to condensing vacuoles was identical to that described above. The inhibitors and incubation conditions to be tested were added immediately post-pulse

292

J. D. JAMIESON

2ot 0

L Antirnycin A

4

4

w

Cy c Io he x imide 5 x I 0-4M

w

FIG.11. Effect of various concentrations of antimycin A on transport of labeled proteins to condensing vacuoles during a 37-minute chase period. The experiment was conducted with 5 X 10-4 .I4 cycloheximide present to uniformly block protein synthesis. 0 2 consumption (0 0) and evolution of 14C02 from '"-labeled palmitate (X-X) are depressed in parallel. 0-0,Transport to ZG fraction a t +37 minutes. From Jamieson and Palade, 196813. Courtesy of the Journal of Cell Biology. TABLE

Ia,b

AND GLYCOLYTIC INHIBITORS ON A. EFFECTOF TEMPERATURE INTRACELLULAR TRANSPORT

Post-pulse incubation conditions

37 Min, 37" 37 Min, 27" 37 Min 17" 37 Min, 4" 37 Min, 4" 37 Min, 37" 37 Min, 37" 37 Min, 37"

Gas phase

Additions

(MI

Reincubation conditions

0 2

-

-

0 2

0 2 0 2 0 2

0 2 0 2 0 2

-

-

F- 10-3 F- 10-2

-

37 Min, 37" -

-

Relative specific activity

(%)

Specific activity (dpm/mg protein)

100.0 26.3 7.5 1.1 57.0

80,000

100.0 106.0 99.0

45,000

293

PROTEINS I N PANCREATIC EXOCRINE CELLS

TABLE I-Continued

B. EFFECT OF NITROGEN, CYANIDE, AND 2,4-DINITROPHENOL ON INTRACELLULAR TRANSPORT^.^ Post-pulse incubation conditions

37 Min, 37" 37 Min, 37" 17 Min, 37" 37 Min, 37" 37 Min, 37 Min, 37 Min, 37 Min, 37 Min, 37 Min, 37 Min, 37 Min,

37" 37" 37" 37" 37" 37" 37" 37"

37 Min, 37" 37 Min, 37" 37 Min, 37" 37 Min, 37" 37 Min, 37" 37 Min, 37"

Gas phase

Additions (MI

-

0 2

N2 NZ

37 Min, 370, 0 2 37 Min, 37", 0 2

Na 0 2 0 2 0 2 0 2 01

0 2 0 2

Oa

0 2 0 2 0 2

0 2

0 2 0 2

Relative specific Reincubation activity (%I conditions

CN- 5 X 10-6 CN- 1 x 10-4 CN-4 x 10-4 CN- 5 x 10-4 CN- 7 x 10-4 CN- 1 x 10-3 C N - 5 x 10-4

DNP 1 X DNP 1 x DNP 5 x DNP I x DNP 5 x

10-6 10-4 10-4 10-3

10-4

-

-

37 Min, 37", no CN-

37 Min, 37", no DNP

100 12 80

Specific activity (dpm/mg protein)

80,Ooo

70 100 98 90 28 22 10 2 111

90,000

100 116 67 20 10 80

47,000

~~

Data from Jamieson and Palttde (1968b). and bSets of pancreatic slices were pulse labeled for 3 minutes with ~-leucine-~H incubated in chase medium for 37 minutes with the additions shown, including 0.5 mM cycloheximide. In reversal experiments, the slices were reincubated after 37-minute chase for a further 37 minutes under the indicated conditions. At the termination of the assay, zymogen granule fractions were isolated from the slices and the protein radioactivity was measured and compared to that in fractions from control, untreated slices. a

and were present throughout the 37-minute post-pulse incubation period; in addition, all assays contained 0.5 m M cycloheximide to inhibit protein synthesis and so provide a uniform baseline of transport. The results of these studies, given in Table I and Fig. 11 show that this segment of the transport pathway is enzymatic, being reversibly inhibited b y Iowering of

294

J. D. JAMIESON

-

the incubation temperature (Q1o 3.9); is not inhibited by compounds which block glycolysis (NaF and iodoacetate) ; but is exquisitely sensitive to any inhibitor that interferes with mitochondria1 energy production. Except for antimycin A, transport block was relieved by removal of the inhibitor. So far, the results indicate that transport from the R E R to condensing vacuoles requires energy, probably as ATP, and that, in the absence of energy, transport is blocked proximal to the condensing vacuoles. But this part of the pathway includes a number of operations including movement of proteins through the RER cisternae, possibly budding off of transitional elements, and translation of Golgi vesicles toward condensing vacuoles. Each of these steps is potentially energy-requiring. To gain further insight into the initial energy-requiring site, radioautographic and cell fractionation procedures were applied to slices which had been blocked immediately post-pulse with antimycin A. From cell fractionation, it was clear that in the presence of the blockers, labeled proteins did not gain access into the smooth microsomal fraction (i.e., into Golgi-derived vesicles recovered in this fraction) a t a time when it was maximally labeled in the controls (Table 11).Radioautograms of similarly treated slices nevertheless showed an accumulation of labeled proteins a t the level of the Golgi peripheral TABLE I1 LABELING OF MICROSOMAL SUBFRACTIONS FROM SLICESINCUBATED POSTPULSE WITH ANTIMYCINAaob

Conditions

3 Min (pulse) +7 Min 7 Min with antimycin A

+

Dpm recovered Dpm in gradient in rough load, total and smooth microsomes microsomes

101 ,260 66,880 73,790

50,430 31,660 37,060

yo Dpm recovered in rough and smooth microsomes 49.8 46.0 50.9

Dpm in smooth microsomes as % rough and smooth microsomes

17.0 43.0 18.8

~~

Data from Jamieson and Palade (1968b). Three sets of pancreatic slices were pulse labeled for 2.5 minutes with l e ~ c i n e - ~ K . At the end of the pulse one set of slices was homogenized for cell fractionation. The remaining two were incubated for a further 7 minutes in chase medium containing a large excess of unlabeled leucine, 5 X M cycloheximidc, and for the antimycin set, 5 X 10-6 M of the drug. After chase incubation, these sets were fractionated. Rough and smooth microsomes were isolated by gradient centrifugation, and the proportion of labeled proteins in the fractions was measured.

295


TABLE I11

I~STRIBUTION OF RADIOAUTOGRAPHIC GRAINSOVER CELLCOMPONENTS IN SLICESINCUBATED POSTPULSE WITH ANTIMYCIN Aash

% Radioautographic grains Chase incubation

Sttbcellulsr components

Pulse 3 min

+7 Min

+I7 Min

+37 Min

+57 Min

Rough endoplasmic reticulum

89.1

50.3 66.8 34.9 24.1 6.2 ?3.1 7.8 6.1 0.4 0.2

39.6 62.8 23.5 29.4 29.9 5.6 6.9 2 .3

38.6 Y6.8 19.7 16.6 35.3 1.6 6.4 6.4 0.5 0.1

37.1 Y8.8 20.4 1 5 .1 19.9 1.8 19.9 4.6 3.1 0

684 1620

395 968

823 884

1447

Golgi complex peripheral regionc

5.0

Condensing vacuoles

1 .o

Zymogen granules

4.4

Acinar lumen

0.2

No. of grains counted

992

0 0

405

Data from Jamieson and Palade (196813). &Setsof pancreatic slices were pulse labeled with leucine-3H for 3 minutes and incubated post-pulse in chase medium containing a large excess of unlabeled leucine, 5 X lo-' M cycloheximide, and for the experimentnls (numbers in italics), 5 X M antimycin A. At the indicated times, the slices were fixed and processed for electron microscopic radioautography and the percent distribution of radioautographic grains was scored. The peripheral region of the Golgi complex is defined as comprising the small vesicles of the Golgi complex and the adjacent zone of transitional elements.

region which includes, it may be recalled, the transitional elements of the RER (Table 111). Taken together, the results indicate that the first energyrequiring site is most likely located a t the level of the transitional elements of the RER, and tentatively we conclude that the energy requirement may be related to the pinching off of the transitional elements. The observation that secretory proteins apparently accumulate at the level of the transitional elements during the block merits comment. First, the finding would indicate that movement of proteins through the ER channels is energy-independent and probably is accomplished by random-walk diffusion. I n fact, if one assumes an intracisternal viscosity of -0.06 poise, then the diffusion time for a secretory protein of average molecwlar weight (-25,000) from the

296

J. D. JAMIESON

most basal ER elements to the transitional elements is only a few seconds. Second, the relative concentration of labeled proteins a t the level of the transitional elements in the blocked state might indicate that the secretory proteins enter into a more positive relationship with the transitional elements than simply being entrapped in their content. For instance, the inner surface of the transitional elements may be provided with specific receptor sites for the exportable products, so ensuring efficient transport even in the face of decreasing intracisternal concentration such as occurs during cycloheximide treatment. Candidates for at least some of the receptors might consist of membrane-bounded glycosylating enzymes of the type mentioned for the addition of polysaccharides to some RNases and DNase. The results given only pinpoint the most proximal energy requiring site, and a t present the techniques are insufficient to determine whether movement of Golgi vesicles (if such occurs) and delivery to condensing vacuoles also require energy. In any case, as seen in Table IV, back diffusion of TABLE IV

EFFECTOF ANTIMYCIN A ON THE CONVERSION OF CONDENSINQ VACUOLES INTO ZYMOGENGRANULE& Radioautographic grains 40 Min 20 Min control 20 Min anti A

+

80 Min

80 Min

20 Min control 60 Min 80 Min anti A anti A

+

20 Min control

40 Min control

Subcellular component

(%)

(%)

(%)

(%I

(%I

(%I

Rough E R Golgi peripheral region Condensing vacuoles Zymogen granules

27.6 18.7 47.0 6.6

16.4 15.4 48.6 19.6

28.3 11 . 9 38.8 20.9

13.3 6.5 16.0 62.4

29.5 10.7 22.6 37.2

57.6 32.7 5.4 4.3


2494

2996

2495

2437

1216

1326

control

Data from Jamieson and Palade (1971a).

* Pancreatic slices were pulse labeled with lei~cine-~H for 4 minutes and incubated for 20 minutes in chase medium to prelabel condensing vacuoles. At this time, 10-6M antimycin A was added to the experimental slices and incubation was continued for a further 20 or 60 minutes. Zero time controls received antimycin a t the end of the pulse and were incubated for 80 minutes with the drug. At the indicated times, the slices were fixed and processed for radioautography.


297

labeled proteins from condensing vacuoles to elements of the Golgi peripheral region appears not to occur, indicating that the process is not easily reversible and that continuously patent channels do not exist between condensing vacuoles and the preceding cell compartments. I n summary, the results indicate that the transport pathway is provided with a n energy-requiring lock or valve located most likely a t the level of the transitional elements of the RER. The opening of this lock establishes a functional connection between two membrane-bounded compartmentsthe cisternae of the RER and the condensing vacuoles-and results in the active transport of macromolecules in bulk between the compartments. This type of active transport differs from active transport in the usual sense where molecules or ions are moved directly across a membrane in association with specific carriers or couplers but is reminiscent of bulk transport of molecules into cells by pinocytosis. Direct evidence for a lock of the type described above is lacking in other cell types. However, the recent morphological studies of Farquhar (1971) on thyrotropic cells of the adenohypophysis show that, after thyroidectomy, storage granule formation by the Golgi complex practically ceases and is accompanied by massive dilatation and engorgement of the ER cisternae leading ultimately to the formation of intracisternal granules. These changes may reflect a relative slowdown or block in the operation of a lock connecting the ER and Golgi compartments. The formation of intracisternal granules in exocrine pancreatic cells under certain physiological states may similarly be secondary to a decreased efficiency in the opening of the lock mentioned above (Palade, 1956). ASPECTSOF CONDENSING VACUOLECONVERSION STEPS4-5. METABOLIC Step 4 consists of the concentration of t,he initially dilute solution of proteins in condensing vacuoles resulting in the formation of mature zymogen granules. Previous studies had shown that this step, like those immediately preceding it, does not depend on continued protein synthesis (Jamieson and Palade, 1968a). We also suggested that condensing vacuole conversion might result from the extrusion of water and electrolytes from the vacuole content to the cell sap, this possibly being mediated by energyrequiring ion pumps akin to those located in the plasmalemma of many cell types (e.g., a Na+-K+ ATPase; Jamieson and Palade, 1967b). To examine this hypothesis directly it would ideally be desirable to study the enzymatic basis of the concentration process on a n isolated fraction of condensing vacuoles. So far, however, it has not been possible to obtain a satisfactory separation of condensing vacuoles from the bulk of the aymogen granule fraction, and we have of necessity assessed the conversion

J. D. JAMIESON

FIG.12. Pancreatic exocrine cell after a 2O-minute chase period following a 4-minute pulse labeling in uitro with leucine-3H. At this time ~ 4 7 %of the radioautographic grains mark condensing vacuoles (arrows). About 7% of the label is associated with zymogen granules ( Z ) . L, acinar lumen. X10,400. From Jamieson and Palade, 1971a. Courtesy of the Journal of Cetl &kiogg.


299

FIG.13. Radioautogram of an exocrine cell incubated post pulse for 20 minutes at which time 10-6 M antimycin A was added and incubation resumed for a further 60 minutes. Radioautographic grains are mainly associated with zymogen granules ( Z ) with some still associated with condensing vacuoles (c).Arrows delineate the periphery of the Golgi region. X11,475. From Jamieson and Palade, 1971a. Courtesy of the Journal of Cell Biology.

300

J. D. JAMIESON

process by radioautography applied to thin sections of intact cells or zymogen granule pellets isolated from slices whose energy production is restricted by the application of appropriate metabolic inhibitors (Jamieson and Palade, 1971a). The plan of the experiments is as follows: Slices were pulse labeled with leucine-%Has usual for -3 minutes, and the wave of labeled proteins was allowed to progress to the condensing vacuoles during a 20minute chase period. During this period only a small proportion of the label reaches zymogen granules. At this time, antimycin A, a t a concentration (0.01 mM) sufficient to block rapidly (in iallyrapid, due most likely to washout of the duct system of the gland and thereafter proceeds a t an approximately linear rate for the next 2 hours (Fig. 20). After degranulation is completed a t 3 hours, the rate of amylase discharge begins to slow and, as seen later, assumes a new steady-state rate in the fully degranulated cell. Because we were interested primarily in an assessment of the route and kinetics of transport of secretory proteins in the hyperstimulated cell,

FIG.19. Electron micrograph of the apical region of an exocrine cell from B slice incubated in vilro for 3 hours with 10-6 M carbamylcholine. The Golgi complex is enlarged in volume and consists of numerous stacked cisternae (Gc) some of which contain electron opaque material on their innermost faces (Gcl). Many small storage granules (sg) and vesicles with an electron opaque content, and frequently surrounded by a coated membrane (arrow), are centrally located in the complex. Transitional elements (tr) and typical Golgi vesicles (Gv) populate the periphery of the Golgi complex. Smooth-surfaced vesicles (av) are found adjacent to the acinar lumen (L) which borders the truncated, rounded apex of the cell. From Jamieson and Palade, 1971b. Courtesy of the Journal of Cell Biology. X 10,600.

