No title

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.

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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

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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

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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

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

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

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

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

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

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<+-ATPase has been approached in three general ways: (1) to define sodium, potassium and ATP sites on both sides of the membrane in order t o elucidate the kinetic mechanisms of ligand interactions and the types of reactions catalyzed by the system; ( 2 ) to define the sequence, i.e., the intermediate steps that participate in the reaction leading to ATP hydrolysis; and (3) to define the mechanism by which cardiac glycosides, specific inhibitors of both the Naf ,I<+-ATPase and the sodium pump, interact with the system. The results of these investigations have clarified, to some extent, the nature of the pump, and have suggested physiological roles for the system. A. Monovalent Cation Activation and Transport

The active transport of sodium out of and potassium into the cell is coupled to the hydrolysis of ATP (Albers, 1967; Glynn, 1964; Hokin and Hokin, 1963b; Judah and Ahmed, 1964; Skou, 1965). 3 sodium (in) 7

3 sodium (out) I

2 potassium (in)+2

potassium (out)

I

ATP-4

+ HzOLAZ)P-3 + P,a + H+

The stoichiometry of the pump (i.e., 3 sodium:2 potassium:l ATP) was

10

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

defined by determining transmembrane potassium and sodium fluxes and by measurement of intracellular ATP hydrolysis in various erythrocyte preparations (Gardos, 1964; Garrahan and Glynn, 1966, l967d; Post and Jolly, 1957; Post et al., 1967; Sen and Post, 1964; Whittam and Ager, 1965). Estimates of three sodium ions extruded per A T P molecule hydrolyzed have also been concluded for the sodium pump in crab and squid axons (Baker, 1965; Baker and Shaw, 1965) and in frog muscle (Dydynska and Harris, 1966; Harris, 1967). Ratios of sodium ions extruded per potassium ions sequestered, however, are difficult to determine in nerve and muscle, because these ratios vary with the conditions of the experiment (Adrian and Slayman, 1966; Keynes, 1965; Rang and Ritchie, 1968). Note that since the hydrolysis of ATP is electrically neutral, the total reaction should be electrogenic because one net positive charge leaves the cell. Although electrogenicity could be nullified by uptake of a proton, there is evidence that the pump may contribute to the membrane potential (Adrian and Slayman, 1966; Carpenter, 1967; Carpenter and Alving, 1968; Chiarandini and Stefani, 1967; I h m e n t o , 1965; Hodgkin and Keynes, 1956; Iierkut and Thomas, 1965; Kernan, 196‘2; Marmor and Gorman, 1970; Mullens and Awad, 1965; Nakajima and Takahashi, 1966; Obara and Grundfest, 1968; Page and Storm, 1965; Senft, 1967; Sokolove and Cooke, 1971; Thomas, 1969; Yonemura and Sata, 1968). The Na+ ,K+-ATPase-transport system is oriented within the membrane and has an apparent molecular weight estimated between 190,000 and 500,000. While a number of different procedures have been employed to estimate molecular weight (Rlizuno et al., 1968; Iiepner and Macey, 1968a, 1969; Nakao et al., 1967,1969; Glossman and Lutz, 1970; Hoogeveen et al,, 1970)) the most recent uses a target theory analysis of radiation inactivation data. Samples are irradiated and the dose to reduce initial enzyme activity to 37% = DIT is calculated. This is related to the molecular weight (RIW) by the equation: RIW = constant + DS7 (Kepner and Macey, 1968a, 1969; Nakao et al., 1967). Since t,he Na+,K+-ATPase is still relatively impure [although Jorgensen et al. (1971) claim a purity of 49% if a molecular weight of 250,000 and a moiecular activity of 12,850 min-1 (“turnover”) are used, and a “purity” of 98% if a molecular weight of 500,000 is used], L‘molecularweight” has little significance a t this time, except that it presents some idea of the complexity of the system. Other techniques for apparent molecular weight determination include ultracentrifugation analysis and gel sieving (Mizuno et al., 1968). Quite recently, Hossler and Rendi (1971), using sucrose density centrifugation of a soluble nucleoside diphosphokinase (NDK) from kidney membranes rich in Na+ ,I<+-ATPase localized three major species with molecular weights of 21,000, 92,000, and 138,000. It seems clear that the Na+,K+-ATPase

THE Na+,K+-ATPare MEMBRANE TRANSPORT SYSTEM

11

consists of subunits, and, as suggested by Hossler and Rendi, the higher molecular weight forms may actually be tetramers and hexamers. The fact that sodium and potassium transport occurs across a membrane indicates that the Na+ ,K+-ATPase is indeed membrane bound. Skou originally showed (1957, 1960) that Na+ ,Kf-ATPase is sedimented with membranes. Subsequent investigations suggested that the membrane enzyme system is the sodium pump (Albers, 1967; Glynn, 1964; Hokin and Hokin, 1963b; Judah and Ahmed, 1964; Skou, 1965; Post and Sen, 1967). The lipoprotein nature of the enzyme is now firmly established (Ahmed and Judah, 1964; Schatzmann, 1962) ; activity of Na+,K+ATPase in fragmented membrane preparations (FMP) is known to require lipids (Askari and Frantantoni, 1964; Emmelot and Bos, 1968; Fenster and Copenhaver, 1967; Formby and Clausen, 1968; Ohnishi and Kawamura, 1964; Tanaka and Abood, 1964; Tanaka and Strickland, 1965; Tanaka and Sakamoto, 1969; Wheeler and Whittam, 1970), perhaps of specific structure, although the exact composition of the required lipids remains controversial. As discussed above, estimations of molecular weight for Na+, K+-ATPase in broken membrane preparations have been made by progressive inactivation of these preparations with varying degrees of irradiation (Kepner and Macey, 1966, 1968a,b; Nakao et al., 1967). As Kepner and Macey (1968a) pointed out, in vacuo irradiation is required for accurate estimates; they determined approximate molecular weights for the pump of 190,000 in guinea pig kidney microsomes and 250,000 in erythrocyte ghosts by use of this technique. The authors also noted that a macromolecule with an assumed density of 1.3 and a molecular weight of 250,000 would correspond to a spherical particle with a diameter of 85 A, a dimension close to that estimated for some biological membranes by electron microscopy (Robertson, 1959). It will subsequently become clear that parts of the Na+, K+-ATPase-transport system must be exposed to the internal and external membrane-water interfaces. This implies that the enzyme system completely penetrates the membrane space. Is such a structure compatible with current concepts of membrane structure? Such an orientation does not appear to be consistent with the Danielli-DavsonRobertson bilayer-unit membrane theory (Robertson, 1959; Danielli and Harvey, 1935; Danielli and Davson, 1935), but this type of relationship is compatible with newer proposals for membrane configurations where proteins may either penetrate the membrane matrix (Lenard and Singer, 1966; Wallach and Zahler, 1966) and/or exist as micelles within the membrane space (Lucy, 1964, 1968). Such configurations could, of course, exist adjacent to bilayered structures of the membrane and, therefore, do not really vitiate the Robertson unit membrane hypothesis. Recent reviews on membrane structure have been presented (Branton and Park, 1968;

12

ARNOLD SCHWARTZ, GEORGE

E. LINDENMAYER, AND JULIUS C. ALLEN

Hendler, 1971; Icorn, 1968a); see also the symposia edited by Chapman (1968) and Jarnefelt (1968). I n conclusion, the sodium pump appears to possess the physical dimensions and characteristics that allow it to reside within or on the membrane and to have surfaces or sites exposed on opposite faces of the membrane. Monovalent cation activation curves for Na+, K+-ATPase in F M P are most simply interpreted by assuming separate activation sites for sodium and potassium (Skou, 1957, 1960). Thus, data suggest the existence of a sodium-activation site with the characteristics of high affinity for sodium and low affinity for potassium and a second site, the potassium-activation site with low affinity for sodium and high affinity for potassium. Potassium competes with sodium for the sodium-activation site and, if successful, inhibits catalysis and transport. Sodium acts in a similar manner a t the potassium-activation site (Skou, 1960, 1962; Green and Taylor, 1964; Hoffman, 1962a,b; Post et al., 1960; Whittam and Ager, 1962). Studies with erythrocyte ghost preparations, which have low permeability to cations and allow independent variations in the internal and external ionic concentrations (Hoffman, 1958; Hoffman et al., 1960; Whittam, 1962), have yielded information concerning the general configuration of the system within the membrane. The pump is asymmetrically oriented so that the sodium-activation site is on the internal surface and the potassiumactivation site is on the external surface of the membrane. Similar results were reported for intact crab nerve preparations (Baker, 1965). This is the obvious orientation consistent with operation of the pump in a forward direction. Intracellular (but not extracellular) ATP is hydrolyzed with release of inorganic phosphate internally (Baker, 1965; Baker et al., 1969; Glynn, 1962; Lark and Letchworth, 1962; Schatzmann, 1964; Sen and Post, 1964; Whittam, 1964). The sodium- and potassium-activation sites may remain separate throughout the transport cycle, or there may be interconversions between the sites within the cycle. Similarly, it is not clear whether sodium and potassium traverse the membrane simultaneously or in sequence. Nonetheless, there appear to be at least three effective monovalent cationic binding states of the system :

13

THE Na+, K+-ATPara MEMBRANE TRANSPORT SYSTEM

The ratios are representative of those necessary for activity or inactivity of FMP as demonstrated by observations of ATP hydrolysis rates. Sodium and potassium can be considered “substrates” of the system in intact transporting systems because they are altered, in the sense of potential energy, by transport. In FMP, however, these ions may be considered as activators of Na+ ,Kf-ATPase for ATP hydrolysis, since no potential energy change is present. Conversely, there is no reason to suspect that ion permeation does not occur in the particulate preparations. Indeed, the similarities between data obtained from FMP and intact transporting systems strongly suggest that the membrane fragments (FMP) or vesicles contain the complete and coupled enzyme-transport complex. In FMP, sodium activation curves (i.e., ATPase activity vs sodium concentrations) deviate from curves predicted by the Michaelis-Menten equation (Dixon and Webb, 1964). The alteration is characterized by lower activities than predicted a t low sodium concentrations. At higher concentrations the discrepancy tends to disappear. Finally a t very high sodium levels, the experimentally observed activities become progressively less than predicted. Potassium markedly alters the degree of deviation, and increasing potassium raises the K , for sodium. At low sodium coneentrations, potassium augments the deviations from the predicted curve while it prevents the changes found at very high sodium levels. The latter appears to involve competition between the ions a t the potassium-activation site and will be discussed below; the former involves competition a t the sodium-activation site (Green and Taylor, 1964; Baker and Connelly, 1966; Post et al., 1960; Whittam, 1964; Whittam and Wheeler, 1961; Skou, 1960), but the observations are not explained by assuming a model for simple competitive inhibition (Green and Taylor, 1964). Rather, the deviation is sigmoidal. Hill plots* of the data have yielded interaction

+

* Hill plots are based on the equation v = V,/[l K/(S)“] where v = the measured initial velocity of the reaction, V, = maximal velocity, n = interaction coefficient, S = activator or substrate concentration, and K = apparent dissociation equilibrium constant; n values of greater than 1 have been used as an indicator of cooperative effects. When this equation is transformed, log [(VJv) - I] = log K - n log (s), a straight line may be plotted to

log

s

(Monod et al., 1963). “Cooperative” signifies that binding of one ligand molecule alters the probability that a second ligand will interact with the system. This alteration is thought to be brought about by allosteric or autosteric transitions in macromolecular conformation. “Allosteric” transition is due to ligand binding to a site which is physically

14

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

coefficient values of greater than 1 (Squires, 1965; Robinson, 1967). Robinson (1970a) reported that these values were highest a t low potassium levels and tended to decrease as the potassium level was raised. Other factors besides cooperativity (e.g., ionic strength, p H), however, produce high n values (Erieden, 1967; Robinson, 1967; Rubin and Changeux, 1966), and reaction mechanisms other than allosteric interactions can produce sigmoidal activation kinetics (Griffin and Brand, 1968; Sweeny and Fisher, 1968). Furthermore, kinetic mechanisms which involve multiple ligand interactions with the system must be considered. In the case of sodium, probably three ions must interact with the pump as a prerequisite for the catalytic-induced hydrolysis of one ATP molecule. Magnesium and calcium compete with sodium for the sodium-activation sites (Judah and Ahmed, 1963, 1964; Judah et al., 1962; Portius and Repke, 1967; Lindenmayer et al., 1971; Lindenmayer and Schwartz, 1970a; Jarnefelt, 1962; Portius and Repke, 1962). This is manifested by a divalent cation-induced increase in the amount of sodium required to half-maximally

remote from the active site. “Autosteric” transition is secondary to ligand binding a t a site which is adjacent to the active site, as defined by Koshland and Neets (1968). For presentation of allosteric models, see Monod et al. (1965), Haber and Koshland (1963), Koshland et al. (1966), and Volkenstein (1969); allosteric models applied to general membrane phenomena (Changeux et al., 1967; Changeux and Podleski, 1968; Changeux and Thiery, 1968) and to transmembrane ion transport (Hill, 1969, 1970; Hill and Chen, 1970a,b,c) have also been presented. Another way of expressing the classical Hill equation is: log,, (Y + l - Y ) = (log,,A) - (nlogIoK),where Y is the fraction of protein sites bound to the ligand, A is the concentration of unbound ligand, and K is the apparent dissociation equilibrium constant. The Hill coefficient n reflects the degree of cooperativity present. When identical ligands interact, the plots of binding or of enzymatic activity vs ligand concentration are sigmoidal, and n > 1 for positive cooperativity.

.-ca.

._ c O

m 0 E

B

m

._ n

Binding ligand

Most allosteric properties are complex and may involve, among numerous factors, electrostatic forces (Epstein, 1971).

THE Na’, K+-ATPare MEMBRANE TRANSPORT SYSTEM

15

activate the enzyme. Epstein and Whittam (1966) found that calcium competed only with magnesium, but the preparation used in this experiment had extremely low activity (1-5 pmoles of Pi per milligram per hour) and a magnesium, sodium, potassium stimulated: magnesium stimulated activity ratio of about 1. Calcium has also been found to increase the Hill interaction coefficients to values greater than 1 (Lindenmayer et al., 1971). These data suggest that calcium may be bound on the internal surface of the cell membrane. If correct, this constitutes a pool of calcium which is sensitive to intracellular sodium levels. Thus, the pool could be released upon sodium influx, which is thought to be characteristic of the action potential, and therefore, could be involved in excitation-contraction coupling (Langer, 1968; Langer and Serena, 1970) and in digitalis-induced inotropism (Besch and Schwartz, 1970). It is of interest in this regard that calcium has recently been shown to “substitute” for sodium in stimulating 3H-labclcd digitalis binding to a cardiac Na+ ,K+-ATPase (Shon et al., 1970; Schwartz et al., 1972). Potassium activation curves also deviate from the prcdicted curve of the Rlichaelis-Menten equation at high potassium levels. Sodium prevents this effectand it is thought to reflect ionic competition for sodium-activation sites as described above. A t low potassium concentrations, sodium raises the amount of potassium required to half-maximally activate the system (Whittam and Ager, 1962). Squires (1965) and Robinson (1967) found that sodium causes the interaction coefficients of the Hill plot to rise above unity for potassium activation (Priestland and Whittam, 1968). Hexum et al. (1970) could not demonstrate this sodium-induced change in the interaction coefficient. Potassium influx and sodium efflux rates in erythrocyte preparations respond in a sigmoidal manner to extracellular potassium (Priestland and Whittam, 1968; Sachs and Welt, 1967; Garrahan and Glynn, 196713). Encrgy utilization in crab nerve does not respond in a sigmoidal manner to extracellular potassium (Baker and Connelly, 1966) ; ion transport in nonmyelinated nerve responds in a hyperbolic manner to extracellular potassium (Rang and Ritchie, 1968). Conversely, glycosidesensitive transport rates in giant axons show a sigmoidal relationship to external potassium (Baker et al., 1969). The reasons for these differences are not clear. Calcium alters potassium activation curves in that the K,,, for potassium is decreased (Lindenmayer et al., 1971). Whether or not this reflects some connection between sodium and potassium activation of the system is not known. It is obvious from the above discussion that pharmacological, physiological, or pathological events which alter divalent cation pools may lead to changes in the activity of the sodium pump. A second modulation factor

16

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

is pH. ATP hydrolysis, catalyzed by Na+,K+-ATPase, has a broad pH optimum centered around pH 7.4. Fujita et al. (1967), however, showed that different ATPase activities could be elicited by varying the pH. Thus, a potassium-induced activity was observed between values of 5 and 6, and a sodium-induced activity was observed between values of 6.5 and 8.5. Both these activities were of low magnitude; the latter but not the former was inhibited by ouabain. Very high sodium plus potassium-induced activities were observed between p H 7.2 and 7.5. This act,ivity was also glycoside-sensitive. It must be concluded, therefore, that pH can alter the pump both quantitatively and qualitatively. To our knowledge, the effects of pH on K,, interaction coefficients and stoichiometric values have not been extensiveIy investigated. Electrostatic forces may also bear on this (see above). This information would allow further insight into the potential responses of the sodium pump to physiological and pharmacological changes. A third factor that may be important in determining the activity of the pump is water structure. It now seems clear that the water layers around membranous elements are more structured than normal water (Ling, 1964,1965,1966,1969a;Ling and Bohr, 1970; Ling and Negendank, 1970). This effect, of course, decreases with increasing distance from macromolecules, but the close approximation of “solid” surfaces in the cell has led to suggestions that most, if not all, intracellular water is structured. The same statement could probably be made for much of the water in the interstitial space. Structured water appears to alter the ionic mobilities of sodium and potassium. It increases the viscosity of movement of these ions and greatly increases the mobility of protons through “tunneling” effects (Kavanau, 1964). These effects have not been extensively investigated with respect to pump activity or as signaling devices between the pump and other subccllular systems. The activity of the sodium pump may also depend on the presence of large macromolecular complexes which exist on the external surface of many membranes. These complexes have ionic binding sites (Katchalsky, 1964) and could presumably control the external environment of the pump membrane with regard to ion concentrations and pH. Similarly, many potential binding sites and a large pH buffering capacity exist on intracellular surfaces. These macromolecular complexes, e.g., mucopolysaccharides, have been implicated in the collagen diseases, atherosclerosis, etc. (Hauss et al., 1970). The “sodium pump,” therefore, probably does not operate on the basis of ions dissolved in normal water; its activity is controlled by non-pump-related binding sites (i.e., by control of ionic concentrations and pH) and by structured water (i.e., by control of ionic mobilities and viscosity). The types of effects each mechanism may have on pump

17

THE Na+, K+-ATPare MEMBRANE TRANSPORT SYSTEM

activity are unclear, but both factors appear to be susceptible to disease or chemical interventions. Several general comments may help to clarify the problem of the molecular mechanism of sodium and potassium transport. First, the net ionic change in the reaction is to remove three sodium ions from a negatively charged environment containing low sodium. These ions are placed in a more positively charged environment of high sodium content. Second, two potassium ions are removed from a positively charged environment of low potassium concentration and placed in a more negatively charged milieu of high potassium concentration. Thermodynamically, therefore, much of the energy from ATP hydrolysis is used to increase the potential energy of the transported ions. Garrahan and Glynn (1967~)calculated the energy changes involved in the operation of the sodium pump at “physiological” conditions (see tabulation). Reactions of energy changes

G (cal)

1. Energy released from the hydrolysis of one ATP

-13,017

molecule 2. Osmotic work required to extrude 3 sodium ions 3. Osmotic work required to take up 2 potassium ions 4. Electrical work required to move one net positive charge out of the cell

+ 4,927 + 4,177 + 207

Net free energy change:

- 3,706

Since the reaction sequence places ions on opposite sides of the membrane, it is logical t o conclude that the molecular pathway for ion movement involves permeation of the ion through the membrane space. Eisenman (1968) summarized five ways in which a monovalent cation could move through this space : (a) free diffusion through the lipid matrix; (b) permeation through membrane pores which are lined by fixed negative charges; (c) permeation through membrane pores which are lined by carbonyl groups; (d) penetration through membrane matrix in the form of a complex with mobile, negatively charged groups; (e) penetration through the matrix in the form of a complex with a mobile, neutral carrier (e.g., a cyclic polypeptide). Eisenman (1968) also pointed out that any of the above mechanisms could be rate-modified by surface-limited (i.e., at the membrane-water interface) systems. Mechanisms (b), (c), (d), and (e) may or may not require total dehydration of the ion as it penetrates the membrane matrix. Another general mechanism was suggested by Jardetzky (1966), who

18

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

+

t

Carrier-cation complex

t +Cation (A)

Unrestricted intramembranol diffusion

I I

m

b

t

+ (B)

FIG.3. (A) Probable physical pathway, based on limitation mechanism, through which cation permeates the membrane space. (B) Carrier system in the absence of the restriction.

proposed that conformational changes in the membrane occur such that a microcosm containing the cation(s) is removed from the environment of one membrane surface and exposed to the environment of the other surface. None of Eisenman’s five possibilities except (a) can be eliminated in the Na+, K+-ATPase transport system. Mechanism (a) is not plausible because of the inherently low monovalent cationic solubilities in hydrophobic environments. It is possible, theoretically, to limit even more the types of mechanisms which may be functional in the sodium pump. It appears unlikely, for example, that transport would be mediated through the transfer of cation to a carrier complex in the membrane matrix, followed by unrestricted diffusion of the complex between the opposite surfaces. Such a

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

19

process would allow lateral diffusion of the complex. All available evidence suggests that active cationic transport, as mediated by the sodium pump, requires the cation t o enter the membrane on one side and to leave the membrane on the opposite side at specific points on the membrane surface. The correct mechanism, therefore, probably defines (i.e., by limitation) the physical pathway by which the cation permeates the membrane space (Fig. 3A). A carrier-system in the absence of this restriction would either require amounts of carrier that are stoichiometrically unrelated to the number of transport sites or be quite inefficicnt (Fig. 3B). A second requirement is that there be a difference in the affinity of the membrane sites for any particular cation on the opposite sides of the membrane, whether the same or different molecular membrane species are involved. It is obvious, upon reflection, that a t one surface the site binds the ion a t low concentration and at the opposite side releases the ion into a medium of high concentration. “Affinity” changes may arise in three ways: (a) the ion is shifted to progressively weaker binding sites as it transverses the membrane; (b) only one site is involved, but the nature of the binding interaction is changed secondary to a change in the state of the site; and (c) the site varies in accessibility. The latter is visualized by assuming that an equilibrium exists for site distribution across the membrane. The unbound-sodium site, for example, may exist predominantly on the internal surface, and the sodium-bound site, at the external surface. Upon dissociation of the sodium ion into the external environment, the unbound site would return to its original internal position. Such a mechanism could favor transport of sodium in an uphill direction. Detailed presentations and discussions of kinetic or descriptive models, applied to the sodium pump, have been presented (Jardetzky, 1966; Barnett, 1970; Caldwell, 1968; Finkelstein, 1964; Glynn and Lew, 1969; Middleton, 1970; Opit and Charnock, 1965; Rapoport, 1970; Stone, 1968; Volkenstein and Fishman, 1970). Any mechanism proposed for the sodium pump must explain reactions other than those realized by forward operation of the pump. One of the most intriguing is a reversal of the pump such that sodium influx and potassium efflux are coupled to phosphorylation of ADP by inorganic phosphate. Garrahan and Glynn (1967~)noted that the free energy available to drive the reaction forward is relatively small (i.e., about 3700 cal). This led to the prediction that under appropriate ionic and product :substrate ratios, the pump should be reversibk. This prediction appears to be satisfied when erythrocyte ghost preparations were used. Ghosts, containing high potassium relativc to sodium and high [ADP][Pi]/[ATP], were exposed to a high sodium and potassium-free environment. Incorporation of inorganic phosphate42 into ATP was observed. This reaction was diminished by

20

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

extracellular ouabain. Potassium efflux under these conditions was observed, and a ratio of efflux to ATP synthesized between 2 and 3 (all ouabain-sensitive) was found (Glynn and Lew, 1970). These results have been confirmed (Lant and Whittam, 1969; Priestland and Whittam, 1969) and downhill movement of both sodium and potassium was observed by Lant et al. (1970). Other reactions catalyzed by the pump are sodium: sodium exchange (Garrahan and Glynn, 1966, 1967a,b,d) and potassium: potassium exchange (Glynn et al., 1970). Actually net synthesis of ATP by the appropriate ionic gradient (or transport) was first shown in mitochondria by Pressman and his colleagues (Cockrell e l al., 1967; Hofer el al., 1966) and by Azsone and Massari (1971) and Rossi and Azzone (1970). B. Mechanism of Energy Transduction

One of the fundamental problems of molecular biology is how energy is transduced from adenosine triphosphate (ATP) to acceptor molecules. Perhaps best understood in this regard are the transphosphorylation reactions between ATP and small molecules (e.g., conversion of glucose to glucose 6-phosphate). But the phosphorylation of ADP driven by electron transport and the dephosphorylation of ATP coupled to osmotic, mechanical, and electrical work are poorly understood, and are consequently the subjects of numerous investigations. Considerable evidence supports the contention that adenosine triphosphate is necessary for function of the sodium pump and may serve as the source of “energy” for the pump (Caldwell, 1956, 1960; Caldwell et al., 1959,1960,1964; Whittam, 1958; Dunham, 1957). Much of the information concerning the mechanism of energy transduction in this system (i.e., the conversion of energy available in ATP into potential energy of the transported ions) has been gained by using both erythrocyte “ghosts” and broken membrane fragments which contain Na+ ,K+-ATPase activity (Albers, 1967; Hokin and Hokin, 1963b; Judah and Ahmed, 1964; Post and Sen, 1965; Glynn, 1968). Examination of ATP activation curves a t constant magnesium levels, magnesium activation curves a t constant ATP concentrations, and Mg-ATP activation curves (i.e., where the Mg:ATP ratio is maintained constant) lead to the conclusion that MgaATP is the “true” substrate for ATP hydrolysis, catalyzed by Na+, K+-ATPase (Hexum et al., 1970). Excess ATP acts as a competitive inhibitor, and excess magnesium leads to noncompetitive inhibition, The K , for ATP in the presence of magnesium plus sodium is markedly sensitive to the presence of potassium.

21

THE Nof, K+-ATPare MEMBRANE TRANSPORT SYSTEM

Post et al. (1965) found K , values for ATP of 0.001, 0.02, and 0.3 mM at the respective potassium concentrations of 0, 0.1, and 16 mM. Squires (1965) observed that sodium and potassium altered the values of the Hill interaction coefficients (n)for ATP activation curves, but Robinson (1967) reported that sodium and potassium did not change these values. The products of ATP hydrolysis, ADP and inorganic phosphate, inhibit the reaction. ADP appears to be a competitive inhibitor of Mg-ATP activation with a K , of 0.45 mM; inorganic phosphate was observed to act noncompetitively with a K , of 23 mdl. These data suggested to Hexum et al. (1970) that the sequence of reaction may involve an ordered release of inorganic phosphate prior to the release of ADP from the membrane system. Baskin and Leslie (1968) reported that Na+ ,I<+-ATPase was more sensitive to inorganic phosphate than ADP (i.e., in terms of concentration) although these authors did not carry out kinetic analysis of their data. It is clear that kinetic approaches to the definition of a reaction mechanism are limited; their main usefulness may be to eliminate certain types of mechanisms as possibilities. Skou (1960) originally demonstrated that there was an ATP-ADP exchange reaction associated with Na+ ,I<+ATPase (later developments with regard to this reaction are discussed below). Since the reaction was independent of the presence of inorganic phosphate, he concluded that a high-energy intermediate, enzymephosphate, was involved in the overall reaction of the pump. Skou accordingly formulated this scheme : E

+ ATP + Mg + Na + K

K E Mg ATP Na

K K EMgATPeE -P+Mg+ADP Ng Na K E-P+E+P,+Na+K Na

K E Mg ATP indicates an enzyme-substrate complex on which Na and I< Na are attached to different sites on the enzyme, “having high affinities for the respective ions.” This scheme (1960) has been changed in form to some extent through the years, but only very recently was there any evidence for a n ATP-enzyme complex (see later). This conclusion stimulated a considerable amount of research, carried out over the past decade, which used the principle of labeling the enzyme system with isotopes. F M P (fragmented membrane preparations) containing relatively high

22

ARNOLD SCHWARTZ, GEORGE E. LINDENMAYER, AND JULIUS

C. ALLEN

Na+ ,K+-ATPase activity, incorporate o rth ~ p h o s p h a te -~from ~ P ylabeled ATP-32P (ATP-32P) (Post and Sen, 1965, 1967; Post et al., 1965; Blostein, 1966; Charnock e t a l . , 1963; Fahn et al., 1968; Gibbs et al., 1965; Hems and Rodnight, 1966; Hokin et al., 1965; Kanazawa et al., 1967; Nagano et al., 1965; Horowicz and Gerber, 1965; Rendi, 1966; Rodnight et al., 1966; Schoner et al., 1966; Skou and Hilberg, 1969). The P 3 P incorporated is only observed after multiple acid washes (i.e., to remove nonbound isotope) and is presumed to be linked to the system “via an acid-stable, covalent bond.” Multiple chloroform-methanol extractions after the acid washes do not remove the label (Bader et al., 1965). This suggests that the labeling is not effected through covalent binding to extractable lipids but, of course, does not eliminate binding to other lipid or proteolipid substances. As predicted by Skou’s original experiments, incorporation required the presence of magnesium. Not predicted by these experiments, however, was the finding that sodium was also required. No other monovalent species could replace sodium, and the response to sodium was saturated a t higher concentrations. Data originally showed that ATP-14C did not incorporate (ie., a t least no evidence was noted for incorporation when the preparations were subjected to acid washes) (Post et al., 1965; Fahn et al., 1968; Rodnight et al., 1966). This again implied that the phosphate moiety was actually transferred to the membrane from ATP. Classic interpretations of enzyme-mediated catalysis are based on the presumed existence of substrate-enzyme complexes (Dixon and Webb, 1964; Westley, 1969). As indicated in Skou’s equations above, the formation of a phosphorylated enzyme should be preceded by a Rlg-dependent synthesis of an ATP-enzyme complex. Earlier reports showed that ATP-14C does not bind via an acid-stable linkage to Na+ ,K+-ATPase preparations (Post et al., 1965; Fahn et al., 1968; Rodnight et al., 1966). These data were recently challenged by Shamoo et al. (1970), who reported the existence of an acid-stable ATP-enzyme complex. Several comments, however, are pertinent with regard to the latter study: (a) the preparation was only 5-25% as active as preparations used where the complex was not found (Post et al., 1965; Fahn et al., 1968; Rodnight et al., 1966); (b) ATP-14C was used (in the original study) a t a concentration of 1 mM; this concentration was found by Rodnight and Lavin (1966) to phosphorylate “nonenzyme” or a t least nonactive residues of the preparation; (c) the amount of sodium-sensitive (i.e., sodium decreased the binding) binding of ATP-I4C was 150 times the amount of that phosphorylated per unit activity of other H per unit activity preparations (Post et al., 1965) or of ~ u a b a i n - ~bound (Schwartz et al., 1968); no evidence was presented which equated this binding to enzyme activity in a quantitative manner; and (d) no evidence

23

THE Na’, K+-ATPare MEMBRANE TRANSPORT SYSTEM

was presented that ATP as opposed to ADP or other derivative was the species bound.* Heinz and Hoffman (1965), and Ngrby and Jensen in Skou’s laboratory (1971) recently demonstrated binding of ATP to Na+, K+-ATPase preparations. The latter study, using a rapid dialysis rate technique, reported ATP binding of about 2 pmoles per unit activity over a range of enzyme activities. This binding did not require magnesium (or at least the latter was much lower than that required for hydrolysis), and the apparent dissociation constant for the complex was increased b y potassium. Also the binding was independent of sodium. Some degree of proportionality was

* In more recent studies (Shamoo and Brodsky, 1971), these investigators employed a rcdriced conccnt>rat,ionof ATP and an increased specific activity of the 1%-labeled ATP ([a-14C]ATP),i n order t,o “increase the detectable binding of ATP to protein.]’ They found t.bat, formation of “E-ATP” is ouabain-inhibitable only in the presence of Mg Na K, while the formation of “E-ATP” in ouabain-treated Na+,K+-ATPase in t.he presencc of Mg alone, is inhibited by Na K, suggesting that about half of the ‘‘F:-A4TP”complcx is an integral part of the ATPase system. The estimated stoichiometric relations a t 0°C among “E-ATP,” “phosphoprotein” and the rate of inorganic phosphate formation show that the turnover of the Na+induced increment of the total “phosphoprotein” (i.e., E-ATP E ,- P E-P etc.) is about, ?.joth that, of E-ATP. These complexities, plus the temperature aspects (0”vs 37°C) await purification of the enzyme, and identification of sites. A more recent communication from this 1abora.tory removes some of these objections but increases the complexity by int,roducing a calcium requirement (Shamoo and Brodsky, 1972). Turtle bladder microsomes were incubated with very low concentrations of ATP (10 p M ) a t O”C, in t,hc presence of magnesium. Addition of sodium and potassium caused a 20% increase in Mg2+-ATPase with no effect on a Ca+-ATPase. Addition of calcium tJothe syst,cm conhining magnesium, sodium, and potassium eliminated the Na+ K+induced increment of ATPase activity. The E-ATP formed was about 10 pmoles per milligram of protein in the presence of 1 mM calcium alone or magnesium alone. The addit,ion of sodium and potassium t,ogcther induced a 32% increase in the calciumdependent format.ion of E-ATP, but no change in the magnesium-dependent formation of IS-ATP. St.udying the synthesis of “l?-P”, thesc workers found that 10 p M ATP32at 0°C cat,alyzed the formation of 10.2 pmolcs of E-P per milligram of protein in the prcscnce of calcium alone and 76 in the presence of magnesium alone, whereas sodium caused a t,ripling of tjhe amount, of calcium-dependent formation of E-P and doubling of magnesium-dependent formation of E-P. Potassium added to the sodium- and calciumcontaining system rcduccd the level of E-P to t,he calcium-dependent level (19.2); atldcd to t,hc sodium and magnesium-containing system, potassium reduced the level to 31, shout half of the magnesium-dependent level. Shamoo and Brodsky feel that their data suggest. t,hst, magnesium, sodium, and pot.assium arc required for the “first intermediary rcsc.t.ionstep.” Calcium apparently forms a stable E-ATP complcx which does not hydrolyzc. Thc sodium-induced increment, of E-P formation may occur in a reaction pathway that. is in parallel to the Na+, K+-ATPase. The E-ATP complex studied was obtained directly from an active hydrolyzing enzyme system and therefore may be of significance in the overall sequencc.

+

+

+

+

+

+

+

24

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

found between the ATP-binding capacity (nmoles/mg protein) and Na+ ,K+-ATPase activity (micromoles per milligram of protein per hour). The authors suggested this series of possible complex formations: E

+ ATP S E - ATP

+ K+ K+ - E K+ - E + ATPSK’ - E - ATP E

E-ATP+K+eK+-E-ATP

Brodsky and Shamoo (1971) suggested that the complex formed between ATP and the “microsomal” protein resembles that expected of a phosphoramido bond. Although this hypothesis is provocative, the data supporting it are scanty and rely chiefly on pH profile. It should be emphasized that the protocols of the experiments by Shamoo and Brodsky (1972) and the studies by NGrby and Jensen (1971) are different, but both results suggest an intermediary membrane-ATP complex. Post et al. (1969) reported indirect evidence of an ATP-enzyme complex. They preincubated microsomes with ATP-321’ and without sodium or magnesium (i.e., a nonphosphorylating condition). The reactions were started by the exposure of the preparations to large amounts of unlabeled ATP with sodium or magnesium (i.e., whichever was lacking) such that incorporation of phosphate was favored. P-32P,as opposed to -31P(nonisotopic), was incorporated into the preparation. This implies that ATP-32P was bound prior to the transphosphorylation reaction. The incorporation of P-32Pfrom ATP-32Pin the presence of magnesium and sodium is quite rapid and was complete within a matter of seconds at low temperatures. Addition of unlabeled AT1’ “chased” the label (i.e., in effect, removed the P-32Pbound to the preparation) by unlabeled phosphate (Post et al., 1965, 1969). This suggested that the phosphorylation step is not irreversible but rather involves a labile linkage susceptible to a turnover type of sequence. If unlabeled ATP plus potassium were added, the rate of the chase was increased by at least a factor of 5 . Furthermore, potassium prevented a buildup of the phosphorylated intermediate if the cation was present in the reaction mixture from the beginning of the experiment. Monovalent cations that can substitute for potassium with respect t o activation of Na+, K+-ATI’ase activity also could substitute for potassium with respect to preventing the accumulation of the phosphorylated enzyme. The sensitivity of the LLchase”reaction to potassium was inversely related to the sodium concentration in the assay medium. Ouabain has two apparent effects on the incorporation reaction. I n the absence of potassium, the glycoside partially inhibits the reaction (Bader

25

THE Na’, K+-ATPase MEMBRANE TRANSPORT SYSTEM

et al., 1970; Charnock, 1969; Post et al., 1965). I n the presence of potassium, ouabain increases the steady-state level of the complex (Post et al., 1965; Rodnight et al., 1966). I t was originally proposed, therefore, that glycosides inhibit both formation and breakdown of the enzyme-phosphate complex. It was subsequently shown, however, that glycosides bind optimally when E-P is present; the glycoside-enzyme-phosphate complex slowly hydrolyzes, leaving the unreactive glycoside-E (Post et al., 1969; Sen el al., 1969). T he amount of phosphorylatcd enzyme formed correlates with Na+,K+ATPase activities of membrane preparations. Thus, Post et al. (1965) found that about 1 pmole of E-P-”P per milligram of protein was formed , of phosphate produced per per unit of ~ n z y m eactivity ( i . ~ . micromoles milligram of protein per hour). Estimates of the turnover number per complex multiplied by the maximal amount of the complex formed yielded close estimatcas of Na+ ,K+-ATPase activity as observed with the preparation. On the basis of these data, the following scheme was suggested by Post et al. in 1965 to describe the general sequence of ATP hydrolysis as catalyzed by Na+, I<+-ATPase : ATP-Mg

-

enzyme

-

+ enzyme

Na+

ADP-Mg

Kf

phosphate

enzyme

+ enzyme

N

phosphate

+ inorganic phosphate

where represents a high-energy bond. The original evidence for a highenergy bond is derived from Skou’s observation (1960) of an ATP-ADP exchange reaction, This particular reaction, however, does not appear to be part of the Na+ ,I<+-ATPase system. The reaction is insensitive t o the effects of sodium (Skou, 1960; Swanson and Stahl, 1966), and it is now clear that sodium is required for the initial phosphorylation reaction. Furthermore, it has been demonstrated that this specific exchange reaction does not possess the same substrate specificity found for Na+, K+-ATPase. Finally, i t has been clearly shown that most (i.e., about 95%) of this exchange reaction can be removed from Na+, K+-ATPase by extraction procedures without altering the ability of the latter to catalyze ATP hydrolysis (Stahl et al., 1966; Stahl, 1967, 1968a). Albers and his colleagues (Fahn et al., 1964, 1966a,b) have, however, found a second type of ATPADP exchange reaction which is closely associated with the “pump” and the Na+ ,K+-ATPase. This reaction requires sodium, but this is demonstrated only under two conditions: (a) a t low magnesium levels compared to those required for optimal Na+,K+-ATPase activity; and (b) in the presence of the sulfhydryl reagents, N-ethylmaleimide or N-butylmaleimide. The latter condition is accomplished in the presence of magnesium concentrations considered optimal for Na+ ,K+-ATPase activity. The exchange

26

ARNOLD SCH WARTZ, GEORGE E. LINDENMAYER, AND JULIUS C. ALLEN

reaction elicited by either (a) or (b) specifically requires adenosine nucleotides; both reactions are inhibited by calcium, ouabain, and potassium. Oligomycin does not inhibit the reaction. These reactions were observed when membrane fragments isolated from Electrophorus electricus were studied. More recently, Stahl (1968b) found that a t low magnesium levels, a sodium-dependent ATP-ADP exchange reaction was present in rat brain preparations. The reaction was inhibited by ouabain or EDTA but was stimulated by oligomycin. The reaction possessed absolute specificity for ATP; the sodium-dependent phosphorylation did not require magnesium. It should be pointed out also that sodium at high concentrations does not inhibit sodium-dependent phosphorylation (Fahn et al., 1968; Post et al., 1965) or the sodium-dependent ATP-ADP exchange reaction (Fahn et al., 1966b). Albers and colleagues (Fahn et al., 1966a,b, 1968; Siege1 and Albers, 1967) found that N-ethylmaleimide (KEM), oligomycin, arsenite, and 2,3-dimercaptoethanol blocked hydrolysis of E-P-32P induced by potassium. This implied that potassium could stimulate the “chase” of one E-PJ2P LLform” but not another. It also was apparent that the potassiuminsensitive form is the complex which mediates the ATP-ADP exchange reaction. These data led to the conclusion that a t least two forms of the complex exist within the turnover cycle of the pump. Magnesium is required to convert the form nearest ATP (i.e., in the native preparations) to the form preceding inorganic phosphate. Post et al. (1969) also reported evidence of two forms: NEM-treated microsomes from guinea pig kidneys were phosphorylated with ATP-32P;E-PJ2P formed under these conditions was labile to ADP (i.e., presumably reflecting an ATP-ADP exchange reaction) but was insensitive to potassium; conversely, native preparations, when phosphorylated, possessed E-P-32Pwhich was resistant to ADP but labile to potassium. We originally showed (and subsequently this has been confirmed by Albers’ group) that pretreatment of the enzyme with ouabain plus magnesium, changed the enzyme in such a way that 32P-labeled inorganic phosphate (Pi-32P),in the absence of any “energy source,” was incorporated into the enzyme preparation (Albers et al., 1968; Inturrisi and Titus, 1970; Lindenmayer et aZ., 1968; Lindenmayer and Schwartz, 1970b). This was surprising, and t o add further to the complexities, potassium in the studies of Albers et al. (1968) did not alter the amount incorporated whereas in our own experiments (Lindenmayer and Schwartz, 1970b) potassium decreased the amount of phosphorylated enzyme. If the terminal step is K+ a E-P

E

b

+

Pi

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

27

and ouabain plus Mg2+promotes reaction (b) from mass action, i t seems reasonable that I<+ would decrease E-PW’ formation. These conflicting results, however, may be due to the sensitivity of the incorporation reaction to potassium and the insensitivity of the hydrolysis of the ouabain-enzyme-phosphate complex to potassium. The rate of 3H-labeled ouabain binding to the enzyme system, stimulated by magnesium and inorganic phosphate, is retarded by potassium (Lindenmayer and Schwartz, 1970b) , but the dissociation of the ouabain-enzyme complex, once having been formed, is insensitive to or retarded by potassium (Allen et al., 1971a). It was originally thought that this complex must be of very low energy and could represent a partial reversal (through ouabain “induction”), of the reaction sequence. Subsequent results suggested that this interpretation is too simple. The electrophoretic patterns of the E-P-”P previously digested by peptic or Pronase treatments, were examined. Profiles of E-PJ2P formed from ATP-32P and Pi-32P(the latter formed in the presence of ouabain) , were identical. Some experiments employed p h o ~ p h a t e - ~and ~ P -33Pprecursors; profiles of the mixture yielded common patterns. These data indicate that the acid-washed preparations contain a chemically identical phosphorylated complex (Chignell and Titus, 1969; Siegel et al., 1969; Sen et al., 1969). Therefore, can E-P formed from ATP and P , be “high energy”? There are three possibilities which infer an affirmative answer : (a) phosphorylysis “stimulates” incorporation of P ;J2P into this form (Siegel et al., 1969); (b) an exchange reaction between P i and E-P exists (Siegel et al., 1969); or (c) the entire protein must be considered in terms of all energy components (Albers et al., 1968; Lindenmayer and Schwartz, 1970b; Schwartz et al., 1968; Siegel et al., 1969). The latter would indicate that energy donated to the system from the ATP is used t o form E-l’, but also alters the conformation of the system. Conversely, E-P formed in the presence of ouabain may be stabilized by a relatively tight binding of ouabain t o the system, presumably through stabilization of a conformer closely related to that induced by ATP. The latter possibility seems consistent with the probable requirement that the system can undergo structural alterations to cause transport. Complete analysis of these data has led Albers et al. (19G8) to propose the following scheme for the turnover cycle (see Fig. 4). Although some facets of this scheme remain to be demonstrated in preparations other than that of the electric eel, as of 1971 the bulk of studies using all sources support this scheme. The complex, ATP-enzyme, although implied, should probably now be added, and the “true” cycle may include states of E-P as yet undiscovered. I n fact, we would amplify the scheme to includc many more conformers. Some evidence challenges this

28

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

CIS

,

trans

FIG.4. Scheme proposed by Albers et al. (1968) for the turnover cycle.

scheme. On the basis of kinetic evidence obtained a t different temperatures, Kanazawa et al. (1967) proposed that a t higher temperatures A T P hydrolysis in the presence of sodium and potassium proceeds through an E-P intermediate, but at low temperatures or in the absence of potassium, hydrolysis proceeds without the intermediate. Skou (1965) raised a second objection : potassium a t 37°C decreased labeling by A T P - T and increased hydrolysis. At O"C, however, potassium decreased labeling but did not stimulate hydrolysis. Other experiments showed that with ITP-32Pat 37"C, potassium decreased labeling and hydrolysis, Skou concluded that the potassium effect on labeling is independent of its effect on hydrolysis and suggested that, in the presence of magnesium, sodium and potassium, nucleotide hydrolysis does not proceed via transfer of phosphate to the membrane. The following counterobjcctions were made, however: (1) Potassium-induced hydrolysis at 0°C may proceed at very low, undetectable rates. Nagano et al. (1967) concluded that E-l'-3zP was chemically identical whether formed at 0°C or at 37°C; these investigators noted that potassium did, in fact, promote hydrolysis of ATP a t 0°C although to a lesser extent than at 37°C. (2) Schoner et al. (1968) showed that potassium does stimulate

THE N o + , K+-ATPase MEMBRANE TRANSPORT SYSTEM

29

hydrolysis of I T P a t 37°C but only a t rates of about 40% compared to ATP hydrolysis. (3) Shamoo and Brodsky (1972) reported that at 0°C in the presence of 0.01 mM ATP, potassium increased (2&25%) hydrolysis of Na+ ,K+-ATPase and decreased labeling (Rlg+Na-induced) by ATPJ2P. Post et al. (1969) more recently reported an experiment which appears to show conclusively that the action of potassium is to stimulate hydrolysis of E-P as opposed to inhibition of the formation of E-PJ2P. Native guinea pig microsomes were exposed to ATP-32P with potassium but in the absence of magnesium or sodium. After preincubation under these conditions, the reaction was initiated by addition of unlabeled ATP with magnesium or sodium, conditions that favor phosphorylation. They found that E-P-32P was formed and that potassium (present throughout the assay) induced the usual breakdown of the complex. By extrapolation to zero time (i.e., time of the second addition), it was shown that potassium could have had no effect on formation of E-P-32P. Conversely, if ADP was added with sodium to the medium preincubated with ATP-32Pand magnesium, in the absence of potassium, formation was inhibited. A considerable number of other investigations were carried out, simultaneously with the above studies, that were designed to determine the chemical nature of the E-P complex. Two likely candidates appeared early. The first mainly resulted from the earlier work of Heald (1957, 1959, 1962) and from the later work of Judah and Ahmed (1962, 1963, 1964; Judah et al., 1962a,b; Ahmed and Judah, 1965a), i.e., phosphorylserine; the second derived from the work of Hokin and Hokin and their colleagues (Hokin and Hokin, 1959, 1960, 1963a, b ; Hokin, 1963), i.e., phosphatidic acid. Much of this work was carried out using intact cellular preparations; Hokin and Hokin (1964) later, examining the possible roles of these groups in homogenates of avian salt glands, found no evidence of a compartment of phosphorylserine or phosphatidic acid which possessed the requisite temporal characteristics for the Na+ ,I(+-ATPase cycle. Glynn et al (1965) could not detect any significant labeling of phospholipids in Na+ ,K+ATPase preparations derived from electric organ membranes, and Rodnight and Lavin (1966) showed that a serine group present in these preparations could be labeled with ATP-32Pbut that this reaction was independent of sodium and required higher concentrations of the nucleotide than that required for sodium-dependent phosphorylation. Skou and Hilberg (1969) also reported two types of labeling: (a) a sodium-independent labeling which was formed in the presence of high ATP levels and had a low turnover rate; and (b) a sodium-dependent labeling which was formed in the presence of lower ATP levels and had a high turnover rate. It should be pointed out that it is assumed that the "unstable" labeling also takes place a t higher

30

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

ATP concentrations but that its detection is masked by the higher “stable” labeling. Nagano et al. (1965) and Hokin et al. (1965) demonstrated that the phosphorylated complex was labile to alkaline treatment and was hydrolyzed, producing Pi-32P; furthermore, the complex was stable between pH values of about 1-3; at 40°C and pH 3.5, hydrolysis is slow enough for determination of the amount hydrolyzed vs time. Examination of these data indicated that hydrolysis adhered to first-order kinetics; this implies that a single chemical species is present (Nagano et al., 1965). Similar p H profiles have been determined by other investigators (Hems and Rodnight, 1966; Glynn et al., 1965). In addition, hydroxylamine stimulated hydrolysis of the complex (Hokin et al., 1965; Nagano el al., 1965), and hydroxylamine is known to act with acyl phosphate linkages to produce hydroxymates : 0

II

E-C-OPOi-

0

+ NHlOH

II

-t

E-C-NHOH

+ HPOi-

The pH profile of lability indicated that phosphate was probably not bonded t o serine, histidine, or thiol groups; the profiles were similar to that found for acetyl phosphate. All these observations, therefore, suggested to these investigators that phosphate was bound to the enzyme through an acyl linkage. Acylphosphatase catalyzed hydrolysis of enzyme-phosphate32P(Hokin et al., 1965). Bader et al. (1965, 1970) and Bond et al. (1972) also concluded that the complex is acyl phosphate because the complex was easily susceptible to methanolysis and ethanolysis and labile to hydroxylamine and molybdate and to divalent cations (Le., magnesium, calcium, and mercuric ions). In addition, acetyl phosphate appears to substitute for ATP in phosphorylating Na+ ,K+-ATPase preparations. Hems and Rodnight (1965) also noted that the complex was easily extracted into methanol. Let us examine the validity and possible importance of the “acyl phosphate” conclusion. If the complex is mediated through an acyl linkage between phosphate and the protein, the number of groups on the protein capable for this interaction are limited: (1) C-terminal carboxyl groups of amino acid residues, (2) L-aspartyl p-carboxyl and (3) L-glutamyl y-carboxyl (y-GP) . Kahlenberg et al. (1967, 1968) reacted [ 2 ,3JH I-N- ( n propyl) hydroxylamine with phosphorylated and nonphosphorylated digests of Na+, Kf-ATPase preparations. Unfortunately, the yield of y-GP was only 2% of the calculated amount (if all the acylphosphate was converted to y-GP hydroxamate). Phosphorylation of a glutamyl peptide is

31

THE No+, K+-ATPare MEMBRANE TRANSPORT SYSTEM

shown : 0

I HO-P-4 l 0

r1:i

H HO -C-CHt-CH,-C

i

H

I

I

H O

I II

N-C-peptide

COOH

The authors claimed that this low yield is due to resistance to enzymatic digestion. It is obvious that more evidence is required for meaningful interpretation. The experiments provided some evidence that the acyl phosphate was an L-glutamyl y-phosphate (7-GP). Although the concept of the reaction sequence as presented in the scheme (see page 28) is supported by considerable evidence, the validity of the suggestion that the E-P is an acyl phosphate remains questionable. Release of phosphate from digests of the membrancs by acyl phosphatase (Hokin et al., 1965) is susceptible to thfi argument that acyl phosphate formation is an artifact of treatment (see below). The fact that potassium-dependent hydrolysis of acetylphosphate (discussed below) is catalyzed by these preparations is not convincing. A more serious objection arises from the studies with hydroxylamine. The classical concept of hydroxylaminolysis is that the hydroxymate formed is irreversible; see reaction on page 30 (Chignell and Titus, 1968). Thus, if the intermediate is an acyl phosphate, exposure of the enzyme to hydroxylamine should lead to inhibition of activity. Fahn et al. (1968) found that hydroxylamine inhibited ATI’ hydrolysis t o about the same extent that it reduced the amount of E-P-32P.Charnock et al. (1967) found that while hydroxylamine caused a breakdown of E-I>-T, it stimulated hydrolysis. They suggested that the hydroxylamine effect is similar to the potassium effect. Schoner et al. (1966) and Chignell and Titus (1966) found that although hydroxylamine does induce hydrolysis of E-P-321’, it neither inhibits nor stimulates ATP hydrolysis. Bader and Broom (1967) and Bader et al. (1970) found that hydroxylamine did inhibit Na+, I<+-ATPase activity but only if low amounts of calcium were present. They reported a K , for hydroxylamine of 20 mM in the presence of 3 x 10-8M calcium; this amount of calcium does not alter the rate of ATP hydrolysis. Although the role of calcium remains unclear, it seems reasonable to suppose that some of the conflicting results discussed above might be due to varying degrees of calcium “contamination” in the different membrane preparations studied. However, Chignell and Titus (1968) found inhibition of deoxycholate-treated microsomes by hydroxylamine, but 0.06 mil4 calcium had no effect on the degree of inhibition. These investigators did note a stimulation of hydrolysis of E-l’-32P, but this was attributed t o ammonium

32

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

contamination of the hydroxylamine solutions. A methylated derivative of hydroxylamine did reduce E-P-32P levels and induced inhibition of ATP hydrolysis, but the effect was reversible, a finding which contradicts predicted irreversibility. It was concluded on the basis of these results that hydroxylamine was a poor probe to use in eliciting the mechanism of Na+, K+-ATPase (Chignell and Titus, 1968). It is conceivable that a covalent acyl linkage is due to acid termination of the reaction and/or washes. The “true” linkage, for example, could be completely electrostatic in nature through sp3 hybridization (Van Wazer, 1958) of the orthophosphate with partial or unequal charge distributions over the four “tetrahedronally placed” oxygen atoms. Acid termination could then cause the following sequence : ATP

+

Enzyme

Acid denaturation

(enzyme --PO:-),

11

M92+

11

K+

(enzyme --PO,+-),

Enzyme

+ PO-:

- - denotes electrostatic

bonds

\

Lo 0

PO,-0 2-

/

I1

- C - -enzyme

Conversion of (enzyme-- PO:-) to the covalent structure would be favored by ATP, magnesium, sodium plus acid reaction (the reactions are usually terminated by trichloroacetic acid, and the pellets are washed about 5 times with acid) ; conversion from (enzyme could be elicited with ouabain plus magnesium and acid reaction. Note that, in the sequence, water would enter into the reaction a t the sodium-dependent step, i.e., at the step where phosphate is transferred to the membrane. I n this regard, Ahmed et al. (1971) recently showed that D20 inhibited Na+,K+-ATPase activity by competing with sodium but not with potassium, an observation that may support the above scheme. A closely related possibility, considered by Alexander and Rodnight (1970), is that an acid-catalyzed migration of phosphoryl groups could occur during acid-induced denaturation. These authors, however, have also provided some evidence that the native com-

THE Na+, K+-ATPare MEMBRANE TRANSPORT SYSTEM

33

plex may be acyl phosphate. Instead of using acid, they terminated the reaction with neutral sodium dodecyl sulfate ( S D S ) . The SDS extracts were precipitated with acetone, and these were labile to hydroxylamine (i.e., hydrolysis of E-P-32P) ; p H profiles of the hydrolysis reaction were also characteristic of an acyl phosphate. Nagano et al. (1965, 1967) found similar results. It should be pointed out, however, that the p H profile would also br compatible with electrostatic linkages, which place the phosphate close to a carboxyl group on the protein. Exposure of the preparations to low p H (i.e., in determining the pH profile) could produce apparent decreases in release rates of phosphate if a portion of the bound phosphate became covalently linked in the presence of the acid medium. Hydroxylamine effects with respect to this complex are difficult to interpret (see above). The difficultics in elucidating a “true intermediate(s) ” are similar to the problems encountered in the mitochondria field. Furthermore, the enzymatic material is heterogeneous and impure, and the functional turnover is extremely rapid which makes intermediary reactions highly labile. These characteristics encourage some individuals to begin looking for “nonchemical intermediates’’ suggestions for mechanisms (e.g., Mitchell, 1966). C. K+-Phosphatase

Judah and his colleagues (1962a; Ahmed and Judah, 1964) reported that membrane preparations contain a system that catalyzes the hydrolysis of p-nitrophenylphosphate (PNPP) in the presence of potassium. Yoshida and his co-workers (1966; Nagai et al., 1966) found that this potassiumphosphatase (K+-Pase) had a similar subcellular distribution to Na+ , K+ATPase. Treatment with NaI increased the activity of both, and ouabain, protarnine, p-chloromereuribenzoate, igrosin, and acetone inhibited both activities. Potassium could be replaced by rubidium, ammonium, and cesium; lithium had little effect. Sodium a t low potassium stimulated activity, but a t higher potassium concentrations it inhibited activity. ATP and ADP inhibited activity presumably by competing with the substrate (p-nitrophenol or acetyl phosphate) ; inorganic phosphate and calcium inhibited activity probably by product inhibition and competition with magnesium, respectively. I<+-Pase catalyzed the hydrolysis of PNPP, carbamyl phosphate, and acetyl phosphate. Bader and Sen (1966) compared the responses of Na*,I<+-ATPase and I<+-Pase to a number of ligands. They reported that the K m for magnesium, rubidium, ammonium, and lithium were identical as was the p H required for optimal response. Similarly, identical K , for ouabain, calcium,

34

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

mercury, and fluoride ions and identical energy of activations were observed for the two systems. ATP acted as a competitive inhibitor for substrates of K+-Pase. The authors suggested, therefore, that K+-Pase was a component of Na+,K+-ATPase. Israel and Titus (1967), however, found a Ki for ouabain of 2 p M , for K+-Pase, and 40 p M for Na+,K+ATPase, using identical ionic conditions for the two assays. They noted that Bader and Sen (1966) used different sodium and potassium levels to test ouabain sensitivity of the two systems. N-Ethylmaleimide was also found to affect the systems differently (i.e., I<+-Pase was less sensitive than Na+, K+-ATPase). They concluded, therefore, that the systems were different. Albers and Koval (1966) found that the sensitivities of the two systems to magnesium were different and concluded that the activities were manifestations of distinct entities. Although ATP acts as a competitive inhibitor of the substrate for K+-Pase, the presence of ATP plus sodium causes stimuIation of this activity (Askari and Koyal, 1968; Inturrisi, 1969; Koyal et al., 1971; Robinson, 1969a, 1970c; Rega et aZ., 1968; Yoshida el al., 1969). Stimulation by ATP plus sodium was found to coincide with a marked reduction in the K , for potassium for K+-Pase (Robinson, 1969a; Rega et al., 1968; Yoshida et al., 1969). ATP alone has been reported to stimulate K+-Pase, but this effect may require calcium (Rega et al., 1968; Pouchan et al., 1969). Hydroxylamine blocks the sodium effect (i.e., in the presence of ATP) (Rega et al., 1968; Robinson, 1971). CTP (Koyal et al., 1971; Robinson, 1970b; Yoshida et al., 1969); ITP and ADP (Koyal et al., 1971; Yoshida et al., 1969) may substitute for ATP; the absolute requirement for sodium was also reported to increase the sensitivity of K+-Pase to ouabain (Yoshida et al., 1969). Robinson (196913, 1970c) constructed a kinetic analysis of some of these observations and found that ATP plus sodium converted the cooperative response to potassium to a positive one. Activity was stimulated and the amount of potassium required to half maximally saturate the system was decreased. CTP plus sodium augmented the K , for PNPP. Robinson also found that nitrophenol was a noncompetitive inhibitor while inorganic phosphate was a competitive inhibitor. These data suggested that there was a sequential release of products; first nitrophenol and second, inorganic phosphate (Robinson, 1970b). Furthermore, it is possible that sodium modifies P N P P activity through interaction at its own sites, and that K+-Pase has coexisting regulatory sites for potassium, sodium, and nudeotides. Oligomycin inhibits the activation induced by ATP and sodium but does not alter the basic K+-Pase activity (Askari and Koyal, 1968; Inturrisi, 1969; Koyal et al., 1971; Robinson, 1969a, 1970b). N-Ethylmaleimide

THE Na+, K+-ATPase MEMBRANE TRANSPORT SYSTEM

35

also blocks ATP plus sodium activation. Askari (1969) and Askari and Koyal (1971) showed that low concentrations but not high concentrations of oligomycin in the presence of sodium activate I<+-Pase. Inturrisi and Titus (1970) reported that PNPP-32Plabeled the microsoma1 preparation in the presence of magnesium and ouabain in a manner similar to P,-nzP labeling of the membrane preparation. No sodium or potassium requirements were found and electrophoresis of membrane digests revealed that PNPP-32Pand P $*P apparently label a common site. Sachs et al. (1967) reported formation of an E-P-32P complex which was stimulated by potassium a t pH 7.8. Robinson (1971) reported that a I<+-stimulated formation of E-P-32P could be observed a t pH 5.0. The complex disappeared a t pH 7.5 and was largely insensitive to hydroxylamine. The reaction was inhibited by ouabain. Robinson concluded that substrates for I<+-Pase phosphorylated one site and that activation by ATP plus sodium occurs secondary to phosphorylation of a second site (Robinson, 1970b,c). Askari and Rao (1969) exposed resealed erythrocyte ghosts to P N P P and potassium and observed a ouabain-sensitive sodium efflux into a sodium-free medium. PNPP hydrolysis was increased with sodium efflux. Sodium efflux into a sodium and potassium containing medium was observed when thc medium contained P N P P and a nucleotide. They suggested that K+-Pase resides on the external membrane surface and functions as the primary ion translocator. The enzyme of the internal surface presumably supplies the physiological substrate. In a more recent study, Askari and Rao (1971) found that the Na+ efflux required in addition t o I<+, P N P P and ATP. Na+ efflux was not stimulated if PNPP were placed inside the ghosts. Ghosts or intact cells were unable to take up P N P P rapidly. These and other studies further suggest that the I<+-dependent phosphatase component of the Na+, K+ATPasc may be on the outside surface of the membrane and that the phosphatase may be “the primary translocator of Na+ and K+.” D. Cardiac Glycoside Inhibition

The Na+ , I<+-ATPase-transport system is closely associated with a cardiac glycoside receptor. Both the sodium pump activity of intact transporting systems and ATP hydrolysis catalyzed by broken membrane preparations (i.e., containing Na+ ,I<+-ATI’ase activity) are inhibited by low concentrations of cardioactive glycosidcs (Kahn and Acheson, 1955; Glynn, 1957; Hoffman, 1962a,b; Portius and Repke, 1962; Post and Jolly, 1957; Schatzmann, 1953; Skou, 1957, 1960). It remains unknown whether

36

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

the glycoside receptor is identicaI to active site(s) on the enzyme, rcsidcs on part, of the enzyme, or is some membrane component closely associated with the pump. Specific detergent treatment of broken membrane fragments yield nonsedimentable, active Na+ ,K+-ATPase preparations (ilIedzihradsky et al., 1967; Swanson et al., 1964) which maintain their scnsitivity to cardiac glycosides ( Lindenmaycr and Schwartz, 1970b; Shirachi et al., 1970), indicating a close physical relationship. I n intact membrane prrparations cardiac glycosides inhibit the hydrolysistransport cycle via intrraction presumably at an cxtrrnal mcmbranc site (Cddwell et al., 1960; Hoffman, 1966; Koeford-Johnscn, 1958; Whittam, 1962, 1964; Diamond, 1962). The potassium-activation site also is located on the external surfacc of thc membrane (Garrahan and Glynn, 1967d; Glynn, 1962; Laris and Letchworth, 1962; Sen and Post, 1964; Whittarn, 1962, 1964), and the glycoside and potassium cffects appcar to be mutually antagonistic for both intact transporting systems and broken membrane preparations (Dunham and Glynn, 1961; Glynn, 1957; Hoffman, 1962a; Schoner et al., 1968). Although it was originally proposed that glycosides and potassium compete for a “potassium-activation site” (Ahmcd and Judah, 1965a,b; Ahmed et al., 1966; Dunham and Glynn, 1961; Auditore and Murray, 1962; Post and Albright, 1959), later experiments demonstrated the complexity of the interaction both on intact and brokcn membrane preparations (Hoffman, 1966). Schatzmann (1965) observed in resealed crythrocytc ghost preparations that potassium could completely overcome the effect of ouabain and that ouabain enhanced the antagonistic effect of sodium against potassium activation of ATPase activity. Rlatsui and Schwartz (1966a) found that ouabain was competitivc with potassium when sodium concentration was held constant in the presence of varying potassium concentrations but that ouabain was noncompetitive with potassium when sodium concentration was varied but the sodium: potassium ratio was hcld constant, a condition which, it was hoped, canceled the inhibitory effect of the sodium on the potassium site, and vice versa. Cardiac glycosides inhibit the Mg2+ Na+ induced formation of E-PW’ from ATPJ2P (Gibbs el al., 1965; Post et al., 1965), as well as the ATP-ADP exchange reaction of E’lectrophorus preparations (Fahn et al., 1966,). Conversely, when the membrane vesicles are exposed to ATP-32P,magnesium, sodium, and potassium (i.e., a condition which prevents buildup of E-P-32P; see above), glycosides cause an apparent increase in the level of the phosphorylated membrane (Post et al., 1965; Shamoo et al., 1970). Ahmed et al. (1966) found that ouabain competes with sodium when thc latter ligand is present in low concentrations. Thus, one could conclude that glycosides act at both the sodium-dependent and the potassium-

+

THE Na+, K+-ATPase MEMBRANE TRANSPORT SYSTEM

37

dependent steps of the ATP hydrolysis sequence. The “steps” or “sites,” however, may not be mutually exclusive, as we will see. Matsui and Schwartz (1967) initially reported studies which used a differentapproach and were designed to delineate the relationships between enzyme and receptor states. They examined glyc~side-~H binding to isolated Na+ ,I<+-ATl’ase preparations, adapting a classical receptor-drug pharmacological approach. In this and subsequent studies (Albers et al., 1968; Matsui and Schwarta, 1968; Tobin and Sen, 1970; Schwartz et al., 1968), it was found that specific. ligands had to be present t o stimulate binding of the labeled glycoside. A t least three ligand conditions were found to yield maximal glycoside binding: (a) ATP plus magnesium plus sodium, (b) magnesium plus inorganic phosphate or acetate, and (c) manganese. It should be recalled that condition (a) with ATPJ*l’ and condition (b) with P,J*P in the presence of ouabain, produce a phosphorylated enzyme system, “E-l’-”I”’, which chromatographically seems to be a single chemical species (Chignell and Titus, 1969; Siege1 et al., 1969). Condition (c) may depend on the presence of endogenous organic or inorganic phosphates, since exogenously added inorganic phosphate increases binding at low manganese (soncentrations (Lindenmayer, 1970) ; magnesium alone also stimulates some glycoside binding (Albers et al., 1968; Matsui and Schwarta, 1968; Schwartz et al., 1968). The relationship between glycoside binding and glycoside-induced inhibition of Naf ,I<+-ATPase was further defined by experiments that showed that inactive glycosides (e.g., hexahydroscillaren), corticosteriods (e.g., prednisolone) and cholesterol were partially or totally ineffective in reducing the specific binding of digoxinJH to the enzyme. A brief explanation of technique might be helpful. The fragmented membrane ATPase preparation is incubated first in the presence of high (lop4 to M ) unlabeled digitalis glycoside (usually ouabain) plus the appropriate ligands. GlycosideJH is then added, and the incubation is continued. The reaction is then terminated by filtration or centrifugation, and the radioactive pellet is counted. An identical reaction is carried out simultaneously, butJ in the absence of excess unlabeled drug. The difference in radioactivity between the two reactions described represents “specific digitalis binding.” The maximal amount of digitalis-3H bound to Na+, I<+-ATPase preparations correlated with the enzyme activity of the preparations (Albers et al., 1968; Hansen, 1971; Schwartz et al., 1968). Thus, Schwartz et al. (1968) found that 1-2 pmoles of glycoside-3H were bound per milligram of protein per unit (i.e., per micromoles of phosphate produced per milligram of protein per hour) of enzyme activity with beef heart preparations. Albers et al. (1968) found a ratio of 2 for electroplax and of 1 for cat brain prepara-

38

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

tions. Other studies have reported ratios of about 1.5 for lamb (Barnett, 1970) and calf brain preparations (Lindenmayer, 1970). These values are close to the ratio of 1 pmole of E-P-32Pformed per unit of enzyme activity as reported by Post et al. (1965). These data initially suggested that digitalis bound to and stabilized a phosphorylated intermediate. Albers et al. (1968) showed that the glycoside-receptor complex was quite stable a t low temperatures and a t higher pH values. As the temperature was raised toward 37"C, or the pH lowered to 6, the complex, however, manifested increasing reversibility. The mechanism by which cardiac glycosides inhibit the Naf ,I<+ATPase-transport system is not completely understood. Originally, it was thought that the glycoside bound to and stabilized the phosphorylated enzyme state (Post et al., 1965; Matsui and Schwartz, 1967). Subsequently, the evidence that glycosides bind (and induce inhibition) when the system is not phosphorylated (Schwartz et al., 1968) led to a modification of this proposaI. Schwartz et al. (1968) suggested that the glycoside does not bind directly to the phosphorylated site but rather to a receptor which is allosterically related to this site. Hoffman, using an intact erythrocyte preparation (1966), earlier suggested that cardiac glycosides inhibit via a n induction of conformational changes. This was based on kinetic evidence that the activating effects of potassium and cesium on transport did not parallel the ability of these two ions to antagonize glycoside interactions with the enzyme system. In this regard, the demonstration that glycosides inhibit the hydrolysis-transport cycle, a transmembrane event, via interaction a t an external membrane site (Caldwell and Keynes, 1959; Caldwell et al., 1960; Hoffman, 1966; Koefoed-Johnsen, 1958; Whittam, 1962, 1964; Diamond, 1962) probably constitutes the most convincing argument that inhibition is an allosteric event. Albers et al. (1968) and Schwartz et al. (1969) found that glycosideinduced inhibition of this system (derived from digitalis-sensitive species but not the rat) was time dependent. The enzyme in the presence of ATP, magnesium, sodium, and potassium catalyzed the hydrolysis of ATE', but the addition of low amounts of glycoside caused a slowing of the hydrolysis rate which progressively became more pronounced with time. Timedependent inhibition appears to be a function of potassium concentrations and is observed for glycoside-induced inhibition of sodium transport by intact transporting systems (Matsui and Schwartz, 196th; Baker and Manil, 1968; Glynn, 1964). Consistent with these observations was the finding that potassium reduced the rate of gly~oside-~H binding but did not change the total amount of the glycoside finally bound (Allen and Schwartz, 1970a,b;Barnett, 1970; Lindenmayer and Schwartz, 1970a,b; Lindenmayer, 1970). This phenomenon could be explained by three mechanisms (Allen

39

THE Na+, K+-ATPase MEMBRANE TRANSPORT SYSTEM

et al., 1970; Lindenmayer, 1970): (1) potassium causes a change in the glycoside-receptor conformation; this change is not directly related to potassium’s effect on enzyme activity; ( 2 ) inorganic phosphate released (from the hydrolysis of ATP) causes a change in receptor conformation which is conducive to glycoside interaction; or (3) the favorable receptor conformation exists within (and is part of) the enzyme turnover cycle. Potassium would complete the cycle, thereby reducing the concentration of this conformer. Allen et al. (1970) showed that the degree of binding was correlated with phosphate produced ; the time-dependent nature of inhibition, however, was insensitive to the presence of excess inorganic phosphate in t,he assay medium. Mechanism (3) seems to be supported by the available evidence. The allosteric hypothesis suggests that glycoside-induced inhibition is secondary to glycoside binding and stabilization of a receptor conformation which is found within the enzyme turnover cycle.

In the cycle scheme x, y, and z represent all intermediate states of conformations of the enzyme and receptor in the turnover cycle; y may or may not represent the predominant form in the presence of ATP, magnesium, and sodium but is the form necessary for glycoside (D) binding (ie., in the presence of ATP). Brackets depict the interrelationships between enzyme state and receptor conformations. The existence of two types of glycosidereceptor complexes, i.e., an easily reversible form (k, k,) and a “difficultto-reverse” form (k3>> 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

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

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

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

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<+-ATPase membrane vesicles. ANS produced significant fluorescence intensity when in a nonaqueous environment, and the fluorescence measured in these experiments essentially reflected that of ANS molecules in such a hydrophobic environment, presumably the matrix of the membrane. Potassium added to a system containing [magnesium sodium ATP] decreased fluorescence; the subsequent addition of ouabain reversed the potassium effect. Inactive glycosides did not induce reversal. Furthermore, heat-inactivated Na+ ,I<+ATPase preparations were completely inactive in changing the fluorescence in the presence or absence of potassium and ouabain. These positive results were due neither to an alteration in the number of ANS binding sites nor to a change in the affinity of these sites for the probe molecules, at least within the limits examined. The conclusion, therefore, is that the ligands, potassium and ouabain, alter the electronic environment “around the probe.’’ (3) Circular dichroism spectra of detergent-treated preparations. Lubroltreated Na+ ,I(+-ATPase preparations yield circular dichroism spectra (Lindenmayer and Schwarta, 1970a; Nagai el ad., 1970) which do not resemble spectra of native membranes (Lenard and Singer, 1966; Wallach and Zahler, 1966); the latter are thought to be due, in part, to electromagnetic distortions of the signal (Urry and Ji, 1968; Urry and Iirivacic,

+

+

THE

N d ,K+-ATPare MEMBRANE TRANSPORT SYSTEM

43

1970; Schneider et al., 1970). Rather, the spectra obtained were typical of those reported for a-helical polypeptides (Greenfield and Fasman, 1969). Ouabain caused a reproducible change in the spectrum when the enzyme was exposed to [magnesium inorganic phosphate] (i.e., a condition which stimz!ates glycoside binding). No effect was observed with heatinactivated preparations or when the active enzyme was exposed to only buffer plus drug (i.e., a nonbinding condition) (Lindenmayer and Schwartz, 1970a). (4) 3H-exchange between the membrane fragments and water. Preliminary results with this technique indicate that active glycosides alter the exchange rate in the presence of an environment conducive for binding; inactive glycosides do not alter the exchange rate, and active preparations are required for the observation. The data obtained with techniques ( 2 ) , ( 3 ) , and (4) may provide the first physical evidence in support of the allosteric hypothesis. If the interpretations are correct, very gross conformational changes must be involved since only about 5-10% of the protein in the samples examined can be relegated to Na+, I<+-ATPase.

+

SULFHYDRYL GROUPSAND ENZYME ACTIVITY Sulfhydryl inhibitors, such as N-ethylmaleimide, p-chloromercuribenzoic acid, inorganic mercury salts, etc., inhibit Na+ ,I<+-ATPase activity (Bresnick and Schwartz, 1968; Schwartz, 1971) indicating a dependency of activity on -SH groups. It is of interest that the Na+,K+-stimulated component is affected to a much greater extent than the ouabain-insensitive component. Any interpretation must await further purification of the enzymatic system.

IV. SOME PHYSIOLOGICAL ASPECTS OF Na+, K+-ATPase A. The Possible Role of the Na+, K+-ATPase Enzyme System in Amino Acid Transport

The interest in nonelectrolyte transport across membrane barriers in a variety of tissues has attracted the attention of numerous investigators for many years. Cori and Cori (1927) and Wilbrandt and Lengyel (1933) first noted that glucose absorption was decreased in animals that had been adrenalectomized. Other investigators found that the decrease in glucose absorption was reversed or prevented by including sodium chloride in the drinking water. In 1958 Riklis and Quastel concluded that sodium ions must be present for glucose absorption to take place. These and many

44

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

other studies have generated significant interest in the role of monovalent cations in the general phenomenon of translocation of a variety of molecules across cell membranes (for example, Wilbrandt and Rosenberg, 1961 ; Bihler and Crane, 1962; Csaky and Thale, 1960; Crane, 1965). It is quite clear that the transport of nonelectrolytes against concentration or electrochemical gradients is dependent in some manner upon sodium and/or potassium asymmetries, as well as on cellular metabolism. In general terms, it does appear that the asymmetric distribution of sodium, in particular, appears to influence the transport of a number of organic molecules (in either a direct or indirect manner). Sodium-dependent transport is undoubtedly influenced by a number of factors. Among these are (1) the inward, downhill sodium gradient somehow influencing the rate of carrier movement; (2) the outward, downhill potassium gradient (reasoning simply, it would appear that intracellular potassium would compete with sodium for the outward movement of the carrier); (3) a t least according to Crane, “The gradient of substrate-carrier affinity is influenced by the equilibrium that is established at a tissue/medium substrate ratio greater than 1. All three of these asymmetries depend ultimately upon the energy-dependent translocation of sodium out of the cell. . .” (Curran, 1965). Crane feels that the sodium-dependent transport in itself is an equilibrating system, not a vectorial biochemical activity. As long as the internal sodium concentration specifically in the region of the “equilibrating carrier” is maintained lower than the medium, transport of nonelectrolytes can take place. It is obvious, therefore, that the Na+ ,K+-ATPase, which is indeed an integral part of the pump which would maintain intracellular sodium a t a level far lower than extracellular sodium might be of importance in the transport of organic materials. A great many test organs have been used to study this phenomenon. Among these are nuclei, lymphocytes, granulocytes, rat diaphragm, tumor cells of various types, red cells, and, in particular, intestinal tissues. The Na+, K+-ATPase is, of course, found in every tissue that carries out some type of active transport process. Since this is not an exhaustive review of the enzyme itself, we refer the reader to any one of a number of comprehensive reviews (for example, Bresnick and Schwartz, 1968; Albers, 1967). Since the intestine has been used by a number of investigators to develop important hypotheses concerning transport, the elucidation of the Na+, I<+-ATPase in this tissue becomes of importance. Taylor (1962) first found an active Na+ ,K+-ATPase in homogenates prepared from intestinal mucosa of the guinea pig. Hirschhorn and Rosenberg (1968) prepared a Na+,K+-ATPase from human jejunal and ileal mucosa and, in fact, found that this enzyme system isolated from patients with cholera and other diarrheal diseases, exhibited a significant depression of activity during the acute phase of the disease

THE N o t , K+-ATPare MEMBRANE TRANSPORT SYSTEM

45

compared to “convalescent” values. Quigley and Gotterer (1969a,b) isolated a Na+ ,I<+-ATPase from rat intestinal mucosa and then proceeded to study its distribution. I t is of interest that the authors found a highly active ATPase in a membrane fraction that was relatively free of brush border, mitochondria, nuclear material and microsomal fraction and concluded that most of the activity was present, therefore, in the plasma membrane. A small, but consistent amount, however, was found in the brush border fraction. The intestinal mucosal cell, being columnar epithelial in type, possesses an apical and basal pole which are morphologically distinct. The apical pole can be isolated in the relatively pure state. The brush border membrane was the fraction employed by Crane and his colleagues for studying sugar transport, and undoubtedly is the site of transport of other biological materials across the intestine. The basal pole, or the remainder of the plasma membrane of the intestinal mucosal cell, has not really been isolated in a pure form and, consequently, its function concerning transport still remains in doubt. In any case, the presence of an active Na+ ,I(+-ATPase in the same tissue that has been employed widely to study organic solute transport, regardless of localization, is highly important since the enzymatic mrchanism for maintaining low intracellular concentration of sodium would, of course, be required to establish any proposal for sodium-dependent solute transport. The amino acid transport story began around 1952 when Christensen and Riggs (1952) and Christensen et al. (1952) found that the uptake of amino acids by Ehrlich ascites cells was inhibited by removing sodium and replacing it with either potassium or choline in the medium. These workers also found that intracellular potassium was decreased, while intracellular sodium was increased in cells that were incubated with glycine for 1-4 hours. I n a series of extensive studies, Christensen and his colleagues (Christensen, 1962) suggested that there may be a common carrier by which potassium, in particular, and the amino acids may be transported; that the energy necessary for the amino acid transport is derived from the potential energy of the potassium gradient. The finding that amino acid uptake was decreased when intracellular potassium was decreased or when extracellular potassium was increased suggested to these investigators that potassium was acting like a “competing amino acid” and that a counterflow of intracellular potassium down its gradient was probably responsible for the amino acid uptake. Thus, the first series of experiments implicated potassium as the major monovalent cation responsible for the transport of amino acids from the medium into the tissue. These investigators quickly realized, however, that, whenever potassium concentrations were altered, sodium ions were altered in the opposite direction, and they did, in fact, realize that the active transport of amino acids could depend on the sodium

46

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

gradient if indeed there was a cotransport of sodium and amino acid on a two-site carrier. However, as is true in all scientific investigations, a hypothesis had to be developed, and the potassium story was selected. This was so mainly because of the finding that pyridoxal, a stimulator of amino acid transport, produced a greater loss of potassium than gain in sodium. The primary importance of a sodium gradient was realized as a result of a t least three studies. Heinz and his colleagues (Iiromphardt et al., 1963; Heinz and Patlak, 1960) found that the uptake of glycine into ascites tumor cells was, in fact, dependent upon sodium. They also found that the free energy in the potassium potential gradient was approximately the same as that in the glycine gradient in the near steady state. Hempling and Hare (1961) found that the rate of free energy expenditure required to maintain the steady state of glycine actually was greater than that made available by the movement of potassium down its electrochemical potential gradient, even if one assumed 100% efficiency of coupling between the fluxes of amino acids and potassium. Heinz and his co-workers found that, if extracellular sodium and potassium were varied independently, there was little or no change in glycine uptake provided potassium was changed a t a constant sodium level; however, glycine uptake decreased significantly when extracellular sodium was decreased at a constant potassium level. About the same time Vidaver (1964) showed a direct interaction of sodium with the substrate carrier. The uptake of glycine by pigeon erythrocyte cells depended on extracellular sodium. Furthermore, the initial velocity of uptake depended on the square of the extracellular sodium concentration. These data convinced Vidaver that two sodiums moved in with each glycine molecule transported. Vidaver later showed that glycine distribution was, in fact, directly dependent on the sodium electrochemical potential gradient and, when this gradient was reversed, an active efflux of intracellular glycine was obtained. Christensen and his colleagues actually reevaluated their own initial work (Wheeler el al., 1965) and concluded that the initial results actually did show evidence for dependence of amino acid uptake on the sodium gradient rather than the potassium gradient. Csaky (1963) also showed that sodium was necessary for the active transport of amino acids across the intestinal membranes, and Schultz and Zalusky (1965) showed that the flux of sodium from the mucosal t o the serosal surface and the short-circuit current increased when alanine was transported and suggested a 1:1 stoichiometry between the amino acid and sodium. This was essentially due to the Michaelis-Menten type of kinetics between the increase of short-circuit current and the alanine concentration on the mucosal side. Schultz and Zalusky (1965) also initially suggested that some type of ternary complex (sodium-amino acid-carrier) is probably involved in the intestinal transport of amino acids and further that the transport is “driven by the

THE Na’, K+-ATPase MEMBRANE TRANSPORT SYSTEM

47

sodium gradient.” As indicated above, the same type of hypothesis essentially has been proposed by Crane and his colleagues for sugar transport in the intestine. How does this phenomenon work and does sodium actually enter the cell along with the amino acid, or does it participate in some type of secondary or tertiary reaction which either induces or accompanies transport? I n 1967, Schafer and Jacquez investigated part of this problem by using intact Erhlich ascites cells. They measured the simultaneous uptake of isotopically labeled a-aminoisobutyric acid (AIB)and 22Naa t a series of different extracellular concentrations) for incubation times of approximately 1 minute at 37°C. The data not only provided direct evidence for a l :l stoichiometry between AIB and sodium uptake, but also provided evidence against the hypothesis that the sodium gradient is the sole source of the energy for the transport of A I B . Using L-phenylalanine, these investigators found a transport that was not dependent upon sodium and, consequently, no increase in sodium uptake accompanied the uptake of phenylalanine. Since both AIB and phenylalanine are neutral amino acids, one should expect that sodium would move along with phenylalanine just as it does with AIB.A more logical interpretation involves the binding of sodium to the carrier involved in AIB transport, but not to the carrier involved with phenylalanine transport and, hence, it is unlikely that sodium is moving independently as a counterion in the transport of amino acids. A simultaneous binding of sodium with amino acids to a carrier or carriers is more likely. Oxender and Christensen (1963a,b ; Christensen, 1969) established a nomenclature for the transport of neutral amino acids and partitioned them into an A and L system. The A system presumably has a high specificity for glycine, alanine, AIB, and methionine, but discriminates against branched-chain amino acids of five or six carbons. The L system has a greater affinity for substrates with appreciable side-chain bulk and length, such as valine and leucine. It also includes methionine within certain limits. One of the major distinctions between the A and L system is that the A system requires sodium and is primarily an active transport system, whereas the L system does not require sodium and may be merely a system for exchange. AIB is an example of an amino acid which enters almost entirely by the A system, whereas phenylalanine is supposed to enter primarily via the L system. Christensen and his colleagues demonstrated that the sodium-dependent portion of the initial flux of uptake of methionine was approximately equal to the portion of the initial flux which was inhibited by AIB. This is good evidence for the existence of two distinct systems. Jacquez and co-workers (1970) recently reexamined the A and L concept. They specifically looked at the temperature dependency of maximal flux, the half-saturation value, and initial transport flux for

48

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

methionine, tryptophan, glycine, alanine, AIB, and phenylalanine transport in Erhlich ascites cells. They found that the maximal flux in transport fell with a decrease in temperature, while the activation energy fell in the range of 11-20 kcal/mole. This is not surprising. The K , (for phenylalanine and tryptophan) decreased with a decrease in temperature; the K , for methionine, glycine, and alanine showed no dependence on temperature, whereas the K , increased for AIB as the temperature dropped. For all the amino acids the K , appeared to be more variable in measurements of initial j h x than maximal flux. Remember that phenylalanine is supposed to enter primarily via the L system, while AIB is an example of an amino acid which enters almost completely by the A system. Christensen and his colleagues reported that, at an extracellular concentration of about 1 mM, only 20% of the initial flux of phenylalanine was mediated by the A system. However, in the experiments reported by Jacquez and his colleagues, at the same extracellular concentration, GO-G5a/, of the initial flux of phenylalanine required sodium and in terms of the maximal flux was indeed sodium dependent. It is entirely possible that differences in techniques might be responsible for the differences in results and this is amply pointed out by Jacquez et al. Are there actually two different types of systems requiring different sites, one primarily in the active transport system (A) , and the other primarily in the exchange system (L)? Johnstone and Scholefield (1965) found that methionine homoexchange was sodium independent, but that approximately one-half of the transport flux was sodium dependent. As Jacquez et al. queried: Is the distinction between the A and L systems really a distinction between transport and exchange and are there really two distinct systems, or perhaps, are they really part of the same system with different subunits? Consequently, there are two hypotheses: The first involves two independent systems (an active transport system that requires sodium and an exchange system that does not require sodium). The second involves only one system, which can either actively transport or exchange. This very same problem is encountered in all systems involving transport, exchange, and leak (see Bresnick and Schwartz, 1968). The solution is not at hand at the present time, but we intuitively feel that a membrane can carry out a variety of functions depending upon the ionic asymmetry. I n terms of modern biology it seems likely that enzymatic functions can be repressed and derepressed as required. If this is so, membrane sites can carry out exchange processes or “active transport” processes by perhaps minute, but significant, alterations in structure. Since none of the systems employed represent homogeneous structures, it is impossible at this time to make definitive conclusions. The studies on amino acid transport in intestinal tissue, tumor cells, red

THE Na’, K+-ATParc MEMBRANE TRANSPORT SYSTEM

49

blood cells, leukocytes, skeletal muscle, and kidney, all recognize that in some way extracellular sodium plays an important role. Those individuals who feel that the energy for transport is derived somehow from ionic gradients, divide the types of mechanisms into two forms, an electrogenic model and a sodium-potassium gradient model, namely an electroneutral “pump.” The electrogenic model assumes that the sodium and amino acid cotransport is itself electroneutral but there must be some kind of contribution from the membrane potential. In the electroneutral sodiumpotassium gradient hypothesis, the sodium gradient alone does not provide all the energy for transport, and additional contributions are thought t o be derived from the potassium distribution. The latter hypothesis appears to be favored by Eddy (1968); but is only a part of the solute or all the solute driven by ion gradients? Is it possible that at least part of the solute movement is coupled to cellular metabolism that does not involve ion transport’? It should be pointed out, however, that suggestions have been made linking ion transport with cellular metabolism (see, for example, Whittam and Willis, 1962, 1963; Whittam, 1961; Whittam and Agar, 1965; Whittam et al. , 1965). Recently, Jacquez and Schafer (1969) reexamined the sodium-potassium gradient hypothesis and compared it to the sodium electrochemical potential gradient hypothesis. They employed Ehrlich ascites cells and studied the uptake of AIB under various ionic conditions. They found that there was an inward transport of AIB against its concentration gradient when the sodium concentration gradient was reversed and also when both sodium and potassium concentration gradients were reversed. They also measured steady-state AIB and ion distribution ratios and measured the energy expenditures required to maintain the concentration of the AIB gradient and compared it, with the energy expenditure calculated to be available from the ion electrochemical potential gradients employing the two-model carrier described. Basically, the results of these investigators make both hypotheses equivocal, and they concluded that the model in which there is a 1 :1 cotransport of sodium and amino acid but no associated potassium movements is inadequate to explain the entire picture. The model in which the carrier operates electroneutrally, that is, returning one potassium for each sodium transported and each AIB brought in, comes “close t o satisfying the d a t a . . . f o r . . . the steady state condition.” The discrepancies are, however, enough to make the investigators doubt that there is a 100% dependency of coupling between ion fluxes and the uptake of amino acid. It should be recalled that Crane (1965) suggested that the sodiumsolute interaction with a carrier system on the membrane really is a type of allosteric effect. When sodium occupies a specific site on the carrier the affinity of this carrier for the transporting solute is increased. But when

50

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

potassium occupies the site, presumably the affinity of the carrier for the solute is reduced. Therefore, active transport of organic materials may be dependent on an inward, downhill sodium gradient as well as an outward, downhill potassium gradient. This, of course, seems to adhere to the sodium-potassium gradient hypothesis described by Jacquez and Shafer although Crane does emphasize the importance of sodium influx and the asymmetry of carrier-solute affinities between the high potassium intracellular side and the low potassium extracellular side and has deemphasized potassium efflux. If the Na+,li+-ATPase enzyme system, and/or if a sodium pump, in some way, is involved in the transport of amino acids and other organic solutes, the specific inhibitor of the pump, ouabain, or any active cardiotonic glycoside, should produce some effect on the solute transport (Bresnick and Schwartz, 1968). In order to examine, in greater detail, the possible role of the Naf ,I<+-ATPase enzyme system, Charalampous (1969) employed KB cells (certified cell line No. 17 of the American type culture collection; epithelium-like and aneuploid cells originally isolated from human carcinoma of the oral cavity, obtained from Dr. Harry Eagle in 1959 and maintained in monolayer cultures) and took advantage of interesting observations concerning impairment of the transport of AIB by inositol deprivation. The experiments were designed to provide information on whether inositol deprivation impairs exchange fluxes in addition to its inhibitory effect on transport fluxes (see above). Furthermore, since inositol-deficient cells failed to increase the steady-state accumulation of AIB in response to increasing concentrations of extracellular sodium it was of interest to study the effect of inositol deprivation on the kinetic parameters of sodium transport as well as on AIB transport. It was of further importance to study the inhibitory action of ouabain on AIB transport both in normal and in inositol-deficient cells. I t had previously been shown by several investigators that transport of AIR as well as other amino acids in various tissues, was inhibited by ouabain (Fox et al., 1964; Rosenberg et al., 1965; Cotlier and Beaty, 1967; Begin and Scholefield, 1964; Bittner and Heinz, 1963; S. G . Schultz and Zalusky, 1965). It should also be emphasized, however, that sodium-dependent amino acid transport in some tissues appears to be insensitive to ouabain a t concentrations which normally cause significant inhibition of the Na+ ,I<+pump as well as of the Na+ ,I<+-ATPase (for example, Parrish and Iiipnis, 1964). The data of Charalampous indicated that inositol deprivation caused a parallel insensitivity of the AIB transport system toward limiting concentrations of extracellular sodium and toward ouabain inhibition with concomitant loss of the ability of these cells to actively transport AIB. However, the evidence was not completely convincing because if there was

.

THE Na+ K+-ATPase MEMBRANE TRANSPORT SYSTEM

51

a direct association between the Na+ ,K+-ATPase and the AIB transport system, omission of sodium from the medium which would lead to an inactivation of Na+,K+-ATPase and pump system, ought to affect AIB transport in the same way as did inositol deprivation. It was shown that when sodium was omitted, the V,,, of AIB uptake was unaffected while ,i the K , increased; inositol deprivation on the other hand, decreased ,T without changing the K,. It appeared likely, therefore, that inositol deprivation affected other mechanisms, one of which was considered to be some type of phosphoinositide, which may be an important structural component of the cell membrane involved in carrier-mediated amino acid transport. The possible roles of potassium and sodium, as well as the Na+,K+ATPase in the transport of amino acids was further evaluated by Charalampous (1971). These studies also employed the IIB cells referred to above and transport of AIB. Studies indicated quite clearly that 0.33 mM ouabain caused a half-maximal inhibition of the Na+ ,K+ pump, a concentration quite consistent with the usual effects of this drug on Na+,K+ATPase. Similar sensitivity was also shown for AIB transport. However, the pump was inhibited maximally within 90 seconds; inhibition of AIB transport was specifically time dependent and was maximal after 30 minutes of exposure of the cells to the inhibitor. Selected inactivation of the Na+ ,K+-ATPase by ouabain or by omission of extracellular potassium was without effect on the rate of AIB influx. The inactivation was not immediate but was time dependent. Subsequent addition of potassium failed, however, to reverse the inactivation, even though the cells were able to concentrate potassium ll-fold to a level greater than 55 mM. The intracellular potassium concentration which protected against inactivation of the AIB transport system was about 30 mM. Extracellular potassium, other than being the source of intracellular potassium, was not required to activate AIB transport. So, when the pump was maximally inhibited by ouabain within 90 seconds of the cell’s exposure to the inhibitor, the influx of AIB proceeded a t its normal rate. These findings are in some variance with those of Bittner and Heinz (1963) , who reported that glycine accumulation in Erhlich ascites tumor cells was inhibited immediately after the addition of ouabain. Discrepancies, however, may reflect different responses of various tissues to the inhibitor. It should be emphasized that selective inactivation of the Na+, Kf-ATPase effected by omission of potassium from the incubation medium, also did not seem to affect AIB transport. These results clearly show that neither extracellular potassium per se nor the functioning of the Na+, K+-ATPase are absolute requirements for active AIB transport in KB cells and probably for a number of amino acid transport systems.

52

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

What is the role, then, of the Na+, I<+-ATPase? The results of Charalampous clearly show that intracellular potassium seems to be essential for maintaining the functional integrity of the transport system. This is not surprising since intracellular potassium is required for the functioning of a variety of intracellular enzyme systems and probably for the maintenance of vital structures required for transport systems. Consequently the role of the Na+,I<+-ATPase is probably not one of a direct interaction with amino acid transport systems. It is likely that this important enzyme system maintains the appropriate ionic milieu, which in turn controls the structural activity of specific transport systems. The finding that low intracellular potassium does lead to an inactivation of an amino acid transport system offers an interesting explanation for the divergent results reported in the literature concerning the inhibition by ouabain of amino acid transport in various tissues (see above). Ouabain certainly inhibits the Na+, I<+-ATPase and leads to depletion of intracellular potassium. What does this event do to amino acid transport? (1) A critical concentration of intracellular potassium may be necessary for maintaining the integrity of the transport system as indicated above; (2) the length of time the cells are maintained a t this critical concentration of intracellular potassium may be vital. Both of these aspects may differ for different tissues, and this can explain why ouabain under some experimental conditions appears to inhibit amino acid transport and under other conditions may not inhibit amino acid transport. This is quite reminiscent of the significant variation in sensitivity of various species to cardiac glycosides, both from the point of view of cardiac contractility and action on N a f , I<+-ATPase, as well as on the sodium-potassium linked pump (Allen and Schwartz, 1969). Charalampous offers some interesting possibilities in attempts to explain the action of the sodium-potassium pump as well as the action of potassium. It is possible that potassium as well as sodium gradients across the membrane, which are maintained by the Naf ,K+-ATPase, “drives” the amino acid transport system [hypothesis discussed above and originally proposed by Riggs et al. (1958)l. It is clear, however, as indicated above, that the operation of the sodium-potassium gradient is probably not an obligatory requirement for transport. Charalampous suggests that the loss of potassium, induced by ouabain would lead to alterations in mitochondria1 function as well as in a t least two enzymes in the glycolytic pathway, phosphofructokinase and pyruvate kinase, and that these effects may be of importance in the transport of amino acids. As stated above, it has been suggested that there is a connection between cellular metabolism, and ion transport. A few comments should be added here concerning the possible function of sodium in the transport of amino acids and other solutes. Does the

THE Na’, K+-ATPase MEMBRANE TRANSPORT SYSTEM

53

interaction of sodium itself with a specific site on the carrier indeed alter in some way the affinity of the carrier for the solute? This in a sense is an extension of the sodium-gradient hypothesis which has been expanded upon by Curran and his colleagues (1970). It should be mentioned at the outset that intestinal tissue, usually rabbit ileum, is employed as the test organ. According t o this concept, the amino acid (or perhaps any organic solute) combines first with the component of the brush border to form a binary complex. This binary complex then can either cross the membrane or rombine with sodium to form a ternary complex, which then crosses the membrane. Kinetic data indicate that the dissociation constant of the binary complex is far greater than that of the ternary complex. Thus, it appears that sodium augments influx because it promotes the formation of a more stable complex. The greater the stability of the ternary complex, with respect to that of the binary complex, the greater will be the stimulatory effect of sodium, a t least according t o the theory. An important feature of this model is the preferred sequence of the formation of the ternary complex, that is the amino acid must combine with the membrane component $rst and then sodium combines with the membrane component, but only after the binary complex has been formed. This model, therefore, suggests that it would be possible to selectively inhibit the binding of sodium and thereby the stimulatory effect of sodium on amino acid transport, without significantly affecting the transport of the amino acid via the binary complex. Frizzell and Schultz (1970), employing the rabbit ileum “test” system, studied the effect of various ions on sodium-dependent alanine influx across the brush border. They found that hydrogen and potassium behave as competitive inhibitors of influx; increasing the concentration of hydrogen or potassium in the mucosal solution was kinetically indistinguishable from decreasing the sodium concentration. In addition, the coupling between alanine and sodium influxes was markedly reduced a t a pH of 2.Fj. With the exception of hydrogen and lithium, none of the monovalent cations significantly affected the carrier-mediated alanine transport in the absence of sodium. This indicates that the inhibitory effects were largely restricted to the sodium-dependent aspect of transport. When the investigators increased H concentration from 0.03 to 3 mM, they found that in the absence of sodium there was no effect on transport but in the presence of sodium there was a marked inhibition of influx. Lithium significantly increased alanine transport in the absence of sodium. Likewise silver and lanthanum also inhibited the sodium-dependent aspect of transport. These data suggest that certain anionic groups possessing a pK, of approximately 4 are probably involved in the interaction between sodium and the amino acid carrier complex. The Curran model (Fig. 5 ) modified t o include the effect

54

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

BRUSH BORDER MUCOSAL SOLUTION

INTERIOR

‘ 4

XAH

t“

X AH

-

p

_1

FIG.5. The Curran model (Curran et al., 1970) of amino acid transport modified to include the cffect of hydrogen on alaiiine influx.

of hydrogen on amino acid influx, is compared to the latest Frizzell and Schultz modification of this model in Fig. 6. The anionic site, X, undergoes a conformational change that alters its cation selectivity. Cf may combine with X to form XC+. Alanine can combine with XC+ to form XAC+ with a K , that does not differ markedly from K1. However, if A combines with X first, a conformational change occurs that makes the anionic site highly selective for Na+. Therefore, the binding of alanine shifts the same site from an H+-preferring site t o a Naf-preferring conformation, in a manner similar t o the Koshland “induced-fit” hypothesis. It is obvious that Frizzell and Schultz prefer a single-site model modification of the sodium-gradient hypothesis of Curran. They did, however, discuss the possible relationship of the Na+,K+ATPase to the entire system. First, they discussed the common features of the pump and amino acid transport which involve ionic selectivity. It is well known, for example, that lithium is capable of substituting to some extent for sodium; it is also well known that potassium and rubidium

55

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

appear to inhibit the sodium-sensitive site on both the pump and sodiumdependent amino acid transport. Finally, Frizzell and Schultz stated that there is a similarity, at least in some respects, between their model and the behavior of the Na+,K+-ATPase. For example, the cation site on the dephospho form of the ATPase appears to be specific for sodium (Albers, 1967; Glynn, 1964, 1966; Glynn and Lew, 1969). “Phosphorylation of a glutamic acid carboxylate group (L. E. Hokin, 1969) initiates. . . some type of conformational change that alters the selectivity of the cation site with K > R b > NH, > Cs > Li > Na. Dephosphorylation of the L-glutamyl-phosphate residue restores sodium specifity.” I n the single-site model of Frizell and Schultz, presumably the binding of alanine would be analogous to the dephosphorylation step. While this is an attractive concept, as indicated in preceding sections, the relationship between phosphorylation, dephosphorylation, glutamyl phosphate, and the binding of various ligands to the complicated enzyme systems and the function of the transport of sodium and potassium is not understood. Christensen and his colleagues have pursued the hypothesis that sodium and neutral amino acids participate as cosubstrates of a specific transport system. They have data that support the concept that sodium may occupy a position a t a specific receptor site involved in transport, which can otherwise be taken by the distal cationic group of a basic amino acid. It appears that sodium and a neutral amino acid of a suitable structure appear to interact as cosubstrates in the following two types of systems: (1) system Ly+ (a system for amino acids with cationic site charge); the preferred substrate appears to be the cationic amino acid; (2) system ASC (the XC’

1tKC

C++A+ X

11 Cf+Nd+XA

-

K, XANCI’

JI

X AC’

FIG.6. Frizzell and Schultz (1970) modification of the Curran model. Cf = either Hf or other alkali cations; K , = dissociation constant of ion-site complex; X = anionic site; A = amino acid.

56

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

sodium-dependent system for alanine, cysteine, and serine and their higher homologs, not serving for N-methylamino acids). Here the preferred substrate apparently is a nonneutral, straight-chain amino acid of 3, 4, or 5 carbon atoms, and sodium is an obligatory cosubstrate. I n a very recent study, Thomas and Christensen (1971) employed a rabbit reticulocyte and a pigeon erythrocyte preparation to study interactions between sodium and various neutral amino acids in a sodium-dependent system that appears to prefer the linear aliphatic and hydroxyaliphatic amino acids. When the side chain was a methyl group, the interaction with sodium was quite strong; the interaction with sodium became weaker when the side chain exceeded the length of the ethyl group. Other alkali metals appeared to be unable to substitute for sodium, and in fact they compete with sodium. A hydroxyl group on carbon number 3 or 4 of the amino acid leads to an even stronger interaction with sodium, but its effect becomes unfavorable if i t is farther removed from the carboxyl group. If the orientation of the hydroxyl group is trans with reference to the carboxyl group, a strong transport-reducing interaction results; a cis orientation, however, lowers the apparent affinity. On the basis of this and other evidence, Thomas and Christensen concluded that the two cosubstrates (sodium and the amino acid) appear to bind in juxtaposition .at the receptor site, and that a substantial portion of the observed interaction occurs directly between them. These investigators feel that this is the “first evidence for the location of the sodium binding site in a transport system specific for sodium and the neutral substrate.” There appears to be a different rate-limiting step for transport of sodium than for the amino acid. Indeed, these investigators indicated that it is an “over-simplification to say that the action of sodium, or t.hat of the substrate amino acid, is simply to increase the stability with which the other substrate is bound into the ternary transport complex [see the Curran model above]; these actions must be understood as kinetic events and not merely as effects on thermodynamic dissociation constants.” In support, one may note that the effect of sodium on lowering the K , of a given substrate amino acid is well correlated with the ability of the latter to serve as a substrate, but not with its apparent affinity for site ASC (Na+dependent alanine, cysteine, and serine). Apparently, the evidence of Thomas and Christensen indicates that only the ternary complex participates in transport. Furthermore, the transport mediator, whatever that may be, cannot recycle unless both cosubstrates are present on both sides of the membrane. The kinetics appear to be best described by a random-order mechanism in which the translocation is not usually rate limiting and in which there is no kinetically preferred pathway to the ternary complex. Since the observed rates of entry of the amino

THE Not, Kt-ATPare

MEMBRANE TRANSPORT SYSTEM

57

acids substrate and sodium differ, there must be two rate-limiting steps, one for each of the two close cosubstrates. These steps cannot be translocation steps for the ternary carrier complex since that step is shared by the two cosubstrates, but must be the subsequent steps of dissociation and replacement. Further, these investigators feel that the transport mediator which becomes loaded with cosubstrates, after becoming oriented to the inner surface of the membrane, may then dissociate and become ‘(reloaded” and, therefore, able t o return by way of three different sequences, or by a statistical combination of these sequences: (1) only the sodium, arising from the outside may be replaced; (2) only the labeled amino acid, arising from the outside may be replaced; (3) both may be replaced in random sequence. The adenyl cyclase system located in the cell membrane, now appears to participate in practically every event associated with transport. Indeed, even in the case of amino acid transport, Harrison and Christensen (1971) now have some preliminary data indicating that the adenyl cyclase system participates in some unknown manner in the sodium-dependent transport of amino acids, but not in the sodium-independent pathway (discussion during Federation Meeting, in Chicago, 1971). They carried out experiments employing young rats which received isotopically labeled-nonmetabolized amino acid analogs (Harrison and Christensen, 1971), selected for specificity to transport systems. After 38 hours, which apparently is a period sufficient for the amino acid to be distributed, glucagon or theophylline, two agents known to increase tissue levels of cyclic AMP, were injected intraperitoneally. Two amino acids typically reactive with a sodiumdependent transport system for neutral amino acids, and one amino acid which interacted with the system for cationic amino acids, showed sharply elevated levels of the amino acids in liver with respect t o the plasma. Levels for the first group also rose in the diaphragm. An amino acid typically reactive with a sodium-insensitive type of transport system exhibited no change in its distribution during this period. This is a rather indirect experiment but nevertheless does suggest that at least some of the amino acids might be transported via a system that in some way is influenced by cyclic AhTP. Recently, some studies (Kypson and Hait, 1971) on the effects of calcium, potassium, and ouabain on the transport of certain amino acids into rabbit atrial tissue have revealed a possible role for calcium, another vital cation frequently mentioned with respect to various types of membrane transport systems. It was found that ouabain in concentrations of lop6 to 10-4M inhibited the incorporation of leucine and tyrosine into total proteins and into various subcellular fractions, whereas calcium, in concentrations of 2.5 and 5 mM, increased the amount of leucine and tyrosine incorporated

58

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

into total proteins as well as into subcellular fractions. The effect of ouabain on the incorporation of leucine was unchanged in media containing different concentrations of calcium, whereas the inhibitory effect of the same concentrations of ouabain on the incorporation of tyrosine was increased in the presence of higher concentrations of calcium, a type of synergistic effect. High concentrations of potassium in the medium (up to 119.6 meq/liter) decreased the amount of leucine incorporated into total proteins; the inhibitory effect of 10-4 M on leucine incorporation was decreased with increased concentrations of potassium, as is typical of the effects of ouabain and K+ on Na+,K+-ATPase. The transport of AIB was inhibited by various concentrations of ouabain and by increasing concentrations of potassium, but was stimulated by higher concentrations of calcium. It appears likely, according to these investigators, that the effects of ouabain, calcium, and potassium on membrane transport are primarily responsible for the various changes in the incorporation of amino acids into atrial tissues. These experiments were stimulated by the original suggestion of Christensen and his colleagues that amino acid transport somehow is coupled to the efflux of potassium and by the experiments of others, who found that increased extracellular potassium inhibited while calcium activated the uptake of tyrosine in rat brain tissue (Kypson and Hait, 1971). It should be pointed out that in these studies the concentration of ouabain M was sufficient to completely block the Na+ ,I<+-ATPase, yet amino acid transport was inhibited by only 83-55%, suggesting that systems other than the Nu+, K+-ATPase involved in the transport process might be influenced by ouabain. Calcium apparently increased the incorporation of leucine and tyrosine, probably by facilitation of the transport of these acids. So it would seem from the heterogeneity of all the studies, employing a variety of different test systems, that there is no direct relationship between the Na+,Ii+-ATPase and the transport of amino acids and other organic solutes. It seems likely that the pump enzyme is indeed involved, but in an indirect manner, perhaps in some way causing vital conformational changes in the membrane leading to specific alterations of cations which in turn affect the affinity or total capacity of specific transport sites. I n our laboratory we have preliminary information that indicates that the Na+, I<+ATPase enzyme system has multiple ligand-binding sites, which can interact with sodium, magnesium, potassium, ATP, and specifically with calcium. Kinetically, we have shown a calcium-sodium type of complex competition, and more recently we have shown that calcium can partially substitute for sodium, in the presence of magnesium and ATP, in stimulating the binding of ouabainJH to Na+,K+-ATPase (Schwartz et al.,

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

59

1972). The latter has been already suggestcd by Repke and his co-workers (Shon et aZ., 1970).

B.

The Possible Relationship between the Na+, K+-ATPase and Sugar Transport

The relationship between ion transport and sugar transport in many ways is similar to the amino acid transport problem described in the preceding section. The sugar transport system is characterized by a number of interesting complexities that require discussion. First, let us reiterate the concept of Crane which attempts to offer a uniform mechanism for all types of organic solute transport (Crane, 1965, 1968). Using the brush border membrane of the epithelial cell of the small intestine as the target transport area for active sugar transport, Crane has suggested that sodium interacts with a specific site and that this interaction increases the affinity of the sugar for a postulated carrier. The carrier system should be capable only of equilibration. An asymmetry of sodium transport is required t o achieve uphill accumulation of sugar, and this is attributed t o a gradient of sodium concentration “downhill” into the cell maintained by the operation of an outwardly directed, energy-dependent sodium pump present a t a different locus in the same membrane. The actual position of the pump is presumably not of prime importance. It is only essential that the local internal sodium concentration in the region of the equilibrating carrier be maintained Eow relative to the medium. So, originally at least, the mobile carrier was represented as possessing two specific binding sites, one for the substrate, namely the sugar, and one for sodium (Fig. 7a,b). Later, Crane did recognize the importance of potassium to this system, and indicated that asymmetric localization of sodium and potassium would be required for the sugar transport. It should be expected from this type of model that ouabain or any cardioactive glycoside would inhibit glucose accumulation simply because it inhibits the sodium pump, which would build up intracellular sodium and prevent sodium depletion. The rate of glucose movement into the serosal compartment would then fall, because the gradient of glucose would be less steep. It should be emphasized, however, that glucose utilization would not necessarily be reduced. The equilibrating system could still allow glucose to enter the cell at a rate exceeding its utilization. The evidence in favor of such a hypothesis is not meager. It has, in fact, been shown that ouabain is a highly effective inhibitor of the active, sodiumdependent transport of sugars and other nonelectrolytes in a variety of

60

ARNOLD SCHWARTZ, GEORGE E. LINDENMAYER, AND

JULIUS C. ALLEN

N0’

> Kt-ATPose?/

SEROSAL BORDER

Nof kept low intmcellularly by operation of No: @-ATPase

a

CELL MEMBRANE

MEDIUM

CELL FLUID

b

FIG.7. (A) Crane’s “unifying” hypothesis for Natdependent nonelectrolyte transport. (B) Crane’s postulated mobile carrier. From Crane (1968).

cells (Csaky et al., 1961; S. Schultz and Curran, 1970). Thus, it certainly is true that sugars can be transported actively, i.e., with the requirement of energy from cellular metabolism across membranes; this is seen predominantly in intestine and in kidney. For example, Kleinzeller and his colleagues have made extensive studies employing kidney cortical cells and a variety of metabolizable and nonmetabolizable sugars and have found a sodium-dependent transport which is specifically inhibited by relatively low concentrations of ouabain (Kleinzeller et al., 1970; Kleinzeller, 1970a,b; Almendares and Kleinzeller, 1971). There is another type of transport, however, that occurs in most, if not all, mammalian cells, particularly in muscle, which is presumably “nonactive” and is generally referred to as mediated transport. The latter concept was originally developed by LeFevre (1948) and Widdas (1954) and by

THE Na’, K’-ATPase

61

MEMBRANE TRANSPORT SYSTEM

Wilbrandt and Rosenberg (1961). A schematic representation is shown in Fig. 8, indicating nonactive transport for sugars, in particular glucose (Park el al., 1968). This system seems to be characterized by six aspects: (1) Saturation kinetics: the initial rate of entry or exit of the sugar approaches the maximum above certain concentrations of the solute. (2) Steroeospecificity : the selectivity of the transport process for certain sugars suggest that a specific combining site is present. (3) The following agents or groups of agents are specific inhibitors of the system: phloretin, phlorizin, lead compounds, and sulfhydryl blocking agents, and dinitrofluorobenxene. (4)Temperature coefficient: the coefficient (Q1O) is approximately 2 in the temperature range of 27-37°C. This value is greater than that for simple diffusion and suggests that the transport processes involves the formation and the deformation of certain chemical bonds. (5) Energy requirements: this transport process specifically cannot utilize metabolic energy. This implies that transport against a concentration gradient is not probable (although not impossible), This type of transport is also freely reversible and tends simply to equilibrate sugar concentrations across the cell membranes. Presumably phosphorylation is not involved, and the free sugar would be the product of transport in either direction. (6) Countertransport: this was first demonstrated by Park and his colleagues (1956). I n the typical type of countertransport experiment, the tissue is perfused with a nonmetabolized type of sugar, such as 3-O-methylglucose, until a substan-

+

+ GX,

GX,

FIG.8. Schematic representation of mediated transport concept. G, glucose; X, unoccupied “carrier”; GX, occupied carrier; subscripts o and i, respectively, on outside surface, on inside surface; reaction closed to equilibrium;-, rate-limiting process. From Park et al. (1968).