320

J. D. JAMIESON

it was of importance to evaluate the rates of protein synthesis under our experimental conditions. Previous studies by others had indicated that secretory stimuli either increased, decreased, or did not change the rates of protein (Webster and Tyor, 1967; Kramer and Poort, 1968) synthesis. In these studies, the stimulants were applied either in vivo, in vitro, or in a combination of situations and to pancreases from animals in various physiological states. I n our experiments, the secretagogues were applied in vitro to slices derived from the pancreases of previously starved animals. Experiments of the type given in Fig. 21 show that secretagogues do not enhance the incorporation of leucine-3H into proteins over a 3-hour period but if anything, slightly depress incorporation. These results were true of carbamylcholine used a t doses from threshold (-lo-’ M ) to those producing maximal secretory responses (-lo-* M ) . While the variations in incorporation rates under stimulation reported by others cannot be satisfactorily explained, they may in part be related to the fact that in some cases the

Hours i n c u b a t i o n

FIQ.20. Discharge of amylase to incubation medium in response to carbamylcholine Controls, 3TC, (0-0) M ) (0-0) or pancreozymin (10 U / d ) (A-A). received no drug; their output is mainly from damaged cells, since it is not blocked by low temperature. From Jamieson and Palade, 1971b. Courtesy of the Journal of Cell Biology.


32 1

Hours incubation

Hours incubation

FIG.21. Effect of carbamylcholine (10-6 M ) and pancreozymin (lOU/ml) on incorporation of leucine-3H into pancreatic slice proteins. Incorporation data are normalized to total slice DNA and include label in the slices and that discharged to the medium. Incorporation is enhanced as the amount of carrier leucine in the medium is increased. In the absence of carrier (0.09 p M leucine) incorporation ceases after 2 hours. 0-0, Control; 0-0, carbamylcholine; A-A, pancreozymin. From Jamieson and Palade, 1971b. Courtesy of the Journal of Cell Biology.

data are normalized to total tissue protein. This can lead to spuriously high specific activity calculations since the stimulated gland can lose up to -40% of its secretory proteins in the course of zymogen granule depletion. I n the present studies the data have been normalized to a constant denominator, tissue DNA, and take into account all labeled proteins synthesized including those discharged to the medium from the stimulated slices. I n addition, in our system the slices are supplied with a complete

322

J. D. JAMIESON

TABLE XI OF RADIOAUTOGRAPHIC GRAINSOVER CELLCOMPONENTS IN DISTRIBUTION PRESTIMULATED PANCREATIC SLICESINCUBATED POSTPULSEWITH CARBAMYLCHOLINE

7'

of Radioautographic grains Chase incubation

3 Min, pulse

+7 Min

+17 Min

+37 Min

+57 Min

Rough endoplasmic reticulum Periphery of the Golgi region Storage granules

90.4 (89.1) 8.7

54.2 (49.5) 35.6

44.7 (38.4) 28.5

37.3 (24.5) 27.0

25.8 (16.2) 18.3

0.9

10.2

26.8

35.7

55.8


1082

1133

1626

914

480

Subcellular component

Data from Jamicson and Palade (1971b). Sets of pancreatic slices were stimula,ted for 3 hours before labeling by incubation in a medium containing M earbamylcholine and 0.04 mM L-1eucineJH. They were then washed with leucine-free medium and kept for 10 minutes at 4°C in a carbamylcholine-free medium containing leucine-3H then pulse labeled for 3 minutes at 37". At the end of the pulse, one set was fixed and the others were further incubated for the times shown under resumed stimu1at)ionin a chase medium containing 4.0 mM I,-leucine-'H and M carbamylcholine. For reference, the percent distribution of grains over the RER in unstimulated slices is shown in parentheses.

supplement of amino acids and an energy source which supports tissue metabolism a t undiminished rates for as long as 12 hours in vitro (unpublished observations). Having established that the rates of protein synthesis remain reasonably constant in the in vitro stimulated slices, we proceeded to examine the efficiency of intracellular transport in cells previously depleted of their store of preformed granules. Again, a combination of electron microscopic radioautography and cell fractionation was employed except that in this case the pulse-labeling with leucine-3H was applied a t the end of a 3-hour prestimulation period with carbamylcholine; the chase medium contained, in addition to a large excess of unlabeled leucine, carbamylcholine a t a dose sufficient to maintain further optimal discharge rates. The radioautographic results are illustrated in Figs. 22-26 and quantitated in Table XI. They indicate that a t the end of the pulse (Fig. 22) the labeled proteins are mainly associated, as expected, with elements of the RER and with time progressively drain from this compartment, first to become associated with the small vesicles in the Golgi peripheral


323

FIG.22. Radioautogram of a pancreat,ic slice preincubated for 3 hours with 10-6 M carbamylcholine, then pulse labeled with Ie~cine-~H for 3 minutes. Grains mark elements of the rough ER (RER). G, Golgi complex. From Jamieson and Palade, 1971b. Courtesy of tlhe Journal o j Cell Biology. X 10,200.

324

J. D. JAMIESON

FIG.23. Pancreatic slice treated as in Fig. 22 but incubated in chase medium for

7 minutes. Label was located over peripheral vesicles (Gv) and proximal cisternae (Gc) of the Golgi complex. Small secretory granules (sg) and adjacent filled Golgi cisternae (arrows) are seen. ly, presumed lysosome. From Jamieson and Palade, 1971b. Courtesy of the Journal of Cell Biology. X 12,750.


325

FIG.24. Prestimulated slice incubated 17 minutes post pulse. Label now is located more distal over filled Golgi cisternae (Gc), with some over small storage granules (sg). Gv, Golgi vesicles; L, duct lumen; id, intercalated duct cell. From Jamieson and Palade, 1971b. Courtesy of the Journal of Cell Biology. X12,750.

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J. D. JAMIESON

FIG.25. Prestimulated slice after 37 minutes of chase incubation. Label is now concentrated over small storage granules (sg). ly, lysosome; Z, mature zymogen granule. From Jamieson and Palade, 1971b. Courtesy of the Journal of Cell Biology. X 11,900.


327

FIG.26. Prestimulated slice after 57 minutes of chase incubation. Label is now mainly located over small storage granules a t the cell apex. The Golgi peripheral elements are largely devoid of label. From Jamieson and Palade, 1971b.Courtesy of the Journal of Cell Biology. X 15,300.

TABLE XI1 EFFECTOF PREINCUBATION WITH CARBAMYLCHOLINJC ON INTRACELLULAR T R A N S P O R ~ . Chase incubation Distribution of Radioactivity

Preincubation

3 Hour control 3 Hour control 3 Hours, 0.01 mM carbamylcholine 3 Hours 0.01 mM carbamylcholine

Homogenate

Rough microsomal fraction

Zymogen granule fraction

Postmicrosomal supernatant

Conditions 4.0mMtleucine-lH

Dpm/U % amylase Dpm

Dpm/U % amylase Dpm

Dpm/U % amylase Dpm

Dpm/U % amylase Dpm

3 Min 3 Min 3 Min

0 Min, pulse 37 Min, control 0 Min, pulse

27,000 26,800 15,500

100 100 100

67,800 23.2 23,700 9 . 3 53,200 18.4

681 0 . 4 16,700 21.8 376 0 . 3

12,800 14.7 9,600 15.8 14,700 16.4

3 Min

37Min,O.O1mM carbamylcholine

18,300

100

23,800

12,900

13,400 17.0

Pulse ~-1eucine-3H

9.0

9.6

Data from Jamieson and Palade (1971b). pancreatic slices were stimulated and labeled as in Table XI. At the end of the pulse, one set each of control and prestimulated slices was fractionated. The remaining control set was incubated for a further 37 minutes in chase medium containing 4.0 mM tleucine-'H, while the remaining prestimulated set was incubated for the same time in the same chase medium containing M carbamylcholine. At the end of the chase, each set of slices was homogenized for cell fractionation. The data are expressed as percent, TCA-precipitable radioactivity recovered in the cell fractions or as specific radioactivity (dpm/unit amylase). The data are meant to show the relative changes of radioactivity in the cell fractions with time. Only the figures for the postmicrosomal supernatant represent complete recovery. a Sets of

.W

2

b

z

329


zone (7-minute chase, Fig. 23), next with the filIed Golgi cisternae (17minute chase, Fig. 24) and finally with the small storage granules centrally located in the complex (37-minute chase, Fig. 2 5 ) . Ultimately the labeled proteins presumably leave the cell by exocytosis of the content of the small storage granules (>57-minute chase, Fig. 26). From the quantitative data given in Table XI it is clear that the rate of drainage of the RER compartment is not accelerated by stimulation compared to the situation in the resting controls (numbers in parenthesis, Table XI). Confirmation of the radioautographic data was obtained by cell fractionation procedures applied in paralleI to controI and prestimulated slices. As seen in Table XI1 neither the rate of loss of labeled protein from the rough microsomal fraction (which is an index of drainage of the RER compartment from which they are derived), nor the rate of accumulation of label in the small storage granules which are recovered in the “zymogen granule fraction” are altered by stimulation. In addition, both the total and specific radioactivity of proteins in the postmicrosomal supernatant remain unchanged in the stimulated cells. Since this fraction represents in part the soluble cytoplasmic matrix and is fully recovered in the cell fractionation scheme, we can also state with reasonable certainty that stimula-

conlrol

pulse

Chose incubotion,min

FIG.27. Assay for total secretory pathway (RER to acinar lumen). Slices were incubated 3 hours in control medium, pulse labeled with leucine-aH, then reincubated for 2 hours with 10-5 M carbamylcholine to initiate secretion. Amylase discharge (0-0) begins without a lag whereas labeled proteins (0-0) begin to appear in the medium after a 20-30-minute lag. From Jamieson and Palade, 1971b. Courtesy of the Journal of Cell Biology.

330

J. D. JAMIESON

tion does not result in a rerouting of secretory proteins through the cell sap as previously proposed by others. More than 80% of the labeled proteins must be in transit through the cell in association with sedimentable cell particulates a t all times and in both physiological states. The source of the labeled proteins in the postmicrosomal supernatant remains unknown, although presumably they come in part from cell particulates ruptured during homogenization and in part from soluble proteins in the duct system of the gland. As a n independent check on the radioautographic and cell fractionation data, a radioassay covering the entire RER-acinar lumen pathway was devised. I n this assay sets of slices were preincubated for 3 hours-without or with carbamylcholine, pulse labeled at the end of this period, then reincubated for various times in medium containing carbamylcholine to initiate discharge in the control slices and maintain it in the prestimulated slices. At various intervals the medium was sampled for output of labeled proteins and amylase. As seen in Figs. 27 and 28, labeled proteins begin to appear in the medium from slices in both conditions after a lag time of 20-30 minutes and accumulate thereafter at linear rates. Evidently both the minimal and average transit times for labeled macromolecules over

0.01 rnM carbamylcholine pulse

Chase incubation,rnin

FIQ.28. As in Fig. 27 except that the slices were incubated both before and after the pulse with 10-6 M carbamylcholine. The lag time for appearance of labeled proteins is again about 20-30 minutes. The absolute output of amylase is substantially less than in Fig. 27 because of discharge of granule contents during the first stimulation period. 0-0, Labeled proteins; 0-0,amylase. From Jamieson and Palade, 1971b. Courtesy of the Journal of Cell Biology.


33 1

the total pathway are the same for slices in the two experimental conditions. However, the net output of amylase from slices stimulated both before and after the pulse is, as expected, considerably smaller than that from slices stimulated only post pulse due to depletion of the pool of zymogen granules in the former during the 3-hour prestimulation period. From the relative specific activities of amylase discharged from the two types of slices it appears that the pool of secretory proteins in zymogen granules in the slices stimulated only post pulse is -6 times larger than that contained in the small storage granules in slices stimulated both before and after the pulse. From these data we conclude that discharge of mature zymogen granules must be random for otherwise, if discharge of old unlabeled granules exclusively preceded that of new, labeled granules the lag time for the appearance of labeled proteins in the medium from slices stimulated only post pulse should be considerably longer in view of the relatively larger pool of secretory proteins contained in the zymogen granules. Finally, it should be mentioned that morphological examination of slices stimulated for 3 hours in the presence of cycloheximide reveals that the entire sequence of events, including dilatation and restitution of the acinar lumen profile, and increase in the volume of the Golgi complex, is identrical to that for cells exposed to the secretagogue alone despite the fact that protein synthesis was blocked by >98% during this time. I n fact, as shown in Fig. 29A and B, two complete discharge cycles covering a 6-hour period can be completed in the absence of protein synthesis. During the first cycle, the cells are progressively depleted of their zymogen granules by the secretagogue, then the block is temporarily relieved to allow the introduction of a new pulse of labeled proteins, and subsequently a second cycle of discharge is induced, this time from granule-depleted cells. The implications of these findings in relation to the dynamics and turnover of intracellular membranes are discussed below. In summary the studies on stimulated exocrine cells indicate that nascent secretory proteins are initially segregated in the cisternae of the RER, are transported to the elements of the Golgi complex, and finally are stored in modified granules prior to discharge. I n general while the pathway followed resembles that already discussed above for the resting cell, in that it involves primarily membrane-bounded compartments, several of the details of the processing of the product differ, First, as indicated by the morphological and radioautographic findings, the cisternal elements of the Golgi complex as well as its small peripheral vesicles are importantly involved in the process. Whereas in the resting exocrine cell (specifically that of the guinea pig) the product appears to bypass the stacks, being transported directly to condensing vacuoles in

332

J. D. JAMIESON

.&

501

-

4 m T

1

2

3

4

5

pulse

Chose incubation, hr

A

revers01 pulse

(30min I

Chase incubation, hr

B FIQ.29. (A) Discharge assay conducted in the presence of cycloheximide (5 X lO-4M). Stimulant and cycloheximide were added immediately post-pulse. 0-0,Carbamylcholine plus cycloheximide; 0-0, carbamylcholine; A-A, cycloheximide; A-A, control. (B) Second wave of induced discharge conducted in the presence of cycloheximide. During the 3-hour preincubation period, the stimulated and nonstimulated slices were treated as in Fig. 29A. Cycloheximide reversal was obtained by a 30-minute wash period in drug-free medium, after which a new cycle of discharge was initiated post pulse. Curves labeled as in (A).