+,

62

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

tial rise in the concentration of intracellular sugar has been achieved. At this point a high concentration of glucose is then added to the medium, which causes a rapid drop in the intracellular 3-O-methylglucose concentration. Since the latter is not metabolizable, it must have been transported out of the cell, although the extracellular concentration was higher than the intracellular concentration. This is countertransport. Presumably glucose would compete favorably for a specific carrier at the external surface of the cell. The ultimate source of energy must be the movement of glucose down its concentration gradient with the carrier gradients mediating the energy transfer. It is fashionable to refer to the carriers as “mobile” although there is no evidence to support this. Presumably the carrier should be accessible t o sugars only at one side of the membrane a t a given time. C. Some Complications of Sugar Transport in Relation to the Na+, K+-ATPare

1. SUGARTRANSPORT IN TISSUES EXEMPLIFIED BY INTESTINE AND BY THE KIDNEY The early experiments designed to show active sugar transport employed intestinal tissues (see discussion above), and defined the process as one of a movement of the sugar against the concentration gradient with the expenditure of metabolic energy. As discussed above, the intestinal absorption of glucose and related sugars appears to be “carrier mediated” and depends on an appropriate gradient of sodium. In the absence of sodium, glucosetype sugars enter the tissue very slowly and are not accumulated against a concentration gradient. The slow entry of the nontransported type of sugar is independent of sodium and is thought to occur by some type of facilitated diffusion (see above). The explanation, however, is not so simple. For example, the entry of D-xylose and L-glucose shows sodium dependence, but transport against a concentration gradient can be demonstrated only under special conditions. Furthermore, the nontransported sugar L-fucose significantly inhibits the transport of some actively transported sugars. A number of so-called nontransported sugars can penetrate the intracellular space (Bihler, 1969). This suggests that the intestinal sugar carrier may have some affinity for compounds that are not necessarily actively transported. It is of interest that the actively transported sugars, @-galactose, 3-methyl-~-glucose, a-methyl-D-glucoside, D-xylose, and L-glucose are all inhibited (that is, the transport across the small intestine) by ouabain and by phlorizin and also require sodium for the transport process. Sodium, in fact, reduced the apparent K , of transport of the actively transported sugars. The so-called nontransported sugars, D-arabi-

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nose, L-arabinose, L-rhamnose, L-mannose, and L-fucose showed MichaelisMenten kinetics, but the K , was not significantly altered by sodium. Furthermore, the V,,, of all the sugars tested was identical and independent of sodium. Inhibition of the entry of nontransported sugars by D-galactose and by phlorizin was shown. The data of Bihler (1969) suggest a “dual specificity of a joint sugar carrier.” Bihler feels that in the absence of sodium the affinity of the carrier for many sugars is low, while in the presence of sodium there is an increase in affinity, but only for some of the sugars. The potential for active transport appears to depend, according to Bihler, on the extent of the activation by sodium and varies with different sugars from very low values to very high values. Consequently, Bihler suggests that even the very slowly penetrating sugars, which are presumably not actively transported, do interact with the active transport system or a t least with similar or the same sites, and that the affinities of the actively transported sugars are increased by sodium to varying degrees. Regardless of these complexities, however, it is clear that (in intestinal tissue) in the case of the actively transported sugars, a specific inhibitor of the Na+, K+-ATPase, ouabain inhibits their transport. This would be in agreement with the general hypothesis of Crane (see above). Kleinzeller and his colleagues have recently made an extensive study of sugar transport in the kidney (Kleinzeller et al., 1970; Kleinzeller, 1970a,b; Almendares and Kleinzeller, 1971), another test organ frequently employed for studies of the active transport of glucose and other sugars and one which is specifically inhibited by ouabain. A comparison of the transport properties of 26 monosaccharides was carried out, and the data suggest that the structures required for active transport are : hemiacetal group, C-3-0-H (in a configuration identical with that of D-glucose) and C-6-0-33; the sodium requirement for active transport is related to the presence of a hydrophilic (-OH or -NH2 group on carbon 2). The kinetic data indicate tremendous complexities and suggest the existence of several pathways for the active sugar transport into renal tubular (proximal) cells. The electrolyte requirement for the active transport of some sugars are as follows: external sodium = 128 mM (this increases the apparent V,,, of D-galactose transport without affecting the transport K,). A significant fraction of the D-galactose transport was found to be sodium independent and also insensitive to 2 m M ouabain. The presence of 0.5 to 7 mM potassium activated the transport, and affected the V,,, rather than the K , of sugar transport. No effect of external potassium on the transport of both 2-deoxyhexoses was observed, however. The absence of calcium, interestingly enough, markedly depressed both the influx and the steady-state accumulation level of all sugars tested. It was concluded that in kidney cortex cells the presence of sodium, both extracellular and intracellular, is not manda-

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tory to bring about an active transport of 2-deoxyhexoses and also a fraction of galactose ;the potassium-induced stimulation of sugar transport appears to be related to the sodium requirement for sugar transport, and finally calcium stimulates some common step in the transport of all sugars tested. Kolinska (1970) also carried out a similar study employing 2-deoxyhexoses and found that external sodium was not required for transport, which is in contrast to the sodium requirement for other monosaccharides. Studying the kinetics of D-galactose, 2-deoxy-D-galactose and a-methybghcoside, Iiolinska developed an even more complicated model than that of Kleinzeller, via. one involving three receptors on the same carrier protein with “mutually more or less interlinked binding sites plus a sodium site on a nearby membrane component. . . .” As mentioned above, ouabain is a highly specific and effective inhibitor of the active-sodium-dependent transport of sugars and other nonelectrolytes in various cells including the kidney cortex. It is attractive to suggest, and indeed it has been suggested (Iileinzeller and Knotkova, 1964), that the action of ouabain is somehow related to an inhibition of the sodium pump and the Naf ,K+-ATPase. On the other hand, as Iileinzeller has pointed out, the sodium-independent active transport of some sugars in kidney cortex cells is insensitive to ouabain. In order to investigate further the mechanism by which ouabain interacts with the sodium-dependent transport system for sugars in kidney cortex cells, Almendaries and Kleinseller (1971), studied the effects of the drug on the active sodium-dependent transport of a-methyl-D-glucoside by slices of rabbit kidney cortex. Ouabain concentrations around 0.02 mdf caused a significant inhibition, with a K , of 0.13 mM. It was found that by increasing external potassium to 25 mM the inhibition induced by 0.02 m M ouabain was abolished. When higher concentrations of ouabain were used, such as 0.5 mM, increasing external potassium was ineffective in abolishing the inhibition of the sodiumdependent transport of the sugar. Preincubation of the kidney slices with ouabain produced an inhibition of both cation and glucoside transport, when the slices were transferred to a ouabain-free medium; 80% or more of the ~ u a b a i n - ~bound H t o the slice “could not be washed out.” This is consistent with the concept that in order for ouabain to produce an inhibition of intact sodium pumps, Na+, K+-ATPase and other sodium-dependent transport systems, the drug must first bind to some receptor. The binding of ouabain by the kidney tissues is of interest since at most concentrations of glycoside (from 1 to 100 g M ) there was a saturable binding component, and this was depressed in the absence of sodium and was abolished by increasing external potassium or by incubating the tissue a t 0°C or by denaturation of the tissue by heating at 80°C. There was also a nonsaturable binding component of ouabain, and this appeared to be

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linearly related to the concentration of the substance in the medium. Kleinzeller computed a LLmaximal’lnumber of ouabain-binding sites in rabbit kidney cortex. This was 6 X 1015sites per gram wet weight with an association constant for ouabain of about 3 X lo5 M-I. It was of interest that, in rat kidney cortex, the value for the maximum number of ouabainbinding sites was a t least one order of magnitude lower than in rabbit. This is entirely consistent with the well known digitalis-insensitivity of rats and with the concept promulgated by Repke (1963) and by ourselves (Allen and Schwartz, 1969) that the sensitivity of isolated Na+ ,K+ATPases to ouabain binding and induced-inhibition is directly related to the sensitivity of the species to digitalis. Thcre was an apparent discrepancy between the Ki for ouabain inhibition of the transport ATPase and for the sodium-dependent transport of the glucoside (the latter was 0.12 mM while the former was approximately 0.02 m M or lower). First, it should be emphasized that ouabain is an extremely complicated inhibitor; the inhibition is temperature dependent and time dependent, and the inhibition induced by the drug in all species except the rat is hyperbolic. Consequently, the use of the phrase “half-maximal inhibitor constant = Ki” is inappropriate. One must specify the time and temperature as well as the type of assay employed. Kleinzeller correctly pointed out that any discrepancies could be due to the “fact that the conditions for measuring the enzyme activity and the transport process in the tissue differ widely, particularly with regard to the concentration of potassium a t the site of action of the cardiac glycoside”. Robinson (1970) suggested that any discrepancy between so-called inhibitory constant for ouabain-induced inhibition of the Na+ ,K+-ATPase and for the sodium-dependent transport of nonelectrolytes in the intestinal mucosa is due to the fact that sodiumdependent transport of solutes may not necessarily be coupled to the Na+ ,K+-ATPase. Because of the aforementioned complexities of assays, however, this conclusion should be applied with caution. On the other hand, because there appears to be some type of relationship between ouabain-induced inhibition of transport of organic materials and the effect of the drug on the Na+, K+-ATPase, this does not necessarily mean that the two are directly correlated. In fact, in the most recent work of Kleinseller and his colleagues, the conclusion is reached that the “membrane ATPase participates (directly or indirectly) in the sodium-dependent transport system for a-methyl-glucoside.” It is attractive to suggest that the Na+ ,K+-ATPase may reflect a more complicated system, perhaps involving not only the maintenance of sodium and potassium concentrations but the control of calcium. For example, Kleinzeller did find (1970b) that the sodium-dependent sugar transport system was stimulated by calcium in a manner quite similar t o the sodium-independent transport

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system. It is possible that, as suggested by a number of investigators, there is a common step involving calcium for both the sodium-dependent and sodium-independent organic solute transport sy6tems. 2. INSULIN-SENSITIVE SYSTEMS

The complexities involved in attempting to link the Na+ ,K+-ATPase with sugar transport are increased when one considers the insulin-sensitive sugar transport systems in muscle, in adipose tissue, and in adipocytes. It is curious that any intervention that results in an inhibition of the sodiumpump or the Na+, K+-ATPase results in a stimulation of transport rather than inhibition, as discussed above. In particular, ouabain or the omission of potassium from the medium, results in stimulation of a variety of sugars in the above systems. Two possible mechanisms for the stimulation have been suggested. One of these, that of Letarte and co-workers (1969), suggests that the insulin-sensitive sugar “carrier” is supposed to bind and cotransport sodium and/or potassium in a manner similar to the carrier which is thought to participate in active sugar transport in the intestine. The second interpretation, put forth by Clausen (1966) and by Bihler and his group (Bihler, 1968, 1970; Bihler and Jeanrenaud, 1970; Bihler and Sawh, 1971; Bihler et al., 1970, 1971), suggests an indirect effect of intracellular sodium and/or potassium on the sugar carrier. The two types of models would lead to different results with respect to the effect of various changes in the ionic milieu. For example, the first model would predict that variations in ionic levels a t one side of the membrane should affect sugar transport from that side without a lag; the effect should be asymmetric, with flux in the opposite direction remaining unaffected. The second hypothesis, or the indirect model, includes a lag period and also accommodates a symmetrical and equal effect on both inside and outside fluxes, such as is observed with insulin. Employing the intact rat hemidiaphragm preparation, fluxes of 3-O-methyl-~-glucose-’4C were measured in this preparation (Bihler and Sawh, 1971). It was found that the stimulation of influx in a potassium-free medium was correlated with the duration of exposure to the specific potassium-free medium and persisted for some time after the transfer of the tissue to a normal medium. The time course of this effect was parallel with that of the concomitant changes in intracellular sodium and potassium levels and was due to an inhibition of the sodium pump. The efflux of the sugar from the tissue was stimulated under all conditions which stimulated influx. The effect of potassium-free medium also showed the same gradual onset and persistence

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after the transfer of the tissue to a normal medium. These effects according to Bihler and Sawh (1971)) are not consistent with the regulation of transport through a binding or cotransport of potassium on the sugar carrier, nor do they support a direct link “between sugar transport and the activity of the sodium pump as such.” Data suggest that the increased sodium, or decreased potassium level in the cell or both caused by sodium pump inhibition, enhances sugar transport in and out of the cell to a n equal extent. In other words, it appears that the inhibition of the sodium pump somehow is indirectly involved in the stimulation of sugar transport in these particular tissues. Some years ago, Kreisberg and Williamson (1964) found a rather interesting effect of ouabain in a perfused intact rat heart preparation, which is quite reminiscent of the recent results of Bihler and his colleagues. Ouabain caused a significant increase in the uptake and subsequent oxidation of glucose by perfused guinea pig and rat hearts to a degree greater than 50% of control. In spite of this, glycogen levels remained constant and, in fact, there was no change in specific activity of glycogen. Of great importance was the fact that the observed changes were entirely dependent on the calcium concentration of the perfusate. Reduction of calcium, for example, abolished the increase in metabolism induced by ouabain. The effects of ouabain, perfused with a buffer containing twice the normal concentration of calcium, was quite similar to that of hearts perfused with buffer containing the normal calcium concentration in ouabain. The effect of the high calcium concentration combined with ouabain was not different from that produced by either alone. The authors concluded that their data were consistent with the concept that the effects of cardiac glycosides on cardiac muscle are dependent on changes in calcium transport or concentration at the site of the contractile elements. Furthermore, the usual types of concentration differences were found with the rat preparation compared t o the guinea pig (i.e., rat is relatively insensitive to digitalis preparations and hence requires much more of the drug to react). Recently, Hoeschen (1971) repeated the experiments, described above, using an isolated perfused rat heart preparation, and found that ouabain did, in fact, enhance glucose uptake and also increase glucose oxidation, while palmitic acid uptake, oxidation, and incorporation into tissue lipids were unaffected by the drug. The concentrations of ouabain used in the experiments were shown to have a significant inotropic effect and the conclusion was that the increase of glucose uptake and oxidation was not due to an antilipolytic effect,nor was it due to any effect of the drug on insulin, but was rather associated with the increased contractility. This again implicates calcium, since the latter was undoubtedly due to a change in calcium metabolism.

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V. OTHER EFFECTS OF CARDIAC GLYCOSIDES ON MEMBRANELINKED FUNCTIONS Antilipolytic Effects of Cardiac Glycosides

Mosinger and Kujalova (1966), originally reported that either potassiumfree medium or ouabain inhibited catecholamine-activated lipolysis in adipose tissue, This suggested that perhaps the influx of norepinephrine across the cell membrane may be related to active cation transport. Consequently, the antilipolytic effects of the cardiac glycosides might be due to an inhibition of active uptake of catecholamine into fat cells. Other investigators have noted that cardiac glycosides inhibit free fatty acid release that was stimulated by ACTH or by cyclic AMP, or by epinephrine. Furthermore, these effects were also mimicked by a potassium-free medium. It is possible, therefore, that cardiac glycosides and potassium-free media exert the inhibitory effects on free fatty acid release by inhibiting the Na+,K+-ATPase associated with active cation transport. Yu et al. (1971) carried out a series of interesting experiments bearing on these findings. The effects of cardiac glycosides and potassium on norepinephrine, theophylline, and dibutyryl cyclic AMP-activated lipolysis in rat epididymal fat pads were studied. In addition, theophylline-inhibited phosphodiesterase activity and the uptake of labeled-norepinephrine were studied. It was found that cardiac glycosides, as well as potassium-free medium, markedly inhibited the catecholamine-induced glycerol release, and also inhibited labeled-norepinephrine uptake. These inhibitory effects appeared to be antagonized by high potassium concentrations. Glycerol release, which was stimulated by theophylline or by dibutyryl cyclic AMP, was also decreased by both cardiac glycosides and a potassium-free medium; whereas theophylline-inhibited phosphodiesterase activity was not influenced by cardiac glycosides. The inhibitory effects of cardiac glycosides on the lipolytic action of theophylline and dibutyryl cyclic AMP were antagonized by high potassium which, of course, suggests that the inhibition of glycerol release by cardiac glycosides may involve cell membrane activity and Na+ ,K+-ATPase. Consequently, these recent studies indicate that the antilipolytic effects exerted by cardiac glycosides occur by altering some membrane function which results in an inhibition of norepinephrine uptake, as well as glycerol release. It would appear that Na+,K+-ATPase is involved. More recent evidence from the same laboratory (Yu et al., 1972) indicates, however, a number of complexities. The inhibitory effects of cardiac glycosides on drug-stimulated glycerol release were compared with those of sulfhydryl reagents and other metabolic inhibitors. A biphasic stimulation of lipolysis by norepinephrine was depressed by 5 X lo-* M

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digitoxin in both minred tissue and isolated fat cell preparations. The norepinephrine-induced glyrerol release also was significantly reduced by PCMBS, NEM, IAA, DNP and by dicoumarol. The addition of sulfhydryl reagents, such as P-merraptoethanol completely reversed the sulfhydryl inhibitor effects, but was unable to antagonize those raused by DNP, dicoumarol, or digitoxin. Cysteine and BAL significantly reduced only the NEM inhibition, but had no significant effect on PCMBS and IAA, and in fact potentiated the antilipolytic effects of DNP, diroumarol and digitorin. The digitoxin-induced inhibition was completely antagonized by sulfhydryl reagents while those of D N P and diroumarol were only partially reversed. These interesting data suggest that rardiar glycosides may exert their antilipolytic effects by a conformational alteration of membrane structure but do not eliminate the possible role of oxidative metabolism. A direct interaction with sulfhydryl groups a t the membrane or the lipase itself is unlikely. The similarity between the effects of cardiac glyrosides and DNP and dicoumarol suggests the aforementioned role for oxidative phosphorylation. However, it should be remembered that both sodium and potassium are required for oxidative phosphorylation. Consequently, an indirect obligatory role for the Na+ ,I(+-ATPase is still probable. OUABAIN-SENSITIVE p-AMINOHIPPURIC (PAH) ACID TRANSPORT

It has recently been demonstrated (Nechay and Chinoy, 1968) that high concentrations of ouabain can inhibit tubular extraction of PAH. While the mechanism is not clear, these authors do suggest that it is not a direct link t o Naf pump inhibition, since there was a differential effect on natriuresis and PAH inhibition. VI. ROLE OF MEMBRANE TRANSPORT IN BlOGENlC AMINE TRANSPORT

Neuronal reuptake of catecholamines and other biogenic amines represents the most important mechanism for the termination of the action of these compounds. Bogdanski and Brodie (1969) have stressed the importance of inorganic electrolytes in the regulation of the storage and accumulation of norepinephrine (NE) by sympathetic nerve endings. These investigators originally employed rat heart slices for their studies, but later used other systems. The original studies showed that sodium, potassium, and calcium all play some important role in the normal storage process. Sodium ion was particularly emphasized and appeared to be an essential requirement for both the accumulation and storage of NE. The effects of

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sodium were antagonized by potassium. A sodium-deficient medium caused release of N E and low extracellular potassium facilitated the uptake in storage, whereas high extracellular potassium antagonized the effect of sodium on both uptake and storage. The efflux of labeled NE was increased in the absence of calcium and was further enhanced by EDTA. Ouabain selectively blocked the uptake more effectively than it increased the efflux. The effects of ouabain appeared to be quite similar to the effects of potassium-free medium, suggesting a relationship between ouabain and the potassium-activated process. These investigators quite early recognized the interesting correlation between their studies and the experiments described for amino acids and sugars (see above) (Bogdanski and Brodie, 1969). In a recent series of experiments (Tissari and Bogdanski, 1972) these investigators developed a model transport carrier mechanism for amines in brain and in periphery which is based upon the Crane hypothesis for sugar transport discussed above. This model relates to the effects of ions in establishing a specific transmembrane gradient in the affinity of a membrane carrier mechanism for amines. The transport of NE and serotonin by nerve endings obeys Michaelis-Menten kinetics. Furthermore, sodium increased the affinity of the membrane carrier mechanism for amines, and high potassium concentrations decreased the affinity in the presence of various concentrations of sodium. The inhibitory effect of ouabain on transport appears to be sodium dependent but is delayed. This is in contrast t o the more rapid effect of ouabain on Na+,K+-ATPase, suggesting, according to these authors, that the inhibition of transport by ouabain is caused indirectly by changes in concentration of intrasynaptosomal ions. However, this conclusion can be criticized in a similar way as the apparent discrepancies noted on amino acid systems and sugar transport carrier mechanisms described above. The Bogdanski group has suggested that the transport of amines may be dependent upon the simultaneous transport of electrolytes which maintain local concentration gradients a t the plasma membrane. It is clear that the inhibitory effect of ouabain on amine transport certainly involves potassium and that low concentrations of potassium facilitate the transport and retention of NE by sympathetic nerve endings in heart slices and in synaptosomes. Potassium-free medium behaves similarly to ouabain. Thus Na+, K+-ATPase appears to “satisfy the energy requirement for transport.” VII. EFFECTS OF PHLORlZlN ON MEMBRANES

As discussed above, a number of studies have suggested a close relationship between active transport of organic solutes and sodium ion transport

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in biological systems. Reviewing briefly, it has been shown in the intestine and in the kidney that active sugar transport requires the simultaneous presence of both the sugar and sodium. The mechanisms had been intensively studied, particularly in the intestine, where i t appears that sodium specifically enhances the affinity of actively transported sugars a t some rate-limiting site or sites. When both sugars and sodium are actively transported across an epithelial cell layer, the sugars and sodium interact in active sugar transport a t one surface, while active sodium transport takes place at an opposite cell surface. Actively transported sugars appear to stimulate sodium transport-independent associated modifications in cell metabolism, and this implies some type of interaction of sugars and sodium at the site of the sodium-potassium pump. Crane and a number of other investigators have pointed out that the low intracellular sodium, which is maintained by active sodium pumping, may provide the driving force for active sugar transport if sugars and sodium share a common carrier. Direct action of the sugars on the sodium pump itself, however, has not been excluded. Phlorizin appears to have a high affinity for the transport system, compared to that of the simple hexoses and pentoses (Alvarado and Crane, 1962; LeFevre and Marshall, 1959; Lotspeich, 1961). Therefore, the search for some direct action of sugars on the Na+,I<+-ATPase, which appears to be important in sugar transport, would involve effects of phlorizin on the Na+,K+-ATPase. This is rather complicated since the molecule appears to have a number of effects. For example, using a Na+ ,K+-ATPase isolated from rabbit kidney, Britten and Blank (1969) found that in concentrations from M to lop3M phloriain inhibited the enzyme at sodium :potassium ratios less than optimal for enzyme activity, whereas stimulation of enzyme activity was noted at sodium:potassium ratios greater than optimal for enzyme activity. Phloriain at concentrations of 10-‘jM to 10-4M appears to be a significant inhibitor of active sugar transport in the kidney and intestine and to facilitate sugar transport in the rat cell. At higher concentrations to 10-3 M ) , phlorizin inhibits oxidative metabolism and causes mitochondria1 swelling (Crane, 1960). Consequently, the results of Britten and Blank suggest that phlorizin might have an effect on the cell membrane to cause a modification of activity similar to what it might do in mitochondria. However, the molecule also may act as a potassium substitute, or to “shift the setting of the enzyme for optimal activity to a higher ratio of sodium to potassium.” Festoff and Appel (1968) suggested a similar effect for diphenylhydantoin. In any event, Britten and Blank concluded that there is an interaction of actively transported sugars with a sodiumpotassium transport system at a level of the sodium-potassium pump. A link between the pump and the sugars would be of importance. In a detailed

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study, Robinson (1969b) found that phlorizin stimulated the potassium-dependent phosphatase activity associated with a rat brain Na+ ,K+ATPase, but, interestingly enough, inhibited the Na+, I(+-ATPase activity. The concentration of phlorizin required for half-maximal stimulation of the phosphatase was 0.05 mM, and the concentration required for halfmaximal inhibition of the ATPase was 0.06 mM. Phloretin, a hydroxylated aromatic compound, also stimulated the phosphatase. With respect to the phosphatase action, 0.03 mM phlorizin decreased the concentration of potassium required for half-maximal activity from 1.92 to 1.17 mM, but it is of interest that it had little effect on V,,,. Sodium inhibited the phosphatase activity and phlorizin increased the concentration for halfmaximal inhibition for sodium from 6 to 12 mM. With respect to the ATPase activity, 0.1 mM phlorizin similarly decreased the concentration required for half-maximal activity for potassium from 0.74 to 0.48 mM and increased the half-maximal inhibitory Concentration for sodium from 5 to 10.5 mM. A Hill plot revealed n > 1 indicating a positive cooperative allosteric response to sodium and this was converted to a negative cooperative response by phlorizin. In addition, phlorizin inhibited the sodiumdependent phosphorylation of the Na+ ,K+-ATPase. Robinson’s data suggest that phlorizin reacts similarly with the Na+ ,K+-ATPase and potassium-dependent phosphatase, perhaps serving as a “heterotropic allosteric modifier to increase the apparent affinity towards potassium, but decrease it towards sodium.” These results are somewhat similar to those described by Britten and Blank. Thus the use of phlorizin represents another tool that suggests that ‘the potassium-dependent phosphatase may represent part of the overall Na+ ,K+-ATPase reaction. The inhibition of sugar transport by phlorizin and phloretin could possibly be mediated in part through an effect on the sodium-dependent complexities of the particular transport system. We have found that phlorizin and phloretin inhibit Na+, K+-ATPase derived from a number of glycoside-sensitive species, but that the inhibition does not resemble that induced by cardiac glycosides. However, Kleinzeller (1971, personal communication) has indicated that phloriain does not affect the distribution of Na+ and K+ or ATP content of renal slices at a concentration (1 mM) which completely inhibits Na+ dependent sugar transport. Thus, complexities are by no means resolved. ACKNOWLEDGMENTS The authors express sincerest appreciation to colleagues in the transport field who so kindly submitted reprints, preprints, and ideas. We thank Dr. M. L. Entman of this Division, who critically reviewed the manuscript, and Mrs. C. Ramey and C. Rollish, who skillfully typed and proofed the copies.

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Biochemical and Clinical Aspects of Sarcoplasmic Reticulum Function* A N T H O N Y MARTONOSI Department o j Biochemistry. St . Louis University School of Medicine. St . Louis. Missouri

I . Introduction . . . . . . . . . . . . . . . . . . I1. The Mechanism of Ca Transport . . . . . . . . . . . . A . Brief Outline of Early Developments . . . . . . . . . B . Sources of Energy for Ca Transport . . . . . . . . . . C . The Phosphoprotein Intermediate . . . . . . . . . . . I). Involvement of Phospholipids . . . . . . . . . . . . E . The Protein Composition of Sarcoplasmic Reticulum Membrane . . F. Conformational Probes of Ca Transport . . . . . . . . . I11. The Regulation of Sarcoplasmic Reticulum Function . . . . . . A . Regulation of Ca Uptake . . . . . . . . . . . . . B . The Release of Ca from Sarcoplasmic Reticulum . . . . . . C . The Ca Permeability of Model Membranes . . . . . . . . IV . The Regulation of Sarcoplasmic Ca2f Concentration in Cardiac Muscle . . A. Sarcoplasmic Reticulum Function in Normal Heart . . . . . B . The Role of Mitochondria in the Regulation of Excitation-Contraction Coupling . . . . . . . . . . . . . . . . . . C . Calcium Uptake by Sarcoplasmic Reticulum Fragments Isolated from Failing Heart . . . . . . . . . . . . . . . D . The Effect of Cardiac Glycosides and Other Drugs on the Ca Uptake of Cardiac Sarcoplasmic Reticulum . . . . . . . . . . E . Na-K-Activated ATPase . . . . . . . . . . . . . F. Mitochondria in Cardiac Failure . . . . . . . . . . . V. Sarcoplasmic Reticulum in Red Skeletal Muscles . . . . . . . VI . The Structure and Function of the Transverse Tubular System and the Triad . . . . . . . . . . . . . . . . . . . . A . The Transverse Tubular System . . . . . . . . . . . B. TheTriad . . . . . . . . . . . . . . . . .

84 86 86 88 90 96 102 107 112 112 114 119 122 122 126 129

131 134 135 136 141 141 149

* Supported by research grant NS07749 from the National Institutes of Health. Grant GB7136 from the National Science Foundation. and a grant-in-aid from the American Heart Association. Inc . The review was completed in July 1971. 83

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VII. The Content of Sarcoplasmic ReticulumTubules . . . . . . VIII. The Sarcoplasmic Reticulum in Iliseases of Skeletal Muscle . . . A. Denervation . . . . . . . . . . . . . . . . B. Muscular Dystrophy . . . . . . . . . . . . . C. Involvement of Sarcoplasmic Reticulum in Other Muscle Disorders D. Myotonia . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . Note Added in Proof, . . . . . . . . . . . . . .

. 151 .

. . . . . .

159 159 166 170 171 175 195

1. INTRODUCTION

Originating from the observations of Marsh (1952) and Bendall (1952, 1953), and based on impressive morphological (Peachey, 1968), physiological (Sandow, 1970), and biochemical developments (Hasseibach, 1964a; Weber, 1966; Ebashi and Endo, 1968; Ebashi et al., 1969; Martonosi, 1971a), the concept of sarcoplasmic reticulum* as the principal regulator of the contraction-relaxation cycle became securely established during the past 20 years. Two distinct membrane-linked functions are involved in this rcgulation. 1. The ATP-mediated accumulation of calcium by sarcoplasmic reticulum (Hasselbach and Makinose, 1961; Ebashi and Lipmann, 1962), which by lowering the free-calcium concentration of the sarcoplasm to levels below lo-' M causes muscle relaxation (Weber et al., 1963). 2. The release of Ca from sarcoplasmic reticulum on excitation (Ridgway and Ashley, 1967; Ashley and Ridgway, 1970; Jobsis and O'Connor, 1966), which by a complex interaction with troponin and related substances (Ebashi and Endo, 1968) initiates muscle contraction.

While a detailed picture of considerable complexity has emerged for the molecular mechanism of the Ca transport process (Martonosi, 1971a), making i t one of the best known active transport systems, the permea-

* The following names are used interchangeably in the literature and in this review: fragmented sarcoplasmic reticulum (FSR), sarcoplasmic reticulum (SR), muscle microsomes, granules, grana, relaxing factor. All these terms generally refer to subcellular fractions isolated from homogenized niuscle by differential centrifugation (80OO-ri0,OOO g for 1 hour). In addition to sarcoplasmic reticulum membranes the preparations are expected to contain elements of surface membranes, T-system tubules, mitochondria1 outer membranes, etc., in varying amounts as contaminant. The following abbreviations are used: EGTA, ethyleneglycol-bis(/3-aminoethylether)-N, "-tetraacetic acid; EPR, electron paramagnetic resonance; ESR, electron spin resonance; TCA, trichloracetic acid.

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bility change underlying the Ca release after depolarization is only now beginning to receive some attention (Duggan and Martonosi, 1970; Martonosi and Feretos, 1964a; Hasselbach et al., 1969). The development in these two areas has been recently reviewed (Ebashi and Endo, 1968; Martonosi, 1971a),and apart from a brief historical introduction we shall confine our attention to the evaluation of the progress made in the last two years. There are several aspects of sarcoplasmic reticulum function which, although of great potential importance, were omitted from earlier accounts. A major part of this review will be devoted to a critical appraisal of information about the possible involvement of sarcoplasmic reticulum in various muscle diseases (cardiac failure, muscular dystrophy, myotonia, denervation atrophy), problems that have generated much interest among clinical biochemists in recent years. Due to the singular concentration of attention on the role of sarcoplasmic reticulum in the regulation of sarcoplasmic Ca2+ concentration, conspicuous gaps developed in our knowledge regarding other possible functions of sarcoplasmic reticulum such as its involvement in the energy metabolism of muscle cell (Fawcett and Revel, 1961; Rosenbluth, 1969). Recent evidence indicates that the sarcoplasmic reticulum may represent a compartment for hexokinase (Karpatkin, 1967), glyceraldehydephosphate dehydrogenase (Fahimi and Karnovsky, 1966), and phosphofructokinase (Aloisi and Margreth, 1967) activities. Other metabolic functions of sarcoplasmic reticulum may include the synthesis of phospholipids and proteins, as suggested b y their turnover in sarcoplasmic reticulum membrane (Martonosi and Halpin, 1972). It is hoped that a brief review of available information on these subjects may generate further interest in the broader metabolic aspects of sarcoplasmic reticulum function, even though some of the discussion will be necessarily speculative. Finally, a detailed biochemical analysis of the function of transverse tubular system and the triad may soon become possible, based on the considerable amount of physiological and morphological information that has accumulated in recent years. A summary of this development provides the necessary perspective for integrating the sarcoplasmic reticulum with the other elements of the excitation-contraction coupling system. The distribution of sarcoplasmic reticulum in the muscle cell and its relationship to the myofibrils, surface membranes, and the T-system tubules bears considerable importance in relation to its physiological function. In order to facilitate the discussion, these morphological relationships are illustrated schematically in Fig. 1. For more detailed presentation of the ultrastructure of sarcoplasmic reticulum the reader is referred t o the excellent collection of papers which appeared in honor of Dr. Emilio Veratti

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FIG.1. Three-dimensional reconstruction of the sarcoplasmic reticulum (SR) of frog sartorius muscle. The membranous tubules are arranged in a delicate pattern with respect to the sarcomer, which is repeated with the striation of myofibrils. A prominent feature of the system is the triad formed by two cisternal enlargements of the sarcoplasmic reticulum which lie adjacent to a T-system tubule. The T system represents the invagination of the surface membrane of muscle cell into the cell interior. In frog sartorius muscle the triads are located a t the level of the Z line. I n several other species the triads are found near the ends of the A bands. Modified from Peachey (1965). Reprinted by permission from Rockefeller University Press.

as a supplement to the Journal of Biophysical and Biochemical Cytology, Volume 10, August 1961, and to more recent reports by Peachey (1965, 1968), Franzini-Armstrong and Porter (1964), Franzini-Armstrong (1970a, 1971), Rayns et al. (1967, 1968), and Rosenbluth (1969).

11. THE MECHANISM OF Ca TRANSPORT A. Brief Outline of Early Developments

Sarcoplasmic reticulum fragments sequester calcium with high affinity in the presence of ATP and Mg (Ebashi, 1960, 1961; Ebashi and Lipmann, 1962), accompanied by the hydrolysis of ATP (Hasselbach and Makinose, 1961, 1963; Makinose and Hasselbach, 1965). For the hydrolysis of each mole of ATP, approximately two Ca atoms are transported (Hasselbach and Makinose, 1963) over a wide range of free calcium concentrations (Weber et al., 1966), and this constancy of

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the Ca/ATP ratio has been intcrpretrd to indicate a close coupling betwcrn Ca transport and ATl’asr activity. The ATI’ mediated Ca uptaka is promoted by Ca precipitating anions such as oxalate (Hassrlbach and nlakinosc, 1961), pyrophosphatc, inorganic phosphate, and fluoridc (RIartonosi and Ferctos, 1964a). On the basis of thrse observations, the Ca accumulation was described by Hasselbach (1964a) as activc transport against a calcium activity gradicnt, which derives its cncrgy from the hydrolysis of ATP through an ATPase enzyme tightly linked to the microsomal membrane. According to Ebashi (Ebashi and Endo, 196S), the ATP-dependent accumulation of Ca reflects Ca binding to mrmbrane-linked cation binding sites, which were made available by the binding of ATP, and the Ca-activated ATI’ hydrolysis has no specific role in the process. This idea was originally based on the very high initial rate of Ca uptake (60 pmoles/ mg per minute) measured by rapid kinetic methods using murexide as Ca indicator (Ohnishi and Ebashi, 1963, 1964; Ebashi and Endo, 1964), which was accompanied by comparatively little ATP hydrolysis (Ebashi and E’amanouchi, 1964; Ebashi and Endo, 1964). The experimental basis of Ebashi’s interpretation of the mechanism of Ca2+ accumulation has been subject to major reevaluation over th e past few years. The initial rate of Ca uptake obtained more recently by similar techniques is 10-30 times less (Harigaya et al., 1968; Harigaya and Schwartz, 1969; Ogawa, 1970) than th at found by Ohnishi and Ebashi and falls within the range of values (1-3 pmoles of Ca per milligram of protein per minute) obtained with conventional methods (Webcr et al., 1966; Sreter, 1969; RIakinose and Hassclbach, 1965). Furthermore, cven a t low ATP concentrations and during the early phasc of the Ca uptake process, trhe number of Ca atoms taken up per mole of ATP hydrolyzed is usually close to 2 , in agreement with the observations of Hasselbach and Rlakinose (1963). While ATP-mediated binding of Ca t o external binding sites on the sarcoplasmic reticulum membrane (Ebashi and Endo, 1968) is not supported by the available evidence, subsequent binding of actively transported Ca to membrane-linked cation binding sites (Carvalho, 1966, 1968a,b; Carvalho and Leo, 1967; Vanderkooi and Rlartonosi, 1971a,b) probably represents an important aspect of the Ca accumulation process. Since the membrane potcntial and the intravcsicular ionized Ca concentration during Ca transport are unknown, the thermodynamic efficiency of the transport system cannot be evaluated reliably. The significance of the observed Ca/ATI’ ratio of 2 (Hasselbach and Makinose, 1963; Weber et al., 1966; Weber, 1966) is doubtful, as i t was measured on microsome preparations in which presumably less than 30% of the vesicles

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were capable of accumulating and retaining Ca (Baskin and Deamer, 1969). This may have resulted from damage suffered during homogenization and isolation, although contamination by inactive membrane fragments originating from mitochondria1 outer membranes, T system, surface membranes, and segments of sarcoplasmic reticulum which do not accumulate Ca, may also have been a contributing factor. In contrast t o Ca2+ transport, Ca activation of microsomal ATPase is a stable property maintained under a variety of conditions that inhibit Ca transport (Martonosi and Feretos, 1963b, 1964b; Martonosi, 1964; Martonosi et al., 1968; Weber, 1971a,b). This may imply that ATP is hydrolyzed by vesicles that do not accumulate Ca. Consequently, the Ca/ATP ratio of Ca transporting particles may be much higher than 2 (Martonosi and Feretos, 196313,1964b). If this is the case, one would expect regularly to obtain Ca/ATP ratios higher than 2 with improved preparations. According to Sreter (1969), the Ca/ATP ratio of white rabbit skeletal muscIe microsomes in the absence of oxalate may be as high as 8-10. Sarcoplasmic reticulum fragments isolated from lobster muscles can accumulate 10.8-24.0 pmoles of Ca per milligram of protein (Baskin, 1971), compared with 6-8 pmoles of Ca per miligram of protein observed in rabbit skeletal muscle microsomes (Sreter, 1969; Hasselbach and Makinose, 1963; Martonosi et al., 1971a). The rate of ATP hydrolysis by lobster and rabbit preparations is similar. This implies again that during Ca2+transport by lobster microsome preparations the Ca/ATP ratio may be significantly higher than 2, although no actual values have been reported.

B.

Sources of Energy for Ca Transporl

In addition t o ATP, other nucleoside triphosphates (Martonosi and Feretos, 1964a; Carsten and Mommaerts, 1964; Makinose and The, 1965; Worsfold and Peter, 1970), acetylphosphate (De Meis, 1969a,b; Pucell and Martonosi, 1969, 1971), carbamylphosphate (Pucell and Martonosi, 1969, 1971), and p-nitrophenylphosphate (Inesi, 1971) may also serve as energy donors for Ca transport. In the presence of 5 mM MgC12, Ca in M ) activates the hydrolysis of ATP low concentration (lo+ to (Hasselbach and Makinose, 1961, 1963; Martonosi and Feretos, 1964b), acetylphosphate (De Meis, 1969b; Pucell and Martonosi, 1971) and p-nitrophenylphosphate (Inesi, 1971). This suggests that a common transport system is involved in all these cases. An unexpected feature of the carbamylphosphatase activity of skeletal muscle microsomes is its apparent independence from the free Ca concen-

SARCOPLASMIC RETICULUM FUNCTION

89

tration of the medium in the range (10-6 to M ) where the ATPase and acetylphosphatase activities are elevated, although carbamylphosphate supports active Ca2+ accumulation. Rubin and Katz found (1967) that in the absence of Na and K, Ca had no effect on the ATPase activity of microsomes during vigorous uptake of Ca. These observations may indicate that the rate of phosphoprotein formation, usually considered to be the Ca-sensitive step of ATP hydrolysis (Makinose, 1969; Yamamoto and Tonomura, 1968; Martonosi et al., 1971a,b; Inesi et al., 1970), may not be rate limiting with carbamylphosphate as substrate or with ATP in the absence of Na and K. Rapid kinetic analysis of the effect of Ca on the formation of phosphoprotein from carbamylphosphate and from ATP in the absence of Na or K would be of considerable interest in explaining these observations. The apparent K , of the transport ATPase for acetylphosphate (Pucell and Martonosi, 1971), carbamylphosphate (Pucell and Martonosi, 1971), and p-nitrophenylphosphate is in the range of 1-2 mM, i.e., about 500-1000 times greater than the K , for ATP. The maximum rate of hydrolysis of nonnucleotide substrates is much less than that of ATP. ATP and ADP are effective competitors of the hydrolysis of acetylphosphate (DeNeis, 196913; Pucell and Martonosi, 1969, 1971) and of p-nitrophenylphosphate (Inesi, 1971) by microsomes. This suggests the involvement of the same active site in the hydrolysis of the three substrates. The following additional evidence suggests that acetylphosphate promotes Ca transport by a mechanism similar to that of ATP (Pucell and Martonosi, 1969,1971) : 1. The formation of similar 32Plabeled phosphoprotein intermediates from 32P-acetylphosphateor 32P-ATP. 2. The occurrence of acetylphosphate-ADP exchange. 3. Similar requirement for Mg and Ca in the hydrolysis of acetylphosphate and ATP. 4. Inhibition of both acetylphosphatase and ATPase activities by treatment of microsomes with phospholipase C.

The dependence of the rate of ATP hydrolysis and of the concentration of phosphoprotein on the ATP concentration is complex, with a secondary substrate-induced activation a t high (0.1-1.0 mM) ATP concentration (Yamamoto and Tonomura, 1967; Inesi et al., 1967, 1970). The increase in the level of phosphoprotein observed a t high 32P-acetylphosphate concentration (Pucell and Martonosi, 1971) is of doubtful significance, as these values are based on small differences in radioactivity. The puzzling inhibition of myofibrillar ATPase activity by 1 ,3-di-

90

ANTHONY MARTONOSI

phosphoglycerate (Ells and Faulltner, 1961) may be explained if 1,3diphosphoglycerate, similarly to acetylphosphate, were to serve as substrate for Ca transport by fragments of sarcoplasmic reticulum entrapped in the myofibrils. Thc disputed relaxing effcets of a-glycerophosphate (Marsh, 1960; Parker, 1961; Bendall, 1960; Lorand and LIolnar, 1962; Takauji and Nagai, 1963) may also be related to the formation of 1,3diphosphoglycerate in the freshly prepared muscle fibers used by RIarsh (1960), although Wcber (1966) has attributed this effect to a possible stimulation of mitochondria1 Ca transport by a-glycerophosphate. C. The Phorphoprotein Intermediate

Microsomcs catalyze a transphosphorylation reaction between nucleoside triphosphates and nucleoside diphosphatcs (Ulbrecht, 1962; Hasselbach and Makinose, 1962; Illakinose, 1966, 1969) according to thc following general mechanism: ATP

+ A*DP F? ADP + A*TP

The dependence of the rate of ATP-ADP exchange on the free Ca2+concentration of the medium is similar to that of the ATPase activity and Ca transport; this suggests that the ATP-ADP exchange represents a partial reaction of ATP hydrolysis. The maximum rate of ATP-ADI’ exchange under optimum conditions exceeds the rate of ATP hydrolysis, and both processes are inhibited by SH group reagents (Hasselbach and Makinose, 1962) or by treatment of microsomes with phospholipase C (Balzer et al., 1968; Martonosi, 19694. A similar transphosphorylation reaction occurs between acetylphosphate and ADP (Pucell and Rlartonosi, 1969, 1971). The connecting link between ATPase activity, ATP-ADP exchange, and Ca2+transport may be the recently discovered phosphoprotein intermediate (Yamamoto and Tonomura, 19G7, 1968; Martonosi, 1967, 1969a; Makinose, 1969). This intermediate was demonstrated after incubation of microsomes (Pucell and Martonosi, 1969, 1971). with ATP-32Por a~etylphosphate-~~P This incubation yielded protein-bound 321’ radioactivity that was retained in the microsomal membranes even after extensive washing with dilute trichloroacetic acid solution, a procedure that removed substrate and reaction products from the system. In the presence of 5 mM MgC12, the steady-state concentration of the phosphoprotein intermediate shows the same dependence upon the free Ca2+ concentration of the medium as the rate of ATP hydrolysis or Ca transport, maximum steady-state concentration (3-5 pmoles per gram of microsomal protein) being reached when

91

SARCOPLASMIC RETICULUM FUNCTION

thc free Ca concentration is about lop5to lop6M (Yamamoto and Tonomura, 1967, 1968; Makinose, 1969; Inesi et al., 1970; Martonosi et al., 1971a,b). This is usually taken to indicate that Ca accelerates the rate of phosphoprotein formation. In the absence of Mg2+, about 100 times greater Ca2+ concentrations (1-5 mM) are required to produce a similar increase in the steady-state concentration of phosphoprotein (Martonosi, 1967, 1969a; Martonosi et al., 1971a,b). The latter effect is probably related t o an inhibition of the decomposition of phosphoprotein, which contributes t o the inhibition of microsomal ATPase activity a t high Ca concentration in the presence of Mg (Martonosi, 1969a; Martonosi et al., 1971a,b). A possible mechanism (Scheme I) of ATP hydrolysis and Ca transport involving the phosphoprotein intermediate may be written as follows: E

+ ATP

(1)

E-ATP ATP

E-ATP

/

+ 2 Ca i? E

(2)

\

2 Ca

ATP

/

us?+

E

i?

\

E-P-2CafADP

(3)

-

(4)

2 Ca

E

N

E*

N

P

- 2 Ca & E*

P

- 2 Ca F?E* + Pi + 2 Ca

P - 2 Ca

(5)

(6)

E*+E

The interaction of enzyme with ATP (step 1) does not require divalent cations (Inesi and Almendares, 1968). The enzyme-ATP complex in the presence of Ca (step 2) is rapidly converted into a phosphoprotein intermediate (step 3), presumably on the outside surface of microsomes, with ADP being released into the medium. This reaction step is readily reversible with formation of ATP from ADP and E P leading to the ATPADP exchange. The hypothetical Ca requirement for the formation of the phosphoprotein is based on the marked dependence of the phosphoprotein conM), centration on medium calcium concentration in the range (lo-’ to where the hydrolysis of ATP and ATP-ADP exchange are markedly activated (Yamamoto and Tonomura, 1967, 1968; Makinose, 1969; Inesi et al., 1970; Martonosi et al., 1971a,b). A similar relationship exists between phosphoprotein concentration and acetylphosphatase activity N

92

ANTHONY MARTONOSI

or acetylphosphate-ADP exchange (Puce11 and Martonosi, 1969, 1971). From these correlations, it appears that a t low ADP concentration the rates of ATP and acetylphosphate hydrolysis are roughly proportional to the concentration of phosphoprotein (V = k. [E PI). When the concentration of ADP (Makinose, 1969) is raised from 0.2 to 1 mM, the rate of ATP-ADP exchange increases, while, as expected, the concentration of phosphoprotein and the liberation of inorganic phosphate are both diminished. Surprisingly, further increase in the concentration of ADP above 1 mM progressively inhibits the ATPase activity, calcium transport, and ATP-ADP exchange, while the concentration of phosphoprotein remains a t a relatively high level (1.5 moles per 106 gm of protein; Makinose, 1969). This has been explained by assuming that ADI' forms an unreactive complex with the phosphoprotein (Makinose, 1969). The requirement for Ca in the formation of phosphoprotein may not be absolute since accumulation of phosphoprotein to levels of 2-3 moles per lo6gm of protein has been observed within 20 seconds a t 2°C in the presence of 5 mM MgClz and 0.5 mM EGTA on microsomes treated with phospholipases C (Clostridium welchii or Bachillus cereus) or phospholipase A (Crotalus terriJicus terrificus) (Martonosi, 1967, 1969a; Martonosi et al., 1971a,b). This implies that a t least on lipid-depleted microsomes the rate of phosphoprotein formation is significant even when the free Ca concentration is as low as lo-* &f, provided R9g is present. The initial reaction of ATP with the membrane which leads to the formation of phosphoprotcin and the ATP-ADP exchange occurs on the outside surface of microsomes, since both processes are regulated by the Ca concentration of the medium, and the functionally important SH groups involved in ATP hydrolysis are located on the outside microsomal surface (Hasselbach and Elfvin, 1967). In the hypothetical mechanism outlined in Scheme I, the transfer of Ca from the outside to the inside surface may involve a conformational change of the phosphorylated Ca carrier (step 4) for which no evidence is as yet available. On the inside membrane surface, the E* P-2 Ca complex undergoes hydrolysis with the release of inorganic orthophosphate and Ca (step 5 ) . The cycle is completed with the return of the carrier to the outside microsomal surface (step 6). When ATP is hydrolyzed in the presence of lead salts, the deposition of lead phosphate precipitate occurs nearly exclusively on the internal membrane surface of microsomes (Ikemoto et al., 1968; Tice and Engel, 1966b). Provided this is not the consequence of active lead transport (Nagai et al., 1965), i t may indicate that, in accordance with Scheme I, the hydrolysis of ATP involves the translocation of phosphate from the outside

-

-

SARCOPLASMIC RETICULUM FUNCTION

93

to the internal membrane surface. Rapid release of phosphate by diffusion across the highly anion-permeable microsomal membrane (Duggan and Martonosi, 1970) may explain the finding (Yamada et al., 1970) th a t only a small amount of the liberated phosphate is retained by the microsomes. The retention of the accumulated calcium in the microsomes is due t o the relative impermeability of microsomal membrane to Ca. The Ca concentration gradient generated by the ATP-dependent Ca uptake may serve as energy source for thc synthesis of ATP from ADP and P, by the reversal of steps 5 to 1 in Scheme I, connected with cfflux of Ca from the microsomes (Makinose, 1971; Makinose and Hassclbach, 1971). The usual rate of Ca release in vitro from particles loaded with 0.1-0.2 pmole Ca per milligram of protein into media containing relatively little ADP or P, is only 0.01-0.06 pmole Ca per milligram of protein per minute (Martonosi and Fcretos, 1964a; Weber et al., 1966; Makinose and Hasselbach, 1965). Addition of ADP, inorganic phosphate, and Mg in the presence of EGTA greatly activates the Ca relcase (Barlogie et al., 1971), and the release of Ca is accompanied by synthesis of ATP from ADP and inorganic phosphate (Makinose and Hasselbach, 1971; Makinose, 1971). Synthesis of ATP was also observed under similar conditions by Ilanazawa et al. (1970), although these authors did not emphasize the connection of ATP synthesis with the efflux of Ca from microsomes. The net ATP formation during calcium release depends on the tightness of microsomal membranes for calcium and may be abolished by treatment of microsomes with ether or phospholipase A (Makinose and Hasselbach, 1971). Uncouplers of mitochondria1 ATP synthesis (azide and dinitrophenol) are ineffective in preventing ATP formation coupled to the release of Ca from microsomes. The release of 2 Ca atoms is accompanied by the synthesis of 1 mole of ATP (Makinose and Hasselbach, 1971) supporting the stoichiometry of 2 Ca t o 1 ATP observed during Ca accumulation. This elegant work provides a further dramatic example, under simpler experimental conditions, of the previously observed conversion of osmotic into chemical energy (Cockrell et al., 1967; Lew et al., 1970). The rate of decomposition of the phosphoprotein intermediate formed in the presence of ATP and Mg, after Ca has been removed from the system with EGTA, is comparable t o the rate of ATP hydrolysis (Yamamot0 and Tonomura, 1968; Inesi et al., 1970). The effect of Mg on the hydrolysis of phosphoprotein is not yet settled. The addition of Mg lowers the concentration of phosphoprotein formed in the presence of Ca (Martonosi, 1969a) in agreement with the about 5-fold increase in the rate of decomposition of phosphoprotein in a Ca-free system when 5 mM MgClz is present (Inesi el al., 1970). Under slightly different conditions, Yamamoto

94

ANTHONY MARTONOSI

and Tonomura (1968) found that removal of Mg had no effect on the rate of phosphoprotein hydrolysis. The chemical nature of the phosphoprotein bond is not fully established. The stability to acid and lability to alkali of the denatured phosphoprotein, together with its sensitivity to hydroxylamine, suggest that it is an acylphosphate (Martonosi, 1967, 1969a; Yamamoto and Tonomura, 1968). However, molybdate does not accelerate the hydrolysis of protein-bound phosphate groups (Makinose, 1969), and the pH stability profile of phosphoprotein is not exactly that of an acylphosphate (Lipmann and Tuttle, 1944; Koshland, 1952). The difference in the pH stability profiles of phosphoprotein in TCA-denatured and salyrgan-treated membranes (Inesi et al., 1970) merely indicates that structural factors may considerably influence the stability and reactivity of the phosphoprotein bond, whatever its chemical nature. The reactivity of the phosphoprotein bond apparently differs in native and TCA-precipitated membranes as the inhibition of ATPase activity, Ca transport, and phosphoprotein formation by hydroxylamine (Martonosi, 1969a) can be readily reversed by removing the reagent (Yamamoto and Tonomura, 1968; Inesi et al., 1970). While these observations do not support hydroxamate formation as the basis of the observed inhibiting effect of hydroxylamine, the hydrolysis of hydroxamate in the course of washing has not been excluded. The acylphosphate character of the postulated intermediate also has been questioned on the ground that the reaction of O-14C-methylhydroxylamine (I mM) with the microsomes is not dependent upon ATP (Inesi et al., 1970). However, the concentration of hydroxylamine used in these experiments was 100-500 times smaller than that required to inhibit ATPase activity and the prolonged incubation (24-48 hours at 3°C) needed to overcome this difference in hydroxyhmine concentration may have permitted the degradation of hydroxamate. The reaction of the denatured phosphoprotein or a peptic phosphopeptide isolated from the phosphoprotein with 2-hydroxy-5-nitrobenaylhydroxylamine yields hydroxamate in amounts expected from the concentration of protein-bound phosphate (Yamamoto el al., 1971). These observations are consistent with the assumption that the protein-bound phosphate in membranes denatured with TCA is an acylphosphate. As the information on the pH stability of phosphoprotein and its reactivity with hydroxylamine or hydrazine (Makinose, 1969) is not likely to provide conclusive evidence for or against the acylphosphate character of the phosphoprotein bond in the native membrane, the question will have to be settled by isolation and direct characterization. The postulated intermediate of the sodium-potassium transport system

SARCOPLASMIC RETICULUM FUNCTION

95

of brain microsomes has been identified as L-glutamyl-7-phosphate by isolation as the stable L-glutamyl-7-propylhydroxamate derivative (Kahlenberg et al., 1967). The effect of hydroxylamine on the Na-I< ATPasc activity corroborates this conclusion, although the evidence is not entirely straightforward (Shamoo, 1971). On the assumption that, the hydrolysis of 1 mole of ATP permits the transport of 2 (or more) Ca atoms across the membrane, the hypothetical calcium carricr should possess multiple calcium binding sites. This is not a likely property of an acylphosphate group. Actually, Ca binding may precede the formation of a phosphoprotcin bond and may not even require the presence of ATI’ (Martonosi, 1964). The apparent independence of Ca binding and phosphoprotein formation suggests that phosphorylation of the membrane ATPase merely initiates a conformational change that leads t o the translocation of calcium previously bound to specific, chemically distinct carrier sites. The possible role of membrane-linked cyclic polypeptides or related I
+

96

ANTHONY MARTONOSI

of the ninhydrin-staining areas (Martonosi et al., 1971b; Martonosi and Halpin, 1971). On chromatography using a Dowex 50- column, the 32P-labeledpeptides emerge shortly after inorganic phosphate but before the elution of the main ninhydrin-staining fractions begins. The absence of a significant ninhydrin reaction in the fractions which contain most of the radioactivity is in agreement with observations made on fingerprints (Martonosi and Halpin, 1971). The 32P-labeled peptide fraction eluted from Dowex 50 column contains three distinct peptides characterized by a high concentration of acidic amino acids (Martonosi and Halpin, 1971). It is tempting to speculate that carboxyl functions of acidic amino acids at the active site of the transport enzyme may constitute a chelating structure of sufficient specificity, affinity, and flexibility to function as the Ca carrier. Phosphorylation of the membrane may influence both the affinity and the polarity of the carrier by altering the relative arrangement of the carboxyl groups. The high affinity Ca binding sites of sarcoplasmic reticulum and mitochondria appear to be different. While the Ca transport of sarcoplasmic reticulum is uninfluenced by osmotic shock or La3+ (Entman et al., 1969e), the high-affiity Ca binding sites of mitochondria are inhibited by La3+ (Mela and Chance, 1968; Lehninger, 1969, 1970, 1971) and are readily released in water-soluble form after treatment with distilled water (Lehninger, 1971). An attractive hypothesis for the formation of calcium specific chelating ring structures from folded segments of collagen and elastin has been proposed recently (Urry, 1971). According to this idea, a specific calciumbinding site of high affinity may arise when short-chain amino acids from membrane proteins are arranged into a structure that bears considerable resemblance to cyclic polypeptide antibiotics. D. Involvement of Phospholipids

Phospholipids are clearly required for the ATPase activity and Ca transport of fragmented sarcoplasmic reticulum membranes (Martonosi, 1963, 1964, 1967, 1968a, 1969a; Finean and Martonosi, 1965; Martonosi et aZ., 1968, 1971,b; Puce11 and Martonosi, 1971a; Meissner and Fleischer 1971). Thus, brief exposure of skeletal muscle microsomes to phospholipase C (C. weZchii) inhibits both ATPase activity and Ca transport together with extensive hydrolysis of membrane lecithin (Martonosi, 1963, 1964; Martonosi et al., 1968). Significant restoration of ATPase activity and Ca transport was observed when micellar dispersions of

97

SARCOPLASMIC RETICULUM FUNCTION

lecithin or lysolecithin preparations were added to the phospholipase C-treated microsomes (Martonosi, 1963, 1964; Martonosi et al., 1968). Similar results were obtained recently with the use of a highly purified phospholipase C preparation obtained from B. cereus. This preparation has broad substrate specificity, hydrolyzing phosphatidylethanolamine and phosphatidylserine as well as lecithin (Martonosi et id.,1971a). Hydrolysis of phosphatidylchoIine by phospholipase C yields diglycerides and phosphorylcholine as reaction products. It was established that neither of these products contributes significantly to the observed effects of phospholipase C on microsomes. Phosphorylcholine being water soluble is readily removed from the microsomal suspension by repeated centrifugation. Diglycerides separate from the microsomal membrane t o form osmiophilic droplets which can be seen under the electron microscope to adhere to the surface of phospholipase C-treated microsomes (Finean and llartonosi, 1965). Hydrolysis of SO-SO% of phosphatidylcholine, representing 60-70% of the microsomal phospholipids, does not influence significantly the three-layered “unit membrane” appearance of microsomal membrane, and the only morphological change known to accompany the removal of phospholipids is the decrease in average microsome diameter (Finean and Nartonosi, 1965). This probably represents an adaptation to the reduced phospholipid content. The ATPase activity and Ca transport are readily reactivated by phospholipids even several days after the treatment of microsomes with phospholipase C; this indicates extraordinary stability of the enzyme in the absence of phospholipids. The reqiiircment for phospholipids in the hydrolysis of ATP is apparently nonspccific as phospholipids and neutral or acidic synthetic detergents are nearly equally effective in restoring the ATPase activity to lipid-depleted microsomes (Martonosi et al., 1968). I t is unlikely that lecithin would serve as an intermediate during ATP hydrolysis as no ATPasc related 32Pincorporation into phospholipids was detected on exposure of microsomes to 32P-ATP. Similarly, microsomes containing V labeled phosphatidylcholine did not release radioactivity during the hydrolysis of unlabeled ATP (Martonosi et al., 1968). The inhibition of ATPase activity and Ca transport in lecithin-depleted microsomes may be related to a requirement for lecithin either in the formation [Eq. ( l ) ]or decomposition [Eq. (a)] of the phosphorylated intermediate: E + ATP-SZP E 32P + ADP (1) N

E

N

32P

+ HzO

$E

+ 3*Pt

(2)

Although inhibition of either step would cause inhibition of ATPase ac-

98

ANTHONY MARTONOSI

tivity, selective inhibition of reaction (1) would lead to a decrease, while that of reaction (2) would lead to an increase in the steady-state concentration of phosphoprotein. The maximum steady-state concentration of the phosphoprotein intermediate in the presence of 5 mM CaC12 or 5 mM MgC12,0.5 mM EGTA and 0.45 mM CaC12,is relatively insensitive to the treatment of microsomes with phospholipase C from either C . welchii (Martonosi, 1967, 1969a; Martonosi et al., 1971a,b) or B. cereus (Martonosi et al., 1971a). This suggests that the inhibition of ATP hydrolysis in lecithin-depleted microsomes is related to a lecithin requirement in reaction (2), the hydrolysis of the phosphoprotein intermediate. These observations were confirmed by Meissner and Fleischer (1971). The formation of phosphoprotein proceeds a t a relatively high rate in lipid-depleted microsomes in the presence of 5 mM MgClz and 0.5-5 mM EGTA even without added calcium, as the phosphoprotein accumulates a t 2°C in less than 10 seconds to steady-state concentrations of 2.0-2.3 moles/lOs gm of protein, a concentration that exceeds by a factor of 10-20 the values obtained under similar conditions with control microsomes (0.14.13 moles/lOs gm of protein; Martonosi et al., 1971a). Comparable observations were made with 32P-labeled acetylphosphate as phosphate donor (Puce11 and Martonosi, 1971). The accumulation of phosphoprotein in lipid-depleted microsomes in the presence of 5 mM MgCl2 and 0.5 mil4 EGTA implies that the rate of phosphoprotein formation is significant even a t a free CaZ+concentration as low as lo-* M , i.e., below the level required for activation of ATP hydrolysis. As the maximum steady-state concentration of phosphoprotein in control microsomes requires the to M free calcium ion, lipid depletion apparently presence of about reduces the dependence of the formation of the phosphoprotein intermediate upon Ca2+. A likely reason for this behavior may be that with the inhibition of phosphoprotein hydrolysis caused by phospholipase C, a relatively slow rate of phosphoprotein synthesis, maintained by Mg2+ alone, is sufficient to allow the intermediate to accumulate. Addition of lecithin or lysolecithin reestablished the normal Ca2+ sensitivity of the steady-state concentration of phosphoprotein, together with the restoration of the ATPase activity (Martonosi et al., 1971a). From these experiments, it seems safe t o conclude th a t the inhibition of ATP hydrolysis caused by treatment of microsomes with phospholipase C is primarily due to the inhibition of phosphoprotein decomposition. Owing t o the high rate of phosphoprotein formation in lipid-depleted microsomes, detection of possible differences in the rate of phosphoprotein formation caused by the removal of phospholipids requires the use of rapid kinetic techniques now under development.

SARCOPLASMIC RETICULUM FUNCTION

99

I n summary, our experiments do not exclude the possibility th a t the primary effect of Ca in promoting ATP hydrolysis is to accelerate phosphoprotein formation. They simply suggest that even with low calcium ion concentrations in the medium the rate of phosphoprotein formation in the medium remains sufficiently high to permit massive accumulation, provided hydrolysis is somewhat inhibited. The phospholipase C isolated from 3. cereus causes nearly complete hydrolysis of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine in microsomal membranes in contrast to the enzyme of C . welchii which did not attack ethanolamine or serine phospholipids and left about 10-20% of the microsomal lecithin undigested. It remains to be determined whether this resistance to the action of phospholipase C is attributable to the presence of an unusually large amount of alkyl-ether phospholipids in sarcoplasmic reticulum membranes (Waku et al., 1971). The inhibited ATPase activity and Ca transport of microsomes treated with phospholipase C of B. cereus is reactivated with lysolecithin. This observation makes unlikely a specific requirement for phosphatidylserine and phosphatidylcthanolamine in these processes (Martonosi et al., 1971a). Phospholipase C enzymes isolated from B. cereus or C. welchii produced similar effects on the steady-state concentration of phosphoprotein. Interestingly, the sphingomyelin content of membranes from sarcoplasmic reticulum is not affected by phospholipase C preparations, even though the enzymes accept micellar dispersions of sphingomyelin as substrate. It is possible that sphingomyelin may occur in microsomal membranes in association with cholesterol; this would alter its susceptibility to phospholipase C action. In contrast to the relatively gentle effects of phospholipase C, treatment of microsomes with phospholipase A (Crotalus terriJicus terrificus) causes the liberation of free fatty acids and lysophosphatides. The detergent effect of liberated free fatty acids increases the permeability of the microsoma1 membrane to Ca, inhibits Ca accumulation and activates the hydrolysis of ATP by microsomes. The steady-state concentration of phosphoprotein is not markedly affected. Fatty acids and lysophosphatides are readily removed from phospholipase A-treated microsomes by repeated washing with lipid-free serum albumin (Fiehn and Hasselbach, 1970). This results in a n inhibition of ATPase activity, while the maximum concentration of phosphoprotein measured with 5 mM CaClz or 5 mM MgC12, 0.5 mM EGTA and 0.45 mA4 CaClz as activators was affected only slightly, if at all (Martonosi et al., 1971a). These observations have been recently confirmed by Meissner and Fleischer (1971). The maximum concentration of phosphoprotein in

100

ANTHONY MARTONOSI

phospholipase A-treated and albumin-washed microsomes in the presence of 5 mM MgClz and 0.5 mM EGTA was 1.7-2.62 moles/lOs gm of protein, as compared with 0.2 mole/106 gm of protein in control microsomes. The inhibition of phosphoprotein formation observed by Fiehn and Hasselbach (1970) after washing of microsomes with serum albumin may have resulted from contamination introduced with serum albumin. Unfortunately no control experiments were reported to ascertain whether this interpretation is correct. Addition of lysolecithin (Martonosi et al., 1971a) or lecithin (Meissner and Fleischer, 1971) to phospholipase A-treated microsomes caused partial restoration of ATPase activity, an observation analogous to earlier findings made with phospholipa,se C. Depletion of the lecithin content of microsomes by phospholipase C inhibits the ATP-ADP exchange and ATPase activity to the same extent while the steady-state concentration of phosphoprotein remains unaltered. On the assumption that the phosphoprotein is a n intermediate in ATP hydrolysis and ATP-ADP exchange, these observations suggest that the absence of phospholipids hinders the access of phosphate acceptors to the phosphoprotein. Extraction of microsomes with acetone:water (9: 1, v/v) mixtures a t low temperature removed nearly all the phospholipids from microsomes, causing irreversible inactivation of all enzymatic functions including the formation of the phosphoprotein intermediate (Martonosi, 1964, 1969a; Martonosi et al., 1968). Since essentially complete removal of phospholipids from microsomes by phospholipase C ( B . cereus) of phospholipase A (C. terri$cus terri$cus) treatment produced only reversible effects, denaturation of enzyme protein probably contributes to the irreversible inactivation that follows acetone extraction. The effects observed with the various phospholipases are not attributable t o contaminating enzymes. Phospholipase C of B. cereus prepared in our laboratory (Donley and Martonosi, 1972) and phospholipase A (C. terriJicus terriJicus) purchased from Boehringer-Mannheim Co. (New York, New York) were devoid of contaminating protease, acid and alkaline phosphatase, neuraminidase, and ribonuclease activities. Commercial preparations of phospholipase C ( C . welchii) obtained from Sigma Chemical Co. (St. Louis, Missouri) contained significant collagenase and neuraminidase contamination. As the effect of the three phospholipases on the various microsomal functions was similar, and purified collagenase and neuraminidase had no effect on the ATPase activity and Ca transport of skeletal muscle microsomes even at high concentrations, it is reasonable to conclude that the observed effects are due to hydrolysis of phospholipids. On the basis of electron microscopic analyses of the dimensions of rat

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skeletal muscle microsomes, we suggested earlier (Martonosi, 1964) that the phospholipid content of sarcoplasmic reticulum membranes may not be sufficient to form a continuous bilayer over the surface of the microsomes. These observations are consistent with a mosaic arrangement of the principal membrane constituents of sarcoplasmic reticulum in which functional areas of the membrane containing the transport ATPase are interspersed with lipid phases arranged in bimolecular layers. The principal support for this view is provided by the electron microscopic study of Deamer and Baskin (1969) which shows globular-presumably protein-structures penetrating across the entire thickness of the sarcoplasmic reticulum membranes. These are thought to represent the Ca transport complex. The localization of the ATPase enzyme in the lipid phase of the membrane agrees with the absolute dependence of ATPase activity on membrane phospholipids and with the relative inaccessibility of the transport ATPase to proteolytic enzymes (Martonosi, 1968a,b). We have reported earlier that electrophoresis on cellulose acetate membranes of microsomes solubilized with cholate-deoxycholate leads to the separation of proteins from phospholipids (Martonosi, 196Sa). A similar separation of proteins and lipids may also be achieved by Sephadex G-150 chromatography or polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (Martonosi et al., 1971b; Martonosi and Halpin, 1971). The fast moving band of lipids is strongly stained with periodateSchiff reagent (Martonosi and Halpin, 1971). This may indicate th a t in addition t o phospholipids and cholesterol, sarcoplasmic reticulum membranes contain significant amounts of glycolipids. The lipid material responsible for the periodate-Schiff reaction is readily extracted from microsomes with acetone:H20 (9: 1, v/v) or ch1oroform:methanol (2: 1, v/v) mixtures. Clycolipids were separated by the method of Weinstein et aE. (1970). The lipids obtained by successive extraction of microsomes with 90% acetone-lO% HzO, followed by dry acetone and finally by ether (2 times each) were evaporated to dryness, The dry residue was suspended in SOYO ethanol and kept a t -20°C overnight. The insoluble material was removed by centrifugation and the ethanol-soluble fraction was concentrated to dryness. Resorcinol assay according to Svennerholm (1957) indicated th a t about 75580% of the resorcinol-positive material remained in the ethanolsoluble fraction and the absorption spectrum of the reaction product a t 480 nm suggested that the principal component responsible for the reaction is not sialic acid, but some hexose. After hydrolysis in 0.1 N Hi304 a t 80°C for 2 hours (Weinstein et al., 1970) only trace amounts of sialic acid were detected in the ethanol-soluble material with the thiobarbituric acid method of Warren (1959). After hydrolysis of the ethanol-soluble

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fraction with 1 N HC1 in sealed tubes a t 100°C for 12 hours, the principal hexose component of the glycolipid was identified as glucose by descending chromatography with butano1:pyridine: HzO (6: 4: 3) as solvent. A minor component migrating with 1.67 times the mobility of glucose (RG = 1) was tentatively identified as N-acetylpcntosamine. Two unidentified trace components with RG = 0.054 and 0.107 were also present. On the basis of the above data, the principal glycolipid fraction is tentatively identified as ceramide hexoside with glucose as the principal hexose component. Saponification of phospholipids by HgClz (Abramson et al., 1965) permits the isolation of ceramide hexoside which is homogeneous by thin-layer chromatography on silica gel G plates with chloroform :methanol: conc. NHdOH (60:35:5, v/v) as solvent. The faint periodate-Schiff reaction observed in the areas of polyacrylamide gel where the protein bands were located may represent a side reaction or the association of a small amount of glycolipids with proteins. E. The Protein Composition of Sarcoplarmic Reticulum Membrane

There are several reasons for caution in the interpretation of electrophoretic data obtained on solubilized membrane proteins. The protein composition of sarcoplasmic reticulum membranes may undergo two types of changes during isolation. (1) Proteins originally not present in the sarcoplasmic reticulum may be adsorbed to the membrane surface or trapped in the interior of the particles during homogenization. Admixture of other types of membranes (mitochondria, surface membrane, T system, nuclei) or hydrolytic enzymes originating from lysosomes may be particularly difficult t o exclude. This could enhance the impression of heterogeneity. (2) On the other hand, the content of the sarcoplasmic reticulum tubules and the loosely linked components of the membrane surface may be lost during fractionation. As a result the protein composition of the isolated particles may appear simpler than is actually the case. In addition, there are significant and sometimes not clearly understood limitations attached to the various methods used for the separation of membrane proteins and for the subsequent analysis of their homogeneity (Kaplan and Criddle, 1971). In view of the possible changes in protein composition during isolation and the frequent occurrence of aggregation or incomplete separation of the membrane proteins during subsequent electrophoresis, it is best t o regard as tentative data obtained so far on the protein composition of sarcoplasmic reticulum . Sarcoplasmic reticulum membranes solubilized with deoxycholate were

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resolved into several distinct bands by electrophoresis on cellulose acetate strips. The separation of the bands was accompanied by inhibition of ATPase activity; electrophoretic recombination of the separated bands after reversal of thc electrodes produced significant restorat>ionof ATPase activity (Martonosi, 196Sa). The demonstrated separation of phospholipids from microsomal proteins during electrophoresis raises the possibility that the inactivation of ATI’ase activity is due to thc removal of lecithin from the enzymatically active lipoprotein. Howcver, addition of micellar phospholipid dispersions to thc elcctrophorctically separated protein bands did not result in reactivation. This suggests that in addition to the known requirement for phospholipids, the ATPasc activity of microsomes also depends upon the interaction of two or more proteins that become separated during electrophoresis with loss of enzyme activity (Martonosi, 1968a). The involvement of a t least two separate proteins in the Na-K activatcd ATI’ase activity of rat kidney microsomes has been suggested by Chignell and Titus (1969). Solubilization and electrophoresis of microsomes in the prescnce of Triton X-100 did not inhibit the ATPase activity, and the microsomal proteins migrated in one broad band, without indication of the separation observed with deoxycholate (hlartonosi, 19GXa). Similar observations have been made recently by R1cl:arland and Inesi (1970). Fractionation of microsomal proteins on polyacrylamide gel with cholate and deoxycholate as solubilizing agents has permitted the resolution of several distinct protcins, which represent about 25% of the protein content of microsomes; a major portion of the proteins, including the ATPase enzyme, did not enter the gel (Rlartonosi, 19GSa). The heterogeneity of the protein composition of sarcoplasmic reticulum membranes was subsequently confirmed by the electrophoresis procedure of Takayama et at. (1966) with phenol-urra-acetic acid as solubilizing agent (Martonosi, 196913; RIacLmnan, 1970; I’anet and Selinger, 1970) and by the method of Shapiro et al. (1967; Shapiro and RIaizel, 1969) in the presence of sodium dodecyl sulfate (Rlartonosi, 196913; hlartonosi and Halpin, 1971). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate permits the entry of essentially all microsomal proteins into the gel under conditions when aggregation of the material is minimized. Electrophoresis of microsomal membrane proteins on polyacrylamide gel after solubilization by sodium dodecyl sulfate in thc absence of reducing agents Icd t o the demonstration of a major protcin constituent (M protein) accounting for over half of thc microsomal proteins (Martonosi, 1969b). In addition several other protein fractions were present in smaller amounts (Martonosi, 1969b; RIartonosi and Halpin, 1971). Thc protein bound 322’

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formed on incubation of microsomes with ATP-32Por acetylpho~phate-~~P was associated with the M protein band which on this basis is assumed to contain part of the ATPase enzyme involved in Ca transport (Martonosi, 1969b). The molecular weight of the M protein is 100,000-106,000, as determined by polyacrylamide gel electrophoresis with proteins of known molecular weight used as reference (Martonosi and Halpin, 1971; Martonosi, 1970). The molecular weight of the M protein is in reasonable agreement with indirect estimates of the equivalent weight of the transport unit that had been derived from Ca binding of the deoxycholate-treated membrane material (Martonosi, 1964), from radiation inactivation measurements (Vegh et al., 1968) and from the maximum steady state concentration of phosphoprotein in the presence of acetylpho~phate-~~P (Pucell and Martonosi, 1971). Prolonged incubation of the microsomal proteins solubilized with sodium dodecyl sulfate in the presence of reducing agents (P-mercaptoethanol or dithiothreitol) causes the dissociation of membrane proteins into subunits of 20,000-60,000 molecular weight which can be readily separated by polyacrylamide gel electrophoresis or Sephadex G-150 chromatography (Martonosi et al., 1971b; Martonosi and Halpin, 1971). There is a definite tendency for the accumulation of preferred sizes of oligomers. Ultrasonic treatment of microsomal membranes for a few minutes in the presence of sodium dodecyl sulfate causes complete dissociation of membrane proteins into subunits even in the absence of reducing agents (Pucell and Martonosi, 1972). This indicates that the contribution of disulfide bonds to the assembly of enzyme protein from its subunits is of lesser importance than previously believed. The maximum concentration of the protein-bound phosphate intermediate formed under optimum conditions from ATP-32P or acetglphos~ h a t e - ~is~about P 0.5-1.0 mole per lo5 gm of protein, i.e., about 1 mole per mole of ATPase enzyme. Since the M protein consists of several subunits, it is likely that only one of the subunits undergoes phosphorylation during ATP hydrolysis. The role of the other subunits in the ATPase activity is unknown. The reversible inactivation of ATPase enzyme during electrophoresis on cellulose acetate in the presence of cholate and deoxycholate (Martonosi, 1968a) could have resulted from a reversible separation of one or more of the subunits, but further evidence is required to substantiate this inference. Mixtures of oligomers prepared from 32Plabeled microsomal membranes by ultrasonic treatment or by prolonged incubation in the presence of reducing agents retain the protein-bound radioactivity primarily associated with proteins of 20,000-30,000 molecular weight, but no definite evidence was obtained for preferential binding of radioactivity to one single phosphoprotein (Pucell and Martonosi, 1972).

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The subunits do not readily reaggregate into polymers of 100,000 molecular weight after the removal of reducing agent and sodium dodecyl sulfate by dialysis. It is possible that ordered reaggregation into intact enzyme may require the presence of phospholipids. Yu and Masoro (1969, 1970) recently reported that proteins of rat sarcoplasmic reticulum membranes dissociate into subunits of 6000SO00 daltons in the presence of sodium dodecyl sulfate and @-mercaptoethanol. They claimed that the subunits contain alanine as C-terminal and glycine as N-terminal groups, migrate as one homogeneous band on electrophoresis, and represent over 90% of the protein content of microsoma1 membranes. It is unlikely that our observations (Martonosi, 1969b; Martonosi and Halpin, 1971; Puce11 and Martonosi, 1972) and those of Rilasoro and Yu (1969; Yu and Masoro, 1970) differ because of species differences either in the composition or subunit association of the proteins of the sarcoplasmic reticulum, sincc in the reviewer’s laboratory similar results were obtained with rat and rabbit microsomes. However, Masoro and Yu (1969) ignored the presence of unresolved protein material (sample 2 in Fig. 4 RIasoro and Yu, 1969) which trailed behind the main band. These higher molecular weight components may be present in amounts comparable t o those observed by us in rabbit sarcoplasmic reticulum preparations treated with sodium dodecyl sulfate and reducing agents. In view of this heterogeneity, it is difficult to accept thc claim that over 90% of the microsomal proteins consists of a single polypeptide of 6,000-10,000 molecular weight. Thc identification of glycinc as thc N-terminal group of the protein subunit (Yu and Masoro, 1970) is questionable as it was based on a yield of about 33% of DNP-glycine and follows a previous claim by the same authors (Uu and Alasoro, 1969) that the N-terminal group is a basic amino acid. The release of alanine by carboxyprptidase A was closely followed by arginine, leucinc, swine, and isolcucine, making the identification of alanine as C-terminal residue also uncertain. The presence of only 15 detectable peptides in tryptic hydrolyxatcs of the microsomal membranc proteins (Yu and R’lasoro, 1970) does not establish conclusively the existence of a single polypeptide of about 600010,000 molecular weight as the bmic subunit of sarcoplasmic reticulum, since in a completely lieterogcneous protclin mixture the peptides originating from t hr nonhomologous portion of the proteins may be sufficiently diluted to make no significant contribution to the observed pattern (Kaplan and Criddle, 1971). In addition to the lJl protein of about 100,000 daltons which probably represents the ATPase enzyme, two fast moving proteins of 51,000 (CI) and 63,000 (C,) molecular weight, respectively, have been observed regu-

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larly on polyacrylamide gel electrophoresis of microsomal material solubilized with sodium dodecyl sulfate in the absence of reducing agents (Martonosi, 1969b; Duggan and Martonosi, 1970; Martonosi and Halpin, 1971). The C1 and Ct proteins are selectively released from microsomal membranes by treatment with EDTA at pH 8.0-9.0 (Duggan and Martonosi, 1970). This indicates that divalent metals may be involved in their interaction with the membrane. The marked increase in the permeability of microsomes to inulin caused by EDTA at alkaline p H may be related in some way to thc detachment of C1 and Cz proteins from the membrane. MacLennan and Wong (1971a,b) suggested that the CI protein may be involved in the binding of accumulated calcium within the microsomes. However, the Ca binding capacity of the C1protein is markedly reduced by 0.1 M BC1 and 5 mM RlgCly, the usual components of the incubation medium used for the measuremrnt of Ca uptake. Furthermore, the C1 protein accounts for only 7% of the protein content of microsomes. It is not certain whether the fractions whose electrophoretic mobility is slower than that of the M band represent aggregates or genuine membrane constituents. Aggregation may be one cause of the occurrence of high molecular wcight components since their amount increases as the microsomes age. On the basis of their molecular weight, they may be dimers or trimers of the M protein. Ammonium sulfate fractionation of microsomes solubilized with cholatedeoxycholate in the presence of sucrose and KC1 represents a simple procedure for the isolation of ATPase from microsomes in enzymatically active and clectrophoretically nearly homogeneous form (Martonosi, 1968a; Rlartonosi and Halpin, 1971). The phospholipid content and composition of the purified ATPase is similar to that of the intact microsomes, although the specific activity increases 1.6 to 2.0-fold. The increase in specific activity is of the expected magnitude since the enzyme protein constitutes about 60-7070 of the protein content of microsomes. A more elaborate purification procedure was reported recently by MacLennan (1970), who used ammonium acetate instead of ammonium sulfate as precipitating agent. The specific ATPase activity of these preparations was higher than of those obtained with the earlier method, although some of the difference in activity may be due to the difference in temperatures a t which the ATPase activities were measured. On removal of deoxycholate, the ATPase enzyme spontaneously reaggregates into vesicular structures (Martonosi, 1968a; MacLennan et a l , 1971) bounded by continuous membrane profiles of approximately 60 A thickness, with the characteristic appearance of a unit membrane when

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examined in thin sections under the electron microscope (MacLennan et al., 1971). Freeze-etched preparations of reaggregated microsomes demonstrate the presence of globular subunits (MacLennan et al., 1971), associated earlier with the ATPase enzyme by Deamer and Baskin (1969). I n accord?.nce with earlier observations (Martonosi, 1968a), the reformed membranes were not disrupted by treatment with phospholipase C (MacLennan et at., 1971). The diglyceride product of the reaction accumulated in the form of osmiophilic droplets which remained attached to the surface of the apparently intact, phospholipid-depleted microsomes (Finean and Martonosi, 1965; MacLennan et al., 1971). F. Conformational Probes of Ca Transport

During the contraction-relaxation cycle, membranes of the sarcoplasmic reticulum probably alternate between several distinct conformational states, each of which has different Ca permeabilities. Localized conformational changes are likely to accompany the operation of the Ca2+pump during relaxation. These changes are probably confined t o the transport lipoprotein complex and result in the active translocation of Ca from the sarcoplasmic side of the membrane to the interior of the tubules. The permeability of the nontransport regions of the membrane to Ca probably remains low, since the rate of Ca release (0.034.06 pmoles Ca per milligram of protein per minute) from sarcoplasmic reticulum fragments previously loaded with Ca is quite slow. The initiation of muscle contraction by the release of calcium from sarcoplasmic reticulum (Jobsis and O’Connor, 1966; Ridgway and Ashley, 1967; Ashley and Ridgway, 1970; Ashley, 1971) almost certainly involves a grneralized conformational change leading to a striking increase in the Ca permeability of sarcoplasmic reticulum membranes. The calculated rate of Ca release in activated muscle (300-3000 pmoles Ca per milligram of sarcoplasmic reticulum protein per minute) is about lo4 to lo5 times greater than the Ca efflux from sarcoplasmic reticulum fragments measured in vitro. Its magnitude is comparable t o that of the Ca flux through surface membranes of skeletal (Bianchi, 1961a) or cardiac (Winegrad, 1961) muscles during depolarization. Although no satisfactory techniques are available to detect conformational changes of either kind, the use of fluorescence probes (Vanderkooi and Martonosi, 1969a,b, 1970, 1971a,b,c; Waggoner and Stryer, 1970; Chance, 1970; Yguerabide and Stryer, 1971), electron spin resonance techniques (RfcConnell and McFarland, 1970; Griffith and Waggoner, 1969; McFarland and McConnell, 1971; Landgraf and Inesi, 1969; Inesi

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and Landgraf, 1970) and X-ray diffraction methods (Levine and Wiikins, 1971; Wilkins et al., 1971) has produced promising results. 1. USE OF ~-ANILINO-~-NAPHTHALENE SULFONATE (ANS) AS STRUCTURAL A N D FUNCTIONAL PROBE ON SARCOPLASMIC RETICULUM MEMBRANES The fluorescence of 8-anilino-1-naphthalene sulfonate is enhanced by skeletal muscle microsomes and micellar dispersions of phospholipids. The magnitude of the enhancement is a unique function of the pH, temperature, and ion composition of the medium, rising with the concentration of cations according to a titration curve (Vanderkooi and Martonosi, 1969a,b). The enhancement of ANS fluorescence by cations is due to the increased binding of ANS to the microsomal membrane, and changes in the quantum yield of ANS fluorescence are of relatively minor significance (Vanderkooi and Martonosi, 1971a). Although the polarization of ANS fluorescence in microsomal membranes is greater than in micellar dispersions of phospholipids, the relatively low polarization in both systems indicates considerable mobility of the probe in the phase of the membrane, in accord with electron spin resonance data on various artificial and natural membrane systems (Iiornberg and McConnell, 1971; McFarland and McConnell, 1971; Tourtellotte et al., 1970). The fluorescence response of ANS in the presence of sarcoplasmic reticulum fragments is not defined exclusively by membrane proteins as claimed by Hasselbach and Heimberg (1970), but phospholipids make a significant and sometimes major contribution to both intensity and polarization of the fluorescence (Vanderkooi and Martonosi, 1971a). Consequently, the probe response cannot be attributed to conformational changes in the membrane proteins without careful assessment of the magnitude of phospholipid contribution. The latter varies with the ion composition, pH, and temperature of the incubation medium (Vanderkooi and Martonosi, 1971a). When fragments of sarcoplasmic reticulum actively accumulate Ca2+, in the absence of Ca precipitating anions, the intensity of ANS fluorescence increases in proportion to the amount of calcium taken up by the microsomes. Aged microsomes that have lost their ability to accumulate Ca in the presence of ATP did not show changes in ANS fluorescence during ATP hydrolysis. A similar correlation was observed between Ca uptake and fluorescence intensity when ATP, ITP, acetylphosphate, or carbamylphosphate were used as energy donors for calcium transport (Vanderkooi and Martonosi, 1971b). Glyceraldehyde-&phosphate, IDP, ADP, inorganic pyrophosphate, and inorganic orthophosphate were ineffective in promoting Ca uptake or fluorescence change. The fluorescence is reduced as Ca is released from the microsomes, either by addition of salyrgan or

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after ATP depletion. Addition of 3 m M oxalate to Ca-loaded microsomes also was found to decrease the intensity of fluorescence (Vanderkooi and Rlartonosi, 1971b). These results clearly establish that the enhancement of ANS fluorescence during Ca accumulation results from changes in the environment of the dye that are brought about when actively transported calcium becomes bound to the membrane (Vanderkooi and Martonosi, 1971b). The close temporal correlation between calcium uptake and the increase in the intensity of fluorescence is in accord with the suggestion (Carvalho, 1966, 1968a,b; Carvalho and Leo, 1967; Carvalho and Mota, 1971) that a large portion (probably more than 80%) of the accumulated calcium is bound to the microsomal membrane. I n these experiments (Vanderkooi and hlartonosi, 1971b) the extravesicular ionized calcium concentration was maintained constant with the aid of ethylene glycol-bis (P-aminoethylether) A', N'-tetraacetic acid (EGTA). It appears therefore that the intravesicular accumulation of Ca causes a change in the fluorescence of ANS initially adsorbed to the outer surface of the microsomes. This observation implies either (a) the effect of Ca bound to the internal surface of the membrane is transmitted to the ANS molecules on the outside, or (b) ANS is relatively rapidly redistributed between the internal and external surfaces of the microsomal membrane. As judged by nuclear magnetic rcsonance (Chance, 1970) and X-ray diffraction studies (Lesslauer et al., 1971), it is likely that ANS is located a t the aqueous edge of the hydrophobic zone of biological membranes (Chance et aE., 1971). Its redistribution between the two membrane surfaces may involve an inversion of the kind suggested for membrane phospholipids (Langmuir, 1938; Deamer and Branton, 1967; Papahadjopoulos and Ohki, 1969; Kornberg and McConnell, 1971), although the rate of redistribution may be much greater than that obtained for lecithin (Kornberg and RIcConnell, 1971) or stearatc (Deamer and Branton, 1967). Binding of cations to membrane proteins and phospholipids causes a similar increase in ANS fluorescence. There is also evidence that Ca promotes energy transfer from proteins to ANS (Vanderkooi and Martonosi, 1971a). Consequently the nature of the Ca binding site within the microsomcs is of considerable interest. This site may be on proteins (Cohen and Selinger, 1969; MacLennan and Wong, 1971a,b) or phospholipids or, probably, on both (Martonosi, 1971a). Thc maximum amount of Ca2+bound to microsomal membranes is similar t o the Ca binding capacity of simple proteins like actin (Martonosi et al., 1964). RIacLennan and Wong (1971a,b) have suggested that the CI protein (Duggan and hlartonosi, 1970; Martonosi and Halpin, 1971; Martonosi

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et al., 1971b), which they named Calsequestrin, alone can account for the binding of a major part of the accumulated calcium. Treatment of microsomes with EDTA a t pH 8.0-9.0 causes the selective and nearly quantitative release of C1 protein from the microsomes (Duggan and Martonosi, 1970). The C1 protein is readily reattached to the surface of microsomes after addition of MgCL or CaC12. The contribution of C1 protein to the binding of transported Ca within the microsomes may be estimated by studying the effect of the selective removal of C1 protein on the passive Ca binding capacity of microsomal membranes. The release of C1 protein from the microsomal membrane with EDTA suggests (Duggan and Martonosi, 1970) that its binding is stabilized by Mg or Ca and that hydrophobic bonds (MacLennan and Wong, 1971b) are not likely to make a major contribution. The active transport of Ca2+by sarcoplasmic reticulum is postulated to involve a conformational change of the hypothetical carrier. The ATPinduced enhancement of fluorescence intensity is not likely to represent such change for the following reasons: (a) No fluorescence change is observed in aged microsomes although they actively transport Ca as judged from the rate of ATP hydrolysis. The absence of Ca accumulation is due to the leakiness of the membrane. (b) Oxalate reduces the magnitude of fluorescence change although the rate of net Ca accumulation actually increases. (c) The fluorescence change is related to the amount of Ca bound to the microsomes whether it follows active transport or passive equilibrium. The increased fluorescenceof ANS-microsome systems caused by monovalent or divalent cations probably reflects the neutralization of electrostatic repulsion, which hinders the binding of ANS to the microsomes. Moreover, there is no strong indication for a genuine conformational change of the Ca transport system associated with changes in fluorescence intensity (Vanderkooi and Martonosi, 1971b). A similar distinction may be of importance in the evaluation of the response of ANS fluorescence to the functional state of mitochondria (Chance, 1970) or giant squid axon (Tasaki et al., 1968; Tasaki, 1970; Conti and Tasaki, 1970), where the assumed alterations of membrane conformation are associated with major changes in the ionic gradients, these changes may suffice to explain the variation in fluorescence intensity. For detection of conformational changes in the Ca carrier, it will be necessary to explore new probes of different specificity. In view of the absolute dependence of ATPase activity and Ca transport on the presence of phospholipids (Martonosi, 1964; Martonosi et al., 1968, 1971a,b), highly lipophilic probes such as 12-(9-anthroyl) octadecanoate (Waggoner and

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Stryer, 1970), N-(2-chloro-6-methoxy-9-acridinyl)-p-aminobenzenesulfonatc (Chance et al., 1971), N , N'-di-(octadecy1)oxacarbocyanine-12-(9anthroy1)stearic acid (Yguerabide and Stryer, 1971) may reflect more closely than ANS the changes in the environment of the transport protein, Chromophoric (Murphy et al., 1970) or fluorescent analogs of ATP (Ward et al., 1969) are likely to react with the active site of the transport system and may selectively respond to changes related to active transport. 2.

ELECTRON

PARAMAGNETIC RESONANCE (EPR) MEASUREMENTS

Although EI'R observations made so far on sarcoplasmic reticulum (Landgraf and Inesi, 1969; Inesi and Landgraf, 1970) do not indicate a relationship of probe response to either Ca uptake or ATPase activity, the large spectrum of possible spin labels now available (McConnell and Rlclcarland, 1970; McFLtrland and McConnell, 1971; Tourtellotte et al., 1970; Huang et al., 1970; Butler et al., 1970; Kornberg and McConnell, 1971) offers promise that EPR spectroscopy may eventually provide useful information on the conformational aspects of the Ca transport process. The EPIt spectrum of sarcoplasmic reticulum membranes covalently labeled with the paramagnetic probe 2,2,6,6-tetramethyl4-isothiocyanate piprridine-l-oxyl (isothiocyanate nitroxide) responds to increasing temperature betwwn 8" and 40°C with a reversible increase in the amplitude of the weakly immobilized component. This indicates increased rotational freedom of the label (Inesi and Landgraf, 1970). These changes may be related to the decrease in intensity and polarization of the ANS fluorescence observed a t elevated temperatures (Vanderkooi and Martonosi, 1969a, 1971a,e), which was accompanied by increased permeability of membranes of the sarcoplasmic reticulum to Ca and inulin (Duggan and Martonosi, 1970; Hassclbach et al., 1969). The EPR spectrum of the membranes of the sarcoplasmic reticulum labeled with 2 , 2 , 6 , 6-tetramcthyl 4-amino-(N-iodoacetamide) piperidine1-oxyl (iodoacetamide nitroxidc) was not influenced by M g , Ca, or chelating agents. However, A T P (0.5-10 mM) ITP and ADP altered the ratio of amplitudes of weakly and tightly imobilized components (Landgraf and Incsi, 1969; Incsi and Landgraf, 1970). Inorganic pyrophosphate, AMP, and cyclic AMP were without effect. The observed effect of ATP on the E P R spectrum is probably not related t o the ATPase activity or Ca transport. This is because RSg is necessary to the effect of ATP on the two functions and because the changes in the E P R spectrum occur at ATP concentrations which are high compared with that required for ATPase activity and Ca transport.

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3. X-RAYDIFFRACTION The potential of X-ray diffraction techniques for the study of membrane structure is now clearly established (Levine and Wilkins, 1971; Wilkins el aZ., 1971). Although the data obtained so far on sarcoplasmic reticulum (Coleman et al., 1969) are difficult t o integrate with existing biochemical and ultrastructural information, further important developments in this area are eagerly awaited, since X-ray diffraction techniques provide an opportunity for conformational studies on systems undisturbed by the various probes used for fluorescence or EPR measurements.

4. CIRCULAR DICHROISM Circular dichroism studies on sarcoplasmic reticulum (Mommaerts, 1967) did not reveal any significant correlation between membrane conformation and Ca transport. I n view of the gross optical inhomogeneity and light-scattering effects associated with membrane suspensions (Urry and Ji, 1968; Ji and Urry, 1969; Urry et al., 1970; Schneider et al., 1970), the limitations of this method for conformational studies on biological membranes may be greater than previously realized. In summary, while considerable progress has seen made in the application of fluorescence, EPR, and X-ray diffraction techniques t o the analysis of the structure and Ca transport function of the membranes of sarcoplasmic reticulum, future advances will require new methods with greater selectivity toward the Ca transport sites. The study of conformational changes related to the Ca release depends upon the development of techniques by which reversible and physiologically relevant changes in the ion permeability of isolated membranes can be induced and detected in uitro. Beyond their potential significance in clarifying the conformational aspects of the mechanism of excitation-contraction coupling, such investigation is expected to provide information of more general nature which may find usefulness on other membrane systems as well.

f l l . THE REGULATION OF SARCOPLASMIC RETICULUM FUNCTION A. Regulation of Ca Uptake

Apart from relatively brief periods of contractile activity, the free Ca concentration of the sarcoplasm is maintained a t levels estimated to be less than 10-7 M, and a major portion of the Ca content of the muscle cell

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is stored within the sarcoplasmic reticulum. Although the ratio of free Ca concentration inside and outside the sarcoplasmic reticulum membrane in relaxed muscle may be of the order of 102-103,the system is so designed that the maintenance of this concentration gradient requires very little energy. It is a common observation that in the absence of Ca precipitating anions the rate of Ca uptake by fragments of sarcoplasmic reticulum rapidly declines as the Ca concentration inside the vesicles increases, even if the extravesicular Ca and ATP concentrations are maintained at optimal levels. A steady-state is established after the accumulation of 0.1-0.2 pmole of Ca per milligram of protein; this represents a balance between Ca influx and outflux. The steady-state Ca-flux is 50-100 times slower than the maximum initial rate of Ca uptake. This suggests that the activity of the Ca pump is inhibited if the intravesicular free Ca concentration M (Makinose and Hasselbach, 1965; Hasselbach et al., exceeds 2 X 1969). Oxalate, by decreasing the intravesicular free calcium concentration, releases this inhibition and permits Ca accumulation to proceed until 8-10 pmoles of Ca per milligram of protein are accumulated, largely in the form of Ca oxalate. The inhibition of Ca flux by elevated intravesicular Ca concentration is accompanied by inhibition of the Ca-activated ATPase activity (Makinose and Hasselbach, 1965; Weber et al., 1966; Weber, 1971a). This provides further support for inferring a close association of ATPase activity with Ca transport. The inhibition of ATPase activity by passive preloading of the vesicles with Ca is in general agreement with this interpretation, although the great variability of the data prevents a quantitative comparison (Weber, 1971a). The inhibition of the ATPase activity of aged microsomes (Martonosi and Feretos, 1964b) and solubilized preparations of microsomal ATPase (Rlartonosi, 1964; Martonosi et al., 1968) by free Ca concentrations exM probably reflects the same phenomenon. Under these ceeding conditions the interior aspect of the Ca transport system is freely accessible to Ca in the medium. These findings reinforce the impression that a high Ca conccntration within the sarcoplasmic reticulum inhibits ATPase activity and Ca transport by a direct effect upon the transport ATPase rather than upon membrane permeability. The inhibition of ATPase activity and the associated Ca flux or Ca-Sr exchange is most readily observed a t low ATP concentrations (8-12 p M ) (Weber et al., 1966). Raising the free ATP concentration to 0.2-0.4 mM produces marked activation of ATP hydrolysis and Ca flux, although the Ca outflow from preloaded vesicles into a Ca-free medium is only slightly affected (Weber, 1971a). Although the steady-state concentrations of

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accumulated Ca or Sr are not influenced markedly at high free ATP concentration, the possibility remains that ATP, by penetrating into the microsomes and complexing somc of the accumulated Ca, decreases the concentration of membrane-bound Ca sufficiently to relieve the inhibition. As the steady-state loading is ultimately defined by the gradient of free calcium concentration between the two sides of the membrane, ATP is not likely to influence steady-state loading. It is interesting that the ATP concentration required to counteract the inhibition of ATPase activity by clcvated intravesicular free Ca concentration causes a noticeable change in thc EPR spectrum of sarcoplasmic reticulum membranes labeled with iodoacetamide nitroxidc (Landgraf and Inesi , 1969; Inesi and Landgraf , 1970). It also produces an anomalous activation of phosphoprotein formation (Inesi et al. , 1970). Previous reports (Yamamoto and Tonomura, 1967, 196s; Inesi el al., 1967) concerning the activation of A T P hydrolysis at high ATP concentrations may have an explanation analogous t o the observation of Weber (1971a) that the inhibitory effect of accumulated calcium on the steady-statc Ca flux is alleviated by relatively high concentration of free ATP. The inhibition of A T P hydrolysis and calcium flux a t elevated intravesicular calcium concentrations provides an cconomical mechanism for the retention of large amounts of calcium within the sarcoplasmic reticulum in relaxed muscle, with minimum ATP utilization. The physiological significance of this effect cannot be evaluated as the free R4g and ATP concentration in the environment of the sarcoplasmic reticulum in living muscle is unknown and no accurate information is available on the amount of Ca stored by the sarcoplasmic reticulum during relaxation.

B.

The Release of Ca from Sarcoplasmic Reticulum

The rate of Ca release from fragments of sarcoplasmic reticulum that had previously been loaded with Ca is about 10-l2 to 3 X 10/13 mole/cm2 linean per second based on data of the average surface area of niicrosomes (I" and Martonosi, 1965; Martonosi, 1964). This value is similar to what has been estimated to be the resting Ca flux through the surface membrane of skeletal (Bianchi, 1961a) or cardiac muscle (Winegrad, 1961) It therefore seems likely that the Ca permeability of sarcoplasmic reticulum fragments in vitro is close to the flux of Ca across sarcoplasmic reticulum membranes in relaxed muscle. The Ca2+release from sarcoplasmic reticulum in electrically stimulated barnacle (Ridgway and Ashley, 1967; Ashley and Ridgway, 1970; Ashley,

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1971) or frog muscle (Jobsis and O'Connor, 1966) appears to be essentially complete in a few milliseconds. This corresponds to rates as high as to mole of Ca released per cm2 of sarcoplasmic reticulum surface area per second. The extra ionic flux which accompanies the clcctrical excitamole/cm2 per second (Tasaki and tion of squid giant axon is 3 t o 6 X Singer, 1966). According to Bianchi (1961a), the Ca influx into skeletal muscle through the surface membrane during depolarization is about 2 X lO-'O molc/cm2 per second. These examples suggest th a t the magnitude of ion fluxes during rest as well as during excitation in various excitable membranes is similar (Katz, 1966) and that a common mechanism may be involved in the regulation of ion permeability of intracellular and surface membranes. Ca efflux from sarcoplasmic reticulum membrane may occur by two distinct mechanisms : 1. Carrier-mediated efflux of Ca. Probably this represents a reversal of the Ca transport process. It accounts for most of the observations made on the rate of in vitro Ca release from reasonably intact fragments of sarcoplasmic reticulum. 2. Diffusion of calcium across the sarcoplasmic reticulum. This may account for the dramatic increase in Ca release during depolarization. The calculated rate of this process is 100-1000 times greater than the maximum rate of carrier mediated Ca flux observed so far in vitro.

The two mechanisms will be discussed in turn. 1. CARRIER-MEDIATED EFFLUX OF CALCIUM

45Caaccumulated by sarcoplasmic reticulum membranes in the absence of oxalate rapidly exchanges with 40Ca (Martonosi and Feretos, 1963b, 1964a; Makinose and Hasselbach, 1965; Weber et al., 1966) added to the medium. The exchange occurs without significant change in the steadystate concentration of Ca within the microsomes. This indicates that the steady state is maintained by a balance between Ca influx and outflux. Similar conclusions were reached with respect to Ca-Sr exchange (Weber et al., 1966; Weber, 1971a). In the presence of ATP and Mg, the steady-state flux of Ca is inhibited by increasing intravesicular Ca (discussed in Section 111,A.) or by decreasing Ca concentration of the medium (Weber et al., 1966; Weber, 1971b). The retention of accumulated calcium by sarcoplasmic reticulum fragments suspended in calcium-free medium containing Mg requires the

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presence of ATP, although the rate of ATP hydrolysis does not exceed that of empty vesicles. Apparently, the mere presence of ATP without undergoing hydrolysis is sufficient to maintain the membrane in a state of relative impermeability with respect to Ca (Weber, 1971b). The net outflow of calcium from fragments of sarcoplasmic reticulum into calcium-free medium increases after removal of Mg (Duggan and Martonosi, 1970) or ATP (Weber et al., 1966; Weber, 1971b; Vanderkooi and Martonosi, 1971b) or after inhibition of the Ca pump with salyrgan (Martonosi and Feretos, 1964a,b). The calcium outflow under these conditions probably represents diffusion across a membrane with somewhat altered Ca permeability characteristics. The rate of Ca release from microsomes previously loaded with calcium into a Ca-free medium containing 5 mM MgClz is greatly enhanced by the addition of 0.05 M ADP and 3 mM inorganic orthophosphate (Barlogie et al., 1971). The Ca release is accompanied by the synthesis of 1mole of ATP for each two calcium atoms released, and the maximum rate of ATP synthesis is about 20% of the maximum rate of ATP utilization during active Ca transport (Makinose and Hasselbach, 1971). ATP synthesis also occurs during the steady-state flux of Ca, provided ADP and inorganic orthophosphate are present. Under these conditions its rate approximates the rate of calcium turnover (Makinose, 1971). These experiments clearly establish that the carrier-mediated efflux Ca represents a reversal of the process of Ca uptake. Further evidence for this is provided by the finding (Barlogie et al., 1971) that the calcium efflux induced by ADP and orthophosphate is inhibited by free calcium in the medium or by the omission of hlg. The medium Ca concentration which produces half-maximal inhibition of Ca efflux is similar to that required for half-maximal activation of Ca2+ transport (Barlogie et al., 1971). The physiological significance of the carrier-mediated efflux of Ca depends on the actual concentration of ADP, P,, ATP, and Ca in the environment of sarcoplasmic reticulum membranes. Its principal role may be to provide fine regulation of sarcoplasmic Ca concentration in relaxed muscle in adaptation to metabolic requirements. The rate of the massive Ca release from sarcoplasmic reticulum during activation exceeds the maximum rate of carrier mediated Ca outflux by a factor of lo2to 10'. For this reason, it seems unlikely that carrier-mediated Ca release significantly contributes to the excitation-contraction coupling. Furthermore, Ca release from sarcoplasmic reticulum during activation is a regenerative process (Ford and Podolsky, 1970; Endo el al., 1970) in the sense that elevated Ca concentration in the sarcoplasm promotes further

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Ca release, while the carrier mediated Ca efflux is inhibited by Ca (Barlogie etal., 1971). 2. THEPASSIVE PERMEABILITY OF SARCOPLASMIC RETICULUM MEMBRANES

The rate of passive Ca release from sarcoplasmic reticulum fragments into calcium-free mcdia is of the order of 10-l2 to 10-13 moles/cm2 per second, i.e., about 50-100 times slower than the maximum initial rate of Ca uptake, and 10-20 times slower than the maximum rate of carriermediated Ca outflux. Even the enhanced rate of Ca release observed a t alkaline pH, elevated temperatures, or in the presence of SH group reagents (Martonosi and Feretos, 1964a; Duggan and Martonosi, 1970) remains several orders of magnitude below the expected rate of calcium release during excitation in vivo. Electrical stimulation of Ca-loaded microsomes is not effective in promoting the release of calcium (Van der Kloot, 1966; Lcc, 1967). It appears from these considerations that isolated sarcoplasmic reticulum membranes are locked in a stable conformation, characterized by low permeability for calcium, which may be similar to the state of sarcoplasmic reticulum membranes in the relaxed muscle. In contrast to the relativc impermeability to calcium, several anions, such as oxalatc, orthophosphate, pyrophosphate, and fluoride readily penetrate through microsomal membranes as evidenced by their potentiating effect on calcium transport and by the Ca-linked accumulation of oxalate in the microsomal particles (Hasselbach and Makinose, 1963; Martonosi and Feretos, 1964a). Direct evidence for the penetration of sucrose, acetate, C1, and EDTA into the microsomal water space was also obtained (Duggan and Martonosi, 1970). It is not known whether this high permeability is the consequence of membrane damage sufferedduring homogenization or represents the natural state of the membrane. If the latter is correct, the content of sarcoplasmic reticulum tubules in vivo would be expected t o contain most of the important metabolic intermediates in exchange with the sarcoplasm. This may be of importance if sarcoplasmic reticulum membranes participate in the various synthetic and energy producing activities for which evidence is beginning to emerge (see Section VII). Fragmented sarcoplasmic reticulum membranes are impermeable to i n ~ l i n - * ~(MW C 5000) or dextran-'4C (MW 15,000-90,000) a t p H 7.0-9.0 with excluded space of 4-5 pl/mg protein (Duggan and Martonosi, 1970). Treatment with EGTA or EDTA (1 mM) at pH 8.0-9.0 increased the permeability of microsomes t o inulin14Cor dextran-I4C, parallel with the

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lowering of the Ca content of the membranes from 20 nmoles per milligram of protein t o 1-3 nrnolcs per milligram of protein. As inulin began to penetrate, the rate of Ca uptake markedly declined with a simultaneous rise in ATPase activity. This indicates that the membranes became leaky for calcium. Proteins, sterols, and phospholipids contribute to the definition of the permeability of microsomal membranes to Ca. The increased permeability of the membranes after treatment with EDTA a t slightly alkaline pH may be attributed to the selective release of C1 and Cz proteins from the microsomes (Duggan and Martonosi, 1970). The molecular weight of these proteins was estimated by polyacrylamide gel electrophoresis to be 51,000 and 63,000, respectively (Martonosi and Halpin, 1971). A major portion of the proteins released from the membrane by treatment with EDTA could be reattached to the microsomes after addition of 1 mM CaC12 in a manner analogous to the binding of ATPase to the surface membrane of Streptococcus faecalis (Abrams, 1965). Brief digestion of membrane proteins with trypsin, subtilisin NOVO,or subtilisin Carlsberg inhibits Ca transport and activates the transport ATPase. This suggests an increased permeability of the membranes for Ca (hlartonosi, 1968b). The passive permeability of microsomal membranes to calcium also increases in the presence of etiocholanolone, 5/3-pregnene-3,20-dione, diethylstilbestrol, progesterone, and bile acids of 5/3 configuration. This leads to inhibition of Ca transport and activation of ATP hydrolysis. A permeability increase as the underlying cause of these effects is suggested by the hemolytic effect of the same steroids a t concentrations similar to those required for the inhibition of Ca transport (Martonosi, 1 9 6 8 ~ ).The membrane-disruptive properties of this class of substances confirm the prediction of Willmer (1961) and point to the importance of phospholipidsterol interactions in the definition of the Ca permeability of biological membranes (see Section 111, C). Treatment of microsomes with digitonin (Martonosi, 196813) or ether (Fiehn and Hasselbach, 1969) also increase the Ca permeability, but the relationship of the permeability change to the removal of cholesterol or cholesterol esters is uncertain. Finally, a major increase in the Ca permeability of microsomal membranes occurred after treatment with phospholipase C (Martonosi, 1964; Martonosi et al., 196s) or phospholipase A (Martonosi et al., 1971a). These observations confirm the expected role of phospholipids. The changes in the Ca permeability of microsomal membranes which follow irreversible modification of membrane composition and structure are primitive imitations of the subtle conformational transitions induced

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by the excitatory stimulus. Future progress will require development of methods by which these transitions can be induced and analyzed in vitro. C. The Ca Permeability of Model Membranes

Hydrolysis of membrane phospholipids with phospholipase C drastically increases the permeability of microsomal membranes to Ca (Martonosi et al., 1968) and inulin (Duggan and Jlartonosi, 1970). The magnitude of the pcrmeability change suggests a major contribution by phospholipids. The role of phospholipids in the barrier function of the membrane for calcium was investigated in greater detail on artificial membrane systems composed of a series of phosphatidylcholine preparations of diverse fatty acid composition (Vanderkooi and Jlartonosi, 1971d). The liposomes used in these experiments were prepared rssentially according to Huang (1969; Huang et al., 1970; Huang and Charlton, 1971) and the measurement of Ca permeability was carried out as described by Bangham and his collaborators (Bangham, 1968; Bangham et d.,1965, 1967; Johnson and Bangham, 1969). The calcium permeability of phospholipid membranes is many orders of magnitude lrss than the Ca permeability of sarcoplasmic reticulum. At 3O"C, the Ca efflux from rgg lecithin vesicles was 8 X 10-ls mole/cm2 per second and 5.3 X 1O-l' mole/cm2 per second from dioleyllecithin in comparison with the rate of passive Ca loss from sarcoplasmic reticulum fragments which is about 3 X 10-13 molr/cm2 per second. The large difference between the rate of Ca release from phosphatidylcholine vesicles and from sarcoplasmic reticulum may result in part from the presence of phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositide in the microsomal membrane. These compounds, by virtue of their charge, are likely to promote the release of Ca in a manner similar to their rffect on the penetration of monovalent cations (Papahadjopoulos and Watkins, 1967). A contribution by membrane proteins t o the permeability may also be considered in view of the dramatic incrrasr in thc Na permeability of phosphatidylserine vesicles upon intrraction with lysozyme, cytochrome c, or poly-L-lysine (Kimelberg and Papahadjopoulos, 1971). The temprrature dependence of the Ca pcrmeability of phosphatidylcholine membranes is strongly influenced by the fatty acid composition. The Ca efflux from dioleyllecithin mcmbrancs increased from 5.3 X lo-'' mole/cm2 per second a t 30°C to 2.1 x 10-l6 molc/cm2 per second a t 50°C. The permeability change between 30" and 50" was less marked with dipalmitoyllecithin, egg lecithin, bovine lecithin, and plant lecithin (Van-

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derkooi and Martonosi, 1971d). The activation energies of Ca release for the various phospholipids ranged from 5 to 10 kcal/mole. I n comparison, the activation energy of diffusion in a water solution would be of the order of 5 kcal/mole. Ca release from dioleyllecithin micelles was inhibited by cholesterol (Vanderkooi and Martonosi, 1971d). This may be related to the known condensing effect of cholesterol on phospholipid bilayers (De Bernard, 1958; Shah and Schulman, 1967a,b; Demel et al., 1967). Cholesterol is known t o inhibit the release of Ii+ and C1- from egg lecithin liposomes (Papahadjopoulos and Watkins, 1967). Addition of macrocyclic polypeptides (valinomycin, tyrocidin, and gramicidin) promotes the release of Ca from liposomes containing egg lecithin, dipalmitoyllecithin, or an equimolar mixture of egg lecithin and cholesterol (Vanderkooi and Martonosi, 1971d). Although a much greater concentration of valinomycin is necessary to promote Ca release than K release (Johnson and Bangham, 1969), significant Ca release occurs with as few as 10-100 molecules of antibiotic per liposome. This is of interest in view of the suggestion that Ca transport in mitochondria may require the participation of a lipid-soluble mobile Ca carrier. However, the evidence for the existence of such a carrier is as yet quite inconclusive (Hull, 1971; Blondin, 1971). Ca transfer across phospholipid bilayers may occur by one of several possible mechanisms (Eisenman, 1968) : 1. Diffusion of Ca2+ through the lipid phase. The solubility of Ca in phospholipids is low. In the case of phosphatidplcholine, binding experiments have provided no unequivocal evidence for lipid-Ca interaction (Hauser and Dawson, 1967; Kimiauka et al., 1967; Kimizuka and Koketsu, 1962; Papahadjopoulos, 1968; Rojas and Tobias, 1965; Shah and Schulman, 1967a,b; Carr and Chang, 1970). Nevertheless Ca diffusion through the lipid phase cannot be entirely excluded (Onsager, 1970), since the liquid nature of phospholipids in biological membranes makes it possible for ion permeation to occur in a manner analogous to the electrical leakage in insulating oils (Eisenman et al., 1967). The temperature dependence of Ca flux can then be explained as resulting from the increased fluidity of the membrane a t elevated temperature which occurs due to the melting of hydrocarbon chains. It is of interest in this regard that the rate of free diffusion of Ca a t 20°C mole/cm2 per second (Kushmerick and in water solution is 7 x Podolsky, 1969) as compared with the observed membrane flux of 8 to 53 x mole/cm2 per second. This difference may arise largely from the sparse solubility of Ca in the lipid phase.

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2. Ca penetration via mobile carrier molecules. For pure phospholipid or phospholipid-cholesterol bilayers, such a mechanism would imply that the phospholipids constituting the bilayer would themselves serve as ion carriers by interacting with ions on one side of the membrane and passing in the form of phospholipid-ion complex to the other side. Pagano and Thompson (1968) have suggested this mechanism t o explain the C1 flux through lecithin bilayers. It may also apply to cation fluxes through phosphatidylcholine and especially phosphatidylserine bilayers (Papahadjopoulos and Ohki, 1969). Exchange of fatty acid molecules between the two layers of an artificial bimolecular lipid leaflet was first observed by Deamer and Branton (1967). Using a spin-labeled analog of phosphatidylcholine, Kornberg and McConnell (1971) estimated th a t the rate of mole/cm2 per second 30°C in egg lecithin this “flip-flop” is 1.7 x liposomes, similar to those used in the present experiments. Since the initial Ca content of the liposomes was 0.0014.003 mole of Ca per mole of phospholipid, the observed rate of Ca outflux (8 X 10-l8 mole/cm2 per second) may well be explained by passage of the phospholipid-Ca complex from the inner to the outer layer of the bimolecular membrane. The activation energy of the flip-flop, 19.4 kcal/mole, is sufficiently close to the activation energy of 45Carelease to support this possibility, without establishing a definite correlation between the two processes. No information is available about the effect of Ca on the rate of flip-flop in phospholipid bilayers, but in stearate bilayers Ca slowed the rate of transverse exchange only moderately (Deamer and Branton, 1967). Because the rate of 45Carelease is independent of the extravesicular Ca2+, Na+, and H+ concentration, exchange diffusion is unlikely to contribute significantly to this process. The Ca permeability of dipalmitoyllecithin membrane abruptly increases a t 4O-5O0C, where melting of the hydrocarbon chains is assumed to occur (Chapman, 1968a,b). The magnitude of this change is far greater than that observed with egg, plant, and bovine lecithin membranes, which contain a greater proportion of unsaturated fatty acids. The possibility remains that as the hydrocarbon layer melts, a fraction of the large dipalmitoyllecithin vesicles fragments and releases their Ca2f content. The fact that calcium is distributed asymmetrically on the two sides of the bilayer may make this fragmentation more likely (Ohki and Papahadjopoulos, 1970). 3. Ca penetration through polar pores. Mueller and Rudin (1967, 1968) have suggested that macrocyclic antibiotics associate t o form a hydrophilic channel across the phospholipid bilayer. This may enhance the ion permeability of synthetic and natural membranes (Mueller and Rudin, 1967, 1968).

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There is also good evidence for a temperature-dependent equilibrium between lamellar bilayer phases and spherical or cylindrical micelles in natural and artificial phospholipid membranes (Luzzati and Husson, 1962; Luzzati, 1968; Stoeckenius, 1962; Benedetti and Emmelot, 1965; Lucy, 1964, 1968; Stocckenius and Engelman, 1969). Cylindrical micelles forming a hydrophilic channel across the bilayer may provide a likely pathway for the penetration of ions across phospholipid bilayers. The relative contribution of the various mechanisms to the observed ion fluxes is unknown. Since in comparison with physiological Ca fluxes the permeability of phosphatidylcholine bilayer membranes is rather small, it would be necessary to postulate a major rearrangement of the lipid phase, triggered by the excitatory stimulus, in order to explain the observed increase in Ca permeability during activation.

IV. THE REGULATION OF SARCOPLASMIC Ca2f CONCENTRATION IN CARDIAC MUSCLE A. Sarcoplarmic Reticulum Function in Normal Heart

It is now generally accepted (Katz, 1967,1970) that the mode of involvement of calcium in the regulation of excitation contraction cycle is similar in skeletal and cardiac muscles. Cardiac myofibrils and actomyosin require calcium for syneresis and ATPase activity (Fanburg et al., 1964; Fanburg, 1964, 1966; Katz, 1967; Katz et al., 1966a,b; Weber et al., 1963a,b, 1964a, 1967; Weber and Herz, 1964; Otsuka et al., 1964) and the dependence of these functions on the free calcium concentration is similar to that of their skeletal counterparts (Weber and Winicur, 1961). Cardiac microsomes are capable of accumulating calcium (Fanburg et al., 1964; Fanburg and Gergely, 1965; Fanburg, 1964; Hasselbach, 1964a,b; Carsten, 1964; Weber et al., 1964a; Inesi et al., 1964) with inhibition of the ATPase activity of cardiac myofibrils, although the rate and extent of calcium accumulation are much less than with white skeletal muscle microsomes. These conclusions have been confirmed with improved preparations (Weber et al., 1967; Katz and Repke, 1967a; Repke and Katz, 1969 Katz et al., 1970), and earlier speculations on the possible involvement of “soluble relaxing factors” in the contraction relaxation cycle of cardiac muscle have proved to be unfounded.