333

shuttle vesicles, in the stimulated cell, concentration of the product begins more proximally on the pathway and is frequently seen within the innermost of the Golgi cisternae and their lateral dilatations. Both in position and in timing, these filled Golgi saccules appear to be equivalent to the condensing vacuoles of the controls. I n these respects, the route of transport and site of concentration in the stimulated exocrine cell are similar to those noted for the majority of endocrine and exocrine cells 80 far examined, including the exocrine pancreas of other species. In addition, the size and shape of the storage granules in the stimulated state differ markedly from those usually observed in resting cells. All these changes, including the increased membrane amount in the Golgi csmplex, may represent a new steady state established by the cell to enable it to concentrate more rapidly its secretory products. I n any event, it is evident that the elements of the Golgi complex and the pattern of concentration and storage are capable of dramatic short-term alterations in response to stimulation. Enlargement of the elements of the Golgi complex in response to stimulation in vivo have been noted by others in the exocrine pancreas (Kern and Kern, 1969; Ribet et al., 1969) and in a number of endocrine cell types (Fawcett et al., 1969).

IV. INTERRELATIONSHIPSOF INTRACELLULAR MEMBRANES DURING THE SECRETORY PROCESS

From the above discussion of the secretory process in the exocrine pancreatic cell, it is clear that all the steps in the sequence are intimately associated with the intracellular membrane systems of the cell. I n addition, although the data so far discussed pertain strictly to the kinetics of transport and discharge of the exportable products, it is clear that the membrane containers themselves are undergoing relocations in concert with movement of their content. This is particularly evident in the case of zymogen granule discharge. Finally, in view of the polarity of transport, we can also surmise that the membrane-membrane interactions that accompany transport and discharge are subject to restrictions. At present little direct evidence is available concerning either the kinetics .of movement of the membrane containers (more specifically their macromolecular constituents) or of their origin and fate during the secretory process. Nevertheless, the data given above indicate that secretory proteins can be transported, concentrated, stored in zymogen granules and discharged from the cell in the virtually complete absence of ongoing protein synthesis for periods of up to -5 hours. This period covers completely the time re-

334

J.

D. JAMIESON

quired for the passage of a wave of labeled proteins through the cell (which takes 60-90 minutes). Our data in general suggest that the synchronous or parallel synthesis of specific couplers, carriers, etc., is not required for the process and in particular indicate that the synthesis of membrane proteins for the containers is not tightly coupled to the handling of the content. The conclusion implies that the cell extensively reutilizes its membranes or macromolecular components thereof during the secretory process (or possesses a large pool of membranes or their macromolecular precursors), possibly via a membrane recirculation scheme of the type originally proposed by Palade (1959). As we have previously suggested (Jamieson and Palade, 1968b, 1971b), two levels of membrane circulation or reutilization in the cell can be envisioned: (1) between the RER and the elements of the Golgi complex and (2) between this complex and the cell surface. The first of these circulatory systems may be represented by the small vesicles of the Golgi periphery which, according to the data discussed above, act as shuttle carriers between the compartments, although the morphological evidence to date cannot definitively rule out the existence of functionally discontinuous channels. Let us assume for the moment, however, that transport over this first link of the pathway is vesicle-mediated. If we then also assume that the concentration of proteins contained in the vesicles is equal to or less than that in the condensing vacuole, and if we assume that the condensing vacuole results either from the coalescence of many small vesicles or by discharge from these vesicles into preexisting empty vacuoles, then in view of the relative surface-to-volume ratios of the two compartments it is clear that a large (-85-fold) excess of membrane should accumulate during condensing vacuole filling. Since this does not occur the excess membrane is either broken down to its molecular constituents and available for reuse or is cycled back, possibly to the transitional elements of the RER. If tubular connections between the compartments are envisaged, then it is not necessary to postulate membrane circulation or movement. Nevertheless, a continued inputJ of membrane to the Golgi complex is required to offset that lost to the forming storage granules, regardless of the method of transport. As will be discussed below membrane input can be postulated to occur via a second level of membrane circulation between the cell surface and the Golgi complex. In any event membrane circulation must be efficient or the precursor pool large, since morphological observations show that the membrane amount and distribution in the Golgi complex is not perceptibly diminished when transport proceeds in the absence of protein synthesis. The morphological evidence for the outgoing link of the second circulatory pathway mentioned above is more secure. In this case the equivalent of the shuttle carrier is the zymogen granule, which clearly moves from the


335

Golgi region to the cell apex, where it contributes its membrane to the cell surface during exocytosis. The return link of the pathway is not yet clear, although, as originally proposed by Palade (1959), it may be mediated by small vesicles which pinch off from the apical plasmalemma and move back into the cell, possibly to the Golgi complex, for reuse. Others have also proposed that a similar membrane circulation from the cell surface may occur in exocrine cells of the rat parotid (Amsterdam et al., 1969) and in the adrenal medulla (Douglas, 1968). Alternatively of course the excess membrane contributed to the cell surface may be disassembled into its macromolecular components and subsequently reutilized (Fawcett, 1962; Hokixi, 1968). In any case, the cell must, possess some mechanism to dispose of the excess of membrane contributed to the cell surface during secretory granule discharge and to replenish that lost] from the Golgi complex during secretory product formation if the membrane balance of the cell is to be maintained. PossibIy the increase in the membrane amount of the Golgi complex following hyperstimulation may reflect the temporary overcompensation of this second circulation system in response to massive granule discharge, although as already mentioned it may simply represent the need to process the exportable products more rapidly. The obvious question which arises in any scheme in which it is postulated that membranes are translocated or circulated is whether or not wholesale mixing of membrane constituents occurs or if, a t the other extreme, the portion of membrane (represented by a vesicle or vacuole) under consideration remains discrete. In the case of the hepatocyte, available enzymatic evidence indicates that membrane translocations are nonrandom; i.e., the membranes of the RER, Golgi elements, and plasmalemma do not mix indiscriminately during intracellular transport of their secreted products (serum proteins and lipoproteins; Ehrenreich, 1969; Siekevitz, 1970). Similar conclusions appear to pertain to the exocrine pancreatic cell. For instance, the lipid composition of the RER (i.e., rough microsomes) is low in cholesterol and sphingomyelin whereas the other smooth membranous elements of the cell (smooth membranous vesicles derived from the Golgi periphery, zymogen granule membranes, and the total plasmalemma) possess substantial and comparable amounts of these two lipids (Meldolesi et al., 1971a). Similarly, the smooth membranous systems of the exocrine cell possess in common, but a t different absolute levels, a group of marker enzymes of the plasmalemmal type (e.g., 5’-nucleotidase, hlg2+-ATPase, and P-leucyl naphthylamidase) whereas these enzyme activities are low or absent in rough microsomes (Meldolesi et al., 1971b). Further, the smooth microsomes are unique in that they possess thiamine pyrophosphatase and ADPase activities although they share in common with rough microsomes two of the electron transport systems (NADH and NADPH-cytochrome c

336

J. D. JAMIESON

reductase). However, since specific additions, deletions, and modifications of the protein and lipid components of the membrane patches may occur during transport,, the conclusion that membranes do not mix during transport must be made with caution. More definite experiments in which the turnover times of specific components of the membrane systems under consideration are measured will help to solve this problem. I n the case of the hepatocyte, however, the turnover times for the phospholipids and proteins of intracellular membranes (Omura et al., 1967) are orders of magnitude longer than those for their contained exportable products, plasma proteins, and lipoproteins. Finally, as mentioned above, the transport sequence is highly polarized, being accomplished b y a well defined, apparently invariant, and ordered sequence of membrane-membrane interactions. This is particularly clear in the case of the zymogen granule which fuses only with that segment of the apical plasmalemma located distal to the junctional elements that seal off the lateral intercellular spaces from the luminal space. It never fuses with the lateral or basal plasmalemma, with which it is often in close proximity. Likewise, Golgi vesicles never appear to transport their contents to the mature zymogen granules nor do condensing vacuoles bypass mature granules and discharge their contents to the acinar lumen. Evidently nothing is known about the factors involved in determining specific membrane-membrane interaction. One possibility is that the regions of prospective membrane fusion possess specific recognition sites. These recognition sites formally might be analogous to those involved in hormone and viral interactions with the plasma membranes, but in this case capable of effecting specific membrane-membrane interactions intracellularly. In support of this is the not infrequent observation in both pancreatic and parotid exocrine cells that the receiving face of the condensing vacuoles and many of the Golgi vesicles, and some of the transitional elements, possess a morphologically recognizable cytoplasmic coat to their limiting membranes. Whether this coat defines LLpatches” of membrane which are in transit, or is a reflection of the recognition site mentioned above is open to question. For the future it will be important to determine what, if any, chemical modifications of intracellular membranes occur in the course of intracellular transport and the attendant membrane fusion and fission. REFERENCES Amsterdam, A,, Ohad, I., and Schramm, M. (1969). J. Cell Biol. 41, 753. Ashley, C. A., and Peters, T. (1969). J . Cell Bid. 43, 237. Babad, H., Ben-Zvi, R., Bdolah, A., and Schramrn, M. (1967). Eur. J. Biochem. 1, 96. Bainton, D. F., and Farquhar, M. G. (1970). J . Cell Bid. 45, 54. Baudhuin, P., Beaufay, H., and deDuve, C. (1965). J . Cell Biol. 26, 219.


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The Movement of Water Across VasopressinSensitive Epithelia RICHARD M . H A Y S Department of Medicine. .4 lbert E i n s t e i n College of Medicine New York New York

.

This paper is dedicated to the memory of Professor Aharon Katchalsky

I . Introduction . . . . . . . . . . . . . . I1. The Pore Enlargement Hypothesis . . . . . . . . . A . Formulation of the Pore Enlargement Hypothesis . . . B . The Dual Barrier Hypothesis . . . . . . . . . C . The Activation Energy for Water Diffusion . . . . . I11. The True Iliffusion Rate of Water across the Luminal Membrane A . The Effect of Unstirred Layers . . . . . . . . . B . Effect of Supporting Layer . . . . . . . . . . C . Discussion . . . . . . . . . . . . . . . D. Contribution of Epithelial Cell Components . . . . . E . L, and the “Sweeping Away” Effect . . . . . . . F. Summary . . . . . . . . . . . . . . . IV . The Activation Energy for Water Diffusion . . . . . . . V. The Solvent Drag Effect . . . . . . . . . . . . A . Early Studies . . . . . . . . . . . . . . B. Effect of the Unstirred Layer . . . . . . . . . VI . Conclusions . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . .

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339 340 341 344 345 346 347 350 353 355 355 356 357 359 359 361 364 365

.

1 INTRODUCTION

It is generally believed that water moves across cell membranes by a process of Poiseuille flow through aqueous channels. or pores. in the mem339

340

RICHARD M. HAYS

brane. The pore hypothesis is based on studies in amphibian skin (KoefoedJohnsen and Ussing, 1953; Andersen and Ussing, 1957) and capillaries (Pappenheimer, 1953). An important application of the pore hypothesis was made by Koefoed-Johnsen and Ussing (1953) to account for the dramatic increase in water flow across vasopressin-sensitive epithelia following stimulation by the hormone. It was proposed that vasopressin enlarged pores in the membrane, permitting an increase of Poiseuille flow, with only a small accompanying increase in the diffusion rate of labeled water. This chapter will review the evidence for the pore enlargement hypothesis and will present recent studies, primarily from the author's laboratory, which provide a basis for an alternative view of water movement across vasopressin-sensitive epithelia, and cell membranes in general.*

II. THE PORE ENLARGEMENT HYPOTHESIS

Let us begin by stating what appears to be an established fact for all epithelia, and for red cells as well: namely, that the net water movement attributable to the process of diffusion accounts for only a fraction of the total osmotic flow of water across the tissue. By diffusion is meant the movement of individual water molecules across the cell membrane. This is experimentally determined by measuring the diffusion rate of isotopically labeled water across the tissue. One may express the diffusion of labeled water (COT)? by the following expression:

where JTis the flow of tritiated water (THO) in moles.cm-2.sec-1 and ACT is the difference in isotope concentration across the membrane. The hydraulic or osmotic flow coefficient (Lp), on the other hand, is the total flow of water moving down a hydrostatic or osmotic gradient, as determined gravimetrically, by dye dilution, or other suitable techniques

L = -J" AP

* For a general review of water movement, see R. E. Forster's article on the transport of water in erythrocytes in Volume 2 of this series. t The term OT wiil be used throughout the text. OT may be converted to the units of LP by multiplying by the molar volume of water (cubic centimeters per mole). A term closely related to OT, K,,,,. THO, denotes the permeability coefficient for water; it will generally be the term used in the discussion of experimental data.

vw,

34 1

THE MOVEMENT OF WATER

TABLE I OSMOTICA N D DIFFUSIONAL NETFLOW I N REPRESENTATIVE TISSUES

(mo1.dyn-*.secc1) Red blood cell Frog gastric mucosa Toad bladder Control Vasopressin

x

21.0 0.2

50 5

0.35 0.60

2.0 80

10'4

2.5 25.0

4.2 16

6.0 133

8.4 41

or (provided the reflection coefficient is 1) : L,

=

AT JV

(3)

where J, is the volume flow in ml.cm-2.sec-1, A p is the difference in hydrostatic pressure, and AT the difference in osmotic pressure across the membrane. If, in a given membrane, water movement proceeded entirely by a process of diffusion, there would be no discrepancy between L, and P w uand ~ the ratio L,/VWuT would be 1 (see Thau et al., 1966). I n Table I, L , and PwuT are compared in three tissues. It is clear that there is a discrepancy between total osmotic flow and flow predicted from diffusion; the discrepancy is 2.5 to 1 in the case of the red cell (Paganelli and Solomon, 1957), and 25 to 1 in the case of frog gastric mucosa (Durbin et al., 1956). Turning to vasopressin-sensitive epithelia, the isolated urinary bladder of the toad provides a particularly striking example of this apparent discrepancy (Hays and Leaf, 1962a). I n the absence of vasopressin, osmotic flow is six times that predicted from diffusion alone. After vasopressin, there is a 40-fold increase in osmotic flow, but only a 70% increase in the diffusion rate of tritiated water, and the ratio L , to vWuTrises to over 100 to 1. In mammalian renal epithelia, vasopressin also increases L , to a far greater extent than PwwT;L , rises 25-fold after hormone in isolated rabbit collecting tubule (Burg et al., 1970) and 7-fold in rat collecting duct (Morgan et al., 1968), while v w u T approximately doubles in both of these tissues. The conclusion drawn from all these studies is that whatever the mechanism responsible for water movement across cell membranes, the process of diffusion plays a very small part, and some nondiffusional mechanism appears to be the dominant one. This appears to be especially true in vasopressin-sensitive tissues.

342

RICHARD M. HAYS

TABLE I1 COMPOUNDS NOT PENETRATIh’G THE TOADBLADDER AFTER VASOPRESSIN” MORERAPIDLY Mean permeability coefficients Ktrsns(10-7 cm-sec-I) Species Inorganic ions Sodium (S to M) Potassium Chloride Organic ions Thiocyanate Methyl sulfate Choline Glycine Organic molecules Formaldehyde Acetanilide Glycerol Sucrose

Before vasopressin

After vasopressin

2.8 26 13

2.9 29 10

8.3 4.8 9.1 2.2

8.6 3.6 9.5 2.6

25 1 927 4.1 8.9

229 917 4.3 5.1

Adapted from Leaf and Hays (1962).