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The low Ca uptake of cardiac muscle microsomes still remains a major problem. This is illustrated by the electron microscopic finding th a t only 5-1Oy0 of the vesicles present in “purified” cardiac microsome preparations showed visible Ca oxalate deposits following Ca accumulation in the presence of oxalate (Baskin and Deamer, 1969). This may be due partly to contamination of the preparation with inactive membrane elements and aggregated proteins. In addition, particles of cardiac sarcoplasmic reticulum appear to be very labile, since during isolation they rapidly lose their ability t o transport Ca (Fanburg et al., 1964). Recent developments in the procedures used for the isolation of cardiac microsomes have been concerned primarily with improvements in the stability of the preparations; elimination of inactive membrane material as yet appears to be a n elusive goal. Addition of &-tocopherol (Inesi et al., 1964) and ascorbate (Carsten, 1964) to protect microsomes from lipid peroxidation (Tappel and Zalkin, 1959, 1960) have not been particularly useful in preserving Ca transport activity during preparation and storage. However, the loss of Ca transport can be prevented by storage of microsomes a t -20°C in the presence of 0.1 M KCI, 0.32 M sucrose, 5 mM histidine a t pH 7.4 (B. Fanburg et al., 1964). Sucrose a t higher concentration (40-4570) protects against the inhibitory effect of 0.8 M urea or 1.0 M LiBr (Repke and Katz, 1969) and retards the spontaneous loss of activity when the material is stored on ice (Repke and Katz, 1969; Carsten, 1967; Fuchs et al., 1968; Fanburg and Gergely, 1965). Lyophilization provides nearly complete protection against loss of activity both in skeletal and cardiac microsome preparations for at least 10 days (Baird and Perry, 1960; Sreter et al., 1970). Dithiothreitol had no effect on the Ca uptake of microsomes isolated from calf hearts (Sreter et al., 1970), although it protected lobster muscle reticulum (Van der Kloot, 1969). Crude cardiac microsome preparations obtained by differential centrifugation are heavily contaminated with mitochondria, cell surface membranes, lysosomes, myofilaments, and adhering sarcoplasmic enzymes (Hulsmans, 1961; Fanburg et al., 1964; Fanburg and Gergely, 1965; Katz and Repke, 1967a,b; Webster and Williams, 1964; Imai et al., 1966; Katz et al., 1970). The amount of contamination varies with the duration and speed of homogenization (Katz et al., 1970), the composition of the medium (Martonosi, 1964, 1968a; Uchida et al., 1965), and the centrifugation schedule (Hulsmans, 1961 ; Imai et al., 1966) ; it presents particularly great problems in tissues such as heart, which contain relatively large numbers of mitochondria and a sparsely developed sarcoplasmic reticulum (Porter and Palade, 1957; Fawcett, 1961 ; Forssmann and Girardier, 1970; Leyton and Sonnenblick, 1971). As a result, preparations obtained in

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different laboratories even from the same species are likely to differ in composition and activity. This necessitates frequent and careful assessment of purity both by electron microscopy and the use of marker enzymes. The application of sucrose density gradient centrifugation (Hasselbach and Makinose, 1963; Seraydarian and Mommaerts, 1965) has resulted in improved preparations of cardiac microsomes as judged by the rate and extent of Ca accumulation (Carsten, 1964; Katz and Repke, 1967a,b; Entman et al., 1969a; Baskin and Deamer, 1969; Suko et al., 1970) and by the reduction of the level of succinate dehydrogenase and fumarase (Suko et al., 1970). However, this improvement may be due in part to the stabilization by sucrose of microsomes and contaminating lysosomal particles (Repke and Katz, 1969). Sucrose density gradient centrifugation in the presence of LiBr, added to reduce the aggregation of particles, increased Ca transport and the Casensitive ATPase activity 2- to 4-fold (Katz et ul., 1970) in a region of the gradient which surprisingly contained only about 5% of the starting protein. Although this method markedly reduces mitochondria1 contamination and greatly increases Ca transport activity, even these preparations may contain as much as 50% inactive protein material (Katz et al., 1970). Relaxation requires the removal of 0.06-0.16 pmole of Ca per gram of tissue from the myofibrils (Harigaya and Schwartz, 1969; Katz et al., 1966b; Weber et al., 1967), presumably by uptake into the sarcoplasmic reticulum. The Ca binding capacity of cardiac sarcoplasmic reticiiliim appears sufficient to perform this function. Human, rabbit, dog, or chicken cardiac microsomes can bind about 26-70 nmoles of Ca per milligram of protein in the absence of Ca precipitating agents (Harigaya and Schwartz, 1969; Weber et al., 1967; Katz and Repke, 1967). If cardiac muscle contains ahout 5 mg of sarcoplasmic reticulum protein per gram of tissue, the calculated calcium capacity is comparable to the physiological need (Weber et al., 1967). These estimates may be in error by as much as an order of magnitude since actual yields of microsomal membranes ranging from 1 to 2.4 mg protein per gram of tissue (Harigaya and Schwartz, 1969; Katz and Repke, 1967a; Weber et al., 1967) could include 90-95% inactive membrane material (Baskin and Deamer, 1969). I n the presence of oxalate as calcium-precipitating agent, dog, human, and rabbit cardiac microsomes accumulate 1.8-3.0 pmoles of Ca per milligram of protein (Harigaya and Schwartz, 1969). This represents a calculated Ca capacity in excess of the total calcium content of the heart. The rather low values of Ca uptake reported by Fanburg et al. (1964)

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may have resulted from postmortem changes in th e calf heart during transfer from the slaughterhouse (Suko et al., 1970). The affinity of cardiac microsomes for Ca is sufficient t o reduce the medium Ca concentration to levels a t which the ATPase activity and syneresis of cardiac or skeletal myofibrils is inhibited (Fanburg et al., 1964; Weber et al., 1964a, 1967). The estimated value of the affinity constant for Ca is 8 X lo5M-' for dog (Katz and Repke, 1969; Katz, 1970) and 2 x lo6 for rabbit cardiac microsomes (Harigaya and Schwartz, 1969). I n comparison, for white skeletal muscle microsomes the reported values range from 4.2 X lo6M-* to 4 X lo7M-' (Ebashi and Endo, 1964; Inesi et al., 1967; Weber et al., 1966; Worsfold and Peter, 1970). The rate of Ca binding b y microsomes isolated from heart is in the range of 0.1-0.35 pmole of Ca2+ per milligram of protein per minute a t 25°C (Fanburg et aE., 1964; Lee, 1965; Weber et aE., 1967; Katz and Repke, 1967a; Suko et al., 1970; Harigaya and Schwartz, 1969), and 0.3-0.5 pmole at 37°C (Suko et aE., 1970). For comparison, the Ca uptake rate of white and red rabbit skeletal muscles was found to be 1.44 and 0.18 pmoles per mg protein per minute, respectively (Harigaya and Schwartz, 1969). The rate in vitro of Ca uptake by cardiac microsomes is about 5-20 times less than the physiological requirement, the actual number depending on the assumptions made about the relaxation rate (Harigaya and Schwartz, 1969; Weber, 1966; A. i'vl. Katz and Repke, 1967a). Usually this discrepancy is attributed to the low yield and poor quality of isolated cardiac microsome preparations and t o the less than optimum conditions used for the measurement of the rate of Ca uptake. Nevertheless, a quantitative evaluation of the contributions of these various factors is necessary before the role of sarcoplasmic reticulum as the principal regulator of contraction-relaxation cycle in heart muscle can be accepted. Lee claimed (1965) that dog cardiac sarcoplasmic reticulum is as potent as its skeletal counterpart with regard to Ca uptake. This conclusion may have been based on technical error since a t 2.5 mM oxalate and 0.5 m M Ca concentrations the solubility product of Ca oxalate is exceeded. Consequently Ca oxalate could have been precipitated in the medium. This would increase apparent Ca uptake. The suggested specific requirement for an ATP regenerating system in the Ca uptake of cardiac mierosomes (Lee, 1965) has not been confirmed (A. M. Katz and Repke, 1967a), and the apparent acceleration of the rate of Ca uptake by inorganic orthophosphate (Lee, 1965) is probably attributable to the precipitation of Ca phosphate within the vesicles (Martonosi and Feretos, 1964a,b; Hasselbach, 1964a,b). The release of Ca from isolated cardiac sarcoplasmic reticulum on

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electrical stimulation in vitro (Lee, 1965) is of doubtful significance (Lee, 1967) since it may have resulted from irreversible side effects (Van der Kloot, 1966). In addition to the sarcoplasmic reticulum, the surface membrane of the muscle cell also transports Ca. The influx of extracellular calcium during the contraction of mammalian heart is a relatively small fraction (perhaps less than 1%) of the Ca required to activate thc contractile material (Winegrad, 1961; Langer, 1968; Sonnenblick and Stam, 1969; E. A. Johnson and Lieberman, 1971). Nevertheless, relaxation must be acpanied by the active extrusion of this calcium across the surface membrane. The process probably involves a Ca transport system which may be similar to that found on the surface membrane of red blood cell (Schatamann, 1966; Schatzmann and Vincenzi, 1969; Wolf, 1970). There are indications that in amphibian muscles this system may be the principal regulator of the contraction-relaxation cycle (Nayler and Merrillees, 1964; Niedergerke, 1963). A similar role may be fulfilled by the surface membrane in amphioxus muscle, which contains very little or no sarcoplasmic reticulum (Peachey, 1961). 8. The Role of Mitochondria in the Regulation of Excitation-Contraction Coupling

There is a definite correlation between the rate of heart beat and the extent of development of sarcoplasmic reticulum in cardiac muscle. The sarcoplasmic reticulum is sparse in the slow turtle atrium (Fawcett and Selby, 1958), but quite extensively developed in the myocardium of bats, animals that have a heart beat of 500-1000 per minute. Most other heart muscles so far analyzed fall between these extremes (Forssmann and Girardier, 1970; Leyton and Sonnenblick, 1971; Nelson and Benson, 1963; Legato et al., 1968; Staley and Benson, 1968; Fawcett and McNutt, 1969). In view of the relatively slow rate of contraction, and the small diameter of the muscle fibers, (Jewett et al., 1971), the sarcoplasmic reticulum of most cardiac muscles appears sufficiently well developed to account for the physiological need. A dominant morphological feature of cardiac muscles in various species is the relative abundance of mitochondria, which in fast-beating hearts appear in regular rows between the myofibrils surrounding, and frequently overshadowing, the relatively thin layer of sarcoplasmic reticulum (Fawcett, 1961). In view of their abundance and their powerful Ca transport system

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(Lehninger et al., 1967; Lehninger, 1970), it did not take long to implicate mitochondria in the regulation of the excitation-contraction cycle (Chance, 1963; B. Fanburg and Gergely, 1965; Lehninger et al., 1967). This possibility is further supported by the fact that the Ca capacity (Slater and Cleland, 1953) and the rate of Ca uptake (Chance, 1965) of heart mitochondria are more than adequate to account for the physiological need and rabbit heart mitochondria are able to lower the medium Ca concentration to about 10-7 M (Weber et al., 1964b), as required for the relaxation of myofibrils (Weber et al., 196313, 1964a). Slater and Cleland (1953) observed that virtually all the Ca content of rat heart muscle (1 pmole/gm) can be recovered in the isolated mitochondria, even though yield may not exceed 30-50% (Cleland and Slater, 1953). This was interpreted to mean that calcium might ‘ h o t be in the sarcosomes in the intact heart but becomes incorporated during the isolation.” Subsequent exprriments by Patriarca and Carafoli (1968) showed that aftcr intraperitoncal injection of 46CaCI2 into rats, most of the radioactivity is found associated with subcellular particles, primarily mitochondria. The specific activity of the mitochondrial calcium was higher than that of any othpr cell fractions. It has been suggested therefore that turnover of the microsomal Ca pool may be too slow to permit it to regulate the contraction-relaxation cycle in heart muscle. Similar observations were made by Horn et al. (1971) on perfused rat heart preparations exposed t o radioactive calcium. However, during tissue homogenization the mitochondria remain intact while the sarcoplasmic reticulum is disrupted and its content released into the medium. On this ground, i t is not surprising that following homogenization little calcium remains associated with the micbrosomal fraction. That calcium may be redistributed during homogenization and fractionation is also suggested by the fact that the mitochondrial fraction preferentially takes u p 45Ca added to the homogenatc at 0”. Localization of large amounts of calcium in the mitochondria in vivo is not compatible with electron microscopic observations on the distribution of calcium in the heart. With the aid of the potassium-pyroantimonate method (Icomnick and Komnick, 1963), dense calcium-pyroantimonate dcposits were observed in the lateral sacs of the sarcoplasmic reticulum of dog papillary muscles and in a sharp band over the I zone of the sarcomere (Legato and Langer, 1969). In both locations the amount of precipitate was greatly diminished by perfusion of the muscles with a Ca-free solution. At the same time tension decreased. After perfusion with 5 m M EDTA or EGTA for 2 minutes, the tension reached zero and the lateral sacs of sarcoplasmic reticulum became strikingly empty of precipitate. These observations are in essential agreement with the suggested distribu-

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tion of calcium in skeletal muscle (Winegrad, 1965a,b, 1970; Costantin et a!., 1965; Podolsky et al., 1970). The established correlations between Ca concentration of the medium, the Ca content of the sarcoplasmic reticulum, and the contractile activity provide strong indication that the sarcoplasmic reticulum is the physiological regulator of contractile activity in heart. Oligomycin (Lardy et al., 195S), a well known inhibitor of mitochondrial ATP synthesis (Lehninger et al., 1967), and of the ATP supported uptake of calcium by mitochondria (Brierley et al., 1963; Lehninger et al., 1963) causes cardiac arrest (Challoner and Steinberg, 1966; Challoner, 1968; Horn et al., 1969) and stops the beating of isolated heart cells (Harary and Slater, 1965). These effects have becn interpreted (Haugaard et al., 1969) as due to the elevated concentration of calcium in the cytoplasm resulting from oligomycin inhibition of mitochondrial Ca transport. However, oligomycin does not inhibit the respiration-supported Ca transport of mitochondria (DeLuca and Engstrom, 1961). Moreover cardiac arrest caused by oligomycin occurs in the relaxed state (Challoner, 1968; Challoner and Steinberg, 1966), a situation incompatible with an elevation of the sarcoplasmic Ca Concentration. Although epinephrine, theophylline, or electrical stimulation of the heart poisoned with oligomycin eventually cause contracture (Horn et al., 1969), this may result from the marked changes in ATP and creatine phosphate concentration observed under these conditions, as expected from earlier observations made with dinitrophenol (Ellis, 1952). In addition, oligomycin is not a specific inhibitor of mitochondrial ion transport, since it also weakly inhibits the Ca transport of sarcoplasmic reticulum (Carsten and Mommaerts, 1964; Fairhurst et al., 1964; Weber et al. , 1966). The functional significance of the frequently observed contacts between mitochondria and T-system tubules (Forssmann and Girardier, 1966) or sarcoplasmic reticulum (Fawcett and Revel, 1961) in cardiac muscle is unclear. They may be related to the relatively large requirement for ATP during Ca transport, It seems doubtful that they would represent regions of excitation-coupling between mitochondria and other membrane systems. A further argument against the possible role of mitochondria in the regulation of contraction-relaxation cycle is that the turnover of accumulated Ca in mitochondria is probably slower than in microsomes (Weber et al., 1964b). It is unlikely that Ca could be released from mitochondria sufficiently fast to account for the rate of contraction. I n summary, the evidence presented so far in favor of mitochondrial participation in the regulation of excitation-contraction coupling is not conclusive.

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C. Calcium Uptake by Sarcoplasmic Reticulum Fragments Isolated from Failing Heart

A possible involvement of sarcoplasmic reticulum in congestive heart failure was suggested on the basis of the diminished in vitro Ca uptake of fragmented sarcoplasmic reticulum obtained from spontaneously failing heart-lung preparations (Gertz et al., 1967). Subsequent experiments with various models of heart failure confirmed these findings but also brought out some significant differences. Progressive pulmonary hypertension induced by ligation of the right pulmonary artery of calves 5-12 weeks after birth causes right ventricular hypertrophy with the development of cardiac failure 6-8 weeks later (Suko et al. , 1970). The rate of Ca transport by microsomes isolated from the right ventricles of the failing heart was 0.224 pmole of Ca per milligram of protein pcr minute compared with the control rate of 0.387 pmole Ca per milligram of protein per minute, measured a t 37°C in the presence of oxalate (Suko et al., 1970). Preparations obtained from the left ventricles of the same hearts showed lesser differences. Surprisingly the Ca uptake capacity, measured after 1 minute of incubation at 25°C in the absence of oxalate, was not significantly different for right ventricular preparations of failing and control hearts; in fact, on prolonged incubation the microsomes from failing hearts accumulated more Ca2+. This raises the possibility that the differences observed in the presence of oxalate may, in part, have resulted from size differences in the vesicles isolated from the two types of hearts. Ouabain 10-7 to 10-4 M had no effect on the oxalate-moderated Ca uptake of microsome preparations isolated from chronically failing hearts a t 25°C (Suko et al., 1970). This is in contrast to the reported effects of ouabain on microsomes from acutely failing hearts from heart-lung preparations (Gertz et at., 1967). When acute cardiac ischemia was induced in dogs maintained on total cardiopulmonary bypass, Ca transport was unchanged in isolated sarcoplasmic reticulum fragments obtained after 15 minutes of occlusion of the anterior descending branch of the left coronary artery (Lee et al., 1967). Ischemia for 60 minutes caused marked inhibition of microsomal Ca transport in the absence of oxalate, although the amount of Ca accumulated with oxalate remained unchanged. Restoration of coronary circulation after 60 minutes of occlusion reversed the inhibition of microsomal Ca transport. After ischemia for 90 minutes, an irreversible inhibition of microsomal Ca transport occurred even in the presence of oxalate. Among the earliest morphological changes occurring in rat hearts following ligation of coronary artery was the swelling and subsequent vesicu-

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ANTHONY MARTONOSI

lation of sarcoplasmic reticulum followed by the enlargement of mitochondria (Bryant et al., 1958). Functional restoration was generally incomplete after 2 hours of cardiac arrest (Michal et al., 1958; Gollan and Nelson, 1957). Microsomes isolated from rat hearts after perfusion for 30 minutes with substrate-free medium (Muir et al., 1970) showed marked inhibition of Ca transport in the absence of oxalate, without comparable changes in contractile force or heart rate (Muir el al., 1970). If glucose was added to the perfusion fluid after 30 minutes, the ability of the isolated microsomes to bind calcium was restored to normal levels. Perfusion for 2 hours with substrate-free medium markedly reduced the contractile force of the heart and resulted in irreversible inhibition of microsomal Ca uptake both with or without oxalate. Since the initial rate of microsomal ATPase activity was elevated a t the time when the inhibition of Ca transport became pronounced, some membrane damage may be suspected to have resulted from perfusion with substrate-free medium, which increases the permeability of microsomes for Ca2+.Accumulation of free fatty acids may contribute to this effect, since the inhibition of microsomal Ca transport by oleate is accompanied by activation of ATPase activity (Martonosi, 1964; Martonosi et al., 1968). Perfusion of isolated heart with a solution containing depresses contractility and increases diastolic free fatty acid (1.7 d) pressure (Henderson et al., 1970). Although it was concluded earlier that perfusion with substrate-free medium decreases the free fatty acid content of the heart (Olson and Hoeschen, 1967) more recent experiments indicate that i t actually increases (Bauer, 1972). A relationship between “heart failure” and the observed changes in microsomal Ca transport has not been established conclusively in any of these experiments. In fact, in the experiments of Muir et al. (1970), the contractile force remains essentially unchanged with maximum inhibition of Ca transport in the absence of oxalate, while the reverse is true in chronically failing calf hearts (Suko et al., 1970). The possibility exists that a t least in some of the reported cases the apparent inhibition of microsoma1 Ca transport occurred during isolation and storage of microsomes and the differences between control and failing hearts would, in this case, reflect differential sensitivity, due perhaps to the presence of metabolic inhibit ors. Since the Ca content of rabbit heart in failure remains normal (It0 et al., 1969), inhibition of Ca transport by sareoplasmic reticulum should prolong relaxation time. Since inhibition of transport raises the free calcium ion concentration in the sarcoplasm, it should also increase the diastolic pressure of the failing heart. No such changes have been reported (Spann et al., 1967; Cove11 et al., 1966). In fact, cardiac failure could arise without a

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131

significant primary biochemical defect in the energy-producing system, the contractile protcins of the myofibrils, or the sarcoplasmic reticulum if the spatial rclationship bctwcen these major systems is disturbed as the result of hypertrophy. Fleckenstein has proposed (1967) that the enlargement of fiber diameter and the increased mass of myofibrils characteristic of cardiac hypertrophy prcvent uniform diffusion of Ca, and therefore full activation of the contractile material. Although the sarcoplasmic reticulum also undergoes hypertrophy (Poche, 1958; Wollcnbcrgcr et al., 1963), this may be insufficient for full compensation (Kaufman et al., 1971). The resulting relative insufficiency of sarcoplasmic reticulum would not be detectable by conventional assay of i n vitro Ca transport. In summary, although cardiac failure has been shown to be accompanied by defective Ca transport of sarcoplasmic reticulum, the physiological characteristics of failing heart are not always those expected to result from such defects, and a direct correlation between the function of the sarcoplasmic reticulum and cardiac failure remains uncertain.

D. The

Effect of Cardiac Glycosides and Other Drugs on the Ca Uptake of Cardiac Sarcoplasmic Reticulum

The relationship between Ca2+ and the effects of digitalis glycosides on heart, first suggested by 0. Loewi in 1918,is now fully established (Langer, 1968; Langer and Serena, 1970). It is also generally accepted that inhibition of the Na-K activated ATPase is the basis of the digitalis effect (Skou, 1964, 1965). Nevertheless, essential details of the mechansim of action of digitalis remain unclear (Baker et al., 1969; Langer, 1970a,b; Langer and Serena, 1970; Besch and Schwartz, 1970; K. Repke, 1964). The Ca concentration of cardiac sarcoplasm appears to be strongly influenced by thc influx of external calcium. This in turn is regulated by the concentration of Na and I<. For this reason, the Ca, Na, and K fluxes in heart muscles and their relation to the excitation-contraction coupling have received considerable attention (Langer, 1968). Low external sodium concentration produces increased force development of the heart (Daly and Clark, 1921). Probably this results from increased uptake of Ca into the muscle cell (Langer, 1964; Luttgau and Niedergerke, 1958). The inward flux of Ca also increases in perfused squid axon if the internal sodium concentration is increased (Baker et al., 1969). There are good reasons to believe that a similar relationship exists between internal Na concentration and the influx of Ca in heart muscle (Langer,

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ANTHONY MARTONOSI

1970a,b; Langer and Serena, 1970). This would explain the staircase phenomenon and the effect of cardiac glycosidcs. The increased Ca influx observed during the development of staircase effect (Langer, 1965) may result from a relative insufficiency of the Na pump (Woodbury, 1963), which permits the accumulation of Na inside the cell a t the expense of a net I< loss (Hajdu, 1953; Langer and Brady, 1966). The positive inotropic effect of cardiac glycosides is probably related to inhibition of the Na-K ATPase (Bcsch et al., 1970). In analogy with observations made on the perfuscd squid axon (Baker et al., 1969), this inhibition leads to an increase in intracellular Na concentration, a loss of I< (Langer and Serena, 1970), and a secondary increase in Ca influx. However, even in cardiac muscle the increased Ca influx per beat represents a small fraction of the Ca2+ required for thc activation of contractile material (Winegrad, 1961). It may be of significance that Ca in small concentration facilitates the release of Ca from sarcoplasmic reticulum. This may explain the regenerative character of the activation process (Ford and Podolsky, 1970; Endo et al., 1970). The facilitation of the release of Ca from sarcoplasmic reticulum by the increased influx of cxternal Ca may contribute to the positive inotropic effect of digitalis glycosides. It is difficult to say whether or to what extent the elevated Na concentration (Palmcr and Posey, 1967) alters directly the Ca2+transport function of sarcoplasmic reticulum. The localization of the Na-I< ATPase enzyme on the surface membrane of cardiac muscle cell is reasonably well established (Wollenberger and Schulze, 1966; Schulze and Wollcnberger, 1969; Stam et al., 1969). On the basis of physiological evidence there is no necessity to postulate a direct action of digitalis glycosides on sarcoplasmic reticulum. Nevertheless, a considerable literature has accumulated in recent years that reports conflicting effects of cardiac glycosidcs on the Ca transport of sarcoplasmic reticulum in vitro. Ouabain and strophantin induced inhibition of Ca uptake by fragmented sarcoplasmic reticulum was observed by Lee and Choi (1966), even though the same group had found strophantin ineffective in earlier experiments (Lee et al., 1965). Ouabain and scveral cardiotonic lactones increased the Ca uptake of cardiac sarcoplasmic reticulum according to Entman et al., (1969a,b). Activation by ouabain has also been reported for cardiac microsomes inhibited with amobarbital (F. N. Briggs et al., 1966; see, however, Chimoskcy and Gergely, 1968). In other laboratories, ouabain or strophantin were found to be without effect on the Ca2+ transport of skeletal or cardiac microsomes (Martonosi and Feretos, 1964a; Van der Iiloot, 1966; Chimoskey and Gergely, 1968; Carsten, 1967; Briggs et al., 1966; Gertz et al., 1967). In view of the small magnitude and variability of the reported effects and the possibility of systematic technical

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errors (Chimoskey and Gergely, 1968), it may be safe to conclude that ouabain has no established influence on the Ca transport of sarcoplasmic reticulum. Epinephrine also increases the influx of external Ca into heart which may be the basis of its positive inotropic action. The claim that epinephrine activates the Ca transport of sarcoplasmic reticulum (Entman et al., 1969c; Shinebourne et al., 1969; Hess et aE., 1968; White and Shinebourne, 1969) is not supported by observations madc in other laboratories (Chimoskey and Gergely, 1968; Dransfeld et al., 1969; Dhalla, 1969; Nayler et al., 1970; Besch et al., 1970; Sulakhe and Dhalla, 1970). It seems unlikely that these differences could be attributed to differences in the microsome preparations, as suggested by Epstcin et al. (1971). Entman et al. (1969d) demonstrated the presence of adenylcyclase activity in cardiac microsome preparations and suggested that epinephrine or glucagon increase the Ca uptake of sarcoplasmic reticulum through stimulation of adenylcyclase (Entman et al., 1969~).In addition t o epinephrine and glucagon being completely without effect on Ca transport (Sulakhe and Dhalla, 1970)) it appears that the adenylcyclase activity is entirely independent of the Ca2+transport function (Sulakhe el al., 1970; Dhalla et al., 1970) and may represent contamination of sarcoplasmic reticulum fragments with surface membrane elements. Earlier, Sutherland et al. (1962) found most of the adenylcyclase activity in fractions sedimenting at low speeds and the activity of microsomes was relatively low. The presence of adenylcyclase in skeletal muscle microsomes has also been observed (Rabinowitz et al., 1965). The reported activation of the Ca transport of cardiac sarcoplasmic reticulum by adenosine 3‘, 5’-cyclic monophosphate (Entman et al., 1969c; Epstein et al., 1971; Entman, 1970; Shinebourne and White, 1970) has not been confirmed (Yu and Triester, 1969; Sulakhe and Dhalla, 1970) and cyclic AnIP appears to have no effect on skeletal muscle microsomes either (Carsten and Mommaerts, 1964; Weber, 1968). I n summary, no reliable evidence is available to indicate a specific action of ouabain, epinephrine, glucagon, or 3’ ,5’-cyclic AMP on the sarcoplasmic reticulum of cardiac muscle and the adenylcyclase activity of cardiac microsome preparations probably has no relationship with the Ca2+transport. Miscellaneous Drug Effects on Cardiac Sarcoplasmic Reticulum. The calcium uptake of cardiac sarcoplasmic reticulum fragments is inhibited by chloroform and sodium pentobarbital (Lain et al., 1968) a t concentrations similar to those producing a negative inotropic effect on contractility. Halothane and ethyl ether inhibit contractility a t concentrations much lower than those required for the inhibition of Ca transport in vitro.

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ANTHONY MARTONOSI

The inhibiting rffect of quinidine on t h r Ca transport of fragmentrd cardiac reticulum (Fuchs et al., 1968) may be morr rasily related to the positivr inotropic rffects of quinidine on t h r hrart (Pruett and Woods, 1967; I<mnedy, 1969) than to th r better known depression of myocardial contractility. E. No-K-Activated ATPase*

Following the early demonstration of the Na-Ii ATPasc in skeletal (Samaha and Gergely, 1965a; 1966) and cardiac musclcs (Skou, 1962; Bonting et al., 1961; Auditore and Murray, 1962; Auditore, 1962; Lce and Yu, 1963; Schwartz and Lascter, 1963, 1964), evidence continues to accumulate supporting the localization of the enzyme in the surface membrane (sarcolemma) of the muscle cell (Wollenberger and Schulze, 1966; Schulze and Wollenberger, 1969; Stam et al., 1969; Sulakhe et al., 1971; Peter, 1970). While the localization of the enzyme in the sarcolemma is well established, the specific Na-I<-ATPase activity and ouabain sensitivity of sarcolemmal preparations recently obtained from cardiac (Stam et al., 1969) and skeletal (Peter, 1970; Sulakhe et al., 1971) muscles, by modification of earlier procedures (Nakao et al., 1965; R'Iatsui and Schwartz, 1966a; McCollester and Semente, 1964; Rosenthal et al., 1965) does not represent a great improvement over earlier preparations (Matsui and Schwartz, 1966a; Samaha and Gcrgely, 19G6). There is a significant body of conclusive evidence to indicate a specific effect of digitalis glycosides on the Na-K-ATPase activity of several tissues (Glynn, 1957, 1964; Skou, 1964, 1965), including hearts from various species (Schwartz, 1962; Rlatsui and Schwartz, 1966a,b, 1968; Allen and Schwartz, 1969; 1970; Schwartz et al., 1968b; I<. Nagai et al., 1970; Lindenmayer and Schwartz, 1970; Allen et al., 1970, 1971). The well known differenccs in the sensitivity of various animal species to cardiac glycosides may be explained by the relativc affinity of their Na-I<-ATPase to the drug (Allen and Schwartz, 1969; Akera et al., 1969, 1970; Akera and Brody, 1971; Repke et al., 1965). The contractile proteins (Katz, 1970), sarcoplasmic reticulum (see above) and mitochondria (Peter, 1970) of heart are not influenced by cardiac glycosides, in spite of numerous claims to the contrary (Stowring et al., 1966). The previously observed binding of ouabain to skeletal muscle microsomes (Dutta el al., 1968a,b) may be attributed to the admixture of surface membranes (Allen et al., 1970; Allen and Schwartz, 1969, 1970; Bcsch et al., 1970; Schwartz el al., 1969).

* See also the chapter by Schwartz, Lindenmeyer, and Allen in this volume.

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135

The introduction of further enzymatic markers (Blomberg and Perlman, 1971) to quantitate the contamination of microsome preparations with surface membranes would be desirable. F. Mitochondria in Cardiac Failure

The dispute about possible impairment of mitochondrial oxidative phosphorylation as an important factor in cardiac failure is gaining momentum. Essentially normal mitochondria1 functions have been observed in failing hearts obtained from dogs (Olson, 1964), guinea pigs (Sobel et al., 1967; Plaut and Gertler, 1959), humans (Chidsey et al., 1966), and cardiomyopathic hamsters (Wrogemann and Blanchaer, 1968; Blanchaer and Wrogemann, 1968). The mitochondrial defects associated with various types of cardiac failure by other authors (Gertler, 1961; Szekeres and Schein, 1959; Schwartz and Lee, 1962; Meerson et al., 1964; Argus et al., 1964; Bing et al., 1964; Fox and Reed, 1966) have been tentatively attributed by Sobel et al. (1967) to several possible technical errors, among which damage due to the liberation of free fatty acids (Van den Bergh, 1967) may be the most important. More recent experiments with improved methods confirmed the OCcurrence of defective oxidative phosphorylation in mitochondria isolated from failing guinea pig hearts (Lindenmayer et al., 1968) and from cardiomyopathic hamsters at a late stage of failure (Lindenmayer et al., 1970; Schwartz et al., 1968a; Lockner et al., 1968; Opie et al., 1964). However, defective mitochondria were isolated from hearts of dying animals with 1-6 hours of life expectancy in the case of guinea pigs (Lindenmayer et al., 1968, 1970) and not more than 48 hours in hamsters (Lindenmayer et al., 1970). The oxidative phosphorylation of mitochondria isolated from hearts in early failure is, if anything, improved (Lindenmayer et al., 1970). Consequently, the defective oxidative phosphorylation in advanced stages of the disease is probably the consequence rather than the origin of the failure and may be caused by the massive circulatory, metabolic, and excretory problems characterizing the terminal stage. It would not be surprising if similar defects of oxidative phosphorylation were to occur in mitochondria isolated from other tissues of these moribund animals. Defective respiration and oxidative phosphorylation were also observed in late stages of dystrophy in mitochondria isolated from skeletal muscles of dystrophic hamsters (Jacobson et al., 1970). Extensive necrotic lesions are frequently observed in skeletal muscles even in fairly young

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ANTHONY MARTONOSI

animals (30-80 days) ; the defective oxidative phosphorylation of mitochondria isolated from these muscles (Lochner and Brink, 1967; Lin et al., 1969) may be corrected by the addition of Mg during the assay (Wrogemann et al., 1970a) or by the removal of Ca with EDTA during the isolation of mitochondria (Wrogcmann et al., 1970b). The apparently normal oxidative phosphorylation observed earlier in mitochondria of young hamsters (Wrogemann and Blanchaer, 1968) may have resulted from a smaller incidence of necrotic lesions in that group. The incidence of necrotic lesions may be related to the elevated level of acid hydrolases in muscle homogenates of dystrophic hamsters (Jacobson et al., 1970) which could contribute to the observed defects in oxidative phosphorylation (Mellow et al., 1967).

V. SARCOPLASMIC RETICULUM IN RED SKELETAL MUSCLES

Fully differentiated mammalian muscles contain several types of muscle fibers (red, white, and intermediate) readily distinguished on the basis of their size and cytological characteristics (Pellegrino and Franzini, 1963; Padykula and Gauthier, 1963; 1967; Gauthier and Padykula, 1966; Edstrom and Nystrom, 1969; Krompecher et al., 1970; Guth and Samaha, 1969; Samaha et al., 1970; Ogata and Murata, 1969; Tice and Engel, 1967; Gauthier, 1969; Romanul, 1964; Schiaffino et al., 1970). The chemical composition and metabolic and contractile properties of the various muscles are determined by the relative contribution of the different types of fibers to the muscle mass. It has been known since the early work of Ranvier (1874) that the rate of contraction and relaxation is faster in white than in red skeletal muscles (Close, 1964, 1965a,b, 1967, 1969). This is in keeping with the primarily tonic or sustaining function of red muscles (such as the maintenance of posture), whereas white muscles are adapted for rapid, forceful activity of shorter duration (Needham, 1926). An interesting example of this functional differentiation has been observed in skipjack tuna. During quiet swimming, electrical activity is continually recorded from red muscles, the white muscles contributing activity only when short bursts of fast swimming take place (Rayner and Keenan, 1967). The differences in the speed of contraction are associated with distinctive metabolic characteristics (Slater, 1960; Beatty et al., 1963; Gutmann, 1967; Short et at., 1969; Mann and Salafsky, 1970; Bar and Blanchaer, 1965; Bocek et al., 1966; Domonkos, 1961; Domonkos and Latzkovits, 1961a,b; Ogata, 1960; Domonkos et al., 1967) and with considerable dif-

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137

ferences in chemical composition and enzymatic makeup (Owens and Angelini, 1970; Heiner and Domonkos, 1970; Kirsten and Kirsten, 1969; Luff and Goldspink, 1970; Gutmann, 1967). The speed of contractile response and the principal metabolic characteristics of white and red muscles are under neural control (Buller et al., 1960a,b; 1969; Buller and Lewis, 1965a,b,c; Mommaerts et al., 1969). A close correlation exists between the speed of muscle shortening and the adenine triphosphatase activity of actomyosin or myosin (Barany, 1967; Sreter et al., 1966; Seidel, 1967; Barany et al., 1965; Maddox and Perry, 1966; Seidel et al., 1964). This suggests that the turnover of ATP at the cross bridges defines the rate of contraction. The rate of Ca release from red muscle microsomes is also much slower than from cardiac or white muscle microsomes (Harigaya and Schwartz, 1969); this implies that the rate of release of activator Ca and the rate of the ensuing response are closely matched. The rate of muscle relaxation is probably determined by the rate of reabsorption of activating Ca2+into the sarcoplasmic reticulum. I n the slowly relaxing red muscles, the sarcoplasmic reticulum is less well developed than in their white counter-parts (Schiaffino et al., 1970; Pellegrino and Franeini, 1963). The relative rate and extent of Ca uptake by fragmented sarcoplasmic reticulum isolated from red muscles (Sreter and Gergely, 1964; Sreter, 1969; Harigaya et al., 1968; Harigaya and Schwarte, 1969; Takauji et al., 1967; Yamamoto et al., 1970; Sreter, 1970; Samaha and Gergely, 196513) is also slower, reflecting the difference in the rate of relaxation between red and white muscles (Close, 1967). In support of the correlation between relaxation rate and the Ca transport capacity of sarcoplasmic reticulum, the twitch characteristics of phasic muscles may be converted to the tonic pattern by inhibitors of the Ca transport of sarcoplasmic reticulum, such as SCN (Zhukow, 1968). Some representative data for the rate of Ca uptake by microsomes isolated from red, white, and cardiac muscles are shown in Table I. A quantitative evaluation of the relationship between the twitch characteristics of the muscle and the Ca transport function of sarcoplasmic reticulum is not yet possible, since we are unable to predict from Ca uptake rates measured in vitro t,he rate of Ca uptake by sarcoplasmic reticulum in living muscle. On the assumption of 1mg of sarcoplasmic reticulum protein per gram of tissue, the reabsorption of 0.1 pmole of Ca per gram of tissue into the sarcoplasmic reticulum during an average relaxation time of about 200 msec would require a Ca uptake rate of about 30 pmole of Ca per milligram of protein per minute. This is about 150 times greater than the rate measured in vitro (0.18 pmole per milligram of protein per minute at 23"C, Harigaya et al., 1968). Even with 5 mg of SR protein per gram of

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ANTHONY MARTONOSI

TABLE I UPTAKEBY MICROSOMES ISOLATEDFROM RED, WHITE, AND RATE OF CALCIUM CARDIAC MUSCLE Maximum amount of Ca taken up

Rate of Ca uptake

(pmoleslmg protein)

(pmoleslmg protein/minute)

Source

No oxalate

Oxalate

No oxalate

Heart, rabbit

0.039

1.83

0.256

Heart, dog

0.075

3.00

Heart, human (failure) Skeletal, rabbit, white Skeletal, rabbit, red Skeletal, rabbit, white Skeletal, rabbit, red Skeletal, human, white Skeletal, human, red

0.045

2.00

0.170

4.80

0.0340.069 1.44

0.058

2 .oo

0.182

0.24

6 .OO

1.80

Harigaya and (1969) Harigaya and (1969) Harigaya and (1969) Harigaya and (1969) Harigaya and (1969) 1.80 Sreter (1969)

0.04

0.3-1.1

0.16

0.16 Sreter (1969)

-

1.01

Heart, dog

0.026

2.32

2.04

Oxalate

Reference Schwartz Schwartz Schwartz Schwartz Schwartz

0.38 Samaha and Gergely (1965a) 0.17 Samaha and Gergely (1965b) 0.06 Kate and Repke (1967a)

tissue and a relaxation time of 400 msec, the Ca uptake would still have to be 3 pmoles of Ca per milligram per minute, or about 15 times greater than the actually observed rate in vitro. This seemingly large discrepancy may arise from the combination of several factors, including (a) low yield of sarcoplasmic reticulum, (b) contamination of isolated preparations by inactive membrane and protein material, (c) rapid inactivation of the Ca transport system during isolaisolation, (d) suboptimal conditions used for the measurement of Ca uptake in vitro, (e) lesser density of Ca transport sites in red muscle. The yield of sarcoplasmic reticulum membranes isolated from red muscles ranges from 0.7 to 1.4 mg protein per gram of wet tissue (Patriarca and Carafoli, 1969; Harigaya et al., 1968; Muscatello et al., 1965), depending on the source of material and the method of isolation. This is somewhat less than the yield of 2.5-4 mg of protein per gram of tissue from white

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139

muscles and is in keeping with the lesser development of sarcoplasmic reticulum in red muscle fibers (Schiaffino et al., 1970). However, red muscle preparations are heavily contaminated by mitochondria and perhaps other membrane elements which do not contribute to the observed Ca transport (Sreter and Gergely, 1964; Gergely et al., 1965; Takauji et al., 1967; Harigaya et al., 1968; Sreter, 1969, 1970) and thereby reduce the specific activity. Red muscle microsome preparations may also be more labile than their white counterparts both during isolation (Harigaya et al., 1968) and storage (Sreter and Gergely, 1964). Consequently, the slower rate of Ca uptake observed in vitro may be misleading. The maximum amount of phosphoprotein formed on incubation of microsomes with ATP-32Pis about eight times less in red than in white muscle preparations (Sreter, 1969). This indicates a lesser density of Ca transport sites in the membrane. It is not known, however, t o what extent these differences can be attributed to contamination by inactive membranes and proteins and to loss of activity during isolation. I n microsome preparations isolated from red muscles large spherical particles predominate (Sreter, 1964; Gergely et al., 1965) in contrast t o the tubular (Martonosi, 1964) or tadpole shape (Martonosi, 1968b) characteristic of white muscle microsomes. On the assumption that the difference in the rate of Ca uptake between red and white microsome preparations is related to the lesser density of Ca transport sites in red microsomal membranes, but that most particles contain a t least a few sites, the maximum amount of Ca accumulated by red muscle microsomes in the presence of oxalate should be equal to or greater than that accumulated by white muscle preparations. This is so because the maximum Ca uptake per milligram of protein in the presence of oxalate is likely t o increase with the volume to surface ratio of microsoma1 particles. As the actual amount of Ca accumulated by red muscle microsomes (1 pmole per milligram of protein) is much less than by white muscle microsomes (6-8 pmoles per milligram of protein) (Sreter, 1969) the number of particles capable of accumulating calcium likely is t o be much smaller in red than in white microsome preparations. I n fact, in red muscle microsome preparations the Ca-accumulating particles may represent less than 20% of the particle population, an estimate in general agreement with the findings of Baskin and Deamer (1969) on cardiac and mixed skeletal muscle microsomes. Although earlier reports emphasized the ineffectiveness of red muscle microsomes in inhibiting the myofibrillar ATPase activity and syneresis (Sreter and Gergely, 1964; Gergely et al., 1965), the relaxing activity of red muscle microsomes was clearly established recently in several laboratories (Takauji et al., 1967; Harigaya et al., 1968; Yamamoto et al., 1970). In view of the known involvement of phospholipids in the Ca transport

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function and Ca permeability of sarcoplasmic reticulum mcmbrancs, differences in the phospholipid composition of red and white muscles may be of importance. The red soleus muscle of mouse (Owens and Angelini, 1970) or monkey (Masoro et al., 1964) contains more total phospholipid but less lecithin than the predominantly whitc mouse extensor digitorum longus or monkcy gastrocnemius. The increased phospholipid content in rcd muscles is principally accounted for in terms of phosphatidylethanolamine, ethanolamine plasmalogen, and cardiolipin, the latter being related to the large number of mitochondria, The higher lecithin and choline plasmalogen content of whitc muscles is probably connected with the well developed sarcoplasmic rcticulum, which contains lecithin as principal phospholipid (Martonosi et al., 1968; Owens and Hughes, 1970). The Ca uptake of microsome prcparations obtained from red muscle is inhibited by caffeinc (Yamamoto et al., 1970), as is also true for mixed and white muscle microsomes (Wcbcr and Hcrz, 1968; Webcr, 1968; Taniguchi and Nagai, 1970; Ogawa, 1970; Fuchs, 1969). It therefore secms plausible t o suppose that caffeine contracturc is caused by the inhibition of sarcoplasmic reticulum Ca transport (Caldwcll and Walstcr, 1963; Sandow, 1965; Gutmann and Sandow, 1965; Sandow and Brust, 1966). This inhibition may also be responsible for the marked effects of caffeine on the radiocaIcium movcments in various muscles (Bianchi, 1961b, 1962, 1963; Frank, 1962; Isaacson and Sandow, 1967a,b). Although these observations tend to provide strong support for the possibility that the sarcoplasmic reticulum plays a similar role in the regulation of the contraction-relaxation cycle in red and white muscles, the abundance of mitochondria and the sparseness of sarcoplasmic reticulum in red muscles raise the possibility that mitochondria significantly contribute to the regulation of sarcoplasmic Ca concentration (Sreter, 1964; Patriarca and Carafoli, 1969; Carafoli et al., 1969; Lehninger, 1970). This possibility is not, so far, supported by conclusive experimental evidence. The preferential uptake of intravenously injected 45Ca into the mitochondrial as opposed to the microsomal fractions isolated from rabbit masseter (red) and adductor magnus (white) muscles, and rat diaphragm (Patriarca and Carafoli, 1969; Carafoli et al., 1969) is inconclusive for several reasons : 1. The sarcoplasmic reticulum is disrupted during homogenization, releasing its content (including Ca) into the medium, while mitochondria are recovered intact. As conditions in the homogcnate favor the respiration-dependent Ca uptake by mitochondria, gross redistribution of calcium is likely to occur.

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2. The ion fluxes across the surfacc membrane are influenced by the ion composition of the medium (Sjodin, 1971; Horowicz et a t , 1970) with coupled effects on the transport of Ca (Langer and Sercna, 1970). Therefore the differences in thc Ca uptakc by surviving rat diaphragms incubated in media favoring the Ca uptakc of either mitochondria or microsomes do not establish the dominant role of mitochondria in the process. 3. The abscnce of oxalate effect on the Ca transport of rat diaphragm (Carafoli et al., 1969) is not surprising, since oxalatc does not penetrate into thc muscle cell. Oxalatc promotes the uptakc of Ca by glycerinated musclc fibers (Hasselbach, 1964b; Elison et al., 1965; Pease et al., 1965; Zebe and Hassclbach, 1966) or muscle fibers from which the surface membrane was rcmovcd (Costantin el al., 1965; Podolsky et al., 1970), and under thesc conditions the deposition of Ca oxalate is confined primarily to the sarcoplasmic reticulum. 4. The inhibitory effect of respiratory inhibitors or uncouplcrs on the Ca uptake of rat diaphragm may reflect simply the cnergy requirement of the Ca transport across the surface membrane, and docs not necessarily implicatc direct involvement of mitochondria in thc Ca transport process. 5. Massivc accumulation of Ca, of the magnitude shown by Carafoli et al. (1969), would have an uncoupling effect on mitochondria (Slater and Cleland, 1953), making the physiological reality of this event doubtful.

Although mitochondria are capable of lowering the free Ca concentration to levels a t which inhibition of myofibrillar activity is expected t o occur (Wcber et al., 1964b; Chance, 1965; Chance et al., 1969; Drahota et al., 1965; Yamamoto el al., 1970) and the mitochondria1 Ca is maintained in a dynamic steady statc (Drahota et al., 1965) it is unlikely that the rate of Ca release from mitochondria would be sufficiently fast t o account for the rate of contraction (Weber et al., 1964b). In summary, there is a possibility that a fraction of the Ca content of red muscle fibers is bound to mitochondria, but its amount is unknown, and a major contribution by mitochondria to the regulation of excitationcontraction coupling seems unlikely.

VI. THE STRUCTURE AND FUNCTION OF THE TRANSVERSE TABULAR SYSTEM AND THE TRIAD A. The Transverse Tubular System

The transverse tubular system is an extension of the surface membrane of the muscle cell (Smith, 1966b; Peachcy, 1968) which conducts the

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excitatory stimulus to the sarcoplasmic reticulum elements deep in the interior of muscle fibers (Huxley, 1964; Sandow, 1970). In frog or toad sartorius muscle the combined surface area of the T system was calculated to be 7-10 times greater than that of the surface membrane (Peachey, 1965; Peachey and Schild, 1968). The extent of development of the T system varies in different muscles even within the same species (Page, 1965) with corresponding variations in the relative area of surface and T-system membranes (Peachey, 1967; Brandt et al., 1965; Smith, 1966a). The continuity of the surface membrane with the T-system tubules is now clearly established in both cardiac (Simpson and Oertelis, 1961, 1962; Nelson and Benson, 1963; Simpson, 1965; Rayns et al., 1967, 1968; Forssmann and Girardier, 1966,1970) and skeletal muscles (Franzini-Armstrong and Porter, 1964; Rayns et al., 1968; Jasper, 1967; Bertaud et al., 1970; McCallister and Hadek, 1970). Beautifully organized rows of T-system openings have been demonstrated by electron microscopy on the surface of frozen-etched myocardial cell membranes of guinea pig (Fig. 2). Even in those skeletal muscles where direct visualization of the continuity of T tubules with the surface membrane proved inconclusive (Peachey, 1965; Walker and Schrodt, 1965; Wolff, 1966), the communication between the content of T tubules and the extracellular medium was established by demonstrating the penetration of permeability markers into the T tubules. Of the several permeability markers tested, ferritin (Huxley, 1964; Page, 1964; Peachey and Schild, 1968), colloidal gold (Page, 1964, Lissamine Rhodamine B 200 (Endo, 1966), serum albumin (Hill, 1964c), neutral thorium dioxide micelles (Birks, 1965), lanthanum micelles (Revel and Karnovsky, 1967; Karnovsky, 1967), peroxidase (Karnovsky, 1965, 1967; Graham and Karnovsky, 1966; B. Eisenberg and Eisenberg, 1968), and ruthenium red (Luft, 1966) proved useful. The formation of a dense precipitate in the T-system tubules of frog muscle following incubation with potassium pyroantimonate indicates that the sodium concentration in the T system is about the same as that of the blood serum (Zadunaisky, 1966; Tice and Engel, 1966a). The sarcoplasmic reticulum contained sodium pyroantimonate precipitate only after the sarcolemma was mechanically removed (Tice and Engel, 1966a). The invaginations of the surface membrane appear quite early during in vitro development of chicken embryo muscles and parallel the evolution of sarcoplasmic reticulum from rough endoplasmic reticulum membranes (Ezerman and Ishikawa, 1967; Schiaffino and Margreth, 1969). The localization of T-system tubules corresponds to the sensitive spots on the surface membrane, where local contraction can be elicited by stimu-

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FIG. 2. Frozen-etched myocardial cell membrane of guinea pig showing the B, or inward-oriented, fracture face. The fiber axis is vertical. Note the prominent array of craterlike T-tubule stumps (sarcolemmal invaginations) through which the frozen extracellular fluid may be seen. The few smaller “craters” are fractured sarcolemmal vesicles. The entire membrane is covered with 9 nm particles. The arrow a t the lower right corner indicates the direction of shadowing. X 25,400. From Rayns et al. (1967). Copyright 1967 by the American Association for the Advancement of Science.

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lation with microelectrodes (Huxley and Taylor, 1958; Huxley, 1959; Huxley, 1964). Longitudinal extensions of the T system are not particularly frequent in frog or toad sartorius muscles (Peachey and Schild, 1968). This observation agrees with the localized nature of contractile response. The recently reported penetration of intravenously injected peroxidase into the terminal cisternae and longitudinal tubules of sarcoplasmic reticulum in frog sartorius and semitendinosus muscles (Rubio and Sperelakis, 1971) may have resulted from mistaken identification of the longitudinal branches of the T system (Peachey, 1965; Peachey and Schild, 1968; B. Eisenberg and Eisenberg, 1968) with the sarcoplasmic reticulum. Local depolarization of the surface membrane in dog myocardial muscle fibers produced either no response or widespread contraction of several sarcomers (Muller, 1966) in contrast to the localized contractions observed on skeletal muscle. Extensive longitudinal branching of T-system tubules revealed by peroxidase staining (Forssmann and Girardier, 1970) provides a satisfactory morphological basis for this behavior. Selective disruption of T-system tubules occurs when frog sartorius muscles incubated in Ringer solution supplemented with 400 mill glycerol are returned to normal Ringer medium (Fujino et al., 1961; Howell and Jenden, 1967; Howell, 1969; Eisenberg and Eisenberg, 1968; Krolenko, 1969). The glycerol-treated fibers, when stimulated electrically, develop normal action potentials without a contractile response; this indicates the disruption of excitation-contraction coupling (Howell, 1969; Gage and Eisenberg, 1967, 1969b; Eiscnberg and Gage, 1967). The glycerol treatment severs the connection of 98% of the T-system tubules with the surface membrane, but causes only occasional and mild damage to the terminal cisternae and fenestrated collar of the sarcoplasmic reticulum (Eisenberg and Eisenberg, 1968). The surface membrane remains largely unaffected as indicated by the apparently normal resting potential (Eisenberg and Gage, 1967; Gage and Eisenberg, 1969b). The Ca efflux from the muscle cell across the surface membrane is probably an active transport process since the ionized Ca concentration of the sarcoplasm is much lower than that of the interstitial fluid or blood plasma (Gilbert and Fenn, 1957). Interestingly, disruption of T-system tubules in frog sartorius, while reducing the surface area of the external membrane to one-fifth or one-tenth of its normal value, had no effect on the rate constant of Ca efflux (Van der Kloot, 1968). This would imply that the T tubules do not participate in Ca extrusion. Caffeine contracture is only slightly affected by the disruption of T-system tubules (Howell and Jenden, 1967; Howell, 1969; Sandow, 1970); this accords with the

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suggested effect of caffeine in promoting Ca release from the sarcoplasmic reticulum (Weber, 1968; Weber and Herz, 1968). The considerable effect of the T system on the electrical properties of muscle can be accurately assessed with the use of glycerol-treated fibers. From impedance measurements, Falk and Fatt concluded (1964) that the capacitance of the T system in frog sartorius muscle (4.1 pF/cm2) is a major component of the total capacitance (6.7 pF/cm2) of the muscle cell. Direct confirmation of these estimates was provided by Gage and Eisenberg (1969a)) who compared the capacitance of muscle fibers with and without T tubules and obtained values that were astonishingly close to the earlier estimates (Falk and Fatt, 1964; Fatt, 1964). The capacitance of various muscles correlates reasonably well with the relative abundance of T-system tubules. The slow frog fibers with meagerly developed T system (Page, 1965) have small capacitance (Adrian and Peachey, 1965). Crab and crayfish (Falk and Fatt, 1964) muscles have high capacitance and an extensively developed T system (Brandt et al., 1965; Peachey, 1967). The contribution of the T system to the capacitance is less than would be expected from its relative surface area. Apart from various technical reasons this may result from a lower capacitance in that portion of T system tubules which participate in junction with the sarcoplasmic reticulum (Gage and Eisenberg, 1969a). A sudden change in extracellular chloride concentration produces a faster change in membrane potential than a similar change in the extracellular potassium (Hodgkin and Horowicz, 1960). By comparing the potassium and chloride conductance of muscle fibers with and without T system tubules Eisenberg and Gage (1969) found that the transverse tubules havc very little, if any, chloride conductance while the potassium conductance is shared between the surface and T system membranes. The relatively slow change in membrane potential following a fast change in extracellular potassium concentration has therefore been tentatively attributed to the slower penetration of N into the T-system tubules. The total conductance of the tubular membrane is much lower than th a t of the surface membrane. This may be of importance in permitting the penetration of excitatory current deep into the muscle fibers. Reliable biochemical information about the composition and enzymatic function of the T-system tubules is not available. Microsomc preparations isolated by conventional methods are likely to contain fragmented T tubules intermixed with surface membranes, mitochondria, and sarcoplasmic reticulum membranes in varying proportions, depending on the muscles from which they were obtained.

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I n the absence of reliable enzymatic markers for T-system tubules, their contribution to the total membrane mass in these preparations is unknown. I n cardiac muscle, the relative abundance of surface membrane invaginations and the sparsely developed sarcoplasmic reticulum (Simpson and Oertelis, 1962; Nelson and Benson, 1963; Page, 1968; Forssmann and Girardier, 1970) have led to the suggestion that crude cardiac microsome preparations may contain not only sarcoplasmic reticulum membranes with contaminating mitochondria, and fibrous proteins, but also appreciable amounts of T-tubule vesicular fragments (Baskin and Deamer, 1969). Although logical, the suggestion rests on indirect and uncertain evidence. It has been observed that only a small fraction (7%) of the particles present in cardiac microsome preparations accumulate Ca oxalate deposits as a result of ATP energized Ca transport. The corresponding value for rabbit skeletal muscle microsomes is 15-25% (Baskin and Deamer, 1969; Ikemoto et al., 1968; Greaser et al., 1969a) and reaches 80% only with lobster microsomes (Baskin, 1971). There are two reasons why it seems unlikely that all or most of the particles devoid of Ca oxalate deposits could originate from T-system tubules: (a) I n whole muscle only the cisternae of sarcoplasmic reticulum accumulate Ca deposits, while the longitudinal tubules and the area of fenestrated collar are free of Ca deposits. It seems reasonable t o assume that vesicular elements derived from these portions will not be able to accumulate Ca (however, see Winegrad, 1970). (b) The total membrane mass of sarcoplasmic reticulum is much greater than that of the T system in most skeletal muscles, yet only about one-fourth of the microsome particles show Ca deposits. The apparent absence of 40 particles from some of the larger vesicular profiles observed in cardiac microsome preparations has been interpreted (Baskin and Deamer, 1969) to indicate that these vesicles may originate from the broad sarcolemmal invaginations of cardiac muscle (Simpson and Oertelis, 196%;Nelson and Bcnson, 1963). However, the absence of 40 d particles may result from uneven negative staining, and according to Imai et aE. (196G) the larger vesicles arc derived from the outer membranes of mitochondria. A t any rate, the size of microsomal particles is of uncertain significance, sinw the effect of homogenization on particle size is unknown. Also, there are large variations in the size of microsomes obtained from different species or even from different muscles within the same species, which are unlikely to originate from varying levels of T-tubule contamination. In view of the uncertainties connected with the identification of the origin of isolated microsomal particles in negatively stained preparations, the use of specific enzyme markers that permit unambiguous quantita-

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tion of the relative amounts of different types of membranes in mixed vesicular preparations assumes great significance. With the aid of Pb(N03)2 as phosphate-precipitating agent, adenosine monophosphate hydrolysis was detected in the T system and the intercalated discs of rat myocardium, but not in the subsarcolemmal cisterns or in the terminal sacs of sarcoplasmic reticulum. With ATP as substrate,the T system was free of enzyme activity, but the lateral elements of the triad and the subsarcolemmal cisterns were intensely stained (Rostgaard and Behnke, 1965). Essner et al. (196ii) and Sommer and Spach (1964) have made similar observations on the distribution of ATPase activity. However, this ATPase activity may not be identical with the Ca transport ATPase (Sommer and Hasselbach, 1967). due to the sensitivity of the latter enzyme to commonly used fixatives. If the possibility of an artifact resulting from fixation and the precipitation of the lead-AMP complex in the T tubules can be excluded, the A3IP-phosphohydrolase activity may serve as enzymatic marker for the T-system tubules. The relatively high acetylcholinesterase activity in fragmented sarcoplasmic reticulum preparations (Martonosi and Feretos, 1963a) probably represents an admixture of surface membrane and T tubules (Davis and Koelle, 1967). The specific activity of the Na+-K+ (Mg*+) ATPase in cardiac sarcolemma preparations is high in comparison with mitochondria and microsomes in agreement with the preferential localization of the Na+I<+(Mg2+)ATPase in the surface membrane and T system (Wollenberger and Schulze, 1966; Schulze and Wollenberger, 1969; Potter et al., 1966). Mitochondria1 contamination in microsome preparations may be readily detected by measurement of cytochrome oxidase, NADH cytochrome c reductase, and succinate-cytochrome c reductase activities (Imai et al., 1966) or the inhibition of ATPase activity by sodium axide (Fanburg and Gergely, 196,i). NADH-cytochrome bg reductase and NADPH-cytochrome c reductase are evenly distributed among the various particulate cell fractions of rabbit skeletal muscle (Imai et al., 1966). The presence of polyanion substances on the surface of T-system tubules was inferred from its staining with ruthenium red (Luft, 1966; MartinezPalomo, 1970). On the basis of the presence of hexosamine in sarcoplasmic reticulum fragments (Seraydarian and Mommaerts, 1965), it was suggested (Philpott and Goldstein, 1967) that these may be mucopolysaccharides or glycoproteins (Birks, 1965). These findings are in line with earlier suggestions (Fatt, 1964; Birks, 1965; Rapoport et al., 1968; Rapoport, 1969) that the transverse tubular system, similarly to the corresponding tubules in cardiac muscle (Sommer and Johnson, 1968a,b), may contain negatively charged material. The presence of such fixed charge may

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reduce the chloride concentration in the T tubule t o such an extent as to explain the nearly zero chloride conductance through the T system Eisenberg and Gage, 1969). I t is not known whether these polyanion substances, if they indeed exist, would remain attached to the T-system membranes after homogenization, but if they do they could serve as convenient markers for the identification of T-system vesicles in mixed microsomal preparations. As an alternative possibility, the surface membrane of the muscle cell could be labeled covalently by nonpenetrating fluorescent or radioactive markers similar to those reported by Maddy (1964), Marinetti and Grey (1967), and Berg (1969). B. The Triad

According t o the generally accepted view the sareolemmal depolarization initiated by the nerve impulse spreads into the interior of the muscle fibers through the T system. It then triggers Ca release from the sarcoplasmic reticulum and thereby activates the contractile material. The transmission of the excitatory stimulus from the T system t o the sarcoplasmic reticulum occurs a t the level of triads, diads (or equivalent structures) which are specialized regions of interactions between the flattened surfaces of the lateral sacs of sarcoplasmic reticulum and the T-system membrane (Figs. 3 and 4). Various aspects of the diverse interpretations previously given t o the fine structure of these junctions (Franzini-Armstrong and Porter, 1964; Huxley, 1964; Revel, 1962; Peachey, 1965; Fahrenbach, 1965; Hoyle, 1965; Walker and Schrodt, 1966, 1968) integrated with new information primarily from the work of Kelly (1969), Franzini-Armstrong (1970a,b), 1971), and Bertaud, Rayns, and Simpson (1970), has yielded a picture of the fine structure of the junctional area that is sufficiently detailed to permit predictions concerning the nature of its function (Franzini-Armstrong, 1970a,b, 1971; Figs. 5 and 6).

FIGS.3 and 4. Longitudinal section a t the periphery of a frog sartorius muscle fiber. Under the sarcolemma are numerous caveolae. Two triads (three arrows) can be followed to the periphery of the fiber. M, mitochondria. Figure 3 X 21,200. Figure 4 is a detail of one of the triads shown in Fig. 3. The T system is a wide, apparently empty, tubule between the two lateral sacs of the triads, belonging to the sarcoplasmic reticulum (SR). Near the periphery of the fiber the T system is narrower. Its opening to the outside is small (arrow). Figure 4 X 107,000. From Franzini-Armstrong (1970a). Reprinted by permission of Rockefeller University Press.

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FIGS.5 and 6. Cross-sectional views of 2 triads. The T-system tubule, in the center, is flanked by two sacs of the sarcoplasmic reticulum. Upper and lower flattened T-system surfaoces are separated from sarcoplasmic reticulum membrane by an approximately 120 A gap. Two distinct clumps of material cross the junctional gap, apparently holding sarcoplasmic reticulum and T system together. From Franzini-Armstrong (1970a). Reprinted by permission from Rockefeller University Press.

In the junctional regions of frog sartorius muscle the facing membranes of the sarcoplasmic reticulum and T system are separated by a gap of 120-150 (Kelly, 1969; Franzini-Armstrong, 1970a). From the surface of the sarcoplasmic reticulum membrane, rows of evenly spaced projections reach out toward the T system surface a t regular intervals. The projections do not come in contact with the T tubules. Rather there remains a gap a t least 50 8 wide, which is crossed by amorphous material with staining characteristics distinctly different from those of the component membranes. Pores of the type suggested by Birks (1965) have not been observed. This of course does not exclude their existence. Tho space between neighboring projections is structureless (Franzini-Armstrong, 1970a; Bertaud et al., 1970), and therc is no indication that the junctional area is sealed off from the sarcoplasm.

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I n different species, the projections are arranged in two (FranziniArmstrong, 1970a) or more (Kelly and Cahill, 1969; Franzini-Armstrong, 1970b) rows, their number being determined by the size and shape of the junctional area without clear relationship with the specd on contraction (Franzini-Armstrong, 1970b). If we assume that electrical coupling occurs only through the area of projections, about 30% of the T system and 3% of the sarcoplasmic reticulum surface would participate in the junction (Franzini-Armstrong, 1970a, 1971). Under these conditions, a capacitativc electrical coupling (Eisenberg and Gage, 1969) between T system and sarcoplasmic reticulum seems unlikely (Franzini-Armstrong, 1970a, 1971). There is, however, sufficient functional and morphological similarity between the triad of muscle (Peachey, 1965; Hoyle, 1965; Hagopian and Spiro, 1967) and the septate junctions of epithelial cells (Wiener et al., 1964; Borek et al., 1969; Loewenstein, 1966) to picturc the triad as a low-resistance pathway. This concept derives support from observations that suggest that small molecular weight substances can penetrate from the T tubules into the lateral cisterns of the sarcoplasmic reticulum. During muscle contraction the triads withstand considerable mechanical stress. This implies that the participating structures are held together strongly. I n fact, in a few instances, structures reminiscent of triads have been observed in fragmented preparations of sarcoplasmic reticulum (Ebashi and Lipman, 1962; Martonosi, 1964; Hasselbach and Elfvin, 1967). The characteristic tadpole-shape of microsomes isolated from mouse skeletal muscle raises the possibility that they may represent relatively intact segments of the sarcoplasmic reticulum (Rlartonosi, 1968b). Approximately .%lo% of the negatively stained tadpole-shaped microsomal particles display a characteristic diff erentiation of the head portion (Rlartonis, 1968b) which may be related to the junctional region. However, no projcctions of the type observed on sectioned material (Franzini-Armstrong, 1970a; Kelly, 1969; Hasselbach and Elfvin, 1967) were evident.

VII. THE CONTENT OF SARCOPLASMIC RETICULUM TUBULES

The cell compartment enclosed by sarcoplasmic reticulum membranes comprises 11-13% of the volume of musclr fibers (Pcachey, 1965; Aloisi and llargreth, 1967; Birks and Davey, 1969) and a large fraction of the extramitochondrial sarcoplasm (Aloisi and Rlargreth, 1967). At least in frog muscle the space enclosed by sarcoplasmic reticulum is clearly divided into four, interconnected by morphologically distinct seg-

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ments, the terminal cisterna, intermediate cisterna, longitudinal tubules, and fenestrated collar (Peachey, 1965). The various segments may have characteristically different functions. According to Winegrad, after muscle activity calcium accumulates primarily in the area of the longitudinal tubules and is then transferred, presumably by diffusion to the terminal cisternae where it is stored during relaxation (Winegrad, 1965a,b, 1968, 1970). Independent evidence for the accumulation of Ca in the terminal cisternae of sarcoplasmic reticulum has been obtained with the use of oxalate on skinned frog muscle fibers (Costantin et at., 1965; Podolsky et al., 1970) and on muscle fibers made permeable to oxalate by brief exposure to glycerol (Hasselbach, 1964b; Pease et al., 1965). The electron dense deposits observed under these conditions in the lateral cisternae were shown to contain Ca by spectroscopic analysis of the X-rays generated by irradiating the deposits with an electron microprobe (Podolsky et al., 1970). No significant amounts of RiIg or Zn were detectable. Some degree of morphological and functional differentiation of the various segments of sarcoplasmic reticulum is recognizable even in fragmented sarcoplasmic reticulum preparations. Sectioned specimens of pelleted microsomal material analyzed under the electron microscope frequently display elongated tubular profiles intermixed with spherical particles (Ebashi and Lipmann, 1962; Martonosi, 1964, 196813; Sreter, 1964). The tubular elements predominate in fresh microsome preparations obtained from white muscles, while in aged microsome preparations or in microsomes obtained from predominantly red muscles most of the particles are spherical (Finean and Martonosi, 1965; Hasselbach and Elfvin, 1967; Sreter, 1964). The tubular profiles in microsome preparations obtained from predominantly white muscles may correspond to the tail portion of the tadpole-shaped particles which are abundant in microsome preparations of mouse (Martonosi, 1968b,d; Ikemoto et al., 1968), barnacle, and lobster muscles (Baskin, 1971) negatively stained with potassium phosphotungstate. They have also been observed, but less frequently, in microsome muscles of rabbit (Martonosi, 1968a; Deamer and Baskin, 1969; Baskin and Deamer, 1969) or pig (Greaser et al., 1969a,b). The absence of tadpole-shaped particles in freeze-etched or freeze-dried preparations (Baskin, 1971;Deamer and Baskin, 1969; Baskin and Deamer, 1969) has led t o the suggestion that the tadpole shape arises from particle shrinkage resulting from the osmotic effect of the nonpenetrating negative stain when the specimen is dried. Since negative staining solutions are usually hypoosmotic, this argument could be used with equal validity to suggest the opposite. It is tempting to consider the possibility that the tadpole shape of iso-

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lated particles, with their characteristic head and tail portions, reflects the division of intact sarcoplasmic reticulum into the cisternal and tubular segments. It is of interest in this context that deposition of calcium oxalate in mouse microsome preparations is confined t o the head portion of the particles (Ikemoto et al., 1968). This is reminiscent of Ca deposition in the cisternae of sarcoplasmic reticulum in skinned or glycerol-treated muscle fibers (Hasselbach, 196413; Costantin et al., 1965; Pease et al., 1965; Podolsky et al., 1970). On the other hand, the Ca accumulating structures in rabbit (Hasselbach and Elfvin, 1967; Deamer and Baskin, 1969; Greaser et al., 1969b; Baskin and Deamcr, 1969), lobster, and barnacle muscles (Baskin, 1971) are largely spherical. The hydrolysis of ATP by skeletal muscle microsome in the presence of Pb(N0,)z is accompanied by the appearance of electron dense lead phosphate precipitate within the vesicles (Ikemoto et al., 1968; Tice and Engel, 1966b; Engel and Tice, 1966; Tice, 1967). In mouse microsomes the lead phosphate deposits are observed in the head portion of tadpole-shaped particles (Ikemoto et al., 1968). Although several reports indicate that the cisternae of sarcoplasmic reticulum are particularly rich in ATPase activity (Essner et al., 1965; Rostgaard and Behnke, 1965), there are reasons to doubt the association of this ATPase activity with Ca transport (Tice, 1967; Sommer and Hasselbach, 1967), since the transport ATPase is markedly inhibited under typical fixation conditions. Additional experiments would be required to clarify the conditions which influence the shape of isolated particles and to determine their relationship to the various elements of sarcoplasmic reticulum in living muscle. The interior of sarcoplasmic reticulum is usually filled with dense material, which in frog (Peachey, 1965; Huxley, 1964; Birks, 1965; FranziniArmstrong, 1970a) and fish muscle (Peachey, 1965) is confined to the terminal cisternae, while in several other species it is distributed throughout the SR. In the best preserved preparations (Franzini-Armstrong, 1970), the material appears as a delicate meshwork in which occasional, small hexagonal structures may be distinguished. Although it has been suggested (Birks, 1965; Peachey, 1968) that the dense material may contain binding sites for the accumulated calcium (Carvalho, 1966, 1968a,b; Carvalho and Leo, 1967), Ca binding is readily demonstrated on isolated microsomes whose interior is free of electron dense deposits and which therefore appear to have lost their tubular content during isolation. Sarcoplasmic reticulum membranes in sectioned fish muscle fixed with osmium tetroxide and embedded in methacrylate showed a characteristic staining with thorium dioxide a t pH 2.0 (Philpott and Goldstein, 1967).

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This has been attributed to the interaction of the positively charged thorium micelles with anionic sites, presumably acid mucopolysaccharides (Revel, 1964) on the sarcoplasmic reticulum membranes. The presence of polyanions, presumably mucopolysacharides, in sarcoplasmic reticulum cisterns (Birks, 1965) is also suggestrd by histochemical staining with mcthylene blue-azure (Richardson et aZ., 1960), or ruthenium red (Luft, 1966; Kelly, 1969) and by the demonstrated presence of hexosamine in isolated sarcoplasmic reticulum preparations (Seraydarian and Mommaerts, 1965). The interior of the sarcoplasmic reticulum is not likely to comniunicate freely with the cell interior as the previously described pores of the membranes in the rcgion of the fenestrated collar (Franzini-Armstrong, 1963) are now viewed as annular fusions of the two membrane layers, which effectively seal the content of sarcoplasmic reticulum from the sarcoplasm (Hagopian and Spiro, 1967; Peachey, 1965; Page, 1965). The permeability of sarcoplasmic reticulum membranes a t the junctionaI surface with the T-system tubules poscs interesting problems (Birks, 1965; Birks and Davey, 1969). The volume of sarcoplasmic reticulum in frog sartorius muscles exposed t o hypertonic sucrose solutions increases from an estimated 11% to about 18% of normal ccll volume (Birks and Davey, 1969) while the muscle volume markedly decreases (Dydynska and Wilkie, 1963; Blinks, 1965). It seems plausible that the osmotic swelling is caused by the penetration of sucrose from the external medium into the sarcoplasmic reticulum. The deviation of electrolyte distribution in frog skeletal muscle from predictions based on the Donnan equilibrium led quite early to suggestions that muscle fibers contain one or more “intracellular” compartments which are accessible to extracellular ions (Steinbach, 1947; Harris, 1958). Harris (1963) demonstrated that muscle chloride is distributed in a space which corresponds to about 15y0of the total cell water. The space occupied by sucrose is consistcntly greater in various muscles than the inulin or albumin space (Tasker et aZ., 1959; Bosler, 1961; Norman et al., 1959). A communication between extracellular medium and sarcoplasmic reticulum (Birks, 1965) is in linc with observations that indicate that the sucrose space increases over several hours of exposure (Tasker et al., 1959; Norman et al., 1959). While sucrose (Birks and Davey, 1969), chloride (Harris, 1963), and probably several other low molecular weight substances seem to enter readily into sarcoplasmic reticulum, larger molecules such as serum albumin (Hill, 1964c), ferritin (Huxley, 1964; Page, 1964; Peachey and Schild, 1968; Forssmann and Girardier, 1966), neutral thorium dioxide micelles (Birks, 1965), lanthanum micelles (Rcvel and Karnovsky, 1967; Karnov-

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sky, 1967), and peroxidase (Karnovsky, 1965, 1967; Graham and Karnovsky, 1966; Sommer and Johnson, 196813; Forssmann and Girardier, 1970) are clearly confined within the T-system tubules. Occasional penetration of ruthenium red (Luft, 1966; Kelly, 1969) or tetanus toxin (Zacks and SheR, 1968) from the T tubules into the lateral sacs of th e triad has been observed, but this may be the result of tissue injury during preparation for electron microscopy. Lissamine Rhodamine B 200 readily penetrates into T-system tubules and is distributed in 1-2oJ, of the fiber volume of frog sartorius muscle (Endo, 1966). As this is greater than the volume of the T system (Hill, 1964c), i t may be assumed that the dye penetrates into an intracellular compartment, presumably the sarcoplasmic reticulum. The swelling of sarcoplasmic reticulum and the shrinkage of the rest of the cell upon exposure to hypertonic sucrose solution persist for a t least 2 hours (Huxley et al., 1963; Birks and Davey, 1969). This suggests that the sucrose-permeability of the sarcoplasmic reticulum membrane in vivo is confined to the relatively small junctional area. Isolated fragments of sarcoplasmic reticulum are readily permeable to sucrose, urea, acetate, C1-, oxalate, inorganic orthophosphate, or inorganic pyrophosphate under conditions which permit the active accumulation and retention of Ca2+ in the vesicle (Duggan and Martonosi, 1970). The ready penetration of microsomal water space by sucrose and various anions establishes the sarcoplasmic reticulum membrane as a much less restrictive permeability barrier than the inner membrane of mitochondria or the surface membrane of muscle cell. If the high permeability is a natural property of the membrane, sarcoplasmic reticulum tubules may contain most of the important metabolic intermediates in equilibrium with the rest of the sarcoplasm. This may be of importance if sarcoplasmic reticulum possesses some of the same enzymatic activities thought to be associated with the endoplasmic reticulum of other tissues (Dallner and Ernster, 1968). There are indications that the sarcoplasmic reticulum may contain various sarcoplasmic, primarily glycolytic, enzymes confined in its interior or bound to its surface, in analogy with the endoplasmic reticulum of other tissues (DcDuvc~et al., 1962; Roodyn, 1965). This possibility must be seriously considered even if most of the glycolytic enzymes appear in the particle-free supcrnatant following the conventional homogenization and isolation procedures (Czok and Bucher, 1960; AIoisi and Margreth, 1967). In contrast with mitochondria, the sarcoplasmic reticulum is certainly disrupted during homogenization and releases its content into the medium. This may be the explanation for the finding that while lactate dehydrogrnase and glycrraldehydephosphate dchydrogenase appear t o be entirely confined to the sarcoplasmic rcticulum cytochemically (Fahimi and Kar-

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FIG.7. Electron micrograph of a portion of one muscle cell from the lobster second antenna. Part of the nucleus is visible at the top, and the rest of the field is occupied by cross sections of myofibrils surrounded by sarcoplastnic reticulum. The myofibrils

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novsky, 1966), the isolated sarcoplasmic reticulum contains relatively small activities of lactate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphohexose isomerase, aldolase, phosphorylase (Margreth et al., 1963), and glyceraldehydephosphate dehydrogenase (Rlartonosi, 1971, unpublished observations). Although UDPG-glycogensynthetase sediments mostly with the sarcoplasmic reticulum (Robbins et al., 1959; hlargreth et al., 1963; Andersson-Cedergren and RiIuscatello, 1963), the enzyme is probably bound to glycogen particles. Sufficient evidence seems to be available, however, to suspect that phosphofructokinase (Margreth, 1963; hlargreth et al., 1963; Aloisi and Margreth, 1967) and hexokinase (Ilarpatkin et aE., 1966; Karpatkin, 1967) are associated with sarcoplasmic reticulum membranes. The intracellular compartmentation of glucose 6-phosphate and triose phosphates in rat diaphragm (Kalant and Beitner, 1971; Landau and Sims, 1967) is in general agreement with this view. About 1% of the ATP-creatine phosphotransferase activity of rabbit skeletal muscle was recovered in the microsomal fraction (Baskin and Deamer, 1970), but the significance of this finding is not clear. A close correlation was observed between postnatal development of sarcoplasmic reticulum (Schiaffino et al., 1970; Schiaffino and Margreth, 1969) and the activity of aldolase, lactate dehydrogenase, and phosphofructokinase in rat skeletal muscle (Aloisi and Margreth, 1967). The potential significance of the relationship between sarcoplasmic reticulum and the glycolytic system becomes obvious in considering the structure of the very fast toadfish gas bladder muscle (Fawcett and Revel, 1961), which requires a stimulation frequency of 300 per second to tetanize. While in most muscles the mitochondria are in close proximity to the myofibrils, permitting effective transfer of ATP to the contractile system during muscle activity, in the toadfish gas bladder the mitochondria are separated by as much as 10 p from the centrally located myofibrils. This raises the possibility that most of their chemical energy is derived from the glycolytic system connected with the sarcoplasmic reticulum (Fawcett and Revel, 1961). A very similar case is the fast-acting remotor muscle of the lobster second antenna (Figs. 7 and S), which contains few mitochondria but a more profuse sarcoplasmic reticulum than any other known muscle (Rosenbluth, 1969). Following the early reports of Caspersson and Thorell (1942) and Eng-

are easily identified by the presence of myofilaments in hexagonal array within them. The small arc-shaped densities a t the edges of some of the myofibrils are dyads. Several mitochondria are also visible. From Rosenbluth (1969). Reprinted with permission of Rockefeller University Press. x 12,600.

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FIG 8. An enlarged detail of sarcoplasmic reticulum (SR) from lobster muscle. It is predominantly agranular and closely resembles the smooth reticulum of steroid-secreting cells. The distinctive features of this particular muscle would probably be best appreciated by a direct comparison with some other muscle. In all other muscles the rnyofibrils occupy a very much higher proportion of the cell and the SR takes up only a very small proportion. From Rosenbluth (1969). Reprinted with permission from Rockefeller University Press. X 58,000.

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strom (1944) who studied the distribution of adenine nucleotides in the muscle cell by ultraviolet microscopy, the autoradiographic analysis of the distribution of tritiated adenine in frog (Hill, 1959, 1960a,b) and toad (Hill, 1964a,b) sartorius muscles provided clear indication that at least 500/, of the adenine nucleotide content of muscle cell is confined in the interfibrillar space at a distinct region of the I band, possibly in the sarcoplasmic reticulum (Hill, 1964a,b). The localization of creatine phosphate is much less certain (Hill, 1962, 1964a). The possible compartmentation of a large fraction of the total adenine and ATP content of the muscle cell in the sarcoplasmic reticulum is in line with its suggested role in energy-generating (Fawcett and Revel, 1961; Rosenbluth, 1969) presumably glycolytic (Aloisi and Margreth, 1967; Karpatkin, 1967) processes. In addition to its established role as the regulator of sarcoplasmic Ca2+ concentration, and its possible participation in glycolytic activity, sarcoplasmic reticulum membranes have been implicated in the synthesis of proteins (Muscatello et al., 1961) and phospholipids (Pennington and Worsfold, 1969) and in the esterification of fatty acids (Stein and Stein, 1968). The presence of acetylcholinesterase in isolated microsomal preparations (Martonosi and Feretos, 1963a; Van der Kloot, 1966; Ulbrecht and Kruckenberg, 1965) is of uncertain significance since it may be due to the contamination by elements of the neuromuscular junction which are rich in acetylcholinesterase activity (Davis and Koelle, 1967). Acetylcholine so far was found to have no effect on the Ca transport of sarcoplasmic reticulum (Martonosi and Feretos, 1964a). Independent evidence would be necessary to establish the localization of adenylcyclase in sarcoplasmic reticulum since the observed adenylcyclase activity in isolated microsome preparations (Entman et al., 1969d; Rabinowitz et al., 1965) may be due to contamination by surface membrane elements.

VIII. THE SARCOPLASMIC RETICULUM IN DISEASES OF SKELETAL MUSCLE A. Denervation

After denervation of rat diaphragm, there is an initial hypertrophy (Miledi and Slater, 1969; Sola and Martin, 1953), which attains its maximum approximately 1 week after section of the phrenic nerve. The increase in muscle weight is accompanied by an increase in the amount of sarco-

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plasmic and contractile proteins (Miledi and Slater, 1969; Stewart, 1955; Buse et at?., 1965; Gutmann et al., 1966; Harris and Manchester, 1966). This is reflected by the increased diameter of the muscle fibers and the larger space occupied by the myofibrils (Miledi and Slater, 1969). Since the mean myofibril size and the average distance between thin and thick filaments remains unchanged, it would appear that the number of myofibrils increases during hypertrophy. Hypertrophy was also observed after denervation of the anterior tibialis muscle of rat (Gutmann, 1960). Within a few days after denervation the maximum tetanic tension developed in response to repetitive electric stimulation (50 per second) markedly decreases. In parallel there occurs a decrease in the rate of development and decline of contractile tension following single stimuli (Miledi and Slater, 1969; Langley and Kato, 1915a,b; Lewis, 1962). It has been proposed (Miledi and Slater, 1969) that the changes in the mechanical response following denervation may result from selective hypertrophy of the predominant smaller fibers (Feng and Lu, 1965), which represent about 60% of the cell population (Padykula and Gauthier, 1967; Gauthier and Padykula, 1966). This proposal is based on the characteristic differences in the rate of contraction between red and white muscles (Buller et al., 1969; Barany and Close, 1971a,b) and on the difference in sensitivity of red and white muscle fibers to denervation (Pellegrino and Franzini, 1963; Feng and Lu, 1965; Bajusz, 1964, 1965; Hogenhuis and Engel, 1965; Engel et al., 1966; Karpati and Engel, 1967). During hypertrophy, accumulation of rough endoplasmic reticulum is a typical finding, in agreement with an increase in protein synthetis (Harris and Manchester, 1966). A few weeks after denervation, atrophy sets in; the muscle fibers are progressively reduced in size, but even in regions where the myofibrils are disintegrating the sarcoplasmic reticulum membranes are still present. The events following denervation of rat soleus and gastrocnemius muscles are slightly different from those described in the case of rat diaphragm. The initial hypertrophy which characterizes the response of the diaphragm to denervation is absent or less pronounced in soleus and gastrocnemius, and consequently the weight of these muscles decreases by 30% a t the first week, 50% a t the second, and close to 60% by the fourth week. After denervation, there is a marked decrease in total protein content (Gutmann and ZelenB, 1962; Hoagland, 1946; Fisher, 1940,1955; Fisher and Ramsey, 1946; Stewart, 1962; Schapira and Dreyfus, 1959; Zak, 1962), Contractile proteins (Fisher, 1940; Fisher and Ramsey, 1946) also decrease in amount. This occurs because protein synthesis decreases (Padieu, 1959; Shapira et al., 1953; Zak and Gutmann, 1960; Goldberg, 1969) and there is increased protein breakdown (Goldberg, 1969; Zak and Gutmann, 1960), in which

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lysosomes (Pellegrino et al., 1957; Tappel et al., 1962) and satellite cells (Mauro, 1961; Katz, 1961) may participate. The degradation of contractile material is accompanied by the disappearance of mitochondria. In contrast to the general destruction of contractile material the sarcoplasmic reticulum appears overdeveloped, particularly in the most affected fibers. In the predominantly white gastrocnemius muscle, the cisternae of the sarcoplusmic reticulum separate into a series of isolated vesicles, and as a consequence the appearance of the muscle approaches that of the red soleus fibers. Particularly striking is the overproduction of T-system tubules, which develop into labyrinthine tubular structures (Miledi and Slater, 1969; Padykula and Gauthier, 1967; Pellegrino and Franzini, 1963) and of pentads, both characteristic of denervated muscle. As the T-system tubules represent invaginations of the surface membrane into the muscle cell, through which influx of extracellular Ca is likely to occur (Winegrad, 1970), the greatly enhanced surface area may result in sufficiently increased Ca influx into the denervated muscle fiber to cause the characteristic fibrillation (Langley and Kato, 1915a,b). It is, of course, equally possible that the fibrillation is connected with changes in the electrical properties of surface membranes (Lenman, 1965; Nicholls, 1956; Albuquerque and McIsaac, 1970; Albuquerque and Thesleff, 1968; Guth, 1968) or the increased sensitivity of surface membrane to acetylcholine (Miledi, 1960; Thesleff, 1960, 1963). In view of the morphological dedifferentiation that follows denervation, it is of interest that the surface membrane of fetal muscle cells also exhibits widespread sensitivity to acetylcholine (Diamond and Miledi, 1961). The proliferation of sarcoplasmic reticulum has been observed also on denervated frog semitendinosus (Muscatello et al., 1965) and pigeon breast muscle (Muscatello and Patriarca, 1968). During the early stages of atrophy in frog semitendinosus muscle, there is a marked increase in the amount of sarcoplasmic reticulum and of mitochondria1 membranes without significant expansion of T system. The incorporation of valine-l4Cinto subcellular membrane fractions increases, indicating an increase in the synthesis of membrane proteins, while the contractile material undergoes progressive atrophy. Denervation of rat gastrocnemius and extensor digitorum longus (Schiaffino and Settembrini, 1970) or psoas (Schiaffino and Margreth, 1969) muscles a t an early stage of development did not prevent the proliferation of sarcoplasmic reticulum, although it had marked effect on the process of differentiation (Engel and Karpati, 1968; ZelenS, 1962). While in normally developing rat gastrocnemius or soleus muscles no myotubes were present at 21 days of age, 42 days after neonatal denervation the muscles still contained many islands with largely nondiff erentiated myotubes.

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The development of the sarcoplasmic reticulum and T-system tubules have been observed also when chick muscle was grown in tissue culture (Eeerman and Ishikawa, 1967; Ishikawa, 1968). Sarcoplasmic reticulum fragments isolated from rat diaphragm (Howell et al., 1966), rat gastrocnemius (Howell et al., 1966; Sreter, 1970), guinea pig gastrocnemius (Brody, 1966), and rabbit gastrocnemius muscles (Nakai, 1968) 10-14 days after denervation accumulate 2-3 times more Ca in the presence of oxalate than microsomes obtained from control or tenotomized muscles. The increased Ca capacity is apparent already 4 days after denervation and reaches maximum in about 2 weeks. Rat soleus muscle microsomes showed less pronounced changes (Sreter, 1970) in comparison with diaphragm (Howell et al., 1966), although both are predominantly red muscles. Increased Ca uptake of human muscle microsomes (Samaha and Gergely, 1965b) was observed in Kugelberg-Welander syndrome and in amyotrophic lateral sclerosis, which involve lesions of motor innervation (Radu et al., 1970). The increase in oxalate-potentiated Ca uptake following denervation is a transient phenomenon. In rabbit gastrocnemius (Nakai, 1968) and in rat gastrocnemius muscles (Sreter, 1970) the values return to normal or below 6 weeks after denervation. Parallel with the increased Ca accumulation, there is a marked increase in the basal ATPase activity of microsomes isolated from rat gastrocnemius muscle. The elevated ATPase activity persists even after the Ca uptake returns to normal levels. The Ca activated component of the total ATPase activity remains unchanged after denervation, as indicated by the constant level of EGTA-inhibited ATPase activity (Sreter, 1970). It is of considerable importance that the rate of Ca outflow from microsomes isolated from denervated muscle is markedly enhanced by EGTA and caffeine (Sreter, 1970). This agrees with the increased sensitivity of denervated mammalian muscles to caffeine contracture (Gutmann and Sandow, 1965). While the Ca capacity of microsomes as measured by the maximum amount of Ca taken up in the presence of oxalate increases after denervation of rat gastrocnemius, the initial rate of Ca uptake measured with microsomes isolated from rat gastrocnemius muscle both in the presence and the absence of oxalate gradually declines. This decline is accompanied by a comparable decrease in the maximum amount of phosphoprotein formed from AT9*P(Sreter, 1970). These findings contrast with those of Nakai (1968), who observed a 1.7fold increase in the rate of Ca accumulation 2 weeks after denervation, although it is not certain that he measured true initial rates. The careful experiments of Sreter (1970) suggest that the increased Ca capacity of

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denervated microsomes does not involve changes in the concentration of Ca transport sites in the membrane or in the rate of operation of Ca pump. The Ca uptake by skeletal muscle microsomes in the presence of oxalate occurs with the precipitation of Ca oxalate within the microsomes. This permits the accumulation of Ca to continue until the vesicles are saturated with Ca oxalate crystals. Therefore, the increased Ca capacity of microsomes following denervation may indicate that the volume available for the deposition of Ca oxalate increases. This may arise from an increase in the average diameter of microsomes isolated from denervated muscle (Sreter, 1970) or from a decrease in the solid content of sarcoplasmic reticulum tubules. No experimental data are available to support this latter alternative. In addition to the increased Ca capacity of isolated membranes, electron microscopic and biochemical evidence indicates a net accumulation of sarcoplasmic reticulum membrane material in denervated frog muscle (Muscatello et al., 1965). In rat gastrocnemius, Sreter (1970) found that the yield of mitochondria and microsomes decreases by at least 50y0 on day 14 after denervation, when the Ca capacity of microsomes reaches its maximum. It appears that different muscles respond to denervation in different ways. Morphological changes following denervation may be taken to indicate a preferential sensitivity of the white muscle fibers to the loss of trophic innervation (Bajusz, 1964; Sreter, 1970). A greater loss of white muscle fibers would not explain the biochemical observations, since this would lead to a decreased Ca uptake in microsomes isolated from denervated white muscles. The increased Ca capacity of microsomes isolated from denervated muscle suggests a dedifferentiation rather than degeneration of white muscle fibers. Since the Ca uptake capacity of microsomes isolated from fetal muscle is quite limited (Holland and Perry, 1969; Fanburg et al., 1968; Szabolcs et al., 1967), the dedifferentiation caused by denervation is not a simple reversal of normal muscle development. An increase in the amount as well as the Ca binding capacity of the sarcoplasmic reticulum may have to be postulated to explain the increased Ca uptake of denervated glycerinated rat diaphragm muscles in vitro (Howell et al., 1966). The dramatic increase in the Ca content of muscle in vivo, following denervation, may be the result of calcification. In rat diaphragm, the increased Ca capacity of sarcoplasmic reticulum develops during hypertrophy which involves accumulation of contractile material. Yet the maximum tetanic tension generated by repetitive electric stimuli (50 per second) markedly decreases following denervation. This suggests that the Ca stored in the sarcoplasmic reticulum is not released in sufficient quantities to saturate the contractile system. A marked decline in

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twitch and tetanus tension was also observed on rat extensor digitorum longus muscle already 24 hours after denervation (Gutmann and Sandow, 1965). Apart from changes in the electrical properties of surface membrane and T-system tubules, this apparent uncoupling between excitation and Ca release may result from the observed separation of cisternae into rows of independent vesicles, with ill-defined relationship to the T-system tubules. Under these conditions, it is likely that the isolated sarcoplasmic reticulum vesicles do not establish contact with the T system and do not release Ca on excitation. The proliferation of transverse tubules may be a compensatory process aimed a t correcting this situation. The surface membranes undergo marked changes following denervation (Albuquerque and McIsaac, 1970; Guth, 1968) among which the appearance of extrajunctional acetylcholine sensitivity (Miledi, 1960 ; Thesleff, 1960, 1963) and increased membrane resistance are of particular importance (Nicholls, 1956). The increased membrane resistance may result from a decrease in K conductance (Ware et al., 1954; Lullman, 1960; Kernan, 1965). In rat extensor digitorum longus, denervation causes the disappearance of miniature end-plate potential in about 24 hours (Albuquerque and McIsaac, 1970). After the end-plate degeneration, the muscle, which is usually resistant to the contracture-producing effect of 20 mM caffeine, readily develops contracture in 5-20 mM caffeine, and the tension developed in caffeine contracture is similar to the average tetanus tension (Gutmann and Sandow, 1965). Similar observations were made on denervated frog muscle (Kuffler, 1943; Gutmann and Sandow, 1965) which is more sensitive to caffeine than mammalian muscles. Procaine blocks the caffeine contracture on denervated rat extensor digitorum longus (Gutmann and Sandow, 1965) as it does on normal frog muscle (Feinstein, 1963). Since the contracture-producing effect of caffeine has been ascribed to its action on the sarcoplasmic reticulum (Weber, 1968; Weber and Hers, 1968), it may be assumed that the increased sensitivity of denervated muscle to caffeine indicates changes in the biochemical properties of sarcoplasmicreticulum membranes. Marked increase in the cholesterol content of isolated microsomes was reported after denervation (Zatti et al., 1969) together with changes in phospholipid composition and increased turnover of membrane lipids (Graff et al., 1965; Bunch et at., 1970). If the cholesterol content of skeletal muscle microsomes originates from contamination by surface membranes (Sanslone et al., 1971), the interpretation of the changes in cholesterol content is uncertain.

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EFFECTS OF CROSS-INNERVATION ON SARCOPLASMIC RETICULUM Mammalian muscles are usually composed of three types of fibers, which are generally referred to as white, red, and intermediate (Padykula and Gauthier, 1967). The fast-twitch muscles contain predominantly white, and the slow-twitch or tonic muscles primarily red fibers, with easily recognizable morphological (Padykula and Gauthier, 1967), biochemical (Gutmann, 1967; Barany et al., 1965; Maddox and Perry, 1966; Locker and Hagyard, 1968; Seidel, 1967; Sreter, 1969; Sreter et al., 1966, 1971; Prewitt and Salafsky, 1967, 1970; Guth et al., 1968; Guth, 1968), and functional differences (Ranvier, 1874; Cooper and Eccles, 1930; Buller et al., 1960a,b; Close, 1967; Buller and Lewis, 1965a,b,c; Fex and Jirmanova, 1969). The isometric contraction characteristics of the various muscles are determined by their motor innervation (Buller et al., 1969; Barany and Close, 1971a,b). If the motor nerves of cat or rat flexor digitorum longus (fast muscle) and soleus (slow muscle) are interchanged, then after reinnervation the rate of shortening of the soleus becomes faster, while that of the flexor digitorum longus becomes slower (Buller et al., 1960a,b; 1969; Close, 1967; Barany and Close, 1971a,b; Mommaerts et al., 1969). This suggests that after cross-innervation the muscle gradually assumes properties dictated by the nerve fibers. This is also supported by characteristic changes in the histological appearance, chemical composition, and enzyme content of cross-innervated fast or slow muscles (Prewitt and Salafsky, 1967, 1970; Dubowitz, 1967a,b; Dubowitz and Newman, 1967; Romanul and Van Der Rfeulen, 1966, 1967; Guth et al., 1968; Guth, 1968). Of particular importance to the observed changes in the rate of shortening which follow cross-innervation are the alterations of the ATPase activity of myosin and of the Ca transport function of sarcoplasmic reticulum (Buller et al., 1969; Mommaerts et al., 1969; Barany and Close, 1971a,b). I n cat flexor digitorum longus muscles cross-innervated with the motor nerve of soleus the decrease in shortening velocity is accompanied by decreased Ca transport of sarcoplasmic reticulum fragments (Mommaerts et al., 1969). The physiological characteristics of the muscle, the ATPase activity of myosin (Buller et al., 1969), and the Ca transport capacity (Mommaerts et al., 1969) of sarcoplasmic reticulum fragments isolated from flexor digitorum longus muscles 6-12 months after cross-innervation with soleus nerve fibers approach the characteristics of soleus. Opposite, but less marked, changes were apparent in soleus muscles cross-innervated with the motor nerve of flexor digitorum longus. The differences in the Ca transport capacity of microsomes from red

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and white muscles may imply differencesin: (a) the density of Ca transport sites on the membrane, (b) the rate of transport process at individual carrier sites, (c) the permeability of microsomal membranes for Ca, (d) the number of actively transporting vesicular fragments in the population of microsomal particles isolated from red and white muscles. It is not known which of these aspects are primarily influenced by motor innervation. 8. Muscular Dystrophy

The various forms of hereditary muscular dystrophies which occur in man, mouse, and chicken were long considered to be primary myopathies, originating from an inherited biochemical abnormality of the muscle cell. While this may still be the case for Bome forms of the disease, recent evidence suggests that a large number (25-70y0) of the muscle fibers of the affected muscles are not functionally innervated by motor nerves in pseudohypertrophic-Duchenne (McComas et al., 1970), myotonic (Campbell et aE., 1970), or limb-girdle type (McComas and Sica, 1970) human dystrophy and in the hereditary dystrophy of mouse (McCornas and Mrozek, 1967; Harris and Wilson, 1971). A muscle fiber was regarded as functionally denervated if it responded to direct (muscle) but not to indirect (nerve) stimulation. It seems unlikely that the denervation in various forms of dystrophy would result from accidental nerve injury since the conduction velocity of motor nerve fibers is usually normal. It is interesting to note that the total number of myelinated nerve fibers in nerves leading t o the dystrophic mouse muscle was only about 40% of the control (Harris and Wilson, 1971) and marked reduction in the number of motor neurons was also observed in the spinal cords of three cases of Duchenne dystrophy (Tomlinson, quoted by McComas et al., 1970). These findings account for such histological characteristics of dystrophic muscle as the presence of abnormal end plates on apparently healthy fibers, muscle fiber atrophy, central nuclei, and connective tissue proliferation, which are known histological consequences of experimental or pathological denervation. Minced skeletal muscle of dystrophic mice transplanted into normal animals regenerates into muscles possessing normal twitch characteristics, while normal mince transplanted into dystrophic mice usually achieves little or no functional regeneration (Salafsky, 1971). This suggests again that the genetic defect does not reside in the muscle cell. The rate of development and decay of isometric tension in cat flexor digitorum longus (fast muscle) is much reduced after denervation, while

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the soleus (slow muscle) is slowed to a lesser extent (Eccles, 1967; Eccles et al., 1962; Guth, 1968). A comparison of normal and dystrophic mouse gastrocnemius indicates that in dystrophy, as after denervation, the gastrocnemius assumes the characteristics of a slow muscle (Eberstein and Sandow, 1963). The rate of rise of tension and especially the rate of relaxation are much slower and prolonged activity causes less fatigue in dystrophic than in control gastrocnemius (Sandow and Brust, 1958, 1962; Brust, 1965). The slow soleus muscle of the mouse is quite resistant to dystrophy, a t least with regard to its contraction characteristics (Brust, 1966, 1969). Essentially similar observations regarding contraction and relaxation kinetics have been made on Duchenne dystrophic human muscles (Botelho et al., 1960, 1961; Green et al., 1961; Desmedt, 1967; Roe et al., 1967). Among other similarities between denervated and dystrophic muscles are the lower resting membrane potential (McComas and Mrozek, 1967; Lenman, 1965), muscle fibrillation potentials (Norris and Chatfield, 1955; McIntyre et al., 1959) altered miniature end plate potentials (McComas and Mossawy, 1965) and diffuse acetylcholine sensitivity (Axelsson and Thesleff, 1959; Thesleff, 1960, 1963) which are also shown by the slow, red soleus fibers (Miledi and ZelenB, 1966; Gutmann, 1967) and by all muscles of cat and rat in the early postnatal period (Gutmann, 1967) when the rate of contraction is slow (Buller et al., 1960a; Buller and Lewis, 1965a,b; Close, 1964). The fetal pattern of LDH isozymes appears in denervation atrophy of rabbit gastrocnemius (Brody, 1964, 1965; Gutmann, 1967; Hogan et al., 1965; Dawson and Romanul, 1964) and in human and avian muscular dystrophies (Wieme and Herpol, 1962; Kaplan and Cahn, 1962; Pearson et al., 1965). There are indications that a t least in chicken dystrophy the differentiation of muscle cells into mature white muscle fibers is impaired (Cosmos, 1966; Cosmos and Butler, 1967). Red and fetal muscles are characterized by sparsely developed sarcoplasmic reticulum. The rate and extent of Ca uptake by microsomes isolated from fetal (Szabolcs et al., 1967; Holland add Perry, 1969; Fanburg et al., 1968) or red muscles (Sreter and Gergely, 1964; Samaha and Gergely, 1965b; Takauji et al., 1967; Harigaya et al., 1968; Sreter, 1969) is much less than that of their adult white counterparts. In view of the structural, biochemical and functional similarities between fetal, red, denervated, and dystrophic muscles, it is not surprising that in chronic denervation atrophy (Sreter, 1970; Nakai, 1968) and in mouse dystrophy (Martonosi, 1968d; Sreter et al., 1964, 1967) or Duchenne-type human muscular dystrophy (Samaha and Gergely, 1969; Peter and Worsfold, 1969a; Sugita et al., 1967), the Ca uptake function of isolated sarcoplasmic reticulum is impaired.

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The biochemical and morphological changes observed in isolated dystrophic muscle microsomes may represent the consequence of early changes of sarcoplasmic reticulum in muscular dystrophy seen by electron microscopy (Van Breemen, 1960; Milhorat et al., 1966; Pearce, 1966). The prolonged relaxation of dystrophic mouse muscle (Sandow and Brust, 1958, 1962, 1965; Roe et at., 1967) does not result from myotonic type repetitive discharges (Conrad and Glaser, 1961; McComas and Mossawy, 1965) and is likely to reflect slower reabsorption of Ca into the sarcoplasmic reticulum during relaxation. It is of significance in this regard that the impaired Ca transport of isolated sarcoplasmic reticulum particles is characteristic of mouse and of Duchenne-type human dystrophy (Samaha and Gergely, 1969; Peter and Worsfold, 1969a) and is not found in myotonic dystrophy (Peter and Worsfold, 1969b; Samaha et al., 1967; Samaha and Gergely, 1969), polymyositis, or alcoholic myopathy (Peter and Worsfold, 1969a), although the number of observations relating to the latter is small and somewhat conflicting (Sugita et al., 1967). The oxidative phosphorylation of muscle mitochondria remains unaffected in mouse dystrophy (Wrogemann and Blanchaer, 1967) and in myotonic (Peter and Worsfold, 1969b) or Duchenne-type (Peter and Worsfold, 1969a) human hystrophy, until massive degeneration of muscle sets in. The Ca deposition observed in the mitochondria of dystrophic muscles (Caulfield, 1966) may contribute to the defects of respiration and oxidative phosphorylation found in these animals (Wrogemann et al., 1970a,b; Jacobson et d.,1970) and to other metabolic changes (Fitzpatrick and Pennington, 1968) observed in mice. The precise nature of the biochemical defect which leads to the impaired Ca uptake of dystrophic muscle microsomes is unknown. The decreased Ca uptake capacity in mitochondria1 as well as in light and heavy microsomal fractions is accompanied by decreased Ca sensitivity of the microsomal ATPase, reflected in the reduced inhibition with EGTA or oxalate (Martonosi, 1968d; Sreter et al., 1967). The Mg activated ATPase activity is unchanged (Martonosi, 1968d) or slightly elevated (Sreter et al., 1967; Sugita et al., 1967). The dystrophic microsomes rapidly lose their Ca transport capacity during storage as was earlier observed for cardiac (B. Fanburg et al., 1964) and red muscle (Harigaya et al., 1968) microsomes. Conflicting reports have appeared concerning the relative sensitivity of control and dystrophic microsomes to treatment with proteolytic enzymes (Sreter et d., 1967; HSUand Kaldor, 1969). Normal mouse muscle microsomes isolated from predominantly white muscle possess a characteristic tadpole shape after negative staining with

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potassium phosphotungstate (Martonosi, 1968b,d). During aging many of the particles lose their tubular projections parallel with a decline of Ca

transport function. Dystrophic mouse microsome preparations contain almost exclusively spherical particles which are quite similar to those obtained from predominantly red or fetal muscles (Martonosi, 1968d; Sreter et aE., 1967). There are characteristic differences in the phospholipid and cholesterol composition of normal and dystrophic microsomes (Godinez et al., 1970; Owens and Hughes, 1970; Hughes, 1965). In view of the involvement of phospholipids in the activity of the Ca transport system and in the regulation of the Ca permeability of microsomes (hilartonosi, 1963; 1964, Martonosi et al., 1968; Duggan and Rlartonosi, 1970), these differences may contribute to the observed changes in Ca transport. The increased sphingomyelin and cholesterol and reduced lecithin and phosphatidylcholine content of dystrophic mouse muscle is largely attributable to changes in the phospholipid content of microsomes. Similar changes have been found in Duchenne type human dystrophy (Schellnack, 1970). There are also changes in the fatty acid and fatty aldehyde content which may be related to the altered fatty acid oxidation and synthesis observed in dystrophic muscle (Lin et al., 1969). The increased cholesterol content of dystrophic muscle is comparable to that observed in denervation atrophy (Zatti et al., 1969), accompanied by changes in the fatty acid composition (Kouvelas and Rilanchester, 1968). The evaluation of the data on the phospholipid composition of subcellular fractions is made difficult by the possibility of lipid exchange during isolation (Wirtz and Zilversmit, 1968; Blok et al., 1971). The phospholipid composition of dystrophic muscle is similar to that of the red soleus. Both contain less lecithin than the extensor digitorum longus, a white muscle (Owens and Angelini, 1970). In view of the distinctive fatty acid content, it is unlikely, however, that dystrophy would represent merely a preferential sparing of red muscle fibers and either a failure of differentiation (Cosmos, 1966; Cosmos and Butler, 1967) or a dedifferentiation to something resembling the fetal state of muscle fiber must be assumed to occur. The elevated cardiolipin content of dystrophic mouse microsomes (Owens and Hughes, 1970) may be indicative of contamination by mitochondria, although electron microscopic analysis of negatively stained material revealed only minor contamination in control and dystrophic mouse microsome preparations (Martonosi, 196Sd). The increased lysolecithin content of dystrophic mouse microsomes (Owens and Hughes, 1970) is probably attributable t o the effect of phospholipases during the lengthy procedure used for the isolation of microsome, since lysolecithin is not detectable in

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fresh muscle. Treatment of microsomes with phospholipase A results in activation of ATPase activity and inhibition of Ca transport (I‘iehn and Hasselbach, 1970; Martonosi et al., 1971a), the types of changes which characterize dystrophic microsomes. As elevated levels of degradative lysosomal enzymes were reported in muscular dystrophy (Weinstock ~t al., 1958; Tappel el al., 196‘2; Zalkin el al., 1962) and denervation atrophy (Hajek et al., 1963; Pellegrino and Franzini, 1963; Weinstock and Lukacs, 1965; Pollack and Bird, 1968), studies on the phospholipases of dystrophic muscle would be of interest. C. Involvement of Sarcoplasmic Reticulum in Other Muscle Disorders

1. MUSCLECONTRACTURE INDUCED BY EXERCISE

The disorder is characterized by painless muscle stiffness with delayed relaxation provoked by 10-15 seconds of strenuous activity (Brody, 1969). The intolerance to exercise is particularly pronounced a t low temperature, creating difficulty of articulation due to stiffness of thc tongue and lips. Brief rest permits the muscles to relax fully after which exericse may be resumed. In contrast to myotonia, tetany or ordinary muscle cramp, therc are no muscle action potentials during the slow relaxation. Although the sarcoplasmic reticulum and T system appear normal under the electron microscope, the rate and extent of Ca2+ uptake by isolated sarcoplasmic reticulum fragments is drastically reduced, without significant change in total ATPase activity. The total phosphorylase activity was also normal in contrast to McArdle’s disease, but 92Oj, of the phosphorylase was active without added AMP. The slow relaxation of muscle and the persistent activation of phosphorylase may be attributable to a selective defect of the Ca uptake function of sarcoplasmic reticulum with elevated levels of sarcoplasmic Ca2+ concentration. 2. HYPOKALEMIC PERIODIC PARALYSIS

The disease usually occurs in young persons in the form of sudden bouts of flaccid muscular weakness associated with a fall in serum potassium. Muscular activity has a protective effect, while rest predisposes to paralysis. Microapplication of Ca2+to skinned fibers prepared from paralyzed muscles of the patients produced prompt contraction followed by relaxation, with a time course comparable to control fibers. These results clearly indicate that electrically inexcitable fibers can be activated by direct application of Ca to the contractile material and that the reabsorption of applied Ca by the sarcoplasmic reticulum occurs a t a normal rate (Engel

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and Lambert, 1969). The failure of excitation-contraction coupling in this disease must therefore bc related to the events lending to the release of Ca from the sarcoplasmic reticulum. Electron microscopy of the affected muscles indicates distensions of the terminal ss)”s of sarcoplasmic reticulum which are filled with granular material (Schutta and Armitage, 1969a; Dunkle et al., 1970). Rluscles from a patient with thyrotoxic hypokalemic periodic paralysis showed massive proliferation of membrane material thought to originate from T-system tubules, together with distension of the terminal sacs of sarcoplasmic reticulum, abnormal mitochondria, and unusual subsarcolemma1 inclusions, indicating a generalized defect of membrane formation in the muscle cell (Schutta and Armitage, 1969b). Tubular proliferations of membrane structures assumed to originate from the sarcoplasmic reticulum werc observed in type I1 fibers of patients with varying ncuromuscular disorders and of some normal individuals with history of chronic drug use (Engel ef al., 1970). 3. hICARDLE’S DISEASE Thc disease is characterized by muscle weakness occurring during ischemic exercise accompanied by subnormal level of lactate in the venous blood, and drcrcase in muscle phosphorylase. The Ca uptake of isolated sarcoplasmic reticulum is normal and the weakness may be due to a defect in the electrical properties of surface membrane (Brody et al., 1970). D. Myotonio

hlyotonia may be defined “as an abnormally dclaycd relaxation of skeletal muscle fibers following a voluntary or induced contraction, which results from a repetitive depolarization of the muscle cell membrane” (Winer et al., 1965). This description characterizes the two principal human forms of the disease, myotonia congenita, and myotonia dystrophica (Adams et al., 1962), as well as the inherited myotonia of goats (Brown and Harvey, 1939) and the various experimental myotonias induced with diazacholesterol (Winer et al., 1965) or 2,4-dichlorophenoxyacetate (Eyzaquirre et al., 1948). 1. MYOTONIAS O F GENETICORIGIN The long bursts of action potentials which accompany the sustained tension implicate the surface membrane in the pathogenesis of various

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myotonias of genetic origin, although, as usual, a defect in the production or utilization of ATP (Kuhn, 1961; Caughey and Myrianthopoulos, 1963) or an impairment of the Ca transport of sarcoplasmic reticulum were also suggested (Seiler et al., 1970). The yield of mitochondria isolated from patients with myotonic dystrophy as well as the rate of respiration, the acceptor ratio, the respiratory control ratio, and ADP/O ratios measured with pyruvate-malate and with with succinate-rotenone as substrates were within the normal range (Peter and Worsfold, 1969b). Sarcoplasmic reticulum fragments isolated from the affected muscles accumulate calcium a t normal (Peter and Worsfold, 1969b; Samaha et al., 1967) or elevated rates (Samaha and Gergely, 1969) without significant change in their Ca capacity (Samaha and Gergely, 1069) or in their ability to lower the medium calcium concentration to levels at which inhibition of myofibrillar ATPase activity occurs (Peter and Worsfold, 1969b). The efficiency of Ca transport expressed as the ratio of initial rate of Ca uptake to the rate of ATP hydrolysis is higher in myotonic than in control microsomes (Samaha and Gergely, 1969). Normal calcium uptake was also observed in sarcoplasmic reticulum fragments isolated from muscles of myotonic goats (Adams and Thompson, 1965). In view of the normal or improved Ca transport of microsomes isolated from myotonic human or goat muscles, the delayed relaxation is not likely to result from impaired calcium uptake of the sarcoplasmic reticulumrather it could be related to repetitive Ca release induced by the myotonic bursts of active potentials. This may constitute the essential difference between the various types of myotonias and the exercise-induced muscle stiffness described by Brody (1969), since in the latter the delayed relaxations is not accompanied by action potentials and could be clearly attributed to the defective calcium transport of sarcoplasmic reticulum.

MYOTONIA INDUCED WITH DIAZOCHOLESTEROL 2. EXPERIMENTAL Chronic administration of 20,25-diazocholesterol to human patients (Winer et aE., 1965), goats (Winer et al., 1965; Burns et al., 1965), or rats (Winer et al., 1966; Briggs and Kuhn, 1968) produces a myotonic response which is similar to those seen in hereditary myotonic disorders. In addition to 20,25-diazocholesterol, several other steroid inhibitors of cholesterol biosynthesis were found effective in inducing myotonia in rats (Winer et al., 1966). Among the effects of these inhibitors were a marked decrease of serum and red blood cell cholesterol and accumulation of desmosterol in serum, red blood cell stroma and muscle tissue (Winer et aE., 1965, 1966;

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Burns et al., 1965). I n addition to a direct effect of the drugs on muscle cell membranes, changes in the cholesterol and desmosterol concentrations are also likely to contribute to the development of myotonic condition. Cholesterol feeding prevented the drug-induced myotonia and even a 10-fold increase in 20,25-diazocholesterol dosage failed to overcome the protective effect of cholesterol. Under these conditions, the desmosterol level of blood plasma remained low. Triparanol, a nonsteroid inhibitor of cholesterol biosynthesis, caused marked accumulation of desmosterol, without producing myotonia. Similarly ineffective was benzyl-N benzylcarbethoxy hydroxamate (W-398), which lowered serum cholesterol without desmosterol accumulation (Winer et al., 1966). Myotonia induced by 25-azacholesterol affects the fast-twitch muscles of rats much more readily than the red slow-twitch muscles (Goodgold and Eberstein, 1968; Eberstein and Goodgold, 1969) and male animals are more susceptible than females (Eberstein and Goodgold, 1969). The greater resistance of slow red muscles to drug-induced myotonia is similar to the known resistance of red muscles to hereditary muscular dystrophy in mice (Brust, 1966) and may be related to the high cholesterol content of red muscle membranes (Froberg, 1967). High serum cholesterol and increased sterol esterification of female rats (Aftergood and Alfin-Slater, 1967) may be the explanation for the sexual differences in drug sensitivity. Although there are no precise data on the cholesterol, desmosterol, and 20,25-diazocholesterol content of muscle cell membranes isolated from animals which were made myotonic with 20 ,25-diazocho1estero1, the accumulation of desmosterol in the muscle tissue is always marked (Winer et al., 1965, 1966; Burns et al., 1965) and may constitute a n important aspect of the disease. I n the muscle tissue of myotonic goats, desmosterol constituted 38-72% of the total sterols, the rest being cholesterol. A dehydrogenated and esterified product of 20 ,25-diazocholesterol was isolated from sarcoplasmic reticulum membranes of rats following chronic peroral application of the drug (Seiler and Kuhn, 1969). The 20,25diazodehydrocholesterol cster is rich in unsaturated fatty acids, especially arachidonic acid. The previously reported changes in the fatty acid composition of muscle lipids after diazocholesterol administration (Kuhn et al., 1968) were attributed to the presence of 20,25-diazodehydrocholesterol esters (Seiler and Kuhn, 1969). The unesterified form of 20,25-diazocholesterol was not detectable in muscle tissue. The preferential accumulation of the csterified form is surprising since over 90% of the membrane cholesterol of the sarcoplasmic reticulum is unesterified (Martonosi, 1968b). The presence of the drug almost exclusively in its esterified form raises

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the possibility that the csterification occurred after homogenization of the muscle, resulting in an altered distribution of the drug among the different particulate fractions. A preferential accumulation of diazocholesterol in the sarcoplasmic reticulum was claimed on the ground that the lipid extract obtained from isolated sarcoplasmic reticulum membranes contained 19 times more diazocholesterol ester than the whole muscle, calculated on the basis of protein (Seiler and Kuhn, 1969). Fragmented sarcoplasmic reticulum isolated from rats with 20,25diazocholesterol-induced myotonia shows essentially normal calcium accumulation (Briggs and Kuhn, 1968; Seiler el al., 1970), similarly to sarcoplasmic reticulum fragments obtained from human cases of myotonia (Samaha et al., 1967; Samaha and Gergely, 1969; Peter and Worsfold, 1969b) or from myotonic goats (Adams and Thompson, 1965). The slight differences between control and myotonic rat microsomes (Seiler et al., 1970) in the rate of Ca exchange and in their ability to inhibit actomyosin ATPase activity would require statistical evaluation to ascertain their significance. Although the rate of Ca efflux from myotonic microsomes clearly excwded the control rate, the use of oxalate as Ca-precipitating agent in these experiments introduces considerable ambiguity in the interpretation. In summary, no conclusive evidence is available indicating the primary involvement of sarcoplasmic reticulum in hereditary myotonias and in myotonias induced by diazocholesterol. The elcctromyographic observations implicating repetitive depolarization of the surface membrane as the cause of the delayed relaxation continue to deserve serious attention. In harmony with the proposed alteration of surface membrane, skin fibroblasts from patients with myotonic muscular dystrophy differ from normal cells in their pattern of growth in tissue culture and contain large amounts of a material with the staining characteristics of acid mucopolysaccharides (Swift and Finegold, 1969). 3. EXPERIMENTAL MYOTONIAINDUCED ACETICACID(2,4-D)

WITH

2,4-DICHLOROPHENOXY

Intravenous injection into rats of small amounts (2 mg) of 2,4-D causes massive generalized myotonia in a few minutes (Bucher, 1946; Eyzaguirre et al., 1948). The repetitive myotonic responses are accentuated by K and acetylcholine and are reduced by quinine, Mg, Ca, or muscular activity in a manner similar to the spontaneously occurring myotonia of man and goat. The slight inhibition of the Ca uptake of isolated rat sarcoplasmic

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reticulum by high concentration (1-10 mM) of 2,4-D in vitro was implicated as the underlying cause of the myotonic condition induced by 2 ,4-D in the living animals (Stein, 1966; Kuhn and Stein, 1966). However, impairment of the Ca2+ uptake function of sarcoplasmic reticulum could hardly explain the protective effect of muscular activity against the myotonic attacks and the in vivo effects are elicited by relatively small doses of 2,4-D, which are ineffective in vitro on isolated sarcoplasmic reticulum. Since the in vivo effect is nearly immediate and the severity of the myotonic condition is accentuated by elevated serum K and obliterated by quinine, Ca2+ or Mg, the general picture is that of a surface membrane effect with primary emphasis on the excitatory aspects of the excitationcontraction coupling process. The evidence so far available does not establish the involvement of sarcoplasmic reticulum. 4. STEROID MYOPATHY

9a-Fluoro-1 lp, 16a, 17a, 21-tetrahydroxy-1 ,4-pregnanediene-3 ,20-dione (Triamcinolone) , used in steroid therapy, causes serious muscle weakness and muscle degeneration as a side effect. Apparently normal mitochondria were isolated from triamcinolone-treated rats, and the calcium accumulation capacity, calcium stimulsted ATPase activity and calcium affinity of sarcoplasmic reticulum fragments from these animals showed no effect of the drug whether applied in vivo or in vitro (Peter et al., 1970). ACKNOWLEDGMENT

I wish to express my thanks to Drs. C. Franzini-Armstrong, L. D. Peachey, D. G. Rayns, and J. Rosenbluth for permission to use their electron micrographs. I am grateful to Mrs. Joann Mitchell for her gracious and excellent help with the manuscript and to Miss Rita A. Halpin, Dr. Jane M. Vanderkooi, Mr. Anthony G. Pucell, Mr. James It. Donley, and Dr. Patrick F. Duggan for their enthusiastic collaboration in various phases of the work. REFERENCES Abrams, A. (1965). J. BioE. Chem. 240, 3675. Abramson, M. B., Norton, W. T., and Katzman, R. (1965). J. Bid. Chem. 240, 2389. Adams, M. J., and Thompson, J. W. (1965). S. Med. J. 53, 1586. Adams, R. D., Denny-Brown, D., and Pearson, C. M. (1962). “Diseases of Muscle.” Harper (Hoeber), New York. Adrian, R. H., and Peachey, L. D. (1965). J . Physiol. (London) 181,324. Aftergood, L., and Alfin-Slater, R. B. (1967). J. Lipid Res. 8, 126. Akera, T.,and Brody, T. M. (1971). J. Pharmacol. Ezp. Ther. 176, 545. Akera, T., Larsen, F. S., and Brody, T. M. (1969). J. Pharmacol. Exp. Ther. 170, 17. Akera, T., Larsen, F. S., and Brody, T. M. (1970). J . Pharmacol. Exp. Ther. 173, 145 Albuquerque, E. X., and MeIsaac, R. J. (1970). Exp. Neurol. 26, 183. Albuquerque, E. X., and Thesleff, S. (1968). Acta Physiol. Seand. 73, 471.

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Flitney, F. W.(1971).The volume of the T-system and its association with the sarcoplasmic reticulum in slow muscle fibres of the frog. J . Physiol. (London) 217, 243. Hess, M. L.,and Briggs, F. N. (1971).The effect of gram negative endotoxin on the calcium uptake activity of sarcoplasmic reticulum isolated from canine myocardium. Biochem. Bzophys. Res. Commun. 45, 917. HSU,Q. S., and Kaldor, G. (1971). Studies on the lipid composition of the fragmented sarcoplasmic reticulum of normal and dystrophic chickens. Proc. Soc. Ezp. Biol. Med. 138, 733. Ikemoto, N.,Bhatnagar, G. M., and Gergely, J. (1971).Fractionation of solubilized sarcoplasmic reticulum. Biochem. Biophys. Res. Commun. 44, 1510. Ikemoto, N., Sreter, F. A., and Gergely, J. (1971).Structural features of the surface of the vesicles for FSR-lack of functional role in Ca2+ uptake and ATPase activity. Arch. Biochem. Biophys. 147, 571. Inesi, G.,and Scarpa, A. (1972).Fast kinetics of ATP dependent Ca2+ uptake by fragmented sarcoplasmic reticulum. Biochemistry 11, 356. Kanazawa, T., Yamada, S., Yamamoto, T., and Tonomura, Y. (1971).Reaction mechanism of the Caa+-dependent ATPase of sarcoplasmic reticulum from skeletal muscle. V. Vectorial requirements for calcium and magnesium ions of three partial reactions of ATPase: Formation and decomposition of a phosphorylated intermediate and ATP-formation from ADP and the intermediate. J . Biochem. (Tokyo) 70, 95. Luff, A. R.,and Atwood, H. L. (1971).Changes in the sarcoplasmic reticulum and transverse tubular system of fast and slow skeletal muscles of the mouse during postnatal development. J . Cell B i d . 51, 369. McFarland, B. H., and Inesi, G. (1970). Studies of solubiliaed sarcoplasmic reticulum. Biochem. Biophys. Res. Commun. 41, 239. McFarland, B. H., and Inesi, G. (1971). Solubilization of sarcoplasmic reticulum with Triton-X-100. Arch. Biochem. Bwphys. 145, 456. Masoro, E.J., and Yu, B. P. (1971).The functioning of the lipids and lipoproteins of sarcotubular membranes in calcium transport. Lipids 6 , 357. Meissner, G., and Fleischer, S. (1971).Characterization of sarcoplasmic reticulum from skeletal muscle. Biochim. Biophys. A d a 241, 356. Meissner, G., and Fleischer, S. (1972).The role of phospholipid in Caa+ stimulated ATPase activity of sarcoplasmic reticulum. Biochim. Biophys. Acta 255, 19. Nakamaru, Y., and Schwartz, A. (1972).The influence of hydrogen ion concentration an calcium binding and release by skeletal muscle sarcoplasmic reticulum. J . Gen. Physiol. 59, 22. Nayler, W. G., McInnes, I., Chipperfield, D., Carson, V., and Kurtz, J. B. (1970). Ventricular function and the calcium-accumulation activity of the sarcoplasmic. reticulum. J . Mol. Cell Cardiol. 1, 307. Panet, R., and Selinger, 2. (1972). Synthesis of ATP coupled to Cae+ release from sarcoplasmic reticulum vesicles. Biochim. Biophys. Acta 255, 34. Panet, R.,Pick, U., and Selinger, 2. (1971).The role of calcium and magnesium in the adenosine triphosphatase reaction of sarcoplasmic reticulum. J . B i d . Chem. 246, 7349. Sanslone, W. R., Bertrand, H. A., Yu, B. P., and Masoro, E. J. (1972).Lipid components of sarcotubular membranes. J . Cell. Physiol. 79, 97. Seelig, J., and Hasselbach, W.(1971). A spin label study of sarcoplasmic vesicles. Eur. J . Biochem. 21, 17.