A. Formulation of the Pore Enlargement Hypothesis

In 1953, Koefoed-Johnsen and Ussing published their important analysis of water flow across the frog skin. They pointed out that in a cell membrane penetrated by pores, total water flow in the presence of an osmotic gradient would proceed by a process of bulk, or Poiseuille flow ; hence : n d L, = -

8qA~

(4)

where n is the number of pores, r the pore radius, q the bulk viscosity coefficient of water, and Ax the thickness of the membrane. The diffusion rate of labeled water, on the other hand, would be expressed by

where D is the self-diffusion coefficientof water. Clearly, in any membrane with pore radii greater than that of the water

343


molecule, Poiseuille flow will be greater tjhan that predicted from diffusion, since the former is a function of r4, and the latter of r2. In addition, a small increase in pore radius, produced by vasopressin, for example, would increase flow far more than diffusion, and widen the discrepancy between flow and diffusion. It seemed reasonable to suggest that vasopressin acted by increasing the radius of aqueous pores in the cell membrane. While earlier workers were careful to avoid giving specific dimensions to these pores, the temptation to do so proved too strong for some. If one writes Eqs. (4)and ( 5 ) as a ratio, a number of common terms cancel out, and one is left with the simple expression:

Since r is the only unknown in this equation, it is possible to estimate TABLE I11 COMPOUNDS PENETRATING THE TOAD BLADDER AFTER VASOPRESSIN~ MORERAPIDLY Mean permeability coefficients K,,,,, (10-7 cm-sec-l) ~

~~

Before vasopressin

After vasopressin

Amides Urea Acetamide Propionamide Butyramide Cyanamide Urethane Dimethyl formamide Nicotinamide Methyl acetamide

26 44 97 132 127 58 1 174 26 87

274 196 215 180 282 639 259 40 242

Water and alcohols Water Methanol Ethanol

944 825 575

1580 913 678

Inorganic ions Sodium (M to S)

36

52

Species

a Adapted from Leaf and Hays (1962). Thiourea has recently been added to this list (see text).

344

RICHARD M. HAYS

pore radius by determining L , and W T experimentally, and expressing them as a ratio. Representative pore radii are shown in column 5 of Table I. 6. The Dual Barrier Hypothesis

If aqueous channels were enlarged by vasopressin to the extent estimated in the toad bladder, one would predict that all small solutes would penetrate the bladder more rapidly in the presence of the hormone. This is not the case; Table I1 (Leaf and Hays, 1962) shows that most small solutes show no change in their permeability coefficients after vasopressin. The amides, thiourea (S. Levine, N. Franki, and R. M. Hays, 1972, unpublished data) and certain alcohols are interesting exceptions to this rule; their permeability increases significantly after vasopressin (Table 111). This holds for the amides irrespective of their mean or cylindrical radii (Hays and Harkness, 1970) and suggests a specific interaction between the membrane and the amide group induced by the hormone. With these exceptions, however, it appears that small solutes continue to be excluded in the face of a large increase in estimated pore radius. To deal with this apparent contradiction, it was proposed for both toad skin (Anderaen and Ussing, 1957) and toad bladder (Leaf and Hays, 1962; Lichtenstein and Leaf, 1966) that the luminal membrane might include LUMINAL MEMBRANE (a) (bl DENSE POROUS

CONTROL

VASOPRESSIN

FIG.1. Schematic representation of the pore enlargement hypothesis of vasopressin action. Hydraulic flow of water (L,) is shown by solid arrows, and diffusional flow (OT) by open arrows. L, and wT are determined by the underlying barrier, both in the control state and in the presence of vasopressin. Vasopressin increases the porosity of the underlying barrier, resulting in Poiseuille flow of water, and a relatively small increase in WT. It has been suggested (Lichtenstein and Leaf, 1966) that vasopressin also increases the permeability of the dense diffusion barrier to urea and sodium, but not to other solutes. From Hays (1968).

345


3.53.43.3

-

0 0

3.2 3.1

-

3.02.9282.7 2.62.5 2.42.32.22.1 L

3.1

I

32

I

3.3

I

3.4

vT

I

3.5

I

3.6

I

.

3.7

1

38

lo3

FIG.2. Arrhenius plot of the temperature dependence for diffusion of THO acrom the toad bladder, in the presence and in the absence of vasopressin. The open and filled circles represent individual determinations of permeability coefficients for tritiated water, plotted against the reciprocal of the absolute temperature. The solid and dashed lines were fitted by the method of least squares; the regression equations are: y = -2.15 z 10.26 (no hormone) and y = -0.90 z 6.35 (following hormone). The difference in calculated activation energies is highly significant ( p < 0,001). With vasopressin (open circles) E = 4.1 kcal/mole; without vasopressin, (closed circles) 9.8 kcal/mole. From Hays and Leaf (1962b).

+

+

two barriers in series-one a fine diffusion barrier, capable of sieving out small solutes, and the second a vasopressin-sensitive barrier, which becomes highly porous in the presence of the hormone and is responsible for the bulk flow of watcr. The dual barrier model is shown in Fig. 1. This appeared to answer the question of how a high degree of membrane selectivity could exist in the presence of large pores. C. Activation Energy for Water Diffusion

There was tl second piece of evidence that appeared to support the pore enlargement hypothesis : the apparent decrease in activation energy for the diffusion of tritiated water across the toad bladder after vasopressin (Hays

346

RICHARD M. HAYS

f VASOPRESSIN

Fro. 3. Schematic representation of water molecules in aqueous pores before and after an increase in pore radius. From Hays and Leaf (1962b).

and Leaf, 1962b). These studies are shown in an Arrhcnius plot (Fig. 2). In the absence of vasopressin, activation energy was high ; after vasopressin, it decreased to 4.1 kcal per mole, approximately the value for the diffusion of water in liquid water (Wang et al., 1953). Thc experiment could be interpreted as shown in Fig. 3. In the absence of vasopressin, channels are small and water molecules are highly bonded to each other and to the membrane; thus activation energy (an index of the extent of hydrogen bonding of water molecules) is high. When the channels enlarge, the water in the central core assumes the properties of liquid water, and activation energy falls.

111. THE TRUE DIFFUSION RATE OF WATER ACROSS THE LUMINAL MEMBRANE

It is important to note that both pieces of evidence supporting the pore enlargement hypothesis (the discrepancy between total osmotic flow and net diffusional flow, and the fall in activation energy), depend on an accurate determination of the rate of diffusion of tritiated water across the luminal membrane of the epithelial cell. This is the membrane that is transformed by vasopressin, as shown by Maffly et al. (1960)’ who found that labeling of intracellular water by 14C urea was significantly increased following vasopressin, if the urea was placed in the luminal bathing medium. Identical results were obtained by Hays and Leaf (1962a) for tritiated water. Civan and Frazier (1968) found that vasopressin decreased the dc resistance of the luminal permeability barricr, accounting for the increased entry of sodium into the cell. While the luminal membrane is therefore the barrier of interest, the measurement of diffusion must be made across the entire thickness of the bladder as it exists in Ringer’s solution. This situation is shown in Fig. 4, which schematically depicts the luminal membrane.


347

In series with this barrier are a number of others, including stagnant or unstirred layers of water, cell cytoplasm and intercellular spaces, and the thick layer of collagen and smooth muscle supporting the epithelial cells. The experiments to follow will show that these “extraneous” barriers greatly retard the diffusion rate of water, but not osmotic flow. Correction for the effect of these barriers results in a completely different picture of water movement across the luminal membrane and the action of vasopressin. A. The Effect of Unstirred layers

It has been recognized for some time that stagnant layers of water in apposition to synthetic and biological membranes can impede the rate of diffusion of molecules moving between the bulk solutions bathing the membrane (Teorell, 1937; Cinsburg and Katchalsky, 1963; Dainty, 1963; Hanai and Haydon, 1966; Cass and Finkelstein, 1967). A study by Dainty and Housc (1966) of the effect of stirring on the diffusion rate of tritiated

FIG.4. Extraneous barriers in an epithelial tissue. The luminal membrane (under magnifying glass) is the membrane transformed by vasopressin. I n series with the membrane are unstirred layers in the bulk solution, cell cytoplasm and organelles, basoIatera1 cell membrane, intercellular channels, and the supporting layer (stippled layer).

348

RICHARD M. HAYS

6ooor

I

I

1

200

400

I 600

I

800

STIRRING SPEED (rpm)

FIG.5. Effect of stirring rate on Ktrana THO. Open symbols are vasopressin-treated bladders; filled synibols are control bladders. Diamond symbols are the earlier values obtained in conventional chambers. Vertical bars are f 1 SE. From Hays and Franki (1970).

water across frog skin showed that wT across the unstimulated skin approximately doubled in the presence of vigorous stirring. There was little effect of stirring on L,. There was, however, no apparent effect of stirring on W T following vasopressin. While the magnitude of the unstirred layer effect in their experiments was small, it led the authors to question the existence of pores in the amphibian skin. Our studies of the unstirred layer effect in the vasoprcssin-treated toad bladder showed a significant relationship between stirring rate and the rate of diffusion of tritiated water (Ke,,,THO) (Hays and Franki, 1970). I n these experiments, conducted in a diffusion chamber, mechanical stirring with Teflon paddles was used, rather than the conventional type of circulation of the bulk fluid by a column of air bubbles (Ussing and Zerahn, 1951). Figure 5 shows the effect of stirring rate on the permeability coefficient of water across the bladder, in the presence and in the absence of vasopressin. In the absence of vasopressin, stirring had little effect. After vasopressin, however, there was a striking increase in diffusion rate as a function of to over stirring speed. The permeability coefficient went from 1 X 5 X cmesec-1, considerably higher than in our original experiments in unstirred chambers, indicated by the diamonds (Hays and Leaf, 1962a). I n contrast to its effect on diffusion rate, stirring had no significant effect on osmotic flow (Table IV). Stirring had no adverse effect on the bladder; the usual rise in potential following vasopressin was seen in these experiments.

349


TABLE IV

EFFECT OF STIRRING SPEEDO N OSMOTIC FLOW (5 PAIRED EXPERIMENTS)= Speed (rpm)

Osmotic flow (ml cm-2 hr-1)

225 800

0.158 f 0.016 (SE) 0.176 f 0.025 (SE)

- -

A = 0.018 i 0.014; p 4

< 0.3

From Hays and Franki (1970).

Unstirred layers, therefore, retard diffusion greatly after vasopressin, but have no appreciable effect on osmotic flow. Correction for the unstirred layer effect yields a value for L , / P w w ~considerably below the value shown in Table I, and therefore reduces the discrepancy between osmotic and diffusional net flow.

FIG.6. Toad bladder, before and after removal of epithelial cells. I n the intact bladder (a), a row of epithelial cells is present along the left border of the tissue. After scraping, the supporting layer is shown (b). Hematoxylin and eosin stain x 180.

350

B.

RICHARD M. HAYS

Effect of Supporting layer

The next extraneous layer to be considered was the layer of collagen and smooth muscle supporting the bladder epithelial cells. This layer, 50-100 p in thickness, is shown in Fig. 6. The question was whether the supporting layer, a thick but highly porous structure, would retard the diffusion rate of water, but not osmotic flow. If this were so, the true value for wT across the epithelial cell layer would be higher than that measured across the intact bladder, further reducing the discrepancy between L , and wT. Earlier experiments with a bilayered synthetic membrane (Hays, 1968) had indicated that when the two layers differed significantly in structure, one layer could be rate-limiting for WT, and the second layer rate-limiting for L,. These experiments will be briefly reviewed. 1. L,

AND W T IN A

BILAYERED SYNTHETIC MEMBRANE

The cellulose acetate desalination membrane, developed by Loeb (1966) and associates, has the structure shown in Fig. 7. It consists of a thin, dense “skin,” approximately 0.25 p in thickness, and a thick supporting layer 1OOp in thickness. When salt or brackish water is forced under pressure against the skin, water flows across the membrane, and over 90% of the salt remains behind. L , and WT measured across the intact membrane were comparable to those shown in Table I for the toad bladder; the values

FIQ.7. Cellulose acetate desalination membrane. A thin skin (a) overlies a thick highly porous layer (b). From Hays (1968).

35 1


TABLE V COEFFICIENTS OF Int,act

DESALINATION MEMBRANE^ Skin removed

Skin

1,795 1.35

70.6 11.86

L, 66.8* 1.19

VHWT

a

From Hays (1968).

All values in (mol.dyn-'.sec-l) X 10".

for the synthetic membrane are shown in columns 1 and 2 of Table V. The ratio L,/PwwTwas 56, and from Eq. ( 6 ) , thc porc radius estimated for the membrane was 25 A. It appeared unlikely that a membrane capable of sieving out salt had a pore radius as large as this, and it was necessary to determine the cocfficients for the skin and supporting layer separately. The skin could be removed by mechanical means, and L , and W T determined across the remaining supporting layer. The results are shown in column two of Table V. L , increased 20-fold, but WT showed virtually no increase. Therefore, the thin skin was rate-limiting for L , and the supporting layer for wT. While no direct measurements could be made for the skin alone, the coefficients for the skin could be estimated from the series barrier equation (Leaf, 1959; Iiatchalsky and Kedem, 1962), which states that the total resistance of a complcx membrane is equal to the sum of the resistances of the separate layers. Thus:

and 1 WT

-

1

1 +-Wdb)

(8)

where (a) and (b) refer to the skin and supporting layer, respectively. Since L , and W T were known for the intact membrane and the supporting layer, these coefficients could bc estimated from Eqs. (7) and (8) for the skin. The values are shown in the last column of Table V. W T across the skin was 10 times greater than that determined for the intact mem~ the brane, while L , was approximately the same. Therefore L , / v W w for skin, which is the critical barrier, was reduced to 7, and the cstimatcd pore radius for the skin was 9 8, a more rensonablc value.

352

2. EFFECT OF SUPPORTING LAYEROF BLADDER ON

RICHARD M. HAYS

WT

The contribution of the supporting layer of the bladder to the resistance to diffusion and flow was determined with the same experimental approach used in the synthetic membrane. The permeability coefficient for tritiated water was determined across the intact bladder, and across the same bladder with the epithelial cells removed by scraping (Fig. 6b). From the series barrier equation, W T across the epithelial layer alone could be estimated. Since it was important to determine wT across the intact bladder and supporting layer in thd absence of unstirred layers, the diffusion rate of water was determined as a function of stirring speed. Teflon impellers, rather than paddles, were used in this experiment; they were positioned close to the bladder, and could therefore be turned a t lower speeds. The results are shown in Fig. 8. By plotting the reciprocals of the values shown in this figure, we obtained the results shown in Fig. 9. The intercepts at the vertical axis give the values for the diffusion rate of water a t infinite stirring speed; that is, in the complete absence of unst#irredlayers. For the intact bladder, Kt,,,,THO is 7.1 X 10-4 cm-sec-I. For the supporting layer, Ktr,,, is 11.3 X cm.sec-'. Thue, more than half the resistance to the diffusion of water resides in the supporting layer. From the series barrier equation, the permeability coefficient for water across the epithelial layer alone becomes 19 X cm-sec-I. Therefore in contrast to earlier experiments with the conventional chamber, in which the diffusion rate of water appeared to increase by only 70% after vasopressin (Table I), the true diffusion rate across the epithelial cell layer increases 14fold. The supporting layer, which offered a significant resistance to wT, offered

FIQ.8. Effect of stirring rate on KtrnnTHO s of intact bladder (filled circles) and supporting layer (open circles). An impeller type of stirring apparatus was used. Vertical bars, f 1 SE.

353


0.30-

0.25-

0 051

0

I 02

I

04

I 06

I

08

I 10

I 12

I 14

1 16

J 1.8

FIG.9. Plot of the reciprocals of the points shown in Fig. 8.

virtually no resistance to L , (Hays and Franki, 1970). Therefore, as in the synthetic membrane, the epithelial cell layer (corresponding to the skin) was rate limiting for osmotic flow. C. Dicussion

At this point, it is useful to consider to what extent the above experiments alter our concept of the action of vasopressin. There is little doubt that the diffusion rate of water rises sharply after hormone. However, the increase in L , is a t least 40-fold,* and while W T across the epithelial layer increases approximately 20-fold, we still fall short of accounting for the entire vasopressin effect by the process of diffusion alone. But it must be kept in mind that the hormone is acting on the thin luminal membrane of the epithelial cell, and that our estimates of W T are for the entire cell thickness. The question then becomes whether barriers in series with the luminal cell membrane (cell cytoplasm, intracellular structures such as endoplasmic reticulum and the large nucleus, the basolateral cell membrane and the intercellular channels) provide significant resistance to diffusion.

* The actual increase in L, is probably greater than 4@fold, owing t o the “sweeping away” effect (see Section 111,E).

354

RICHARD M. HAYS

FIG.10. Electron micrograph of toad bladder epithelial cells. A mitochondria-rich cell occupies most of the field. Granular cells are on either side. The lunlinal surface is a t the top of the picture; the large nucleus is a t the bottom. mv, microvilli; g, granules; m, mitochondria; jc, junctional complexes; cf, convoluted folds; d, desmosomes. From Hays et al. (1965). X 13,200.


355

D. Contribution of Epithelial Cell Components

It is generally assumed that the thin layer of cell cytoplasm provides a negligible resistance to the diffusion of water. If the path length (Ax) for the cytoplasm of the epithelial cell is taken as l o p , and the cytoplasm is assumed to have the properties of bulk water, then the permeability coefficient for water diffusing across the cytoplasm will be very high, in the neighborhood of 240 X lo-* cm-sec-’. Since Ka,,,THO across the intact epithelial cell, including the luminal membrane, is 19 X lo-* cm-sec-l, the cytoplasmic resistance to diffusion, expressed as a reciprocal of the cytowould be negligible. plasmic Ktrane, However, it, is probably erroneous to consider the cell cytoplasm as simply a thin film of water. The toad bladder epithelial cell (Fig. lo), like all epithelial cells, is filled with structures including endoplasmic reticulum, mitochondria, granules, and a large nucleus. Diffusion may be greatly impeded by these intracellular structures. Osmotic flow, on the other hand, would be relatively less hindered, since spaces exist between the structures. To the extent that these elements provide a real resistance to diffusion,the diffusion rate of water across the luminal membrane would be higher than that across the entire epithelial cell. Thus, we can write: 1 1 1 (9) wT(epithelia1 layer) wT(lumina1 membrane) -k wT(intracellu1ar)

This treatment of the problem is completely hypothetical, of course. It also neglects the possible retarding effects of the basolateral cell membrane and the intercellular spaces on diffusion. Other workers have also proposed a rate-limiting role for cell cytoplasm for the diffusion of water and solutes; the reader is referred to the recent review of the problem by Fenichel and Horowitz (1969). Schafer and Andreoli (1972) have recently presented experimental evidence in vasopressin-treated, isolated rabbit cortical collecting tubules that cytoplasm, basolateral cell membranes and basement membrane offer a significant resistance to water diffusion. Using 5-hydroxyindole, a lipophilic molecule whose penetration of the cell appeared not to be limited by the luminal membrane, these workers estimated that the diffusional resistance of structures beyond the luminal membrane was 15-25 times that predicted for an equivalent thickness ( 6 ~ of ) water. A resistance of this magnitude is considerably greater than that required for the toad bladder epithelial cell to achieve the total increase in W T required for the “diffusional” model of vasopressin action. E. L, and the “Sweeping Away” Effect

Dainty (1963) called attention to the fact that osmotic flow across a membrane has the effect of concentrating the solution on one side of the

356

RICHARD M. HAYS

membrane, and diluting the solution on the other side. This has been termed the “sweeping away” effect. As a result of this phenomenon, osmotic flow in the steady state is significantly lower than a t the commencement of flow (zero time). L,, which is a measure of the flow per osmotic driving force, will be underestimated as a result. This has been shown to be the case in giant algal cells (Barry and Hope, 1969), and in rabbit gallbladder (Wright et al., 1972). We have obtained an estimate of the “sweeping away” effect in toad bladders pretreated with vasopressin by rapid serial determinations of net water movement across the bladder following the institution of an osmotic gradient (R. 1LI. Hays and N. Franki, 1972, unpublished observations). By extrapolation, we determined net water loss a t zero time, and found that it was three times that in the steady state. Thus, as an approximation, the increase in L, following vasopressin is three times that shown in Table I, or 120-fold. F. Summary

This section began with the question of the relative roles of diffusion and Poiseuille flow in the action of vasopressin. Previous experiments in a conventional diffusion chamber suggested that diffusion contributed only a small fraction of the total osmotic flow, since L , increased 40-fold, and W T only 70% in the presence of hormone. This discrepancy could be explained if vasopressin increased the radius of aqueous channels in the membrane. Recent studies have changed our picture of hormone action in two ways. First, L , at zero time appears to increase to an even greater extent than 40-fold, when one takes into account the rapid dissipation of the osmotic gradient due to the “sweeping away” effect. An increase of 120-fold may be closer to the truth. Second, the increase in W T has been greatly underestimated. Consideration of the retarding effects of extraneous layers in series with the luminal membrane permits the following estimates of the extent to which W T increases after vasopressin: (1) unstirred layers: approximately 7-fold ; (2) supporting layer : approximately %fold. Taking these two barriers into account, from Fig. 9, wT increases across the epithelial cells approximately 19-fold. If we are to attribute the 120-fold increase in water flow to a process of diffusion, rather than pore enlargement, it is necessary to postulate an additional 6-fold resistance t o diffusion across the cell cytoplasm, basolateral membrane, and intercellular space. While experimental evidence does exist for such cellular constraints for diffusion in other tissues, a direct estimate of their role in the toad bladder epithelial cell has not been made. Until this is done, it is reasonable to conclude that water diffusion increases sharply following vasopressin, and may eventually be shown to be solely responsible for the increase in osmotic

357


flow. If this is the case, vasopressin would increase the number, rather than the size of sites for water diffusion in th r luminal mrmbrane. We may now turn to a consideration of the physical properties of these membrane sites. The concluding portion of this chaptrr will deal with the activation rnergy for water diffusion, and the solvent drag effect. Evidence will be presented that unstirrcd layers have important effects on both phenomena.

IV. THE ACTIVATION ENERGY FOR WATER DIFFUSION

The apparent fall in activation energy ( E A ) for water diffusion following vasopressin (Fig. 2) supported the pore enlargement hypothesis. However, since the estimate of EA depends on an accurate determination of Kt,,,,THO, the problem was restudied (Hays et al., 1971) with mechanical stirring to determine to what extent unstirred layers had entered into our earlier calculations. Table VI shows the effect of stirring rate on the measured activation energy for water diffusion across th r intact toad bladder. These experiments wrre performed with paired half bladders, one in the cold, and onr a t room temperature. At the lowest stirring speed, EA is relatively low; as stirring rate increases, E A increases, indicating that our original value of 4.1 kcal-mole-' after vasopressin really represented the activation rnergy for diffusion across unstirred layers of water, rather than across the luminal membrane. Using the series barrier equation, and the experimental protocol outlined in Section III,B, we were able to estimate EA across the epithelial layer alone before and after vasopressin. The results are shown in Table VII. EA is high both in the absence and presence TABLE VI

EFFECTOF STIRRING SPEEDO N E A INTACT,VASOPRESSIN-TREATED BLADDERSQ

OF

Stirring speed (rpm) 60 (6) 128 (6) 580 (7) 800 (8) ~~

~~

a

EA (kcal-mole-') 6.1 7.0 8.5 9.3

f 0.9

f 0.7

f0.7 f 0.8

~

From Hays et al. (1971).

(SE)

TABLE VII THO DIFFUSION ACROSS INTACT BLADDER, SUPPORTINQ LAYER,AND EPITHELIAL LAYEW

K trans, room temperature (em-sec-l X lo7) Vasopressin Absent (10) Present (7)

Intact

Supp.b

Epith.c

Intact

Supp.b

Epith."

En, epithelial (kcal*mole-*)

436 f 49 2140 f 132

4734 f 212 5000 f 300

486 f 60 4046 f 482

1107 + 60 4610 f 361

6968 i 977 8421 f 828

1332 i 86 10644 f 1688

11.7 i 1 . 4 10.6 f 1 . 1

From Hays el al. (1971). Supporting layer. e K,,,,, calculated from series barrier equation for epithelial layer. a

b

THE MOVEMENT

OF WATER

359

+ VASOPRESSIN

FIG.11. Current view of action of vasopressin, in which the number, rather than the size, of aqueous channels is increased. The extent of water bonding is unchanged.

of vasopressin, and the difference between the two values is not significant. This finding is consistent with the view that vasopressin increases the number, rather than the size of aqueous channels in the membrane. This is shown diagrammatically in Fig. 11. Since the geometry of the individual channels does not change, the extent of water bonding would not change, and activation energy before and after vasopressin would stay the same. Further, the opening of many more small channels or sites is consistent with our finding of a large increase in Kt,,,,THO following vasopressin. Finally, the finding that these new channels need not be large would account for the observation (Table 11) that penetration of the bladder by most small solutes does not increase following vasopressin.

V. THE SOLVENT DRAG EFFECT

The statement that there is no increase in the size of aqueous channels following Vasopressin gives no answer to an important question: What is the exact size of these channels? We may go even further and ask whether channels larger than a single water molecule exist in the membrane, and whether bulk flow occurs at all. These questions relate directly to the phenomenon of solvent drag. A. Early Studies

An important part of the evidence for the existence of aqueous channels large enough to permit bulk flow was provided by the observation of Andersen and Ussing (1957) of the solvent drag effect. They reasoned that bulk flow of water through channels would accelerate the movement of

360

RICHARD M. HAYS

I

0.8 0.7

UREA (THEORETICAL

0.6 FLUX RATIO 0.5 J In 1 JZ 0.4

UREA :(OBSERVED) WATER (OBSERVED)

0.3 0.2 0 .I

0

-0.1

1

0

I

50

1

I

I

200

250

I

I00

150

A,

(pl/cm2/hr)

FIG. 12. Effect of water movement (Aw) on the flux ratio of urea. W-labeled urea was used to measure unidirectional flux, J!,and "N-labeled urea to measure simultaneously the opposing unidirectional flux, Jz.Net movements of water were induced by an osmotic gradient across the bladder wall in the presence of vasopressin. In the absence of net movements of water, the unidirectional fluxes for water and urea, respectively, were equal in the two directions across the bladder as indicated by intersection with the ordinate at the origin. The equations for the lines which best represent the observations for water and urea are: y = 0.0016 z 0.028, y = 0.0023 z - 0.0194, respectively. The theoretical regression for urea is also indicated. The shaded area includes twice the standard error of the slope for the urea regression. From Leaf and Hays (1962).

+

small molecules in the direction of flow, and slow down their movement in the opposite direction. The magnitude of this asymmetry could be predicted from their expression for the flux ratio of an uncharged substance:

Here, the logarithm of the ratio of the permeability coefficients of water molecules or solutes moving in opposite directions (Kin and flout) is proportional to net water flow. The other terms in the equation are constants: D is the free diffusion coefficient of the substance, A the fractional area of the membrane open to diffusion, xo the thickness of the membrane, and x

36 1

THC MOVEMENT OF WATER

the distance of the membrane from one boundary. The equation predicts a linear relationship between the logarithm of the flux ratio and the rate of net water transfer. Further, in a porous membrane, the slopes of the regression lines for a solute and for water should be inversely proportional to their free diffusion coefficients. The logarithm of the flux ratios of water, acetamide, and thiourea across the toad skin was shown experimentally to have this linear relationship to net water flow, confirming the prediction of these workers. The solvent drag effect, therefore, appeared to establish the presence of aqueous channels, open to water and small solutes, in which bulk flow occurs. In experiments on toad bladder, using similar chambers in which bubbling provided the stirring, Leaf and Hays (1962) were able to show a solvent drag effect for 14C- and 16N-labeledurea (Fig. 12). Here an asymmetry of movement of urea was seen, although the slope of the regression line for urea was below the theoretical slope predicted from the data on water. B. Effect of the Unctirred layer

At this point, it is important to consider the data from which this figure was made. Table VIII shows the apparent unidirectional flux of tritiated water across the bladder, the net water flux, and, by subtraction, the unidirectional water flux in the opposite direction. The flux ratio for water and its logarithm are shown, and, finally the predicated logarithm of the flux ratio for urea at the same net water flow. We may now ask what these data would look like if we substituted the true value for unidirectional water flux across the epithelial cells, some 19 times higher than its value before vasopressin. Table I X shows a recalculation of the data, where unidirectional flux is now 6840 pl- cm-2 hr-l.

-

TABLE VIII

FLUXRATIOSFOR WATERAND UREAACROSS THE TOAD BLADDER (CONVENTIONAL CHAMBERS) Flux (pl/cmz/hr)

J," -

m-+s

net

s+m

717

186

531

a

Ratio of m

-+

s to s

-+

Jz

1.35 m flux.