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Seiler, D., and Hasselbach, W. (1971). Essential fatty acid deficiency and the activity of the sarcoplasmic calcium pump. Eur. J . Biochem. 21, 385. Somlyo, A. P., Devine, C. E., Somlyo, A. V., and North, S. R. (1971). Sarcoplasmic reticulum and the temperature-dependent contraction of smooth muscle in calciumfree solutions. J. Cell Biol. 51, 722. Somlyo, A. V., and Somlyo, A. P. (1971). Strontium accumulation by sarcoplasmic reticulum and mitochondria in vascular smooth muscle. Science 174, 955. Suko, J. (1971). Alterations of Caz+ uptake and Ca2+-activated ATPase of cardiac sarcoplasmic reticulum in hyper- and hypothyroidism. Biochim.Biophys. Acta 252, 324. Sulakhe, P. V., McNamara, D. B., and Dhalla, N. S. (1971). Excitation-contraction coupling in heart. VIII. Influence of adenine nucleotides on calcium binding by subcellular fractions of rat heart. J. Biochem. (Tokyo) 70, 571. Takagi, A. (1971). Lipid composition of sarcoplasmic reticulum of human skeletal muscle. Biochim. Biophys. Acla 248, 12. Winegrad, S. (1971). Studies of cardiac muscle with a high permeability to calcium produced by treatment with ethylenediaminetetracetic acid. J. Gen. Physiol. 58, 71. Yamada, S., Yamamoto, T., Kanazawa, T., and Tonomura, Y. (1971). Reaction mechanism of the Ca2+-dependent ATPase of sarcoplasmic reticulum from skeletal muscle. VI. Co-operative transition of ATPase activity during the initial phase. J . Biochem. (Tokyo) 70, 279.

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The Role of Periaxonal and Perineuronal Spaces in Modifying Ionic Flow Across Neural Membranes* W. J . ADELMAN, J R . and Y . PALTI Laboratory of Biophysics, National Institute of Neurological Diseases and Stroke, National Institutes of Health, U.S. Public Health Service, Bethesda, Maryland, and Department of Physiology and Biophysics, The Aba Khoushy School of Medicine, Israel Institute of Technology, Haifa, Israel

I. Introduction . . . . . . . . . . . . . . . . . . 11. External Potassium Ion Accumulation . . . . . . . . . . . A. Accumulation Associated with Axonal Activity . . . . . . . B. Accumulation Associated with Membrane Currents during Voltage Clamping . . . . . . . . . . . . . . . . . . C. Accumulation Associated with Neuronal Activity . . . . . . D. Accumulation Associated with Brain Activity . . . . . . . 111. Significance of Potassium Ion Accumulation for Axon and Neuron Behavior . . . . . . . . . . . . . . . . . . . IV. Significance of Potassium Ion Accumulation in Brain Behavior . . . . Appendix A: Model for Ion Accumulation in Periaxonal Space . . . . . Appendix B: Calculation of [K.] Changes upon Voltage Clamping the Squid . . . . . . . . . . . . . . . . . . . . Giant Axon Appendix C: Reconstruction of a Membrane Action Potential . . . . . . References . . . . . . . . . . . . . . . . . . . . .

199 201 201

203 210 215 220 223 226 229 23 1 233

1. INTRODUCTION

The composition of the ionic environment has a marked effect on the behavior of cells in general, and of neurons and axons in particular. It

* This article was prepared at Bethesda, Maryland, with sponsorship of the National Institutes of Health. Reproduction for the purposes of the United States Government is permitted. 199

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has generally been assumed that the ionic composition external to the cell membrane is identical or very similar to that of the bulk solution (cf. Kuffler and Potter, 1964). This assumption is probably justified in systems where cell membrane ionic fluxes or currents are very small or negligible. However, it was first suggested by Hodgkin and Huxley (1952b, p. 493) that the [K] close to the axolemma is different from that in the external solution. Frankenhaeuser and Hodgkin (1956) demonstrated that the apparent potassium concentration in the medium immediately outside the squid axon membrane increases during repetitive firing of nerve impulses. From phenomenological indices, Frankenhaeuser and Hodgkin concluded that, during a repetitive spike train, the potassium concentration in the periaxonal space (Geren and Schmitt, 1953, 1954) increases from 10 mM to over 20 mM a t firing rates of up to 100/second. This accumulation was explained as the result of the difference between potassium transference numbers through the axolemma and the Schwann layer. A good correlation was found between the experimental results and those predicted by a model of a multicompartmental system in which ion accumulation occurs during current flow. Using a similar model (Appendix A) and numerical methods, Adelman and Palti (1969b, 1972) computed the changes in potassium ion concentration in the medium just outside the squid giant axon membrane, [KJ, during voltage clamp pulses (see Appendix B). Upon complete membrane depolarization, [KB]was found to increase from its normal value of 10 mM to over 100 mM with time constants on the order to 10 msec. During hyperpolarization, [K,] decreased. However, the rates of decline were much slower than the rates of increase, since under hyperpolarizing conditions membrane conductances and thus membrane currents are much smaller than during depolarization. In general, these computations indicated that either accumulation or depletion of [K,] could be theoretically predicted to occur as a function of the direction, amplitude, and duration of the membrane potential changes which determine the currents flowing across the axon membrane. Potassium ion accumulation is found in other neural systems with anatomical features having the properties of constrained extracellular spaces. In vertebrates, nonmyelinated fibers are found in Schwann cell invaginations (Nageotte, 1922; Gasser, 1955; Hess, 1956; Robertson, 1957a). Repetitive activity in mammalian nonmyelinated axons results in an increase in the potassium ion concentration in a region just external to the excitable membrane (Ritchie and Straub, 1957). Orkand, Nicholls, and Kuffler (1966), Baylor and Nicholls (1969a), and others, have presented evidence suggesting that the potassium ion concentrations external to the membranes of neurons and glial cells of Necturus and the leech, respectively,

PERIAXONAL AND PERINEURONAL SPACES

20 1

are markedly altered during repetitive firing. Barry and Hope (1969a) have theoretically analyzed the changes in ion concentration, potential differences, and volume flow in multicompartmental membrane systems where the ion transference numbers are different a t the various interfaces between the subcpmpartments. They also demonstrated experimentalIy (Barry and Hope, 196%) that such changes occur when an electric current is passed through the membranes of Cham cells. This article examines the evidence for ionic concentration changes in spaces and layers adjacent to excitable membranes and presents a general model system '(Appendix A) for the analysis of such phenomena. I n addition, the significance of these effects on the behavior of a variety of neural elements is discussed. II. EXTERNAL POTASSIUM ION ACCUMULATION A. Accumulation Associated with Axonal Activity

When a neuron or axon is excited to fire an impulse, the membrane potassium conductance increases manyfold. As a result there is a transient flow of potassium ions down their electrochemical gradient, i.e., outward. The quantity of potassium ions which flows out of 1 cm2 of membrane during a single impulse has been estimated to be about 4 X 10-l2 M in squid axons (Shanes, 1951, 1954; Keynes, 1951; Keynes and Lewis, 1951), about 10W2 M in unmyelinated mammalian fibers (Keynes and Ritchie, 1965), and about lo-'* M in leech neurons (Baylor and Nicholls, 1969a). The increase in potassium ion concentration in the external space due to this efflux was first evaluated theoretically and experimentally in squid axons by Frankenhaeuser and Hodgkin (1956). They used the amplitude of the action potential undershoot as a measure of [K,] changes. Their measurement was based on the finding that during this period the nerve membrane is very sensitive to potassium ions and therefore acts very much like a potassium ion electrode. This high sensitivity to potassium ions during the undershoot is due to the fact that the membrane potassium conductance is very high while the membrane sodium conductance has already been attenuated by the inactivation process (Hodgkin and Huxley, 1952~).As the rate of change of E Mat the peak of the undershoot is zero, the net membrane current should also be zero, and the Goldman relationship (Goldman, 1943; Hodgkin and Katz, 1949) may be applied to estimate the membrane potential. Thus, since at the minimum of the undershoot the membrane permeability t o potassium ions exceeds that to all other ions, one expects the membrane potential to be mainly a function of the gradient of potassium ions across the membrane.

2 02

W. J. ADELMAN, JR. AND Y. PALTI

Using these criteria, Frankenhaeuser and Hodgkin (1956) concluded that after a single impulse the change in the apparent external potassium concentration, which they assumed to reflect [KJ, was only 1-2 mM. However, during a train of impulses potassium ions began to accumulate externally. The time constant for this accumulation process was on the order of hundreds of milliseconds. The final [K,] values obtained were directly proportional to the rate of firing. K. concentrations rose to steadystate values of about 20 mil4 at firing rates of about 100/second. These experimental data were found by Frankenhaeuser and Hodgkin to be best fitted by predictions based on a multicompartmental model with a space thickness of 280 A (cf. Appendix A). Ion concentrations in the immediate proximity of the membrane may also change as a result of a slow and incomplete diffusion of ions away from the membrane surface and its immediate neighborhood, This poor mixing results in a so-called “unstirred layer” external to the membrane in which concentrations may differ from those of the bulk solution, The kinetics of ion accumulation or depletion in such an unstirred layer can be treated by mathematical models similar to those of a multicompartmental system. Because the multicompartmental model fits the experimental results better than the unstirred layer model, Frankenhaeuser and Hodgkin (1956) rejected the unstirred layer model. Greengard and Straub (1958) in their study of the afterpotentials of mammalian nonmyelinated C fibers suggested that the negative afterpotential results from a transient increase in the external potassium concentration in the space between the excitable membrane and the Schwann cell. They stated that an increase of about 3.3 mM in [Ka]would be sufficient to account for the initial amplitude of the negative afterpotential and that this would correspond to a net potassium efflux of 4.9 X mole/cm2 per impulse. As the negative afterpotential declined with a time constant of about 150 msec, Greengard and Straub suggested that the external barrier was highly impermeable to potassium ions. They suggested that there are additional external barriers to potassium than the Schwann cells. These workers considered the positive afterpotential as being representative of an active uptake of potassium ions by the axons. They cited as evidence for this active process the Qlo of 2.0 for the positive afterpotential, which was compared with an expected Qlo of 1.0 for a passive flux process. The additional external barrier proposed by Greengard and Straub (1958) may be made up in part of a highly hydrated gel (Robertson, 1957b), as well as the dense-staining material often referred to as the basement membrane (Gasser, 1955; Robertson, 1957a). In 1960, Narahashi and Yamasaki presented evidence that the negative

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afterpotential in cockroach axons can also be interpreted as resulting from an accumulation of potassium ions in a space in the immediate vicinity of the external surface of the nerve membrane. The rise in [KJ was estimated to range between 0.9 and 3.0 mM. 6. Accumulation Associated with Membrane Currents during Voltage Clamping

While the evidence for the effect of potassium ion accumulation in the periaxonal space on membrane parameters of various preparations is indirect, somewhat more direct evidence for these effects may be obtained from squid axon behavior under voltage clamp conditions. For the past 20 years, the voltage clamp method has been used t o obtain accurate determinations of membrane conductance parameters under a variety of conditions. Potassium current, I K , is given by (Hodgkin and Huxley, 1952d) IK = gKn4(EM

- EK)

(1)

Since, for the membrane, E K is a function of [KJ, not of bulk solution [IZ],IK is affected by depletion or accumulation of potassium ions in the space. To account for the discrepancy between predicted and experimental changes in membrane potential induced by changing external potassium ion concentration, Hodgkin and Huxley (1952b) suggested that possibly “the potassium concentration in the immediate vicinity of the surface membrane is not the same as that in the external solution.” This hypothesis was also advanced by Hodgkin, Huxley, and Katz (1952) t o explain the slow decline in outward potassium currents with strong depolarizations of voltage clamped squid axons. Normally, when the temperature was low, the delayed outward potassium ion current rose to a maintained steady-state value during mild depolarizations in the voltage clamp. In order to support their explanation of the changes in the undershoot associated with repetitive firing, F’rankenhaeuser and Hodgkin (see Fig. 14 in Frankenhaeuser and Hodgkin, 1956) referred to membrane current records obtained in 1949 during voltage clamp experiments on Loligo axons. These records indicated that the decline in steady-state potassium current was a function of pulse duration and amplitude. They interpreted this result by assuming that a diffusion barrier is found between the axolemma and the bulk solution. As a result, potassium ion flow from the axoplasm to the periaxonal space leads to accumulation of potassium ions in this space. This accumulation reduces the potassium ion driving force

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across the axon membrane and decreases the reversal potential for the potassium current. Let us analyze this phenomenon in detail. When a membrane is depolarized (El) in the voltage clamp for long periods (about 10 msec), the sodium conductance is completely inactivated while the potassium conductance remains high. Under these conditions, when the potential is stepped back to a more polarized value (E2), the membrane potassium conductance turns off as parameter n tends toward a new value with time constant rn. As a result, after the initial step change due to the potential step, the potassium current turns off. This decaying potassium current has been termed the “tail” current (Hodgkin and Huxley, 195213). Since this tail current is a potassium current, it is given by: Itail

= IK = gK(E?d -

ER)

(2)

The initial value of the tail current is determined by the product of the potassium conductance just before the step in membrane potential (at El) and the difference between the membrane potential after the step and the value of the potassium reversal potential (Ez - EK).Thus, when (Ez - E K )is negative the tail current is inward; when the two potentials are equal, the tail current is zero. For a nerve a t rest, EK is about -90 mV. Therefore, when the membrane is first depolarized and then repolarized again to the resting potential (- 60 mV), the tail current should initially be outward and decline with a time constant of about 5 msec. Frankenhaeuser and Hodgkin (1956) found that after prolonged depolarizations of about 30 msec the tail current was inward. They interpreted this finding as a result of the reversal of the sign of (EM - EK)as potassium ions accumulated in the space and as EK became less negative. These experiments have been confirmed and extended (Adelman and Palti, 1972; Y. Palti, W. J. Adelman, Jr., and J. P. Senft, 1972, in preparation). These workers found that tail currents following very short depolarizing pulses were outward; following longer depolarizing pulses they were inward, having reversed at some finite pulse duration. In a similar manner, it was shown that E K is a function of the amplitude of the potassium conductance. In order to measure the time course of the potassium ion accumulation for different depolwizing voltage clamp pulses, Y. Palti, W. J. Adelman, Jr., and J. P. Senft (1972, in preparation) had to determine the values of tail currents for depolarizing steps of short duration (about 0.5-1.0 msec). However, at these times sodium conductance does not reach complete inactivation, so that the tail current is determined by both the sodium and potassium conductances. Therefore, in order to obtain pure potassium current tails the sodium current was eliminated. This was done by replacing

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' I

Fra. 1 . Membrane potassium currents, Zg, recorded from a voltaged clamped squid giant axon. The currents were elicited by stepping membrane potential, EM,from the resting value of -60 mV to a value of E l = 0 mV and then, 3 msec later, to: EZ= -54 mV (A), Es = -69 mV (B), and Ez = -94 mV (C). The curves show the steplike changes in ZK elicited by stepping E Mfrom the value of E l to El and the tail current which follows. The continuous thin line gives the zero current level. Note that when Ez = -54 mV the potassium tail current is outward, indicating that E1 is more positive than EL<.When EX = -69 mV the tail current is almost zero, indicating that EK = Ez. When Et = -94 mV the current is inward, indicating that Et is more negative than EK.

sodium ion by an impermeable ion or by the use of a drug such as tetrodotoxin (TTX) which specifically inhibits the sodium conductance without influencing the potassium conductance (cf. Narahashi, 1971 ; Cuervo and Adelman, 1970). Figure 1 illustrates a typical example of tail currents recorded from a voltage clamped axon. In Fig. 1A the tail current was generated upon stepping the membrane potential, EM, first to a value El = 0 mV and then to a new value Ez = -54 mV. In this case E2 was more positive than EK (normally -80 to -90 mV). Therefore, the tail I K which is given by: IK =

BKn4(E2- E K )

(3)

was positive, i.e., outward, as expected from the previous discussion. In Fig. lC, E2 = -94 mV so that (E2- E K ) was negative. It can be seen

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W. J. ADELMAN, JR. AND Y. PALTI

that the tail current is inward in Fig. 1C. When Ez = -69 mV = E K , the tail current was negligible (Fig. 1B). Since in the above case both IN^ and IL were negligible (IL = 10 pA/cm2), the recorded membrane current reflected I K . The curve relating the instantaneous values of the tail currents t o Ez was linear. The zero crossover point of this curve was taken t o correspond to the value of E K a t the time of the end of El (i.e., at tl). For any t l , the instantaneous current-voltage relation between I t a i l and Ez was practically linear (cf. Fig. 12, in Hodgkin and Huxley, 195213). This linear relation may be interpreted, on the basis of Eq. (3), as being due t o a constancy of both j i ~ and n ~ EK for any specific t l . Therefore, the observed changes in the slope and zero crossover point of the relation with increasing t l must have been due to a dependency of a t least one of the three parameters (OK, n, or 4 ~on) 11. Let us accept for the moment the Hodgkin and Huxley definition of j i K as a constant. Since r,, is relatively large, n changes slowly upon a step in membrane potential. Therefore, immediately after EMwas stepped from E l to Ez, i.e., a t the time I t a i l was determined, the value of n was still typical of E l rather than Ez. Thus the value of n4 during the initial phase of the tail was independent of EZ, and can be taken to be the same for all the points on the graph. Since QKn4is constant, EK becomes a direct function of t l . Since it was EK which determined the zero cros~overof the instantaneous current-voltage relations (see Eq. a), the shifts of the zero crossover points were used to determine the shifts of E K with increasing tl. N.Palti, W. J. Adelman, and J. P. Sneft, (1972, in preparation) showed that the attenuation of I K with time was not accompanied by decrease in g K . This was demonstrated by the increase in the slope of the line relating the instantaneous value of the tail current versus E M as tl increased (tl from 0 to 10 msec). The slope of the line for t l = 50 msec was equal to the slope of the line for tl = 10 msec; this indicates that the potassium conductance had become saturated. From similar data Palti and co-workers concluded that the increase and eventual saturation of the potassium conductance was in agreement with the Hodgkin and Huxley (1952d) axon model. The data indicated that the conductance decreased slightly only after very long pulsing (tl = 200 msec) . Thus, the droop of I K with pulse durations up to 20 msec must have been due to a change in only EK.These changes in E K reflected [KJ values up to 100 mM. The potassium current reversal potential, E K ,is usually assumed to be given, to a good approximation, by the potassium ion equilibrium potential, VK (Hodgkin and Huxley, 1952a) :

V K = (RT/ZF) ln([Kont]/[KiJ)

(4)

Therefore, the observed shift of E K toward a more depolarized membrane

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PERIAXONAL AND PERINEURONAL SPACES

-100

00

-50

-

+$-

-20

-

[Ksl mM 20

I

- I0 0

40

5

10

15

L,

20

25

30

50

10

msec

FIQ.2. Changes in the potassium reversal potential of the squid axon membrane as a function of duration ( t , ) of a depolarising prepulse. E K was determined from the zero current crossover point of curves relating the amplitude of the instantaneous current (obtained by stepping membrane potential in the voltage clamp from a depolarizing value, El, to a value more negative than E2) and the membrane potential El. Values of El varied between -4 mV and +6 mV. The different symbols represent values obtained from 7 different axons. The curves (solid lines) represent computed values of E g resulting from increases in [KJ during depolarization (see Eq. 15, Appendix A). The method of computation is described in Appendix B. In the computation El = 0 and PK,= lo-'. Space width was 100 d in the lower curve, 200 d in the middle curve, and 280 d in the upper curve. The curve which originates a t the lower left is the computed [KB]corresponding to the EK change for a lOOb space. From the scale for the [K.]'s shown at the right in the figure, note that EK changes with time constants of the order of 5-10 msec, [K.] reaches values of more than 80 mM, and Sfold increase.

potential value implies that either [Kout] increases or [Ki,] decreases. However, [Ki,] is about 300 mM; for external ASW, [Kout]is 10 mM. Thus, for example, a 20 mV change in EK could be due either to a 190 mM change in [Ki,] or to a change of only 12 m M in [KOut]a t the external surface of the axon. Adelman and Palti (1972) concluded on the basis of this consideration and from the predictions of their multicompartmental model (see Appendix A), that external [K] rather than internal [K] changes. Typical shifts of the apparent Eg (experimentally determined on the basis of the zero current crossover point) as a function of time in seven axons are illustrated in Fig. 2. In order to account for these changes in apparent [Kout],Adelman and Palti (1972) assumed that potassium ions accumulated in the periaxonal space. Therefore, the apparent [KOut]was represented by the potassium ion concentration in the space, [K,]. The corresponding values of [KB]computed for [K,] = [KO]by means of equation 4 (assuming that the effective change is not in the internal axoplasm*), are given on

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W. J. ADELMAN, JR. A N D Y. PALTI

the right of Fig. 2. The changes of E K and [IL] were exponential, with a time constant of about 10 msec. For depolarizations of about 60 mV the apparent [KJ approached values on the order of 100 mM (a 10-fold increase). The experimental values were seen to agree well with predictions based on the multicompartmental model (Appendix A) when the space thickness was taken as 200-300 A (Adelman and Palti, 1972). Adelman and Palti (1972) also demonstrated that, for a given duration of membrane depolarization ( 2 1 = 29 msec), the value of E K is directly related to the magnitude of the potassium current density just before the end of El. The value of E K was determined from the potential for the zero current crossover point of the curve relating the instantaneous value of the tail current to membrane potential, El. For example, E K changed from -70 mV (when El = -50 mV) to -20 mV (when El = +SO mV). Thus, the magnitudes of potassium ion accumulation in the space and the E K shift were shown to be proportional to the current and degree of depolarization during the pulse. Previously, we discussed the droop or attenuation of I Kduring the course of relatively long depolarizations in terms of a change in [K] a t the axolemma surface. This phenomenon, which is difficult to demonstrate a t low (0-4°C) temperatures, becomes quite pronounced and fast in Loligo pealei axons when the temperature is raised about 10°C. Similar attenuation of potassium currents was observed by Villegas (1971) in axons of Doryteuthis plei a t about 2OoC, the natural temperature of the sea water in which these Caribbean squid live. Figure 3 illustrates a computed IK for a given membrane depolarization The dashed line of Fig. 3 plots computed membrane current when the potassium ion accumulation a t the axon surface is ignored. The dotted line plots computed membrane current when potassium ion accumulation a t the external surface of the axon is considered in the computation (see Appendix B). The figure clearly shows that when [I<J changes are introduced into the Hodgkin-Huxley equations a much better fit to the experimental data is obtained. Palti and his co-workers (in preparation) also determined experimental and computed membrane currents for a higher temperature. The computed results again were shown to fit the experimental data better when [KJ changes were considered than when they were ignored. In addition, Palti and his co-workers tested the assumption of an external diffusion barrier in the following manner. Axons were exposed to both *When the membrane potential was clamped to the Et value given in Fig. 2. IK was on the order of 1 mA/cm*. Such a current density flowing out of a 500 p axon for 10 msec would change the average [Kin]by less than 0.1 mM.

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PERIAXONAL A N D PERINEURONAL SPACES

hypertonic and hypotonic external solutions in an effort to shrink or swell both the Schwann cells and the giant axon. I n this way the periaxonal space could be either enlarged or made smaller. The kinetics of potassium ion accumulation were measured in both cases and the results were again compared with model predictions. It was concluded that, in accord with model predictions, potassium ion accumulation was altered in the right direction and by the appropriate magnitude by the physical alterations brought about in the periaxonal space. Up to this point we have considered evidence for accumulation of potassium ions in the periaxonal space during membrane depolarizations of biogenic or external origins. It would seem logical to assume that if the current direction is reversed, i.e., when a hyperpolarizing current flows through the membrane, the potassium ion concentration a t the external surface of the membrane [KJ should decrease. Although no direct evidence for such a phenomenon has been found, Adelman and Palti (1969b) reported an effect indicating that this is indeed the case. The inward sodium 2-

I -

0-

-I

-

-2

1 ~~

0

12

24

36

40

msec

FIG.3. Experimental and computed axon membrane currents generated by stepping

EM from a holding potential value of E M = -60 mV to E M = 0 mV. Temperature = 3°C. -, Experimental membrane current record (axon 70-65); -, computed membrane

..

current curve generated by means of the Hodgkin-Huxley (1952d) equations; . . , computed membrane current curve generated by means of the Hodgkin-Huxley equations modified to include the effects of changes in [K.] (see Appendix C). Ordinate gives arbitrary current scale. Maximal IK was normalized t o 2 units for all curves. Only the experimental inward sodium current is illustrated. Note the droop in the experimental curve and in the computed curve which includes the [KB]effect. Note also that while the tail predicted by the Hodgkin-Huxley equation is outward, the experimental current is inward in agreement with the predicted Eg shift.

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W. J. ADELMAN, JR. AND Y. PALTI

current initiated upon depolarization of squid axon membrane was shown to be an inverse function of external [K] (Adelman and Palti, 1969a). Therefore, when other conditions are kept constant, this inward sodium current can serve as a measure of external potassium concentration. When an axon is hyperpolarized, sodium inactivation approaches zero within about 10 msec (Hodgkin and Huxley, 1952c,d). However, Adelman and Palti (196913) found that if hyperpolarization is continued for much longer spans, the values of peak inward sodium current slowly increase, with time constants on the order of 100 msec. Using the multicompartmental model (Appendix A), Adelman and Palti (196913) computed the changes in [K,] during membrane hyperpolarization (see Appendix B). [Ks] was found to decrease with time constants in the order of 100 msec. When the effect of external potassium concentration on sodium current was considered (Adelman and Palti, 1969a), the predicted decrease in [KJ had the right time course and was of the right amplitude to account for the experimental increase in the sodium current. C. Accumulation Associated with Neuronal Activity

On the basis of the Frankenhaeuser and Hodgkin (1956) findings on squid giant axons, numerous workers in the CNS and other fields began a search for similar phenomena in other systems, Since neurons are separated from e2ch other or from satellite cells, such as glia, by aqueous spaces about 150 A in width (Kuffler and Potter, 1964; Coggeshall and Fawcett, 1964; Orkand et al., 1966), one might expect that during repetitive firing potassium ions would accumulate in these spaces in a manner similar to that demonstrated for the corresponding squid axon spaces. Orkand et al. (1966) investigated this possibility, using the resting potential of amphibian glial cells as an indicator of changes in potassium ion concentrations in the extracellular clefts or spaces. The method was based on the finding that the glial membrane potential is very sensitive to the extracellular potassium ion concentration (Kuffler et al., 1966). They found that during repetitive firing of nerve impulses the glial cells depolarize slowly. This depolarization, which was proportional to the rate and duration of stimulation of neighboring neurons, was interpreted as resulting from potassium ion accumulation in the perineuronal spaces during activity. This conclusion was reached after careful evaluation and rejection of the other possible depolarizing mechanisms. Upon firing at rates of lO-lOO/second, potassium ion concentration was calculated to increase from the normal value of 3 mM to over 20 mM with time constants on the order to 100 msec. The rates of decline of potas-

PERIAXONAL A N D PERINEURONAL SPACES

21 1

sium ion concentration were found to be much slower; the time constant of the decline was on the order of seconds. I n 1969, Baylor and Nicholls (1969a,b) confirmed the potassium ion accumulation by applying the Frankenhaeuser and Hodgkin method (1956) to the leech nerve. They used the values of the membrane potential during the undershoot of the nerve action potential to estimate the external potassium ion concentration. They found that after a single impulse the amplitude of the undershoot of a second action potential was decreased by a factor corresponding to a peak increase of external potassium ion concentration of about 0.8 mM. The excess of potassium declined exponentially with a time constant of about 100 msec. During trains of impulses, [KJ increased with a time constant of about 100 msec to values of 10-20 mM, i.e., more than double the prestimulation concentration. The fact that leech glial cells actually act as barriers that restrict potassium ion flow and thus lead to potassium ion accumulation in the neuronglial cell spaces was demonstrated by Baylor and Nicholls (1969a). By removing the glial cells around neurons they were able to eliminate the changes in the action potential undershoot associated with the neuron activity. It thus seems that the undershoot changes, which were shown t o reflect, [K,] changes, depend on the presence of an intact glial cell barrier. This finding serves as an additional justification for use of the multicompartmental model in describing these phenomena. Electrical recordings have been made from cortical glial cells in the sigmoid gyri of cats by Grossman, Whiteside, and Hampton (1969). These authors demonstrated that these glial cells were not directly excitable by intracellular depolarization. However, these cortical inexcitable cells would show slow depolarizations of from 1 to 5 mV during spindle bursts of the electrocorticogram. These depolarizations also occurred whenever thalomocortical volleys of impulses were evoked by stimulation of the nucleus ventralis lateralis (VL) of the thalamus (8/second). The time constant of the glial cell depolarization was estimated to be about 100 msec. VL stimulation at 50/second produced a maximum glial cell depolarization of 18 mV in 4-5 seconds. Grossman et al. (1969) concluded that these glial cell depolarizations were the result of I<, accumulation in perineuronal spaces. They calculated that the 18 mV depolarization was the equivalent of a [KO]increase of about 3.1 meq/l. This corresponded to a raise in [KO]from a normal value of 3.0 meq/l to an experimental value of 6.1 meq/l. The implication of the above findings for neuron-neuron and neuronglial cell interactions will be discussed later. Nicholls and Baylor (1968) and Baylor and Nicholls (1969b) reported that during hyperpolarization of leech CNS neurons resulting from a

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preceding train of impulses, the sensitivity of the membrane potential to small increments of external potassium ion concentration was enhanced by a factor of about three. This hyperpolarization was not accompanied by a decrease in [KJ, or by an increase in total membrane resistance and could not be mimicked by applying potassium ions to a neuron hyperpolarized by passing a current into its cell body. Therefore, Baylor and Nicholls rejected explanations of this phenomenon which were based on electrodiff usion-membrane conductance changes. They suggested that, when the extracellular potassium ion concentration is increased, a membrane pump transports ions into the cell a t a higher rate and thereby becomes less electronegative, i.e., more neutral. However, they suggested this explanation only as a remote possibility. When the changes of [KJ are taken into account, the hypersensitivity of the membrane potential to external potassium ions can be explained by passive ionic mechanisms. During hyperpolarization, when the membrane is hypersensitive to potassium ions, the membrane conductances differ from those of a resting membrane. To compute the conductance changes associated with hyperpolarizations of up to 30 mV, i.e., those found during the hypersensitive period, we shall assume that the membranes of leech CNS neurons behave, a t least qualitatively, like the membrane of the squid axon. Under this assumption we can use the Hodgkin and Huxley (1952d) equations that describe the potassium and sodium membrane conductances as functions of membrane potential. Because the rate of change of potential during the hyperpolarizing period is very slow with respect to the rates of change of the sodium and potassium conductances, g N a and gK, we can use the steadystate ionic conductance relationships of Hodgkin and Huxley (1952d) to compute membrane conductances. These are : gK =

(5)

gKn:

gNa = gNamLh,

(6)

where g K and g~~ are constants representing the maximal ion conductances and

+ + Pm) +

n m = an/(a, m , = a,/(a, hm

= ah/(ah

Pn)

Ph)

The a’s and p’s are voltage-dependent rate constants. Substituting the proper values of membrane potential in the equations, which give the voltage dependency of the a’s and P’s, we can solve for n,, moo,and h,. Substituting these in Eqs. (5) and (6), we can compute the ratio between the potassium and sodium conductances. This ratio has the same value as

213

PERIAXONAL AND PERINEURONAL SPACES

TABLE I PREDICTED CHANGESIN MEMBRANEPOTENTIAL (AEY) COMPUTEDFOR Two DIFFERENT INCREMENTS OF EXTERNALPOTASSIUM CONCENTRATION AS FUNCTIONS OF INITIAL MEMBRANE POTENTIAL"

A E M ( ~ Vfor ) [KO] increase of EM (mV) -60 -70 -80 -100

PK/PN& 35.4 104 294 1520

1mM

2mM

0.4 1.7 2.0 2.3

1.7 2.9 3.9 4.2

The P K / P N *permeability ratio was computed for each membrane potential by means of the

Hodgkin-Huxley (1952d) equations. AEM was computed for each E M by the Goldman, Hodgkin, and Katz relationship (see text) using the appropriate permeability ratio and external potassium concentration (resting concentration is 10 mM). The value of internal potassium concentration was taken as 274 mM. Note that the greater the hyperpolarization of the membrane, the greater is its sensitivity to changes in external potassium concentration.

the potassium-sodium permeability ratio, Px/PNa. Table I gives some computed permeability ratios for different membrane potentials. It is seen that a s the membrane hyperpolarizes, the P K / P N ~ratio increases. We can now compute the predicted membrane potential, as external potassium concentration increases above the normal value of 10 mM (for the squid), by using the Goldman-Hodgkin and Katz relationship (Goldman, 1943; Hodgkin and Kata, 1949) :

Chloride and other ion effects are neglected as a first approximation. Baylor and Nicholls (1969b) found the hyperpolarization of leech neurons to be due to a change in activity of an electrogenic pump. They also concluded that it was not accompanied by a decrease in [KJ, such a s is usually observed when neurons are hyperpolarized by the passage of an

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inward electric current. Therefore we can regard the membrane potential as the sum of a Nernst-Planck type diffusion potential and of a pumpgenerated potential. In a system of this kind, the Goldman, Hodgkin, and Katz relationship predicts the effects of ion concentration changes only on that part of the membrane potential determined by passive diffusion (Schwartz, 1971). Note however that g K and g N s are functions of the total membrane potential. The effect, if any, of changes in the potassium concentration on the activity of the electrogenic pump is unknown and we will assume that it is small, a t least as far as EM is concerned. Following Schwartz (1971), we can compute the changes in E Mwith increasing external potassium concentration by using Eq. 4, which bears only on a part of E M .Table 1 gives the changes in E M predicted by Eq. 7 when the external potassium concentration increases by 1 and 2 mM. The.computa~ from Table I under the assumption tion made use of the P K / P Nratios that for such small concentrations and EM changes the changes in ratios are relatively small. In Table I we see that when the membrane is hyperpolarized it becomes more sensitive to an increase in external potassium concentration, [KO]. For example, when E M = -60 mV a 2 mM increase in [KO] results in the same potential change as a 1 mM increase when E M = -70 mV. Thus, the predictions are in general agreement with experimental results. The increased sensitivity of the membrane to an elevation of external potassium ions during hyperpolarization is thus, most probably, the result of the relative increase in potassium ion conductance which makes the membrane a better “potassium ion electrode.” The reason that the hypersensitivity could not be mimicked by applying potassium ions to a neuron hyperpolarized by the passage of current becomes obvious when one considers the effects of such a current on potassium ion concentration in the space. It has been shown (Adelman and Palti, 1969b) that when a squid axon membrane is hyperpolarized by an inward going electric current, the potassium ion concentration in the space decreased with time constants on the order of 100 msec. [K,] decreased from values of 10-30 mM to practically zero in 200 msec. When potassium ion concentration is increased in the bulk solution under experimental conditions, the potassium ion concentration in the space increases relatively slowly (seconds). Therefore, the rate of removal of potassium ions from the space is almost equal to its rate of entrance, and the actual change in potassium ion concentration external to the neuron surface is very small. Thus, the actual changes in membrane potential obtained by applying potassium ions to a neuron hyperpolarized by an external current are small in comparison with those found in a neuron hyperpolarized by the action of an electrogenic pump (Baylor and Nicholls, 1969b). On the basis of their data, Baylor and Nicholls (1969b) concluded that there is no appreci-

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215

able decrease in [KJ during the hyperpolarization phase which follows a train of neuronal impulses. Trachtenberg and Pollen (1970) have measured the membrane time constant of neocortical glial cells and have found a value of about 385 psec. Measurements of the membrane capacity gave values of from 0.8 to 2.0 pF/cm2. From these values, they calculated membrane resistances of 200-500 ohm cm2. On the basis of this rather low value of membrane resistance, they assumed that glial cells are very permeable to ions, particularly K. In this regard, they envisioned glial cells as perineuronal potassium ion buffers, capable of preventing large increases in [K,] which might adversely affect neuronal function. The significance of this hypothesis will be discussed later.

D. Accumulation Atsociuted with Bruin Activity

1. SPREADING DEPRESSION

Another example of a neural effect which probably results from potassium ion accumulation has been described by Grafstein (1956, 1963), and Brinley, Kandel, and Marshall (1960b). These reports describe an efflux of potassium ions from brain neurons during spreading depression in the cerebral cortex of the cat and rabbit. The spreading depression which is accompanied by a slow potentia1 change can most probably be explained by an increase of potassium ion concentration in the spaces outside the neurolemma. Brinley et al. estimated the extracellular potassium ion concentration change to be a t least 7- to 8-fold. Local application of potassiumrich Tyrode’s solution was found to induce spreading depression, but the concentrations required under these conditions were about twice as high i.e., about 15 times normal. BureS, Buregovh, and Kfivhnek (1960) reviewed the literature pertaining to those agents that can induce spreading cortical depression. Among these are KC1, a variety of amino acids, strophanthin and metabolic inhibitors such as 2,4-dinitrophenolJ NaCN, and NaF. These effects can be blocked by the addition of divalent ions to the solutions containing the initiating agents. This review also gave support to the finding that the efflux of K from the cortical surface increased during spreading depression. An argument was presented for concluding that this K efflux was of intracellular origin. These authors also showed that certain metabolic changes were related to spreading depression. Brain glycogen and glucose decrease about 30% whereas brain lactic acid increases by about 100%. Creatine phosphate levels also decreased during spreading depression. It was concluded

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that these changes in metabolite concentrations were probably only a result of the increased metabolic effort necessary for membrane potential restoration. Freygang and Landau (1955) simultaneously measured cerebral cortex resistance and electrical activity in cat brains. They found that spreading cortical depression produces a 10-20% decrease in total cortical resistivity. This effect was most marked in the superficial layers of the cortex. Freygang and Landau suggested that these changes resulted from the depolarization of neural structures and the swelling of brain cells. This hypothesis is consistent with an electrolyte shift between brain cells and extracellular spaces during spreading depression which is accompanied by a significant water flow into the cells. Recently Fifkova and Van Harreveld (1970) presented evidence that glutamate is released into extracellular spaces during spreading depression in isolated chicken retinas. They stated that cellular depolarization resulting from extracellular glutamate accumulation would increase I<+release and add to the potassium accumulation process proposed by Grafstein (1956). 2.

EPILEPTIFORM SEIZURES

In 1870 Hulings Jackson (reprinted 1931) stated that in animals and man “convulsions result from excessive discharges of nerve cells.” I n 1947, Colfer and Essex determined by chemical analysis the concentrations of total electrolyte sodium and potassium in cerebral cortical neurons of rats and rabbits. These analyses were performed so as to follow the changes in brain electrolytes accompanying or following experimentally induced convulsions. Colfer and Essex found that either RIetrazol or electroshock convulsions in rabbits produced a consistent loss of brain cell potassium and an increase in intracellular sodium. About 50% of rats subjected to audiogenic convulsions exhibited similar shifts in brain electrolytes. The total electrolyte analyses of cortical tissue indicated that there was a possible accumulation of potassium in the brain interstitial fluid during and following such convulsions. In 1957 Feldberg and Sherwood were able to induce seizures in cats by injecting potassium ions into the cerebral ventricles. Tower (1960) has marshaled the evidence indicating that one of the basic precipitating causes of seizures in brains with epileptogenic lesions is a change of the ionic concentration in the extracellular spaces of the brain. In 1956, Granit and Phillips described a type of neuronal activity in the cerebellum which they named an “inactivation process.” Inactivation processes are marked by a surface-positive electrographic wave with sud-

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den bursts of neuronal activity. These bursts eventually cease and lead to a marked pause in spontaneous activity. One of the more interesting phenomena associated with the development and decline of an epileptic discharge is that it is recurrent, alternating between an active or ictal period and a less active or interictal period. During the ictal period, the tonic discharge becomes intermittent and enters a clonic phase. h$latsumoto and Ajmone Marsan (1964b) recorded intracelluIar potentials from cells in the anterior and posterior sigmoid gyri in the brains of cats that were activated to show epileptiform discharges following local cortical application of penicillin (1964a). They noticed that the clonic phase was marked by an increased repolarization of the membrane potential toward values recorded in the preictal period. The rate of appearance of large depolarizing waves decreased, but the duration of after polarizations increased. They suggested that the discharge was gradually inactivated and that this inactivation contributed to the discharge cessation. Long-lasting depolarizations, rather than true hyperpolarizing factors, were invoked to account for this inactivation. It has been known for some time that the hippocampus region of the brains of various mammals is very sensitive to seizure discharges (Gibbs and Gibbs, 1936; Green and Shimamoto, 1953). In 1961, Green and Maxwell, on the basis of an electron microscopical study, concluded that the dendrites of hippocampal neurons are separated from each other by extracellular spaces of only 100 A. These authors suggested that this small perineuronal space might accumulate potassium ions during and following sustained neuronal activity such that the raised extracellular potassium aided and maintained neuronal depolarization. Inactivation processes were aIso seen in the hippocampus (von Euler and Green, 1960a,b). Green, Maxwell, and Petsche (1961) noted that the inactivation process only occurred when the brain cells fired rapidly. On the average, the interspike intervals were usually about 10 msec (the “critical” period) in order for the process to be initiated. Once a few action potentials were fired a t frequencies above 100/sec there followed a general cellular depolarization and an accelerating burst of action potentials. Green et al. (1961) concluded that, “in the hippocampus, if ionic flow is significantly restricted by limited available space and close approximation of neuronal membranes, local variations in the ionic batteries of the neuronal membranes must occur (Frankenhaeuser and Hodgkin, 1956) and these conditions may vary with the particular conditions a t any locus, not only f2r the active neuron but for adjacent neuronal membranes a hundred or so Angstrom units away.’’ Tower (1965) has discussed a t length some of the difficulties in estimating functional extracellular spaces in brain tissue by tracer distribution tech-

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niques and electron microscopy. This article is important in that it suggests that some caution must be taken when attempting to define the dimensions of the perineuronal space in which phenomenological [KO]values are distributed. Kandel and Spencer (1961) have examined the relationship between the characteristics of afterpotentials in cat hippocampal neurons and repetitive firing. Microelectrode recording from these neurons (Kandel et al., 1961) revealed that, in contrast to motor neurons, hippocampal neurons are marked by an absence of a hyperpolarizing afterpotential, the presence of a depolarizing afterpotential and the tendency to fire in high-frequency bursts. Three classes of repetitive discharges were recorded : (1) brief bursts of 2-4 spikes with a spike frequency of up t o 400/second, (2) moderateduration bursts of 5-8 spikes with an underlying depolarization lasting 20-40 msec, (3) prolonged bursts lasting almost 100 msec with the major portion of the train exhibiting abortive spikes riding on top of a severe depolarization of from 20 to 30 mV. The moderately bursting and prolonged bursting neurons showed a few rather high amplitude short duration initial spikes which gave way to shorter, broader, and lower amplitude spikes. In the prolonged bursting neurons these later spikes were termed abortive as little more than a membrane potential oscillation was seen. Kandel and Spencer (1961) were able to demonstrate that the depolarizing afterpotential existed whether the spikes occurred spontaneously or were initiated by direct stimulation. They proposed a number of explanations for these afterpotentials. One of these invoked a perineuronal K accumulation mechanism such as has been described previously for the squid giant axon. They claimed that their experiments were compatible with this mechanism for the following reasons: (a) repetitive firing produces a cumulative depolarizing afterpotential, (b) spike height decreases as the depolarizing afterpotential increases, (c) the depolarizing afterpotential declines exponentially, and (d) the time constant of this exponential decay is independent of the number of previous repetitive spikes. In addition, they cited the anatomical evidence of Green and Maxwell (1959) that in the hippocampus cell bodies and dendritic processes are separated from other cells and processes by spaces of about 150 b. This constrained perineuronal space was considered to accumulate K ions with each discharge, thus raising [KB]to the level where neuronal depolarization occurred. In addition they suggested that the sodium conductance mechanism was inactivated by the potassium mediated depolarization. In this way they were able to account for spike attenuation throughout the repetitive discharge. However, Van Harreveld, Crowell, and Malhotra (1965) have claimed that rapid freezing techniques for nondestructive tissue fixation allow electron micrographs to be obtained which show interneuronal spaces

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219

in brain tissues with somewhat larger dimensions than Green and his collaborators have found. These spaces are several hundred angstroms wider. Previously, Van Harreveld and Schad6 (1960) had argued for a larger extracellular space than accepted by most electron microscopists. Sawa, Maruyama, and Iiaji (1963) recorded intracellular potential changes in hippocampal neurons during electrically induced seizures. They explained sustained seizures by invoking reverberating circuits and a general blockage of inhibitory synaptic transmission. Reverberation was postulated as being continuous until the normal inhibition was released from blockade. Zuckermann and Glaser (1968) were able to induce epileptiform activity in the hippocampus regions of cat brains by localized ventricular perfusion of high potassium solutions into the cerebrospinal fluid (CSF). They were able t o raise the CSF [K] from two to six times normal. They stated that this activity was the result of a combination of the effects of potassium depolarization on excitatory and inhibitory processes. I n accord with Tower’s hypothesis (1960), they stated: “The convulsive effect of single shocks delivered during the latent period of high K+-CSF perfusion suggests that if abnormal concentrations of K+ occur in the extracellular spaces of the hippocampus, trivial superimposed stimuli, ordinarily without any epileptic potential, might be able to induce a seizure.” Zuckermann and Glaser (1970) perfused the inferior horns of the lateral ventricles of cat’s brains with high [K] CSF solutions such that the dorsal hippocampi of these brains were constantly bathed in these solutions. Latent epileptogenic foci had previously been established in the hippocampal regions by the injection of irritants such as cobalt powder or alumina cream. While the latent foci did not result in epileptic discharges per se, subsequent perfusion with the high [K] CSF solution induced seizures. High [K] solutions also produced seizures in brains not pretreated with irritants, but the required concentration of potassium ions was higher and the latency for the appearance of the effect was longer. These authors suggested the possible existence of barriers between the CSF and hippocampal neurons. As the latencies for the onset of seizures were decreased and the percentage of successfully induced seizures was increased in animals with latent foci, it was concluded that these postulated protective barriers are lowered by the irritants or the foci such that equilibration of [K.] with [KCSF]occurs more rapidly and more efficiently. The possible anatomical basis for such protective barriers was suggested as being either perineuronal macromolecules or glial cells. In the discussion of his work on electrically induced seizure activity in the hippocampus of the cat, Zuckermann (1971) stated that as “an increase in extracellular [K:] following synaptic activation can be well demon-

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strated, it becomes quite likely that such a mechanism might be a major one in explaining an epileptogenic build-up.” Recently, Prince (1971) has been able to make recordings from glial cells in the cerebral cortex of the cat and demonstrate that these cells depolarized in synchrony with electrically induced epileptiform discharge activity. He was able to calculate that during these epileptiform discharges [KO]increased by as much as 9.6 meq/liter, and that in some constrained perineuronal spaces [K.] might increase by over 30 meq/liter. The most compelling evidence for potassium accumulation in brain interstitial space during epileptiform seizures has been presented by Fertziger and Ranck (1970). These workers used the Brinley, Kandel, and Marshall (1960a) method for estimating changes in [KO] by loading the brains of cats and rats with radioactive 42K and using the efflux of this isotope as an index of [K.] alterations. During and after either electrically induced or Metrazol-induced seizures, the 42Kefflux increased from two t o nine times the resting level. They calculated that this represented a potassium efflux of about 5 X 10-l2 mole/cm2 per impulse. By assuming that this efflux was into a perineuronal space of 300 A thickness, they predicted that each action potential would raise [K,] by 2 mM. Inasmuch as a large number of adjacent neurons can be taken as being simultaneously active, the effect on the general level of interstitial [K] should be profound.

111. SIGNIFICANCE OF POTASSIUM ION ACCUMULATION FOR AXON AND NEURON BEHAVIOR

The possible effects of extracellular potassium ion accumulation on neuron and glial cell activity have been discussed by numerous workers (e.g., Brinley et al., 1960b; Kuffler and Potter, 1964; Orkand et al., 1966; Nicholls and Baylor, 1968; Baylor and Nicholls, 1969a,b; Fertziger and Ranck, 1970; Prince, 1971). While a small increase in external [K] has relatively little effect on the membrane resting potential of leech neurons, it significantly affects their spontaneous activity and firing rates (Baylor and Nicholls, 1969a). This effect is probably the result of membrane depolarizations which are sufficient to influence the release of transmitter and the threshold of postsynaptic membranes. Furthermore, under a variety of conditions the effects of small changes in potassium ion concentration on membrane potential are amplified so that they result in changes in neuron activity patterns (Baylor and Nicholls, 1969b). The effect of potassium ion accumulation may also be partly due to the elevated sensitivity of the neuron membrane potential to external [K] during the hyper-

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22 1

polarization which follows a train of impulses or hyperpolarizations associated with the activity of other central neurons and sensory cells (in the leech). Such hyperpolarization affects the configurations of action potentials, thresholds, synaptic potentials, and the amounts of transmitter released, and may even block axon conduction. Therefore, since the potassium ion accumulation in perineuronal spaces markedly affects the magnitude of such hyperpoiarizations, it can be expected to affect all these membrane phenomena and may thus have a significant effect on neuron signaling by influencing frequency of firing, etc. Moreover, potassium ions liberated by one neuron may affect the membranes of numerous adjacent neurons. The magnitude of this neuron-neuron interaction may also depend on whether the neurons had previously been active. Thus it seems that the increases in [I<,] that occur physiologically are sufficiently large (Nicholls and Baylor, 1968) to influence, for example, the integrative function of a neuron that is recovering from previous activity. This is a nonsynaptic, potassium ion mediated mechanism. Since external [K] has been found to have marked effects on sodium pumping in rabbit nonmyelinated neurons (Rang and Ritchie, 1968) and since it also attenuates the sodium current generated when squid axons are depolarized in the voltage clamp (Adelman and Palti, 1969a), it would seem that [KB]changes play a major role in modulating neuronal activity. This dependence of I N s on [KO]has been shown to be mediated through a direct effect of potassium ions on parameter h (Adelman and Palti, 1969a). Thus, under a variety of conditions, one can expect a change in practically all the membrane electrical parameters as a result of [K] changes in the medium just external to the axonal membrane. Wright, Coleman, and Adelman (1955) correlated a gradual loss of excitability of lobster axons with a rise in extracellular potassium ions resulting from repetitive activity. Since increasing external potassium ion concentration was shown to attenuate g N a by decreasing the h parameter, it is possible that changes in [K,]are an integral part of sodium inactivation as defined by Hodgkin and Huxley (Palti and Adelman, 1969). In fact, in an axon perfused with dextrose and Tris C1buffer internally and zero [K] ASW externally, sodium currents are only slightly inactivated (Adelman et al., 1965). It was shown by Adelman and Palti (1969a) that the rate constants of sodium inactivation, ffh and P h , vary as a function of external [K]. a h was given as:

As a first approximation (see Adelman and Palti, 1969a, Fig. 7), the ratios between the values of Cyh at various [Ko]’sare independent of E M .On this

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W. J. ADELMAN, JR. A N D Y. PALTI

basis, we may assume that a h is practically independent of EM and is dependent only on [KO] or actually on [KJ. The apparent voltage dependency of a h , as given by the Hodgkin-Huxley equations, results from the voltage dependency of the potassium currents, which load or unload the space with potassium. Thus, we can substitute for E Many particular voltage value and arrive a t a general equation. Let us choose E M = -60 mV and substitute in Eq. (8). We arrive a t the following general equation for ah (Yh

=

0.126 - 0.065 log [KO]

(9)

With this assumption, Eq. (9) can be used with the other Hodgkin-Huxley equations to describe the axon behavior by substituting values of [K.] for [KO]. In contrast t o a h , o h was found to be dependent on [KO] only for membrane hyperpolarizations. For depolarizations, it remained dependent only on voltage (see Fig. 10 in Adelman and Palti, 1969a). Thus, it may be that the o h factor actually represents two separate factors, one of which is voltage dependent and another which is [K.] dependent. Note that the above derivation, and the conclusions which follow it, hold only for membrane depolarizations. During relatively strong membrane hyperpolarization, the immediate high rates of change of parameter h are not adequately described by the potassium ion-dependent rate constants. For a discussion of this point, see Adelman and Palti (1969b). Palti and his co-workers were able to reconstruct the membrane action potential by means of the Hodgkin-Huxley method (1952d) using the above relation for (Yh and the Adelman and Palti (1969a) [K] dependent equation for o h . I n this computation (see Appendix C) Palti et al. (1972) incorporated the various effectsof external [K], as manifested by [KJ, on membrane parameters ( a h , o h , E M ,and E K ) .Axon membrane responses reconstructed by the use of the standard Hodgkin and Huxley equations were compared with reconstructions using Hodgkin and Huxley equations modified to include the effect of the changes in [KJ, as described above. As previously noted by Hodgkin and Huxley (1952d), the unmodified reconstruction resulted in a marked deviation of the reconstructed action potential from the experimentally recorded response. This deviation occurred near the foot of the action potential. The deviation almost completely disappeared when the [Ks] changes were incorporated into the nerve equations. Reconstructed and recorded responses superposed quite well.. Therefore, the assumptions that potassium ions accumulate a t the external surface of axons and neurons and that the sodium inactivation rate constant is a function of [K.], not of EM, seem to lead to valid predic-

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tions. Note that for this case the membrane was never strongly hyperpolarized, so that &, was actually only a function of EM. Thus, one may assume that at least one of the two processes of the so-called sodium inactivation represented by the f f h rate constant actually reflects the effect of interferenre with the sodium “channel” by the potassium ions in the periaxonal space. The nature of this interferenre, which may be due to competition of potassium ions with sodium ions for sodium sites or to noncompetitive inhibition, must be determined experimentally. IV. SIGNIFICANCE OF POTASSIUM ION ACCUMULATION IN BRAIN BEHAVIOR

Some previously unexplained phenomena reported in the mammalian

CNS can be explained on the basis of potassium ion accumulation in perineuronal spaces. An example of a phenomenon which was not attributed to potassium ion accumulation but which could be satisfactorily explained on this basis is the slow depolarizing responses recorded from “idle” cells in the cat cerebral cortex. Iiarahashi and Goldring (1966) recorded intracellularly from these “idle” cells and found that they would depolarize by as much as 20 mV. Whenever a direct cortical response was elicited by electrical stimulation of the surface of the cortex, it was found the “idle” cells depolarized in synchrony with the negative shift of the surface steady potential accompanying spreading cortical depression. This synchrony was most apparent in recordings from “idle” cells in the cortical surface. If we consider the theory of Grafstein (1936) and the evidence of Brinley, Kandel, and Marshall (1960b) that spreading depression advances because of a general large efflux of potassium ions from depolarized neurons at the front of the spreading wave of depression, then we can assume that the “idle” cells’ membrane potentials reflect changes in the concentration of extracellular potassium ions in their surroundings. Thus the “idle” cells in the cat cortex behave similarly to glial cells surrounding neurons in the leech ganglion. Recently, Sypert, Oakley, and Ward (1970) have performed a singleunit analysis of propagated seizure in the pericrueiate (motor cortex) gyri of the cat. They found the tonic phase of a propagated seizure was marked by depolarizing waves which progressively enlarged and lengthened. Conromitant with these waves were high frequency bursts of action potentials which gradually decreased in amplitude until a sustained depolarization was reached. These authors concluded that the neurons were inactivated (Granit and Phillips, 19.56; von Euler and Green, 1960a,b; Green et al., 1961; RIatsumoto and Ajmone Narsan, 1964b) at the transi-

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tion from the tonic to the cIonic phase. Verification of the inactivation of the spike generator was obtained from the failure of antidromic invasion of these cells. They likened this inactivation to classical sodium inactivation (Hodgkin and Huxley, 1 9 5 2 ~ ). As Adelman and Palti (1969a) have presented evidence that increased external potassium leads to enhanced inactivation of the sodium conductance in squid axon, it is tempting to speculate that the inactivation seen in the work of Sypert et al. (1970) may have a similar basis. I n other words, both the depolarization per se and the accumulation of potassium ions may have additive effects in contributing to and sustaining the inactivation. Previously, we discussed the work of Trachtenberg and Pollen (1970) on glial cells. I n a subsequent paper, Pollen and Trachtenberg (1970) considered the relation of glial cells and [Ka] changes to the possible mechanism for focal epilepsy. These authors stated that epileptigenic foci are marked by intense fibrillary gliosis bordering neuronal tissue. They made reference t o the finding (Tower, 1960) that brain slices of scar tissue from epileptic patients were different from slices from normal brain in that they fail t o extrude Na+ and take up Kf. Pollen and Trachtenberg suggested that these abnormal cells formed a more impermeable perineuronal barrier to potassium diffusion away from the perineuronal space. This condition should result in very rapid rises in [K,] with neuronal activity and should be contributory to neuronal hyperexcitability and to epileptigenesis. In this regard, it is interesting to note that Fertziger and Ranck (1970) have taken the position that [K,] accumulation in cerebral cortical perineuronal spaces is of causal significance in the development of epileptiform seizures. These authors proposed a regenerative cycle relating [K,] accumulation to the ictal period of an epileptiform discharge. This model is based on some previous speculations of Green (1964). I n essence, the model proposes that a t some time and by some unstated mechanism there is a rise in the interstitial [K] sufficient to depolarize brain neurons as the potassium equilibrium potential is made more positive. They also propose that, as the neuron membrane is depolarized, chloride permeability is increased, as is the influx of chloride ions into the neuron. Thus, the chloride equilibrium potential is made more positive. These equilibrium potential shifts should make the reversal potential for the IPSP less negative and thus the efficacy of postsynaptic inhibition should be reduced. Combined with the general increase in excitability concomitant with mild depolarization, the general level of neuron firing rate should increase. This should further increase [IL], adding to thc regenerative nature of the process. Eventually, neuron depolarization should become profound enough to result in a depolarizing or cathodal blockage bringing the discharge to an end. Figure 4 illustrates the Fertziger-Ranck cycle. These authors caution

PERIAXONAL A N D PERINEURONAL SPACES

225

‘ t

Firing rate)

FIG. 4. Regenerative consequences of accumulation of interstitial K+. IPSP, inhibitory postsynaptic potential. From Fertziger and Ranck (1970). [The authors acknowledge with thanks permission granted by Drs. Allen P. Fertziger and James B. Ranck, Jr., and Academic Press to reproduce this figure from Experimental Neurology 26, 581 (1970).]

that this hypothesis says nothing about either the initial increase in neuronal excitability or the initial rise in [I<.]. They also state that the underlying pathology, the interictal episodes, and the spread of a seizure are not incorporated in the scheme. The Fertziger-Ranck cycle relates primarily to the regenerative all-ornone aspect of the initiation of epileptiform seizures. Up to some value of [I<.] the process is only localized or subthreshold, i.e., factors such as potassium diffusion out of the space and K influx into the neurons are more effective than those factors contributing to [Ka]accumulation. According to the scheme, a t some threshold [Ka]value, the mechanism should become regenerative and initiate a seizure. In the words of Prince (1971), “Neurons exposed to increasing [Kt] would tonically depolarize during the active phase of each cycle and repolarize during quiet phases. . . . It would still be necessary to postulate an inter-neuronal network to explain latency shifts, and sudden on-off switching of epileptigenesis. However, the decremental-incremental changes in latency (excitability) might be explained by the effect of gradually increasing concentrations of KO(initially increasing transmitter release and later blocking conduction in fibers) .” It has been known for many years (Merritt and Putnam, 1938) that the drug diphenylhydantoin (DPH) is useful in relieving and reducing the frequency of attacks of epilepsy. Only recently has a basic neurophysiological membrane mechanism been demonstrated for a D P H effect on peripheral nerve. Lipicky et aE. (1972) have demonstrated that DPH blocks the sodium conductance in the squid giant axon a t dosages in the 10-100 & range. At these dosages, the resting potential is not affected, and the potassium conductance is only slightly depressed. By decreasing the sodium conductance, D P H should reduce the excitability of nerve. It is also inter-

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esting to note that Fertziger et al. (1971) have demonstrated that 100 pM DPH stimulates potassium uptake by lobster axons. These authors have shown that the DPH-stimulated potassium influx is abolished by 2,4dinitrophenol, a metabolic inhibitor. These results suggest that DPH has a stimulatory effect on active ion transport by nerve, thereby reducing the extracellular potassium concentration. I t is interesting to note that Woodbury (1955) has presented evidence for DPH protecting against seizure discharges in rats on the basis of its action in stimulating the sodium pump in brain cells. The recent work by Lipicky et aE. (1972) and Fertziger et al. (1971) may provide us with the possible mechanism whereby DPH acts to reduce the incidence of epileptiform seizures. By both decreasing neuronal excitability and increasing active extrusion of sodium and uptake of potassium by both nerve and glial cells, DPH should help in reducing the effectiveness of the K, accumulation phenomenon discussed previously as of causal significance in the generation of epileptiform seizures. As these data are preliminary, the above-mentioned hypothesis must be considered as tentative. The existence of interstitial macromolecular barriers and of glial cell barriers external to the neurolemma have been postulated by Greengard and Straub (1958) for unmyelinated axons, and by Zuckermann and Glaser (1970) for hippocampal neurons. Therefore, it seems likely that the external barrier or multicompartmental model presented in Appendix A may be generally useful in describing ion accumulation phenomena in perineuronal extracellular spaces. APPENDIX A Model for Ion Accumulation in Periaxonal Space

1. GENERALCONSIDERATIONS

Let us consider a neuron or an axon as a system consisting of a relatively large volume, which we will call the cell, of any arbitrary shape completely surrounded by a very thin semipermeable layer. This layer, to be called the membrane, may be identified with either the axolemma or the neurolemma. External to the cell membrane is a thin layer (about 100-200 A in thickness) of aqueous medium to be called the space, surrounded by a boundary which restricts ionic flow from the space to the bulk solution. This boundary, to be called the external barrier, may be identified with the Schwann cell sheath, the external basement membrane, collagen fibers, and/or a variety of glial cells. External to the barrier is the bulk external solution.