J,

In JZ

0.30

J1

In - urea Jz

0.51

362

RICHARD M. HAYS

TABLE IX

FLUXRATIOSFOR WATERAND ACETAMIDE ACROSS THE TOAD BLADDER (MECHANICAL STIRRING) Flux (@l/cm*/hr) m+s

Net

JI

Ji -

s+m

Ji

In - acet-

In -

Jz

Jz

J2

amide 6840

186

6654

1.03

0.03

0.05

Net water movement, which is unaffected by stirring, is the same as in the earlier experiments, but unidirectional flux in the opposite direction is almost equal to the m to s flux, and the logarithm of the flux ratio is close to zero. Therefore, the expected asymmetry of a small solute, such as urea or acetamide, would be quite small and probably undetectable. Figure 13 is a solvent drag experiment across the toad bladder, in which tritiated and I4C-labeled acetamide were used, and in which mechanical stirring was provided. Even at high water flows, no asymmetry can be seen, and the solvent drag effect could not be demonstrated. There was no difficulty in showing asymmetry for acetamide in the conventional bubble chambers (Fig. 14), and asymmetry could also be demonstrated in the mechanically 0.3

0

0 0

0.1 In

0

J ’ o ---------------------8’-””-’ JZ 0

0

-0.1 -

0

-0.2I

I

I

363


r

0.7

AW (pl.cm-2 hr-1)

FIG.14. Effect of water movement on the flux ratio of 3H- and 14C-labeledacetamide in a conventional bubble chamber. A significant ( p < 0.02) relationship could be demonstrated between the logarithm of J , / J 2and Aw. The equation for the line is: y = 0.0016 z - 0.09.

stirred chamber when the scraped supporting layer, a porous membrane, was substituted for the intact bladder (Fig. 15). We would conclude that the unstirred layers in the earlier type of chamber contributed heavily to the apparent solvent drag effect. This finding was unexpected, since urea and acetamide do not move nearly as fast as water, and the unstirred layer effect would not be expected to be this significant. However, it is entirely possible that the older chambers actually create unstirred layers, which are thick enough to be important for these solutes. It is reasonable to conclude from the calculations in Table I X and the experiments shown in Fig. 13 that if solvent drag exists a t all, it should be, and has been, difficult to detect in the toad bladder. The question must be left open, and it is appropriate to consider the possibility that water movement is independent of amide movement, and indeed, of the movement of all small solutes. It is of interest to mention in this connection the recent studies of Handler and co-workers (1969) on the toad bladder, and Macey and Farmer (1970) on the red cell. Both workers have found that certain inhibitors (cycloheximide in the case of the toad bladder, and phloretin in the case of the red cell) can dissociate the movement of urea and water across these tissues, decreasing urea movement, but leaving wat,er flow

364

RICHARD M. HAYS

09r

080706 -

I

0

100

I

I

200 300 A W ( )II. cm-'. h i ' )

I 400

FIG.15. Effect of water movement on flux ratio of aH- and W-labeled acetamide across supporting layer in mechanically stirred chamber. Albumin was used to create an osmotic gradient in these experiments.The regression is significant ( p < 0.02; y = 0.0019 z 0.07).

+

unaffected. Current studies with phloretin in our laboratory (S. Levine, N. Franki, and R. M. Hays, 1972, unpublished data) indicate that, as in the red cell, urea movement across the bladder is strikingly inhibited, while water movement is unimpaired. This supports our view that water and urea move through different pathways in the membrane.

VI. CONCLUSIONS

Extraneous layers of a vasopressin-sensitive epithelium, the urinary bladder of the toad, retard the diffusion rate of tritiated water to a significant extent. When this retarding effect is taken into account, a number of conclusions can be drawn about the effects of vasopressin on the structure of the luminal membrane of the epithelial cell. First, the diffusion rate of water increases dramatically following vasopressin, possibly enough to

365


account fully for the increase in water flow. Second, the activation energy for diffusion remains high following hormone treatment. This indicates that water is moving through the membrane in a highly bonded state and that there may be no change in the physical properties of the aqueous pathway. Vasopressin appears to increase the number, rather than the size of sites for water movement across the membrane. Finally, it cannot be said with certainty whether the membrane sites are channels, or simply points in the membrane through which individual water molecules can move. There now is a question about the existence of solvent drag, but this does not necessarily mean that there are no aqueous channels. Channels may be present, but may admit only water. If the water were highly structured (icelike), as the activation energy studies suggest, solutes might well be excluded. I n any case, whether we are dealing with channels, or diffusion sites, they are apparently small enough to explain the selectivity of the membrane toward small solutes. To what extent can these findings be applied to other epithelia? They may be applicable to many, including gut, frog skin, and renal tubule. It appears likely that water diffuses across the cell membranes of these tissues a t a faster rate than has been recognized, and that attention to unstirred layers and the complexity of epithelial structure will lead to a new picture of water movement through the cell. ACKNOWLEDGMENTS

I am indebted to Mr. Nicholas Franki, Mr. Roy Soberman, and Miss Dorit Caliph for expert technical assistance. I also wish to express my gratitude t o Dr. Alexander Leaf, who set me to work on this problem in my fellowship years, and to Dr. Ora Kedem, of the Weizmann Institute of Science. The experimental work described in this chapter was supported in part by Grants HE-05928, AM-03858, and HE-13979 from the USPHS, and 14-01-001-1759 from the Office of Saline Water, US Department of the Interior. REFERENCES Andersen, B., and Ussing, H. H. (1987). Acta Physiol. Scand. 39, 228. Barry, P. II., and Hope, A. B. (1969). Biophys. J. 9, 700. Burg, M., H e h a n , S., Grantham, J., and Orloff, J. (1970) I n “Urea and the Kidney” (B. Schmidt-Nielsen, ed.), pp. 193-199. Exerpta Med. Found., Amsterdam. Cass, A., and Finkelstein, A. (1967). J. Gen. Physiol. 50, 1765. Civan, M. M., and Frazier, H. (1968). J. Gen. Physiol. 51, 589. Dainty, J. (1963). Advan. Bot. Res. 1, 279. Dainty, J., and House, C. R. (1966). J. Physiol. (London) 185, 172. Durbin, R. P., Frank, H., and Solomon, A. K. (1956). J. Gen. Physiol. 39, 535. Fenichel, I. R., and Horowitz, S. B. (1969). I n “Biological Membranes” (R. M. Dowben, ed.), pp. 177-221. Little, Brown, Boston, Massachusetts. Forster, R. E. (1971). Cum. Top. Membranes Trunsp. 2, 41.

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RICHARD M. HAYS

Ginsburg, B. Z., and Katchalsky, A. (1963). J. Gen. Physiol. 47,403. Hanai, T., and Haydon, D. A. (1966). J. Theor. Biol. 11, 370. Handler, J. S., Sugita, M., Preston, A. J., and Orloff, J. (1969). Proc. 4th Int. Congr. Nephrol., 1969 Abstracts, No. 1, p. 257. Hays, R. M. (1968). J. Gen. Physiot. 51,385. Hays, R. M., and Franki, N. (1970). J. Membrane Biol. 2,263. Hays, R. M., and Harkness, S. H. (1970). In “Urea and the Kidney” (B. SchmidtNielsen, ed.), pp. 149-158. Excerpta Med. Found., Amsterdam. Hays, R. M., and Leaf, A. (1962a). J. Gen. Physiol. 45,905. Hays, R. M., and Leaf, A. (1962b) J. Gen.Physiol. 45,933. Hays, R. M., Singer, B., and Malamed S. (1965) J. Cell Biol. 25, 195. Hays, R. M., Franki, N., and Soberman, R. (1971). J. Clin. Invest. 50, 1016. Katchalsky, A., and Kedem, 0. (1962). Biophys. J. 2, 63. Koefoed-Johnsen, V., and Ussing, H. H. (1953). Acta Physiol. Scand. 28, 60. Leaf, A. (1959). J. Cell. Comp. Physiol. 54, 103. Leaf, A., and Hays, R. M. (1962). J. Gen. Physiol. 45, 921. Lichtenstein, N. S., and Leaf, A. (1966). Ann. N.Y. Acad. Sci. 137, 556. Loeb, S. (1966). Desalination 1, 35. Macey, R. I., and Farmer, R. E. L. (1970). Biochim. Biophys. Acta 211, 104. Maffly, R. H., Hays, R. M., Landin, E., and Leaf, A. (1960). J. Clin. Invest. 39,630. Morgan, T., Sakai, F., and Berliner, R. W. (1968). Amer. J. Physiol. 214, 574. Paganelli, C. V., and Solomon, A. K. (1957). J. Gen. Physiol. 41, 259. Pappenheimer, J. R. (1953). Physiol. Rev. 33, 387. Schafer, J. A., and Andreoli, T. (1972). J. Clin. Invest. 51, 1264. Teorell, T. (1937) Trans. Faraday Soe. 33, 1020. Thau, G., Bloch, R., and Kedem, 0. (1966). Desalination 1, 129. Ussing, H. H., and Zerahn, K. (1951). Acta Physiol. Scand. 23, 110. Wang, J. H., Robinson, C. V., and Edelman, I. S. (1953). J . Amer. Chem. SOC.15,466. Wright, E. M., Smulders, A. P., and Tormey, J. McD. (1972). J. Membrane Biol. 7, 164.

Active Transport of Potassium and Other Alkali Metals by the Isolated Midgut of the Silkworm WILLIAM R . HARVEY and K A R L ZERAHN Department of Biology. Temple University. Philadelphia. Pennsylvania and Institute of Biological Chemistry A . University of Copenhagen. Denmark

I. Introduction . . . . . . . . . . . . . . . . . . A. Transepithelial K Pumps . . . . . . . . . . . . . B. The Midgut Transport System . . . . . . . . . . . I1. Methods . . . . . . . . . . . . . . . . . . . I11. Active K-Transport . . . . . . . . . . . . . . . . A . Midgut Potential . . . . . . . . . . . . . . . B . Short-circuit Current . . . . . . . . . . . . . . C. Agreement between K Flux and I,, . . . . . . . . . . IV. Influence of [K] on P D and I., . . . . . . . . . . . . . A. [ K I a n d P D . . . . . . . . . . . . . . . . . B . [K] and I., . . . . . . . . . . . . . . . . . C . Interpretation of [K]. PD. and I,, Relationships . . . . . . V. Coupling of K-Transport to Metabolism . . . . . . . . . . A . Ratio of K-Transport to O2 Uptake . . . . . . . . . . B . Dependence on Temperature . . . . . . . . . . . . C . ATPase . . . . . . . . . . . . . . . . . . VI . Transport of Other Alkali Metal Ions and Other Substances . . . . A . Rubidium . . . . . . . . . . . . . . . . . . B . Cesium . . . . . . . . . . . . . . . . . . C. Sodium . . . . . . . . . . . . . . . . . . D . Lithium . . . . . . . . . . . . . . . . . . E . Ammonium . . . . . . . . . . . . . . . . . F. Hydrogen Ions . . . . . . . . . . . . . . . . G . Amino Acid Absorption . . . . . . . . . . . . . . VII . Competition between Alkali Metal Ions . . . . . . . . . . A . Competition for Overall Transport Mechanism between Alkali Metal Ions . . . . . . . . . . . . . . . . . B . Uptake of Sodium by the Midgut . . . . . . . . . . C . Competition Sequence for Uptake of Alkali Ions . . . . . . D . Role of Calcium in Competition . . . . . . . . . . .

368 369 372 375 378 378 378 379 379 380 382 3x3 384 384 385 3x5 386 386 386 386 387 387 387 388 388 389 390 391 392 367

368

WILLIAM R. HARVEY AND KARL ZERAHN

VIII. Route of Ion Transport . . . . . . . . . . . . . . . 393 A. Structure of the Midgut . . . . . . . . . . . . . 393 B. Microelectrode Potential Profiles . . . . . . . . . . . 393 C. Background for Kinetic Studies . . . . . . . . . . . 395 D. Rationale for Kinetic Studies . . . . . . . . . . . . 395 E. LagTime . . . . . . . . . . . . . . . . . 396 F. Midgut Potassium . . . . . . . . . . . . . . . 397 G. Kinetic Equation . . . . . . . . . . . . . . . 397 H. Kinetic Models for Transport Route . . . . . . . . . . 398 I. Model 6 (Variable Transport Pool Model) . . . . . . . . 403 J. Pool Location . . . . . . . . . . . . . . . . 406 K. Summary of Transport Route . . . . . . . . . . . . 408 References . . . . . . . . . . . . . . . . . . 409

I. INTRODUCTION

Active transport of cations through the membranes of single cells has been studied extensively and has been used as a model for the study of active transport across epithelia. However, there are important differences between the two cases. The rate of transport, expressed on a basis of gross surface area, is a t least an order of magnitude larger across epithelia than across cell membranes (Table I). Unlike the plasma membranes of single cells, which usually do not show regional transport differences, the apical, basal and even the lateral surfaces of epithelial cells may differ in their transport properties. One surface may be impermeable to ions and the other surface may not, even though they may look the same in electron micrographs. Traditionally epithelia have been viewed as a collection of cells which have been joined to form a barrier but which retain Na-K exchange pumps in their plasma membranes. During active transport across epithelia it has been assumed that ions enter the cells from one side of the epithelium and leave them from the other side by some combination of passive diffusion and the action of the Na-K exchange pump. However, Loewenstein (1966) and others have shown that several properties not present in single cells appear as cells are joined to form an epithelium. For example, there are electrical and chemical connections between epithelial cells which do not exist between single cells. Therefore it does not follow that the active transport properties of epithelia can be deduced from the properties of single cells. Epithelia may have ways of performing transport which are quite different from the ways used by single cells. One major difference is that, although transport can be only into or out of single cells or parts of them, transport across epithelia could go either through cells or between them. A second major difference is that, aIthough cellular tranpsort almost invari-

369

TRANSPORT OF ALKALI METALS BY MIDGUT

TABLE I ACTIVETRANSPORT OF CATIONS I N SINGLE CELLS A N D EPITHELIA Flux Preparation Frog muscle Squid axon Frog skin Silkworm midgut

( p eq/cmZ/hr)

References

1.6 X Keynes (1954) 1.4 X lo-* Hodgkin and Keynes (1955) 1 Ussing and Zerahn (1951) 40 Harvey and Zerahn (1969)

ably depends on the Na-K pump, transport across epithelia utilizes a variety of ion pumps. Therefore we shall consider several routes which transport across epithelia may follow and not just the pathway through the cells. Moreover, we shall restrict our analysis to the transport of potassium and other alkali metal ions across the isolated silkworm midgut. A. Transepithelial K Pumps

As was just pointed out, a consequence of using the ceIluIar Na-K pump as a model for active transepithelial ion transport has been the emphasis of sodium transport and the relegation of potassium transport to a secondary role. Although Ramsay had demonstrated active K transport in the insect Malpighian tubules as early as 1953 and Harvey and Nedergaard had demonstrated in 1964 that active K transport in the isolated and shortcircuited insect midgut is independent of sodium, the opinion persisted that the important pumps are sodium pumps. I n 1969 Keynes discussed ion transport across epithelia in the most comprehensive review since that by Ussing in 1960. Keynes emphasized that besides the cellular Na-K pump there are several other types of ion pumps in epithelia. He suggested the recognition of five types of epithelial ion pump. In his classification potassium pumps from several insect epithelia, such as the Malpighian tubules, the midgut, and the labial glands, were included together with the K-pump in the salt gland of the desert iguana (Templeton, 1964) and possibly with that in the stria vascularis of the mammalian inner ear (reviewed by Johnstone, 1967) and were designated as ‘(typeV” pumps. TO this list we can now add the K-pump in the salivary gland of the fly Calliphora (Oschman and Berridge, 1970). According to Keynes the type V pump “transports I Li and Cs. This inversion also has no similarity to the sequence for size of the unhydrated ions or to the mobility of the ions. In summary, the only significant active transport taking place in Ca-free solutions which contain 16 m M K and 16 m M of any of the other alkali metal ions is the K (or Rb) transport. The sequence for the transport of these ions under these circumstances is K > R b >> Cs > Na = Li. The molarity of the alkali metal ions in the midgut tissue equilibrated with these same solutions follows the sequence K > R b >> Na > Li > Cs. The result has some bearing on the route of ion transport (see Section VIII, I). One can ask how the cells could be passively permeable to the alkali metal ions from the blood-side, could transport K but neither Nrt nor Cs out of the cells to the lumen-side, and yet be able to keep the cellular concentration of Na and Cs below that of K. D. Role of Calcium in Competition

Weinberg (cited in Harvey and Wood, 1972) showed that Ca inhibited the Na-transport whereas Mg had no systematic effects. Although Ca and Mg were not tested separately in the competition experiments just reviewed, Ca rather than Mg seems to be the critical ion. Ca does affect the uptake of ions by the midgut (Harvey and Wood, 1972)) but the mechanism of the Ca inhibition of the transport of alkali metal ions is unknown. The inhibition is not a simple one because the inhibitory effect of calcium from the lumen-side is approximately the same as that from the blood-side (Zerahn, 1971a).


393

VIII. ROUTE OF ION TRANSPORT A. Structure of the Midgut

Anderson and Harvey described the structure and the ultrastructure of the midgut (see Fig. 4) and were able to locate the pump in the one-cellthick epithelium (1966). They showed that the epithelium is composed of large columnar cells along with somewhat smaller goblet cells whose apical surface is invaginated to form a goblet cavity. The plasma membrane lining the goblet cavity is folded outward to form microvillus-like projections, each containing a large mitochondrion. On the inner leaflet of the plasma membrane of these projections are spike-like units. The close association of these units with the mitochondria in the projections led Anderson and Harvey to suggest, in accordance with Gupta and Berridge (1966) that the units may be associated with active K-transport. Accordingly, the pump would be located in the apical plasma membranes of the goblet cells. However, a basal location for the pump was not ruled out because the basal plasma membranes of the columnar cells possess numerous infoldings in intimate association with mitochondria resembling the arrangement found in many Na-transporting tissues. Therefore, Anderson and Harvey were unable to choose between an apical and a basal location for the pump on the evidence available to them. 6. Microelectrode Potential Profller

Wood, Farrand, and Harvey (1969) used microelectrodes to measure the potential difference between the bathing solutions and the interior of the midgut cells. With the midgut bathed in 32 mM K (32-K-S) and showing the spontaneous midgut potential, they found that, as the microelectrode was advanced into the tissue from the blood-side, it abruptly became 28 mV negative with respect to the blood-side solution. When the microelectrode was moved further the potential changed but little, then suddenly the full midgut potential was recorded. The midgut potential and the positive step disappeared when the tissue was deprived of oxygen, but the small negative step between tissue and blood-side remained. They interpreted this result to mean that the small negative step is caused by a passive process, whereas the large positive step from the tissue to the lumen-side solution is caused by an active process. This interpretation is supported by their finding that when the potassium concentration on the blood-side was decreased from 32 mM K to 2 m M K, the tissue negativity changed from -25 t o -75 mV, whereas the positive step was little affected.

394


FIG.4. A schematic representation of the cell types comprising the epithelium of the midgut of a mature fifth-instar larva of Hyalophora cecropia. MVC, microvilli of the columnar cells; CA, canal formed by the villuslike units derived from the larger protoplasmic projections (PJ) of the apical portion of the goblet cell; FMV, fine filaments within the microvilli of the columnar cell, ZA, zonula adhaerens; ZO, zonula occludens; MV2. microvilli; MT, microtubules; ER, endoplasmic reticulum; GC, cavity of goblet cell; GC’, Golgi complex of columnar cell; NC, nucleus of columnar cell; MVl, mitochondria-filled cytoplasmic projections that line the major portion of the cavity of the goblet cell; MC, mitochondria of columnar cell; GC*, Golgi complex of goblet cell; NG, nucleus of goblet cell; BIF, basal infoldings of columnar cell; BL, basement lamina; MS, muscle fiber; BOF, basal podocytelike extensions of the goblet cell; LOF, lateral evaginations of the goblet cell; NT, nucleus of tracheolar cell; T, tracheole. From Anderson and Harvey (1966) with permission from the Journal of Cell Biology.


395

They concluded that the negativity is in the epithelial cells although they could not determine in which type of cell. Lassen (1971) points out that cells may be damaged by microelectrodes and that only the potential recorded during the first few milliseconds after penetration is valid. However, the midgut cells are large (40 by >60 p ) , and the measured potential differences are large (cell as much as 180 mV negative to the lumen-side), stable for more than 20 minutes, and consistent, so the values may have but little error. Wood, Farrand, and Harvey argue that these data support a localization of the K-pump on the apical plasma membrane (see also Harvey, 1968). However, this conclusion does not help in choosing between the six models discussed in Sections VIII, H and I below, because all six models are consistent with the conclusion that the pump is located on the apical plasma membrane or on some structure electrically continuous with it. The results from the microelectrode studies show clearly and decisively that there is no electrogenic active transport of potassium or of any other substance across the basal plasma membrane into the cells. C. Background for Kinetic Studies

Kinetic studies of transport across the frog skin were initiated by Hoshiko and Ussing (1960) and Andersen and Zerahn (1963). Rather than confirming a route through the cells, these studies led to the hypothesis that in the frog skin Na-transport follows a non-mixing route possibly along the outside surfaces of the cells (Cereijido and Rotunno, 1968; Zerahn, 1969, unpublished results). The comparative simplicity of the midgut structure i.e., large cells of just two types arranged in a one-cell-thick epithelium, led Harvey and Zerahn (1969) to initiate kinetic studies of the K-transport through the midgut. D. Rationale for Kinetic Studies

The problem is to find ways of choosing between a mixing pathway and a non-mixing pathway. A mixing pathway is one in which the ions being transported mix with the bulk of the tissue K whereas a non-mixing pathway is one in which the ions being transported pass between the cells or along some structure such as the endoplasmic reticulum or the microtubules within cells and do not mix with the bulk K of the cytoplasm. A delay was found between the time a t which isotope is added to the blood-side solution and the time a t which it reaches a constant rate of appearance in the lumen-side solution. The delay must be caused by one or more pools somewhere in the transport route. Experimentally, then, the problem is to determine whether the pool is a transport pool, i.e., one before the pump, or

396


a transported pool, i.e., one after the pump (terminology after Andersen and Zerahn, 1963; Zerahn, 1969, unpublished results). E. Lag Time

1. DEFINITION OF LAGTIME

To measure the lag time 42Kis injected into the blood-side compartment. The appearance of labeled K on the lumen-side is plotted against time. The intercept of the extrapolated linear portion of this influx curve with the time axis is defined as the lag time (after Andersen and Zerahn, 1963; illustrated by Fig. 5). 2. CONSTANCY OF LAGTIME

For the midgut perfused as a sphere the lag time was constant for every midgut preparation studied and ranged from 2 to 4 minutes. The lag time was independent of the flux, supply of oxygen, concentration of K in the

2.0mM KHCO,

32-K-S

IC

L 4

TIME (min)

8

12

FIG.5. The time course of 42K-movement from blood-side to lumen of an isolated midgut is plotted for a representative experiment in which the gut is equilibrated with 32-K-S, with 2 mM-KHC03 (ordinate expanded 10 X), and again with 32-K-S. The lag time is estimated by extrapolating the steady-state line to the abscissa. Although the flux varied from 119 to 6.2 and back to 49 peq of K per hour, the lag time was virtually unchanged. From Harvey and Zerahn (1969) with permission from the Journal of Experimental Biology.


397

bathing solutions, and the PD. The K-transport rate could be reduced as much as 100-fold with only minor effects on the lag time (Harvey and Zerahn, 1969). Harvey and Wood (1972) reported a similar independence of the lag time on K concentration and oxygen tension using the flat sheet preparation. This constancy of the lag time means that the pool size must be proportional to the flux rate. The crucial question then becomes: Is the pool whose size is proportional to the flux rate a transport pool or a transported pool? F. Midgut Potassium

1. EFFECT OF EXTERNAL [K] ON TOTAL GUT K

The midgut tissue was equilibrated with 32, 20, 10, 6, and 2.4 m M K in the closed chamber. Then the K content was determined by flame spectrophotometry and corrected for K in adherent solution and extracellular space. The K content (peq of K per gram wet weight of gut) was 65 in 32 m M K but dropped only to about 45 in 20 mM K and was constant with further decrease in [K] in the bathing solution even though there was Na present to exchange with the K. 2. EXCHANGE OF MIDGUT K WITH 42KIN BLOOD-SIDE SOLUTION

A reasonable estimate of the exchange between 42Kand unlabeled K was possible in low K solutions. The midgut was perfused as a sphere in the chamber for 10 minutes. Then the gut was removed from the solution and rinsed briefly with 260 mM sucrose, and the specific activity was determined after 12 minutes in all. The midgut specific activity was 18% of that in the blood-side solution so it was considered a reasonable approximation to divide this value by 12 and obtain a value of 2% per minute for the exchange rate in 2 mM K. In 32 mM K (high [K]) the total direct labeling of gut K with 42Kfrom the blood-side solution amounted to as much as 70% of the blood-side specific activity in about 12 minutes. However, it would be necessary to correct for the high specific activity of K in the midgut to determine the exchange rate per minute. G. Kinetic Equation

The expcrimcntal facts which have just been reviewed are that the lag time is short and constant, that the total midgut K is relatively constant and that the exchange between midgut K and blood-side solution is small

398


relative to the flux in low K solutions. Several models for the movement of K through the gut were proposed and evaluated against these and other experimental data (Fig. 6 ) . To aid in this evaluation Harvey and Zerahn (1969) derived a kinetic equation with assumptions valid for the midgut following the treatment of Ussing and Zerahn (1951) and Solomon (1964) (see Scheme 1). Blood- side

Gut cells

Lumen

SCHEME 1

Labeled K enters the midgut from the blood-side solution at the rate, a A, mixes completely with gut K, and leaves the gut from this side a t the rate a, where A is the active K-flux toward the lumen, and a is the passive movement between midgut and blood-side solution in excess of the flux. The flux from the lumen to the gut cells is assumed to be so small that it can be neglected. Assume no change in the amount of K in the gut, So (peq). The amount of 42Kin the midgut a t time t is S L(peq). Taking the specific activity of 42Kin the blood-side solution to be unity, the rate of change of 42Kin the midgut is given by

+

dSi

=

(a

S1 + A)& - (a + A)& so

(1)

Calculating the time for 75% mixing of cell K with blood-side 42K,i.e., taking S t = 0.75 Soand integrating we obtain: t75 =

1.4 So a + A

-

Although this equation is written for the 75% mixing time, ing times would change only the value of the constant.

t76,

other mix-

H. Kinetic Models for Transport Route

1. MODEL1 (MIXINGWITH TOTAL K MODEL)

Model 1 (Fig. 6.1) is the simplest, most testable model possible. So is taken to be the total cellular K, and a is assumed to be zero. Calculating

399


Bloodside

Lumenside

@ Cells

n

Bloodside

Cells

Lumenside

" - *

3.

FIG.6. Diagrams illustrating models of the route of ion transport through the isolated passive fluxes equal to A by--+; and midgut. Active fluxes, A , are represented by-; passive exchange not involved in transepithelial flux by =:=. Transport pools (before the pump) are designated by Pt, and transported pools (after the pump) are designated by P d . In model 1 (Section VIII, H, 1 ) all the midgut K is involved in a transport pool the size of which does not vary; there is no passive exchange between cells and bathing compartments; connections between cells are irrelevant; and either type of cell can transport ions actively. I n model 2 (Section VIII, H, 2) the conditions of model 1 all obtain and in addition there is a n exchange, a, between celIs and blood-side compartment, hut no exchange between cells and lumen-side compartment. In model 3 (Section VIII, H, 3) each cell can behave like the cells of Model 2, but the cells are not coupled electrochemically and all the cells are not involved in transport all the time. The size of the transport pool varies directly with the active flux because the number of cells transporting K varies in this way. In model 4 (Section VIII, H, 4) only the goblet cells are involved in the active flux; there is no electrochemical coupling between cells; a small transport pool within the goblet cells is assumed, but a transported pool which varies directly with the transport rate and which is located on the lumen-side is implied as well. In model 5 (Section VIII, H, 5 ) a non-mixing pathway between or through the cells is postulated; most of the cell K is not in the pool; and a transported pool which varies directly with the transport rate is implied. In model 6 (VIII, I) all the epithelial cells are involved in transport and all are electrochemically coupled; a transport pool which varies directly with the transport rate and which is located within the cells is postulated, as is a nonexchangeable fraction of the cell K. Although a model such as this could accommodate an ion pump in all the cells, the pump is restricted to the goblet cells on the basis of structural and electrochemical evidence.

400


t in minutes for So = 5 peq K (the amount of K contained in a gut weighing 100 mg wet weight) and for A = 5 peqjhour (the K flux for 2 mM K on the blood-side) we obtain: t

=

5 1.4 X - = 84 minutes 5/60

The lag time predicted by this model is therefore much longer than that observed in low [K]. Furthermore, the model predicts either that the lag time should be inversely proportional to A if So is constant or that So should vary with A. Since neither the lag time nor So vary with A, this simplest model must be rejected.

2. MODEL2 (MIXINGWITH TOTAL K PLUS EXCHANGE MODEL)

In this model (Fig. 6.2) a represents the exchange of labeled K on the blood-side for unlabeled gut K, or other gut cation, or simple diffusion of 42K into the midgut cells. We know that such an exchange occurs because after 12 minutes in 32-K-S (32 m M K) the gut is a t least 70% equilibrated with blood-side K. The exchange between lumen-side and cells would have to be small to account for the large observed net K-flux toward the lumen and was measured to be about O.l%/minute and therefore was neglected. Harvey and Zerahn calculated the exchange with the blood-side that would be required to obtain a lag time of 3 minutes in 2 m M K in which the flux rate is about 5 peqjhour. For 75% mixing of cell K with blood-side 42K, for So = 5 peg K, and for A = 5 peq/hour; and taking the measured lag time, t = 3 minutes, Harvey and Zerahn obtained for a : a = 1.4

35)

(s~/t) - A = 1.4 X (

- - = 2.25 peq/min

o:

Thus, when A is only 0.08 peq/minute, a would have to be 2.25 peq/minute to yield 75% mixing in 3 minutes. This calculated value a is faster than the measured exchange, determined to be 2%/minute X 5 peq I(, which amounts to 0.1 peq/minute or 22 times less than that required by model 2 for a lag time of 3 minutes. Therefore, Harvey and Zerahn rejected model 2.

Specijk Activity of Gut K and Lumen K . Evidence that the short-circuit current and the transport of 42K-labeledpotassium agree within a few percent was reviewed in Section 111, C. This agreement between current and 42Kflux shows that when the steady state is obtained after about 5 minutes (but see Section VIII, I, 2) the actively transported K appearing on the lumen-side must have a specific activity approaching 100% of that of


401

blood-side K. At this time the K in all the pools directly on the transport pathway must have this same 100% specific activity. However, after 5 minutes the specific activity of the midgut K in 2.4 mM. K was found to be only 8% of the blood-side K (see Table V of Harvey and Zerahn, 1969), a value far less than the specific activity of the K appearing a t the lumenside. Although it was not possible to short-circuit the midgut accurately a t this low K concentration, the agreement between the net flux and the current was reasonable in 4 mM K (Table IV). These results support the kinetic evidence in rejecting models 1 and 2. Even though there is an appreciable exchange of blood-side 42Kwith total gut K, it is not large enough in solutions with low [K] to account for the constant lag time; and it does not label the gut fast enough to provide a K source with sufficiently high specific activity to account for the agreement between K-flux and current. 3. MODEL3 (HETEROGENEOUS CELLPOPULATION MODEL)

In models 1 and 2 it was assumed that all the cells behaved in the same way. In model 3 (Fig. 6.3) we express the possibility that the number of cells transporting K is proportional to the K-flux. If this were the case we would obtain a constant lag time because the size of the pool (i.e., the number of cells involved in the transport) would be directly proportional to the flux rate. Therefore, there would be a large transport pool in high [K] and a small transport pool in low [K]. Against this model are the observations that all cells of the same type seem to be structurally equivalent, are the same age, and are surrounded by the same medium. Furthermore, no difference in electrical potential of the individual midgut cells is found when the gut is punctured with microelectrodes, whether under normal conditions or with the flux inhibited by lack of oxygen or by low K concentration (Wood et al., 1969). These observations render model 3 implausible, but do not allow one to reject it. 4. MODEL4 (GOBLETCELLMODEL)

This model (Fig. 6.4) assumes that only a fraction of the epithelial K is taking part in the transport process. For example, the goblet cells alone might perform the K-transport as suggested on structural arguments by Anderson and Harvey (1966) (see Section VIII, A). There is only about one goblet cell for every four columnar cells, and the amount of cytoplasm in each goblet cell is far less than half of the amount in each columnar cell (Wood, 1972). The concentration of K in the goblet cells is not known, but it could be lower than the mean K concentration of the gut even if it is difficult to conceive it to be close to zero. The actual So,if restricted to the goblet cells, would be much smaller than the total gut K so that a

402


treatment like that in model 2 might be valid for the goblet cells alone. Model 4 would thus postulate a transport pool, but one so small that changes in its size would not be detectable. It also implies a variable, transported pool to account for the constant lag time. Although low blood-side [K] does not decrease gut K significantly (Section VIII, F, l ) , the amount of K in the goblet cells may be so small that changes may escape detection. Therefore, restricting the transport route to the goblet cells would both satisfactorily explain the constant lag time and account for the low specific activity of the midgut. However, it is difficult to visualize the goblet cells being chemically uncoupled from the columnar cells, as implied by this model and yet being electrically coupled as implied by the results of Wood et al. (1969; see Keynes, 1969, p. 248). 5. MODEL5 (NoN-MIXING MODEL) Models 1 and 2, which assume that all the gut K is involved in the transport, were discarded. Model 3, which assumes that a variable part of the epithelial cell K is involved, cannot be discarded on available evidence but is hard to test (but’see Section VIII, I). Model 4, which assumes that a small constant part of the epithelial cell K is involved and suggests a route through the goblet cells, cannot be rejected but presents the coupling problem mentioned a t the end of Section VIII, H, 4. Models 1-3 are transport pool models in that they all assume that the pool is before the pump and that there is mixing between K in transport and some or all of the cell K. Model 4 implies both a transport and a transported pool. Harvey and Zerahn suggested a fifth model (Fig. 6.5) which is a transported pool model. According to model 5 the transport route may pass through the midgut without mixing in K in transport with any of the cell K. No special intracellular pathway was suggested, but there is abundant endoplasmie reticulum and there are numerous microtubules in the midgut cells. Such non-mixing intracellular transport of numerous substances along the microtubules of nerve cells is well known (see Dahlstrom, 1971, for references). The route may follow the cell surfaces. The route may even be placed for a short distance between adjoining cells through the tight junctions. Model 5 assumes that the transport pool is so small as to be negligible. Although Harvey and Zerahn did not explicitly discuss it, this model implies that the pool causing the lag time is a transported pool (see Section VII1,D). The constancy of the lag time would then require that the size of this transported pool vary directly with the flux rate. Since this transported pool would be on the lumen-side of the apical plasma membrane diffusing toward the lumen, its size would vary with the flux rate according to Fick’s law.


403

I. Model 6 (Variable Transport Pool Model) Harvey and Wood (1972) proposed a model (Fig. 6.6) in which, like models 1 and 2, all the cells are involved in a transport pool but in which, like model 3, the size of the transport pool varies directly with the flux rate. However, unlike model 3, which assumes that the variation in transport pool size is due to a variation in the number of cells involved, model 6 assumes that the transport pool size varies because the K content in the cytoplasmic matrix varies. To make this assumption plausible, they suggested that the [K] in the cytoplasmic matrix (ground cytoplasm; see De Robertis, Nowinski, and Saez, 1970) of all the epithelial cells varies directly with the flux rate. Therefore model 6 implies that there is a n exchangeable fraction of cell K (the fraction in the cytoplasmic matrix) and a non-exchangeable fraction of cell K (for example, in such compartments as the mitochondria and nuclei). 1. RATIONALE FOR MODEL 6

The constancy of the lag time requires a large pool in high [K] and a small pool in low [K]. The question is whether this variable pool is a transported pool, as implied by models 4 and 5 but not explicitly stated by Harvey and Zerahn, or whether it is a transport pool, as implied by model 6 and proposed by Harvey and Wood. The argument for model 6 is that (1) the lag time in high [K] is long, (2) the 75% mixing time, required by the kinetic equation, is even longer, (3) the corresponding pool size in high [K] is large, (4)the only compartment in the midgut that is big enough to contain a pool this large is the epithelium itself, (5) the pump is located in the apical plasma membrane of the epithelial cells on electrical and structural grounds, and therefore (6) the pool must be a transport pool located before the pump. 2. LAGTIMEIN HIGH[K]

Wood (1972) developed a suggestion by Maddrell that the lag time measurement must take into account the decay in the I S c .Wood argued that because the pumping rate decays with time the achievement of isotopic steady state is signaled not by a constant influx (the classical definition), but by an influx that is decaying a t the same rate as is the I s c .Wood (1972) developed a method for correcting the influx for decay in I,, and used the correction to demonstrate a lag time of about 9 minutes for 86Rb-

404


influx across the A . pernyi midgut in the flat sheet preparation. Harvey and Wood (1972) thcn presented evidence that after 120 minutes of equilibration, when the decay in I,, had bccomc negligible, the lag time for 42Kinflux across the H . cecropia midgut mas now 9 minutes with only small current corrections necessary in the flat-sheet preparation. They also pointed out that Eq. (2) actually requires thc 75% mixing time, which is about 1.4 times the lag time, and for the flat-sheet preparation amounts to about 13 minutes. This long mixing time corresponded to a pool size amounting to about 66% of the midgut K. They argued that this amount of K could be placed nowhere else in the midgut but in the cells (but see Section VIII, J). One may ask whether the method for correcting the influx for current decay is valid. The corrections require accurate values for the I,, and for the steady-state flux as well as a demonstration that the efflux is constant. These values are only approximately known for the sphere, so it is not advisable to use the corrections in this preparation. Moreover, the lag times measured in the sphere experiments do not seem to vary with the limited current decay rate as much as anticipated from the flat sheet results. The rather steep slope of the influx time-course curve (e.g., Fig, 5) does not indicate any long lag time for the spherical preparation. It is beyond the scope of this review to assess the validity of the corrections for the flat sheet. It is also difficult to decide whether the lag time for the sphere can be compared validly with the lag time for the flat sheet. 3. POOLSIZEIN HIGH[K]

A major purpose for studying the lag time is to determine the size of the pool, i.e., the amount in microequivalents of labeled K used to label the midgut to a value that will allow the flux to the lumen-side to be constant. The pool size can be calculated from the lag time and flux (Andersen and Zerahn, 1963). An alternative method is shown in Fig. 7. The timecourse of the influx usualIy becomes constant after a certain time. The area LL (labeled level) approximately represents the actual amount of labeled K missing from the lumen-side solution and causing the delay in attaining a constant influx. Thus a pool size equal to zero would give a curve following the ordinate, then. parallel to the abscissa. The area between this theoretical curve and the measured curve represents the amount of labeled K missing, i.e., the pool size. The pool size calculated in this way for the sphere was found to be approximately equal to the pool size calculated from the lag time (Table X). I n the mean a deviation of 13% was found between the pool sizes calculated by these two methods. Zerahn argues that this small deviation


405

/

I5

I0

Y

a rs l

i

5 w

TIME (min)

FIG.7. Comparison of two methods for calculating the pool size from influx kinetics. The lag time is obtained by extrapolating the time course of accumulation of K (crosses) to the time axis as described in Section VIII, E, and the pool size, So,is calculated from the corresponding intercept on the ordinate. Alternatively, the pool size is given directly from the influx time course (filled circles) by the area LL. In the absence of a pool the influx time-course would follow the ordinate and abruptly yield a straight line parallel to the abscissa. The effect of a pool in the transport route is to delay the attainment of a constant influx rate by an amount of time described by the influx curve. The curve can be approximated by the dotted line oblique to the ordinate with the result that a trapezoid is formed whose area, LL, is a good approximation of the pool size. This area represents the labeled level which the tissue must attain before a constant influx can be measured by taking samples from the lumen-side solution. For these data LL is given by: (5.6 1.0)/2 x 71/60 = 3.9 geq K, whereas the pool size from the lag time of 3.6 min is given by 71 X 3.6/60 = 4.3 peq K.

+

indicates strongly that the pool size and therefore the lag time reported for the sphere are correct within 15%. The pool sizc was found to be closely proportional to the A ux. The proportionality is consistent with a transported pool in which the labclcd K has already passed the transport mechanism arid is on its way passively diffusing out of the tissue t o the lumen-side solution, but it is also consistent with a variable transport pool within the cells (see Section VIII, J for a discussion of pool location). The main point to be made, however, is that despite the controversy over the length of thc lag time and the size of the pool, there is now general ngreement among Harvey, Wood, and Zerahn that the pool size is large (4 or more peq of Ti per 100 mg of gut) when the midgut is bathed in high [K] and exhibits a large flux.

406


TABLE X POOL SIZESCALCULATED FROM LAGTIME AND

FROM

FLUXTIMECOURSE" Pool sizes (/*eq)

-

Date 5-13-66 5-17-66 6-3A-70 6-3B-70 6-4-70 6-18-66 6-9-66 6-18-66

a

[K] (mM) 32 32 32 32 32 32 74 2

PD (mV)

Lag time (min)

Flux (peq/ hour)

From lag time

From flux curve

Ratio 1ag:flux x 100

1.9 3.6 2.4 2.2 3.0 2.8 2.8 3.1

56 71 170 112 116 30 80 7

2.0 4.3 6.8 4.2 6.0 1.6 3.5 0.36

2.8 3.9

72 110 68 83 88 106 94 77 87

0 0

0 0 0 100 0

-

10.0

5.1 6.8 1.5 3.7 0.47 Mean Value

W.R. Harvey and K. Zerahn, (1970), unpublished results.

4. POOL SIZEIN Low [K]

No accurate data are presently available regarding the I,, in 2 m M KHCOI because of the difficulty of short-circuiting the midgut in a solution with such low conductivity. However, Harvey and Zerahn measured a lag time of about 2 4 minutes for the flux from blood-side to lumen-side and a flux of about 5 peqlhour in this solution. Whether the pool size is calculated directly from the flux curve or by the graphical method of Fig. 7, its magnitude is less than 0.5 peq of K. J. Pool Location 1. POOLLOCATION IN HIGH[K]

From Table X we see that the total pool in the midgut may amount to 6 peq of K or more per 100 mg in high [K]. Where is this pool located? The stirring on the blood-side is so fast that the size of the unstirred layer should be small. The K pool might be in the cells as a transport pool or it might be in the extracellular space open to the lumen-side as a transported


407

pool. There really is no way to decide from present influx kinetics, and we must turn to chemical determinations of K in the midgut. Harvey and Zerahn (1969) (see Section VIII, F) reported that there are about 6 peq of K in a 100 mg of midgut tissue. If the pool is a transported pool in the extracellular space on the lumenside, then its 6 peq must be added to this 6 peq in the cells to yield a total amount of midgut K (cellular K plus extracellular K as determined by a flame photometer) approaching 12 peq/100 mg during transport. Where are the 6 peg of missing K? It is likely that a transported pool would be removed before the midgut K can be determined chemically. Before the midgut can be removed from the chamber the short-circuiting is stopped, and with the full PD the transport rate will be only 30-5070 of that in the short-circuited midgut, with a correspondingly smaller transported pool. Furthermore, the midguts were washed 1 minute in 260 m M sucrose and this procedure would remove a large part of any transported pool. To make the K determinations properly, they should be made much faster. If on the other hand, the pool is a transport pool in the cells then we need expect t o find only 6 peq K/100 mg of midgut tissue during transport. Since this is the amount of I< we do indeed find, it is tempting to conclude that the pool is a transport pool. However, if the lag time is 9 minutes, is constant, and is the same for flat sheet and spherical preparation, then for the influx determination of Fig. 5 in which the influx was 120 peq/hour the pool size would be 18 peg of K, which is more K than is present in a gut weighing 100 mg. The data used in this estimate were not all obtained from the same preparation. Nevertheless, we emphasize that until a serious attempt is made to measure the extracellular K during transport no definitive conclusion can be reached regarding pool location in high [K] solutions.

2. POOL LOCATION IN Low [I

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