227

PERIAXONAL A N D PERINEURONAL SPACES

Let an electric current, of either biogenic or external origin, flow from the cell t o the external solution or vice versa. If ion concentrations a t the two sides of the membrane are different or the transference numbers of an ion through the membrane and the external barrier are different, a different fraction of the total current will be carried by the specific ion into and out of the space. Thus the concentration of the ion in the space will change during current flow. The change in ion concentration will depend on the width of the space, on the direction, duration, amplitude, and rate of change of the current, and on the difference in the ionic transference numbers. After termination of an imposed or natural current, the concentration in the space will tend to return to its original value. However, as the return is exponential, a considerable length of time may pass before a new steady state is reached. Since the multicompartmental model yields predictions quantitatively closer to experimental observations than an “unstirred layer” model, we shall use this approach. 2. MULTICOMPARTMENTAL MODEL In the model system described above, the potassium ion concentration (mole/cm3) in the space, [K.], a t any time, depends on the initial steadystate potassium concentration, [K,,], and the integral, over time, of the potassium ion fluxes into and out of the space. Thus the increase or decrease of [K,] resulting from a difference between inflow and outflow of potassium ions from the space is given by:

dS[K,]/dt

=

(Mf(

+ Mff)/O

(10)

where e is the space thickness (cm), M f ( and Mff are the net fluxes (mole. cm-2.sec-1) of potassium ions between the cell and the space and between the space and the external solution, respectively. Flow into the space and potassium ion accumulation in the space are defined as having positive signs. Let us evaluate the terms M f ( and M f f .The net flux of potassium ions driven by diffusion and electrical forces through the membrane is given by (Hodgkin and Huxley, 1952b) :

Mf( = @ ( E M - EK)/F

(11)

where g K is the membrane potassium conductance (ohm-’), EMis the potential difference across the membrane (volts), EK is the reversal potential of the potassium current, and F = 96,500 C. per mole. Using the Hodgkin and Huxley (1952d) equations, gK can be solved as a function of both EM and time. However, note that gK has been shown to be a function

228

W. J. ADELMAN, JR. A N D Y. PALTI

of external potassium concentration (Adelman and Palti, 1969b). Even though we are mostly concerned with depolarizations, note that, during hyperpolarization, potassium current is carried mainly through the leakage conductance, so that under these conditions, g K is practically independent of time. The term M E can be considered as being the sum of two fluxes, one driven by diffusion forces, Md, and the other by electric forces, Me; thus: M"K - Me f Md (12) Assuming that a t any given moment potassium ion concentration in the 100-300 A-thick space is constant, the diffusion of potassium ions from the space to the external solution is given by (Frankenhaeuser and Hodgkin, 1956): Md = - 6[K,]*Pg,

(13)

where of the external barrier potassium permeability (cmasec-I). The magnitude of the potassium ion flux carried by an electric current flowing across the external barrier is given by (Adelman and Palti, 1969b):

Me = -IM*tK/F (14) where I Mis the total membrane current calculated as a function of E Mand time, and t K is the transport number of potassium ions in the external solution used. For any set of elements (i.e., resistances or conductances) in series, the current flow in the path can be determined by the current flow through any one of the series elements. Since we can measure the net current flow through the membrane ( l ~ )it, follows that the same net flow can be used to obtain the flow across the external barrier. Note that for inward membrane currents, M e signifies an influx of potassium ions from the external solution to the space. For outward membrane currents M e signifies an efflux of potassium ions from the space to the external solution. From Eqs. (10) through (14) we obtain:

Since PKs has been evaluated experimentally (Frankenhaeuser and Hodgkin, 1956) and the values of gK, I M , and E K have been determined experimentally as a function of [ K ] (Adelman and Palti, 1969b), Eq. 15 can be solved for dlKJ (see Appendix B). Thus, [K,] can be evahated as a function of 8, El and time (Palti et al., 1972). This multicompartmental model is more general than the Frankenhaeuser and Hodgkin (1956) model. The Frankenhaeuser and Hodgkin model is restricted t o [KB]changes during the falling phase of the spike

PERIAXONAL A N D PERINEURONAL SPACES

229

when the following conditions may be assumed as first approximations t o prevail: outward potassium ion flow through the excitable membrane is large compared with potassium ion flow through the external barrier, and inward potassium ion flow through the excitable membrane is small compared with transfer through the external barrier. These constraints were necessary to allow the Frankenhaeuser-Hodgkin equations to be solved analytically. The Adelman-Palti model given above does not require the assumptions and approximations made by Frankenhaeuser and Hodgkin, as i t is based on a general set of equations that can be solved numerically. Therefore, the model given here can be applied to almost any axonal or neuronal system possessing a constrained extracellular space, whether it is resting, active, or subjected to the effects of external current flow. APPENDIX B Calculation of [Ks] Changes upon Voltage Clamping the Squid Giant Axon

All the constants of Eq. (15) (EM, t ~P, K ~F ,, 0) are known or have been evaluated experimentally (see above). To solve the equation we must also determine the values of the variables, g K , E K , I M . Since the membrane potential, EM,is known, the potassium conductance, g K , is given, as a function of time, by the Hodgkin and Huxley equations (1952d). However, the potassium equilibrium potential varies with time since [K,] was shown to be a function of both potential and time. Thus for each instant in time the value of EK must be computed using the Nernst relationship:

by substituting the changing values of [K,]. Note that now both g K and E K ,and thus IK,vary in time. The effect of the potassium concentration in the space, [K,], is also reflected in another term of Eq. (15), I M ,since this term, the total membrane current, includes the sodium current. As shown by Adelman and Palti (1969a,b) the sodium inactivation rate constants f f h and P h are affected by external potassium concentration. Therefore, the Adelman and Palti equations for f f h and Oh, which account for the potassium concentration effect on the rate constants, are used. After making the above corrections for the [K,] changes, in the values of EK,g N a , gK, and g,, (the sodium, potassium, and total membrane con-

2 30

W. J. ADELMAN, JR. AND Y. PALTI

ductances) are determined for the initial conditions and subsequent increments of time, by the numerical methods and a digital computer program described by Palti (1971a). The appropriate values are substituted in Eq. (15), and the equation is solved numerically by means of a digital

READ CONSTANTS 6 INITIAL CONDITIONS

~

GO, TAKE

ANOTHER PULSE

SET POTENTIAL VALllE TO THAT < OP THE PULSE REQUIRED AT THE NEXT POINT

COMPUTE THE CHANGE IN [Ks] USING EQUATION 15 FOR EM AT

SAMPLING POIW

'Lm. a,, En

AS GIVEN BY HODGKIN

COMPUTE ALPHAH 6 BETAH AS FUNCTION OF [Ksl ah-0.126-0.065 log [K,]

I

T 1

I

6 HUXLEY

COMPUTE THE OTHER ALPHA'S 6 BETA'S BY HODCKIN 6 HLKLEY FOR THIS POINT

A COMPUTE

T ~ ,T ~ ,T ~ ,

h,,

n_

m-3

FOR

f CALCULATE EK

I

I

COMPUTE m. n, h FOR THIS POINT

I

THE SAMPLING POINT

FIG.5. Flow chart of computer program for the solution of membrane currents in the voltage clamped squid axon. The program incorporates the changes in [K.] and their effects on the membrane conductances and currents into the Hodgkin-Huxley (1952d) and Adelman and Palti (1969a) equations. For further details see Appendix B.

PERIAXONAL AND PERINEURONAL SPACES

23 1

computer. The solution is now repeated after increasing time in Eq. (15) by a small step, At. This process is continued a t each new iteration, using the values computed in the previous one as its initial points. Thus 6[K,] and from it the total potassium concentration in the space, [I<.], are determined as a function of time. Figure 5 gives the flow chart for this solution.* The time course of changes in [IL]have been computed as a function of time when the membrane is depolarized (see Fig. 6 in Adelman and Palti, 1972). The [K,] reaches values of over 100 mM with time constants of 10 msec or more. It was shown that the narrower the space the faster is the potassium accumulation; the higher the external barrier potassium permeability, the lower is the steady-state value of [KJ. The time course of the depletion of potassium ions from the space when the membrane is hyperpolarized also has been computed (see Fig. 8 in Adelman and Palti, 1969b). Again the rate of change of [IiJ was shown to be inversely proportional to the width of the space, and potassium depletion is more pronounced when the PK$is higher. The concentration was shown to decrease with time constants on the order of 100 msec, final values sometimes being as low as 25% of the initial concentration.

APPENDIX C Reconstruction of a Membrane Action Potential

Appendix B gives the analytical solution for determination of the [K.] changes in a voltage clamped squid giant axon. Since membrane potential is constant under these conditions, the voltage-dependent terms of Eq. (15) have a fixed value that can be determined analytically. If the restriction of EMt o a constant value is removed, we must solve numerically for the changes of these parameters (IM,g K , etc.) as a function of potential and time. The numerical method and a digital computer program for this problem were described by Palti (1971b,c). The incorporation of this program into the program which solves Eq. (15) (see Appendix A) is described by the flow chart of Fig, 6. The combined programt enables one to determine the E Mchanges during and after a stimulus or during natural activity.

* Listing of the full Fortran IV program, including the Calcomp subroutines, for the solution of [K.] and other parameter changes may be obtained from the authors. Listing of the full Fortran IV program, including the Calcomp subroutines, for the reconstruction of the membrane action potential may be obtained from the authors.

232 232

W.J. J.ADELMAN, ADELMAN,JR.JR.A AN ND DY.Y.PALTI PALTI W.

(7) \I / f READ CONSTANTS h I N I T I A L CONDITIONS

YES

COMPUTE ALL ALPHA'S h BETA'S FOR AT SAMPLING POINT

+=

INCLUDED?

4

N

NUMBER PRESET O EQUAL VALUE T O

YES

t

0

I

r--L

COMPUTE TXE STEP CHANGE IN

I

ah* 'h'

I

COMPUTE ALPHAH 6 BETAH AS FUNCTION OF [Ks]

Urn* 'me % * En

q;0.126-0.065

Log [Ks)

USING EQUATION 15.

AS GIVEN BY HODGKIN h HUXLEY

B- I K, 1/ (32.5 [ K, 1+18 5)

c, INCLUDED?

I

ALPHA'S h BETA'S BY HODGKIN h HUXLEY FOR "RIS P O I N T

COMPUTE CURRENTS BY HODGKIN h HUXLEY EQ. COMPUTE T,,, 2

h,,

I&,n,

T ~ ,K,,,

FOR

t CALCULATE EK

THE SAMPLING POINT

FIG.6. Flow chart of computer program for the solution of axon membrane parameters; E M ,m, n, h, IN&,etc., as a function of time. The program incorporates the computed [K.] changes and their effects on membrane parameters into the Hodgkin-Huxley (1952d) and Adelman and Palti (1969a) equations. For further details see Appendix C.

Note that that in in this this general general treatment treatment the the [K8] [ I Q changes changes affect affect sodium sodium and and Note [I<,] changes. potassium conductances and currents, which further affect the potassium conductances and currents, which further affect the [I<,] changes. Also note note that that since since the the changes changes in in membrane membrane potential potential are are functions functions of of Also membrane current, the [K,] affects the membrane potential changes. membrane current, the [K,]affects the membrane potential changes.

PERIAXONAL AND PERINEURONAL SPACES

233

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Properties of the Isolated Nerve Endings* GEORGINA RODRfGUEZ de LORES ARNAIZ and EDUARDO De ROBERTIS Instituto de AnatomZa General y Embriologla. Facdtad de Medicina-Universidad de Buenos Aires Buenos Aires. Argentina

I . Introduction . . . . . . . . . . . . . . I1. Isolation of Nerve Endings and Their Limiting Membrane . I11. Chemical Composition . . . . . . . . . . . A. Content of Active Substances and Related Enzymes . B . Cation-Stimulated Phosphohydrolases . . . . . C . Proteins and Ribonucleic Acid . . . . . . . . D . Lipids . . . . . . . . . . . . . . . IV. Immunological Properties of Isolated Nerve Endings (INE) V. Osmotic Properties of the I N E . . . . . . . . . VI . Synthesis of High-Energy Compounds . . . . . . A. Glycolysis . . . . . . . . . . . . . . B . Respiration . . . . . . . . . . . . . C . Hexose Monophosphate Pathway . . . . . . . VII . Metabolism of Amino Acids . . . . . . . . . VIII . Metabolism of Phospholipids . . . . . . . . . I X . Amino Acid Uptake and Protein Synthesis . . . . . X . Uptake Mechanisms Related to the Transmitter Function . A. Choline . . . . . . . . . . . . . . B . GABA . . . . . . . . . . . . . . . C. Tryptophan . . . . . . . . . . . . . D . Norepinephrine . . . . . . . . . . . . XI . Ion Permeability . . . . . . . . . . . . . A. Sodium . . . . . . . . . . . . . . B . Potassium . . . . . . . . . . . . . . C. Calcium . . . . . . . . . . . . . . . D . Anions . . . . . . . . . . . . . . . XI1. Concluding Remarks . . . . . . . . . . . . References . . . . . . . . . . . . . . .

. . . . 238 . . . . 239

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246 248 . . . . 249 . . . . 250 . . . . 251 . . . . 252 . . . . 252 . . . . 253 . . . . 254 . . . . 255 . . . . 256 . . . . 258 . . . . 259 . . . . 259 . . . . 260 . . . . 260 . . . . 261 . . . . 262 . . . . 262 . . . . 263 . . . . 263 . . . . 266 . . . . 266 . . . . 268

* This work has been supported by Grants of the Consejo Nacional de Investigaciones Cientificas y T6cnicas, Argentina, and National Institutes of Health, 5 RO1 NS 06953-05, NEUA

.

237

238

GEORGINA RODRIGUEZ do LORES ARNAIZ AND EDUARDO Do ROBERTIS

1. INTRODUCTION

The complex neuronal networks that characterize the central nervous system (CNS) and constitute the structural substratum for its multiple functions are established by special zones of contacts between neurons, the so-called synapses (i.e., synaptic regions, synaptic junctions). At the synapse a nerve ending, generally representing an enlargement of a fine ramification of the axon of one neuron, enters in contact either with a dendrite, the somata or even with the axon of another neuron. The use of the electron microscope permitted the discovery of the synaptic vesicle as the most conspicuous structural component of the nerve ending (De Robertis and Bennett, 1954, 1955), and, after its isolation, it was demonstrated to contain the transmitter substances (De Robertis et al., 1963). These vesicles and the presence of pre- and postsynaptic differentiations of the synaptic membranes suggested the existence of still other types of functional interactions between neurons. Thus dendrodendrite synapses (Rall et al., 1966) and reciprocal and serial types of synapses have been described (see Dowling and Boycott, 1966). The electron microscope study of the CNS, as well as the cell fractionation methods which will be mentioned below, have permitted us to estimate that the synaptic regions represent the greatest volume of the gray regions of the brain. For example, from classical studies, it was known that, in the cerebral cortex, neuronal perikarya represent only about 5% of the volume and another 5% is contributed by the glial cells. Electron microscope observations show that in the rest of the cortex the nerve endings and the fine dendrites represent about 60% of the volume, and the axons 10-15% (Nafstad and Blackstad, 1966). The above considerations stress how important cell fractionation methods have become in the last ten years as applied to the study of the brain, especially in relation to the isolation of the synaptic region. It will be shown below that by gentle procedures of homogenization and by differential centrifugation it is possible to separate rather intact nerve endings to which are attached some of the postsynaptic structures. The isolation of the nerve endings has simplified the problem of tackling the complex structural and biochemical organization of the brain by allowing direct study of this most important part of the neuronal circuitry. It will be shown here that the so-called isolated nerve ehdings (INE) provide a means of studying i n vitro the properties of the synaptic region of the neuron. These isolated particlea, surrounded by a membrane, are able to carry many of the functions that have been previously recognized in brain slices (see Harvey and McIlwain, 1969). For example, they are able to produce high energy compounds through glycolysis and oxidative

PROPERTIES OF THE ISOLATED NERVE ENDINGS

239

phosphorylation, and they synthesize different types of small molecules and even of larger molecules, e.g., lipids, proteins, and polysaccharides. They have the ability to accumulate or extrude ions, to transport different types of molecules by passive, active, or facilitated mechanisms. I n the process of chemical transmission, it is generally thought that transport of mono- and divalent ions takes place a t the synaptic region and that some of these ions are involved in the release of the transmitters. The isolated nerve endings provide a direct approach in learning about the sequence of reactions involved in the entrance of precursors, the synthesis of transmitters, and the triggering mechanisms involved in the release of the transmitter a t the arrival of the nerve impulse. Another fundamental line of research opened by these isolation studies, but which will not be considered here, concerns the binding of drugs that may act at receptor sites localized in the postsynaptic membrane. These studies have led to the isolation of specific receptor proteins that interact with the transmitters (see De Robertis, 1971). The action of drugs affecting the synaptic components of the CNS has also been reviewed (Rodriguez de Lores Arnaiz and De Robertis, 1972).

II. ISOLATION OF NERVE ENDINGS AND THEIR LIMITING MEMBRANE

Throughout this article the pame isolated nerve ending (INE) is used to designate the particle that comprises the nerve terminal detached from the axon, together with the subsynaptic membrane and other structures that remain adherent to the presynaptic ending. This denomination should be considered synonymous with pinched of-nerve ending, nerve ending particle, and synaptosome, used by various authors. Our objections to the use of the term synaptosome, which is becoming the most popular in the literature, were given in a previous article (De Robertis and Rodriguez de Lores Arnaiz, 1969). The electron micrograph of Fig. 1, corresponding to a slice of cerebra1 cortex incubated for 2 hours in Krebs-Ringer a t 37"C, illustrates very clearly the ultrastructure of the synaptic region with fine axons, enlarged nerve endings containing the synaptic vesicles, and synaptic contacts with fine dendrites (arrows). The incubation has produced a n enlargement of the extracellular space and thus a better recognition of the contours of the synaptic junctions. The axons are about 0.15 p wide, and the nerve endings are of the order of 0.7-1 1.1. It is thus understandable that with mild shearing forces it is possible to detach the ending from the axon and also a portion of the dendrite with the subsynaptic structures. The identity of these

240

GEORGINA RODRIGUEZ de LORES ARNAIZ AND EDUARDO De ROBERTIS

FIQ.1. Electron micrograph of the neuropil region of a slice of rat cerebral cortex after incubation for 2 hours in Krebs-Ringer. Ax, axon containing neurotubules; e, nerve ending filled with synaptic vesicles; d, dendrite; es, extracellular space; mi, mitochondria. Arrows indicate synaptic contacts; a thick arrow shows the point at which the axon enlarges to form the nerve ending. From Lunt and Lapetina (1970b). X 45,000.

PROPERTIES OF THE ISOLATED NERVE ENDINGS

241

FIG.2. Electron micrograph of isolated nerve endings from cat cerebral cortex separated by Ficoll gradient. e, nerve ending; d, dendrite; mi, mitochondria. Arrows indicate synaptic contacts. From De Robertis el al. (1966b). X50,OOO.

242 GEORGINA RODRIGUEZ de LORES ARNAIZ AND EDUARDO De ROBERTIS

h

--

s

+

N

o o - - -

r n a O O N d

d

8

FIG.3. Fractionation techniques used for the separation of nerve endings and parts of the synaptic region.

PROPERTIES OF THE ISOLATED NERVE ENDINGS

243

structures with those isolated is clearly visible in Fig. 2, corresponding to I N E which have been incubated for 30 minutes a t 37°C. The electron micrograph shows that the membrane of the ending has been resealed a t the point of detachment. On the other hand, the subsynaptic membrane, with the subsynaptic web and a portion of the dendrite, remains attached to the ending but without a continuous membrane. Different methods for the separation of the INE may be employed. Independently (see Gray and Whittaker, 1962; De Robertis et al., 1962a), our laboratory and that of Cambridge demonstrated that the crude mitochondrial fraction separated from brain contained INE that could be separated by a sucrose gradient centrifugation. Figure 3 shows the general sequence of the methods developed in our laboratory for separation of nerve endings and also to subfractionate the synaptic region into the nerveending membranes, the junctional complexes (which comprise the two synaptic membranes), and the synaptic vesicles, which was first achieved in 1962 (De Robertis et al., 196213). More details on these techniques and on those used by Whittaker et al., (1964) are discussed by De Robertis and Rodriguez de Lores Arnaiz (1969). Ficoll gradients have also been used to separate the I N E from the crude mitochondria1 fraction (Kurokawa et al., 1965; Alberici et al., 1965; AbdelLatif, 1966). This method has the advantage of using isosmotic solutions and of giving good preservation of the I N E (Fig. 2). However, the separation from other structures is not as clear-cut as that obtained with discontinuous sucrose gradients. Zonal rotors with continuous sucrose gradients (Cotman et al., 1968a; Mahler et al., 1970) or isosmotic Ficoll-sucrose gradients (Day et al., 1971) have also been employed to separate the INE and membranes. A, combination of a discontinuous sucrose gradient and a continuous cesium chloride gradient has been used for the same purpose (Kornguth et al., 1969, 1971). Other interesting new methods for the separation of I N E and other subcellular fractions of brain involve the use of Millipore filters (Baldessarini and Vogt, 1971) and free-flow electrophoresis (Ryan et al., 1971). Isolation of the Nerve-Ending Membranes

The study of the chemical and enzymatic composition of the nerveending membrane is of primary importance in understanding neuronal so-caIled function. Being a continuation of the axon membrane-the axolemma-t.he limiting membrane of the nerve ending serves to regulate the passage of ions, metabolites, and other small molecules, thus main-

244

GEORGINA RODRIGUEZ de LORES ARNAIZ AND EDUARDO De ROBERTIS

taining a special milieu for the metabolic activities of this compartment. Across this membrane ionic gradients originate the resting potential and ionic fluxes are enhanced with the passage of the action potential into the terminal. The introduction of osmotic shock of the nerve endings to separate the synaptic vesicles (De Robertis et al., 196213, 1963) has also been employed to separate nerve-ending membranes (De Robertis et al., 1966a; Rodrfguez de Lores Arnaiz et al., 1967). In the method used in this laboratory, fract,ions MI 0.9, MI 1.0, and MI 1.2 contain nerve-ending membranes (see Fig. 3). These various fractions were separated by their specific gravities, which essentially depend on the lipid :protein ratio (Lapetina et al., 1968). These membranes are also characterized by differences in the content of gangliosides (Lapetina et al., 1967), by the distribution of a group of membrane-bound enzymes (see Table 11) and by their different binding capacity to drugs (see De Robertis, 1971). In the method of Whittaker et al. (1964) for synaptic vesicles, membranes also are separated.

111. CHEMICAL COMPOSITION

A. Content of Active Substances and Related Enzymes

The isolation of the nerve endings permitted the introduction of methods of quantitative cytochemistry and the correlation of the neurochemical results with the submicroscopic organization of the synaptic region. Furthermore, it became possible directly to assay active substances that are important in chemical transmission. This made it possible to demonstrate the presence in the I N E of high concentrations of the different biogenic amines, e.g., acetylcholine (De Robertis et al., 1962a; Gray and Whittaker, 1962; Ryall, 1964), 5-hydroxytryptamine (Michaelson and Whittaker, 1963; Zieher and De Robertis, 1963; Maynert et al., 1964), catecholamines (Zieher and De Robertis, 1964; see De Robertis, 1966; Whittaker, 1966a), and histamine (Kataoka and De Robertis, 1967; Kuhar et al., 1971b). Substance P was also found in the I N E (Inouye et al., 1963; Ryall, 1964). Although only a relatively small percentage of gamma aminobutyric acid (GABA) is recovered particle-bound, this amino acid, which is present only in nerve tissue, was recovered in fractions of I N E (Weinstein et al., 1963; Ryall, 1964; Mangan and Whittaker, 1966; Neal and Iversen, 1969). The study of the enzymes related to the metabolism of the active substances has also contributed to the knowledge of the chemical machinery for

TABLE I DISTRIBUTION OF THE BIOGENIC AMINES AND SOMERELATEDENZYMES IN ISOLATED NERVEENDING (A-E) SUBMITOCHONDRIAL FRACTIONS ISOLATED BY GRADIENT CENTRIFUGATION AS IN FIG. 30 Submitochondrial fraction Myelin Compound

A

Aminergic nerve endings B

C

E

Data from

0.67 0.76

0.27 0.48

De Robertis et al. (1962a) Zieher and De Robertis (1963) Zieher and De Robertis (1964) Zieher and De Robertis (1964) Kataoka and De Robertis (1967)

Acetylcholine 5-Hydroxytryptamine

0.24 0.61

2.02 0.78

Norepinephrine

0.32

2.05

1.66

0.77

0.72

Dopamine

0.79

1.85

1.13

0.91

0.71

Histamine

0.72

2.70

1.56

0.44

0.70

Choline acetylase Acetylcholinesterase 5-Hydroxytryptophan decarboxylase Monoamine oxidase

0.10 Oi 15 0.05

1.88 2.24 1.05

Enzymes related to amines 0.98 1.oo 2.99 0.94 2.05 1.22

0.59 0.58 0.26

-

-

Glutamic acid decarboxylase

0.02

0.49

GABA aminotransferase

0.15

0.11

1.16

2.28

Enzymes related to GABA 1.22 2.00

0.40

1.10

m =! rn

2

Mitochondria

Amines 4.11 2.17

0.29

2rn

v,

Nonaminergic nerve endings D

0.17

W 'D

8.00

-I

De Robertis et al. (1963) De Robertis et al. (1962a) Rodriguez de Lores Arnaiz and De Robertis (1964) Rodriguez de Lores Arnaiz and De Robertis (1962) Salganicoff and De Robertis (1963) Salganicoff and De Robertis (1963)

a Results are expressed as the relative specific concentration of amine or of enzymatic activity recovered, divided by the percentage of protein recovered.

-fIn rn

?4 L

rn

m

d

4

E Z

$

246

GEORGINA RODRIGUEZ de LORES ARNAIZ AND EDUARDO De ROBERTIS

synaptic transmission. The enzymes related to the acetylcholine system, i.e., acetylcholinesterase and choline acetyltransferase, were found asso ciated with the nerve ending which is rich in acetylcholine, i.e., fraction C in Fig. 3 (De Robertis et al., 1962a, 1963). Also the enzymes related to the norepinephrine and 5-hydroxytryptamine metabolism are found in the INE. Both 3 ,4-dihydroxyphenylalanine or hydroxytryptophan decarboxylase (Rodriguez de Lores Arnaiz and De Robertis, 1964) and catechol-0methyltransferase (Alberici et al., 1965), as well as tyrosine hydroxylase (McGeer et al., 1965) were reported to he present in the INE. I n our laboratory, fraction D of I N E (Fig. 3) was found to be rich in glutamic acid decarboxylase, a n enzyme that catalyzes the synthesis of GABA (Salganicoff and De Robertis, 1963, 1965). This finding led us to the interpretation that the D fraction of I N E contained mainly inhibitory nerve endings. Table I shows the distribution of biogenic amines and some enzymes related to them and to GABA. It should be noted that monoamine oxidase and GABA aminotransferase, two catabolizing enzymes, are associated with mitochondria. The separation of different types of INE from striatum, midbrain, and hypothalamus has been investigated with the use of continuous sucrose gradients and incomplete equilibrium centrifugation. Slices were incubated with labeled GABA, catechoIamines, or 5-hydroxytryptamine and then submitted to fractionation. It was observed that the GABA-containing I N E were separated in a lighter fraction than those containing exogenous norepinephrine or 5-hydroxytryptamine (Kuhar et al., 1971a). Morphological differences between the lighter and denser I N E were observed. The latter had more intrasynaptic mitochondria and postsynaptic membrane attachments (Gfeller et al., 1971). Similar studies have been carried out for the localization of histamine (Kuhar et al., 1971b) and glutamic acid in I N E (Wofsey et al., 1971). B. Cation-Stimulated Phosphohydrolases

To establish the localization of these enzymes is of great importance because of their well known involvement in sodium and potassium transport. I n different tissues, membrane fractions contain Na+-K+ stimulated ATPase (see Skou, 1965; Siege1 and Albers, 1970). I n nerve endings an ATPase stimulated by Na+-K+ has been demonstrated in the I N E of various mammals (Tanaka and Abood, 1964; Kurokawa et al., 1965; Hosie, 1965; Albers et al., 1965; Bradford et al., 1966; Abdel-Latif et al., 1967; and others). I n the I N E the localization of this enzyme is strictly a t the membrane,

W V

0

i

-

rn

TABLE I1

v,

DISTRIBUTION OF VARIOUS ENZYMES AND BINDINGCAPACITY FOR [I4C] DIMETHYL d-TUBOCURARINE ([14C]DMTC)..

a -

a

5rn 0

Subfraction

Structure

AChE

Na+-K+ ATPase

p-Nitrophenyl phosphatase

Glutamine synthetase

Adenyl cyclase

Phosphodiesterase

Monoamine [14C]DMTC oxidase

5

rn

4 M10.8 M10.9

M1 1.0 MI 1.2 Mlp

b

Myelin Nerve-ending membranes Nerve-ending membranes Nerve-ending membranes Mitochondria

1.68 3.22

1.37 2.28

0.56 2.41

0.66 0.96

1.06 2.04

1.32 1.77

0.44 0.33

2.14 4.16

2.13

3.16

1.39

2.04

2.46

2.68

0.23

6.88

0.98

1.40

2.53

1.74

1.95

1.03

0.65

3.00

0.15

0.17

0.30

0.73

0.31

0.43

1.56

1.60

Data from De Robertis et al. (1966a, 1967) and Rodriguez de Lores Arnaiz et al. (1967). The results are expressed as in Table I. Fractions are indicated in Fig. 3.

P z

0,

248

GEORGINA RODRIGUEZ de LORES ARNAIZ AND EDUARDO De ROBERTIS

where it is found together with acetylcholinesterase, K+ p-nitrophenyl phosphatase, glutamine synthetase (Rodriguez de Lores Arnaiz et al., 1967), and adenyl cyclase (De Robertis et al., 1967) (Table 11). Nerve ending membranes isolated by zonal centrifugation also showed a high specific activity for Na+-K+ ATPase (Cotman et al., 1968a). C. Proteins and Ribonucleic Acid

In membranes of the nerve endings the structural proteins were studied with acrylamide gel electrophoresis. A pattern showing some difference with other brain membranes was observed (Cotman and Mahler, 1967; Cotman et al., 196813). The saline-soluble and the detergent-soluble proteins from subcellular fractions of the brain were compared. The most complex pattern was observed in the nerve-ending fraction and the cellsoluble fraction. In the INE there were a t least two proteins which did not occur in other particulate fractions (Davies, 1970). The presence of microtubular protein in the I N E was suggested by studies of binding with colchicine and precipitation with vinblastine as markers. Some of this protein is soluble, but some is bound to membranes in the I N E (Feit and Barondes, 1970). After [14C]leucineinjection the labeled soluble protein appeared in the nerve endings after considerable delay. This was interpreted as being due to the fact that they had to be transported by axonal flow to the terminal (Barondes, 1964, 1966). A rapid transport of fucosyl glycoproteins to the nerve ending has been found by the intracerebral injection of [3H]fucose.On the other hand, [14C]glucosamineis apparently incorporated by nerve endings in a macromolecular form (Zatz and Barondes, 1971). The problems related to a possible participation of the I N E in protein synthesis will be considered below. Studying the turnover of proteins after intracerebral injection of [3H] leucine in different subcellular brain fractions, yon Hungen et al. (1968) found a half-life value of 20-22 days for all of them. Further studies from our laboratory have demonstrated that there are differences in half-lives among the subcellular fractions. For example, while the synaptic vesicIes showed a value of 20 days, the nerve-ending membranes doubled this figure (44 days). These findings suggest a different turnover rate for proteins in different brain membranes (Rodriguez de Lores Arnaiz et al., 1971). Ribonucleic acid has been reported to be associated with membranes of the I N E (Bal&zsand Cocks, 1967; Austin and Morgan, 1967). The incorporation of RNA precursors in the I N E has a much shorter delay than that for proteins (Balhzs and Cocks, 1967).

PROPERTIES OF THE ISOLATED NERVE ENDINGS

249

D. Lipids

Lipids are essential structural components in the membranes of the CNS; some lipids seem to be involved in various membranous functions, among them the transport of ions (see Hokin, 1969). A study of the lipid composition of nerve endings was done by Seminario et al. (1964) and Eichberg et al. (1964). With the availability of techniques for the separation of different membranes, i t could be found that each one has a specific lipid pattern (Lapetina el al., 1968). This was reflected especially in the molar ratio of the different lipid species, i .e., phospholipids :cholesterol :galactolipids, as well as in the content of proteolipids. A high concentration of gangliosides was found preferentially in the acetylcholinesterase-rich nerve-ending membranes (Lapetina et al., 1967). The finding of a high content of gangliosides in membranes was also reported by Whittaker (1966b), Wiegant (1967), and others. Studies in embryonic chicken brain have shown that most of both sialyland galactosyltransferases involved in the biosynthesis of gangliosides were associated with the nerve-ending fraction (Den and Kaufman, 1968). Subcellular fractionation studies of the human brain have indicated that ganglioside sialidase, which is the enzyme that initiates the degradation of gangliosides by splitting off sialic acid, was mainly recovered in the nerveending fraction (ohman, 1971). After intraperitoneal administration of [32P]orthophosphate,it was found that the turnover of the various phospholipids occurred a t different rates according to their nature and subcellular localization (Mandel and Nussbaum, 1966), whereas after intracranial administration there were no differencesamong the various membrane fractions (Abdel-Latif and Abood, 1965; Lapetina el al., 1969a). With different labeled precursors, a different half-life was demonstrated for the different parts of the phospholipid molecule in the total homogenate (Lapetina et al., 1969b). A more complete study, extended to subcellular fractions including the nerve endings, also indicated that the metabolism of phospholipids is a heterogeneous process and that the various parts of the phospholipid molecule may be interchanged or metabolized a t different rates (Abdel-Latif and Smith, 1970). Intraventricular administration of [Me-14C]cholineled to a study of the turnover of phosphatidylcholine in the different membranes. The halflives for synaptic vesicles was 30 days while in nerve-ending membranes it was 60 days. The long half-lives found for this phospholipid suggests that it may be primarily a structural entity (Lapetina et al., 1970). Similar results demonstrating the faster turnover of phosphatidylcholine in synap-

250

GEORGINA RODRIGUEZ de LORES ARNAIZ AND EDUARDO De ROBERTIS

Pr 20 PC 3 0

Pr 41 PC 27

Pr 44 PC 61 Pr 50

FIG.4. Diagram of an isolated nerve ending in which the half-lives of protein (Pr) and phosphatidylcholine (PC) for the various synaptic components are indicated. Data from Rodriguez de Lores Arnaiz et al. (1971) and Lapetina et al. (1970).

tic vesicles than in membranes were found in slices of cerebral cortex incubated with [Me-l'C]choline (Lunt and Lapetina, 1970b). Figure 4 shows a diagram of an I N E in which the half-lives for protein and phosphatidylcholine are indicated. The different half-lives obtained with the use of leucine and choline indicated that the protein and lipid components turn over independentIy in the various synaptic components.

IV. IMMUNOLOGICAL PROPERTIES OF ISOLATED NERVE ENDINGS (INE)

Antisera were prepared by injecting I N E (fraction C of Fig. 3) from rabbit (homologous system) or cat (heterologous system) into rabbits (De Robertis et al., 196613). It was also produced by injecting a rather pure fraction of isolated nerve-ending membranes (fraction MI 1.0 of Fig. 3) (De Robertis et al., 1968). The effect of both antisera was studied on INE from the cat cerebral cortex separated on a Ficoll gradient (Fig. 2). I n the presence of the antiserum against I N E and complement the nerve endings showed various degrees of lysis and disintegration. There was swelling with clumping and disappearance of synaptic vesicles and sometimes only empty IN E , still containing mitochondria, were observed. With antiserum against nerveending membranes plus complement, the cytolytic effect was slightly different. Many I N E showed loss of synaptic vesicles and a rather empty axoplasm. Some of the vesicles were seen lying free between the IN E .

PROPERTIES OF THE ISOLATED NERVE E N D I N G S

25 1

Discontinuities in the nerve-ending membrane and even larger breaks were observed. This work was complemented with an electrophysiological study of the effect of the antiserum on the synaptic transmission of mollusc neurons (Wald et al., 1968). Antisera against I N E from guinea pig cerebellum and cerebral cortex were produced, and a y-globulin coupled with fluorescein was prepared. The fluorescent antibody was shown to stain discrete regions in the neuropil regions of the cerebellar and cerebral cortex. However the low resolving power of the light microscope did not permit establishing precise relationship between these stained regions and the nerve endings (Kornguth et al., 1969). V. OSMOTIC PROPERTIES OF THE I N E

Morphological and biochemical evidence on the structure of the I N E given above indicated the integrity of this isolated particle. In other words, separation of the nerve endings from their attachment to the axon led to the resealing of the nerve ending membrane and the reformation of a self-contained structure (Fig. 2). Most of what will be said in the rest of this article confirms the integrity of the I N E as a functional unit. One way to study this problem is to record the change in volume of the particle when it is suspended in media of different osmotic pressures. As in the case of erythrocytes, mitochondria, muscle fibers, and so forth, the I N E may be shown to behave as a n osmometer. The membrane of the nerve ending is permeable to different substances. When they enter into the I N E they carry water, thus producing a change in volume. The rate of this change is an indication of the rate of entry of the substance. The osmotic properties have been studied using light scattering techniques (Keen and White, 1970). Because the refractive index of the I N E is presumably lower than that of mitochondria the changes in volume could not be followed in sucrose solutions but were very evident in solutions of NaCl of increasing osmolarity. The change in volume could be reversed by restoring the tonicity of the medium. The permeability of the I N E to nonelectrolytes followed the order: glucose<
252

GEORGINA RODRIGUEZ de LORES ARNAIZ AND EDWARD0 De ROBERTIS

and KC1 and apparently impermeable to Ca2+, Mg2+, oxalate, PO:- and SO:- ions (Keen and White, 1970). Gramicidin was found to increase the permeability of the I N E to Na+ and K+ ions as in other membrane-bound structures (Keen and White, 1971). The osmotically sensitive Na+ and K+ contained within the I N E was measured. When the nerve endings were passed through a Sephadex column equilibrated with an isoosmotic solution they retained these small ions; upon elution with hyposmotic solutions the Na+ and I<+ were released. The degree of osmotic shock needed for this release was similar to that produced upon the occluded lactic dehydrogenase (Rlarchbanks, 1967). When the experiments were carried out at low temperature (5”C), it was found that the rates of Na+ and K+ uptake into the osmotically sensitive compartment were equal to the rate of loss of these ions. This indicates that under such conditions there is no active transport. I t was concluded that the osmotically sensitive compartment represents the inner volume of the INE, which is separated from the outside environment b y a membrane having many of the general properties of biological membranes (Marchbanks, 1967).

VI. SYNTHESIS OF HIGH-ENERGY COMPOUNDS

In previous sections we have considered some aspects of the structural and biochemical complexity of the nerve ending and the synaptic region as a whole. Special emphasis was put on the content of transmitter substances, the enzymes involved in their synthesis and degradation and those involved in the metabolism of certain physiologically active amino acids. Here we shall consider rather the metabolic properties of the I N E in relation to the production of high-energy compounds and their involvement in the active transport of ions and other substances across the nerve-ending membrane. A. Glycolysis

When methods for the separation of I N E became available (De Robertis et al., 1962a; Gray and Whittaker, 1962), it was possible to demonstrate that the glycolytic activity in the crude mitochondrial fraction of the brain resided not in the free mitochondria, but in the nerve endings, which represent a considerable portion of the so-called “mitochondrial fraction.” Using gradients, it was found that the glycolytic cycle followed the distribution of the I N E while the oxidative phosphorylation accompanied the

253

PROPERTIES OF THE ISOLATED NERVE ENDINGS

free mitochondria. However, hexokinase also followed the latter fraction. This indicates that this brain enzyme was probably associated with the mitochondria1 membrane (Tanaka and Abood, 1963). B. Respiration

Several workers have demonstrated that, when incubated into proper media, the INE behave as small whole cells actively respiring and being capable of generating considerable amounts of ATP and phosphocreatine. The I N E remained metabolically active in spite of the several hours of exposure to the low temperature, hypertonic sucrose, and anaerobic conditions needed for their separation. The addition of 10 m M glucose to a Krebs-Ringer medium at, 37°C stimulated oxygen uptake about 3-fold. The I N E showed a rapid and linear respiration with glucose and pyruvate as substrates whereas mitochondria, in a medium lacking Na+ and high in

TABLE I11 EFFECTOF INCREASING PHOSPHATE ON RESPIRATION, ATP, PHOSPHOCREATINE LEVELSI N SYNAPTOSOMES'

Concentration (mM) Preparation

Synaptosomes Synaptosomes Synaptosomes Synaptosomes Synaptosomes iodoacetate and cyanide Cortex slice

+

Phosphate

Sodium

Respiration (nmoles 0 2 / 100 mg protein/hr)

1.2 21.2 41.2 1.2 1.2

124 164 204 164 124

67 68 81 65 3.3

1.2

124

72

ATP (nmoles/ 100mg protein/hr)

AND

Phosphocreatine (nmoles/ 100 mg protein/hr)

223 355 352 210 20

339 842 810 350

-

1010

1400

Data from Bradford (1969). Synaptosomes or cortex slices were incubated a t 37°C in media of basic Krebs-Tris composition adjusted to pH 7.4 with NaOH and containing 10 m M glucose and 2.8 m M Ca. When NaH2P04 was added, Ca was omitted; ionic composition was otherwise unchanged. 4

254

GEORGINA RODRIGUEZ de LORES ARNAIZ AND EDUARDO De ROBERTIS

K+ and phosphate, showed respiration only with pyruvate (Bradford, 1969). The respiratory activity was grossly altered after exposure of I N E to hypotonic media and other treatments that may injure their integrity. The high endogenous respiration of the I N E suggests the presence within them of pools of metabolites readily available together with the enzymes and cofactors needed for the oxidation of glucose. Table I11 shows the effect of increasing concentration of phosphate on the respiration, ATP, and phosphocreatine levels of INE. The effect of poisoning with iodoacetate and cyanide, which cause a considerable decrease in all these parameters, is also indicated. Other interesting findings are that glutamate is an effective substrate for free mitochondria, but not for the nerve endings (Bradford, 1969). Respiration of I N E was not changed by active substances, such as acetylcholine, glutamate, and catecholamines, nor by reserpine and phenobarbitone (Balfour and Gilbert, 1971). To achieve maximal oxygen uptake, a certain optimal concentration of Naf and K+ in the medium is needed. This effect is similar to that produced on the activity of the Na+ and K+ activated ATPase. Also in both cases ouabain has a marked inhibitory effect (Appel et al., 1969). Electrical pulses applied to I N E produced a n immediate and rapid large increase in glycolytic and respiratory rates; the extent of the response was similar in magnitude to that found in stimulated cortical slices (Bradford, 1970b). Morphological studies revealed that the I N E had not changed substantially. Only a decrease in the number of complex vesicles was observed after stimulation (Jones and Bradford, 1971). The transport of glucose into the I N E seems to involve its phosphorylation; however, the kinetic characteristics differ from those in other brain permeability barriers (Diamond and Fishman, 1971). C. Hexose MonophosphatePathway

Under certain experimental conditions involving short periods of incubation, it was demonstrated that the hexose monophosphate pathway was active in I N E and could be stimulated by addition of norepinephrine, 5-hydroxytryptamine1 and acetylcholine. This alternative route of glucose metabolism was also stimulated b y artificial electron acceptors which enhance the conversion of the C-1 of glucose to CO, but practically have no effect on the C-6 conversion. This effect results in a marked increase in C-1:C-6 ratio. The hexose monophosphate pathway in I N E could serve to generate the NADPH needed in numerous metabolic reactions (Appel and Parrot, 1970).

PROPERTIES OF THE ISOLATED NERVE ENDINGS

255

VII. METABOLISM OF AMINO ACIDS

Well known is the capacity of whole brain in vivo and of brain slices in vitro to convert glucose into glutamate, aspartate, glutamine, GABA, and other amino acids (see Quastel, 1969), and in Section 111, A we reported on the subcellular localization of the enzymes related to such metabolic pathways (see Salganicoff and De Robertis, 1965). When related to the INE, such study is of particular interest because some of the amino acids synthesized may exert an excitatory or an inhibitory action in synaptic transmission. It is also evident that another consequence of the metabolism of glucose would be the maintenance of ionic gradients across the nerve ending membranes and the production of an electrical potential. It has been demonstrated that the amino acids produced from glucose, particularly glutamate, aspartate, and GABA, are not released to the incubation medium and are retained within the IN E . These may later be released by exposure to hypotonic media. The metabolic pattern of I N E incubated in the presence of [U-14C]glucoseand in a medium with high Naf and low K+ is strikingly similar to that previously demonstrated in brain slices (Beloff-Chain et al., 1955). The radioactivity appears mainly in CO,, lactate, aspartate, glutamate, glutamine, alanine, and GABA. However, with both glucose and glutamate the conversion into glutamine was lower than for the other amino acids (Bradford and Thomas, 1969). This finding may be correlated with the fact that glutamine synthetase is mainly in microsomes (Salganicoff and De Robertis, 1965). However although glutamate decarboxylase is present in the I N E (Salganicoff and De Robertis, 1963, 1965; Balhzs et al., 1966), the amount of GABA produced in vitro is relatively small. Possible explanations of this discrepancy are given by Bradford and Thomas (1969). Since, according to previous studies, it is possible to prepare a nerve-ending fraction rich in enzymes of the glutamate cycle and poor in the acetylcholine system (De Robertis et al., 1962a), further studies of the metabolism of glutamate in I N E may be rewarding. Of particular interest is the work of Bradford (1970a), who demonstrated that electrical stimulation causes the preferential release to the medium of certain amino acids, particularly glutamate, aspartate, and GABA (Fig. 5). A similar pattern of amino acid release occurred from stimulated cortical slices but the proportion was lower and of the same magnitude for all free amino acids. High Kf concentration in the incubation medium evoked a similar pattern of response from the INE, including the release of physiologically active amino acids (Bradford, 1970b). This effect was interpreted as resulting from the depolarization of the transmembrane potential generated by the I N E (Bradford, 1970a, b).

256

GEORGINA RODRIGUEZ de LORES ARNAIZ AND EDUARDO De ROBERTIS

100

90

90

80 70

70 I

I/

II

6otII

JO

10

FIQ.5. Chromatogram of amino acids released into the medium from isolated nerve endings. A, electrically stimulated sample; B, control sample. Amino acids: 1, arginine; 2, ammonia; 3, glycine; 4, tryptophan; 5, histidine; 6, GABA; 7, phenylalanine; 8, tyrosine; 9, norleucine; 10, leucine; 11, isoleucine; 12, valine; 13, alanine; 14, glycine; 15. glutamate: 16. serine: 17. alutamine: 18. amartate. From Bradford (1970a).

VIII. METABOLISM OF PHOSPHOLIPIDS

The INE from rat brain when incubated under optimum conditions of oxidative phosphorylation rapidly incorporate S2P-labeledorthophosphate into nucleotides, phosphoproteins, and phospholipids (Abdel-Lat,if et al., 1968). The labeling of phospholipids and phosphoproteins is closely related to the production of 32P-labeledATP, which may originate from the intraterminal mitochondria, but in addition may result from the glycolytic system present in the ending axoplasm. More than 90% of the total radioactivity was found in the more metabolically active phospholipids, such a s phosphatidic acid, phosphatidylinositol, and the polyphosphoinositides,

257

PROPERTIES OF THE ISOLATED NERVE ENDINGS

which constitute less than 9% of the total phospholipids of the INE (AbdelLatif et al., 1968). The metabolism of phospholipids of the I N E from rat cerebral cortex was also studied using [ l-14C]glycerol and [Me-14C]cholineas precursors (Lunt and Lapetina, 1970a). It was confirmed that the incorporation into phosphatidylinositol was very much faster than into phosphatidylethanolamine and phosphatidylcholine, which are the two major phospholipids of the nerve endings (Fig. 6). These findings are in agreement with several previous suggestions in the literature that phosphatidylinositol may play a special role in synaptic transmission. Glycolipids have also been marked by the use of radioactive monosaccharides (Bosmann and Hemsworth, 1970).

550 500 450

400 c

a

pl

\i .-c

>E .-0 $ a

350

300

c

250

c

.-c

g

200

I

a cn

I50 I00

50

0

I

0

10

20

30

40

50

60

Incubation time (min)

FIG.6. Incorporation of [l-14C]glycerolinto phospholipids of isolated nerve endings. 0 , Phosphatidylcholine; A, phosphatidylinositol; 0, phoshatidyl ethanolamine; SR, specific radioactivity. From Lunt and Lapetina (1970a).

258

GEORGINA RODRIGUEZ de LORES ARNAIZ AND EDUARDO De ROBERTIS

IX. AMINO ACID UPTAKE AND PROTEIN SYNTHESIS

As well demonstrated in peripheral nerves and also in the CNS (Barondes, 1964, 1966), proteins flow from the perikarya to the nerve endings; however, the possibility of a local protein synt,hesis cannot be discarded. The advantage of the I N E fractions is that they may be prepared rather uncontaminated from other protein-synthesizing systems. The literature on this subject is a t present rather controversial, but it seems well established that the intraterminal mitochondrion is the most important contributor to the local protein synthesis of the I N E ; the role in protein synthesis of the other components of this particle appear rather unclear. A study of the incorporation of [14C]leucineinto the proteins of the submitochondrial fractions of the guinea pig cerebral cortex showed that the free mitochondria were labeled a t a much faster rate than the I N E (Bachelard, 1966). Using I N E separated in Ficoll, Morgan and Austin (1968) found that [14C]leucinewas incorporated into proteins of the axoplasm, mitochondria, and membranes but not in synaptic vesicles. This synthesis was not stimulated by ATP or by a n ATP-generating system. They concluded that the nerve ending contained two protein-synthesizing systems-one probabIy mitochondrial and sensitive to ChIoramphenicol, and the other nonmitochondrial and inhibited by cycloheximide. Autilio et al. (1968) arrived at essentially similar conclusions. Gordon and Deanin (1968) sustained that the protein synthesis in I N E occurs exclusively in the intraterminal mitochondria and that brain mitochondrial protein synthesis-at variance with mitochondria of other tissues-were resistant to chloramphenicol and sensitive to acetoxycycloheximide ; a similar sensitivity to antibiotics was also reported by Goldberg (1971). Using different isolation techniques, Bosmann and Hemsworth (1970) found that leucine, glycine, and to a lesser extent aspartic acid were incorporated into proteins in the intraterminal mitochondria of the I N E and that both chloramphenicol and cycloheximide inhibited protein (and glycoprotein) synthesis. Protein synthesis in nerve endings has also been studied with the use of autoradiography a t the electron microscope level after intracerebral injection (Droz and Barondes, 1969) or incubation in vitro (Cotman and Taylor, 1971). For the incorporation of labeled amino acids into proteins, the ionic content of the medium appears to be critical. Thus the presence of 100 m M Naf and 10 mM K+ results in 4-fold stimulation over a control in sucrose (in the absence of both Na and K). The role of the high-energy compounds produced by mitochondria in protein synthesis is demonstrated by inhibition with oligomycin and dinitrophenol (Autilio et al., 1968). The effect of

PROPERTIES OF THE ISOLATED NERVE ENDINGS

259

ionic stimulation on amino acid uptake was found to be related to the function of the Na+-K+ activated ATPase which is present in the nerveending membrane (see Section 111, B). Appel et d.(1969) reported a relationship between the activity of this enzyme, oxygen uptake, potassium transport,, and synthesis of proteins. A tight coupling of all these functions was suggested. Hypertonicity of the medium produced inhibition of protein synthesis, but this was reversible ; on the contrary, hypotonicity, by damaging the nerve-ending membrane, produced a n irreversible deterioration of protein synthesis (Morgan and Austin, 1969). X. UPTAKE MECHANISMS RELATED TO THE TRANSMITTER FUNCTION

As mentioned in Section 111, A the synthesis, storage, and release of transmitter substances is one of the main functions of nerve endings. The different biogenic amines and the amino acids that are released at the synaptic cleft may be taken up again and reutilized as such. Products of their degradation may also enter the terminal to be used by the synthesizing enzymes. I n other cases, precursors may enter directly through the nerve-ending membrane to find their way in the different metabolic pathways that will achieve the synthesis and storage of the transmitter within the synaptic vesicles. A. Choline

Acetylcholine is degraded by acetylcholinesterase at the synaptic cleft, but the choline moiety may be reutilized, after being taken up b y the nerve ending. Labeled choline put into the medium is rapidly incorporated into the INE, and a substantial part of it remains unaltered within the nerve ending, as may be demonstrated chromatographically after osmotic shock. This suggests that choline is taken across the limiting membrane into the inner compartment of the INE. Choline influx shows two components: one that is linear with increasing concentrations, and another that tends to saturate. The uptake of choline is activated by sodium ions (Marchbanks, 1968). According to Diamond and Kennedy (1969) , the first component is nonspecific, and the one exhibiting saturation kinetics is specific. The latter was competitively inhibited with hemicholinium-3 while the other was unaffected. Furthermore only about 5% of the choline was found to be transformed into acetylcholine within the I NE . In addition to choline and acetylcholine, the radioactivity was found as phosphorylcholine and betaine. The small fraction of [L4C]acetylcholinesynthesized was found mainly in the soluble compartment, and less within synaptic

260

GEORGINA RODRIGUEZ da LORES ARNAIZ AND EDUARDO De ROBERTIS

vesicles separated by osmotic shock. One possible explanation is that the recently stored vesicles are more unstable and do not resist the drastic isolation procedure (Marchbanks, 1969). More recently Haga (1971), using small concentrations of [14C]choline in the medium found that a considerable portion (50%) was converted into [14C]acetylcholinewithin the INE. It was found that sodium ions had an important effect in the uptake of choline and the synthesis of acetylcholine. A significant part of the acetylcholine synthesized in these experiments was found to be rapidly released by increasing the K+ concentration in the medium. After intracerebral injection of [14C]hemicholiniumit was demonstrated that it penetrates through cell membranes including nerve endings and mitochondria. Thus the effect of this drug may not be only by competition for the entrance of choline a t the nerve-ending surface. The possibility that hemicholinium could be acetylated instead of the natural substrate has been proposed (Rodriguez de Lores Arnaiz et al., 1970). 0. GA0A

This amino acid, which is characteristic of the CNS and acts as an inhibitor in synaptic transmission (see Roberts, 1968), may in part be taken up again into the nerve endings. When labeled GABA was used, both INE and mitochondria accumulated it (Varon et al., 1965). The mechanism of uptake is both energy independent and dependent; in other words, some accumulation occurs in the cold, and this increases a t 30°C. GABA accumulation at 30°C is stimulated by pyruvate, but not by glucose, and is inhibited by metabolic poisons. The uptake of GABA depends on the presence of electrolytes, particularly sodium, and does not occur in a sucrose solution. The influence of the Na+ and K+ ATPase on this uptake is suggested by the inhibitory effect of ouabain (Kuriyama et al., 1969). The property of certain nerve endings to accumulate exogenous GABA may be used cytochemically. Slices of brain were incubated with 13H] GABA and then sectioned and processed for radioautography a t the electron microscope level (Bloom and Iversen, 1971). The number of nerve endings accumulating [ SH]GABAwas estimated in sections of the cerebral cortex and in homogenates. In both cases between 27% and 30y0 of the terminals were found to accumulate this inhibitory transmitter. C. Tryptophan

In nerve endings producing 5-hydroxytryptamine, the rate of synthesis of the transmitter is determined by the intraterminal concentration of

PROPERTIES OF THE ISOLATED NERVE ENDINGS

26 1

L-tryptophan. This level will be determined by the balance between its influx and efflux across the nerve-ending membrane and by its rate of utilization within the terminal (Grahame-Smith and Parfitt, 1970). The uptake of L-tryptophan into the I N E is rapid, temperature-dependent and partially inhibited by cyanide, 2-deoxy-~-g~ucose, and ouabain. This process is probably mediated by a carrier stereospecific system, since the D isomer is a poor inhibitor of the uptake of the L isomer. The uptake process was competitively inhibited by L-phenylalanine, but was independent of the concentration of Na+ in the medium. Kinetic studies indicated the presence of a saturable carrier transport system already present in the rat brain a t birth. Using I N E preloaded with L-tryptophan, evidence for countertransport or facilitated diffusion was found. Most of the L-tryptophan taken up remained as such within the I N E and, as in other uptake mechanisms, this was dependent of the integrity of the nerve-ending membrane. In the synthesis of 5-HT the rate-limiting step is the 5-hydroxylation of the L- tryptophan present within the nerve ending. For these reasons, these studies may be relevant to understanding some congenital mental defects caused by metabolic diseases, such as phenylketonuria and hyperleucinemia (Grahame-Smith and Parfitt, 1970). D. Norepinephrine

Nerve endings isolated in sucrose achieve optimal uptake of norepinephrine in the presence of Na+ and K+ a t physiological concentrations. I t has been postulated that these ions may be related to the formation of ATP-metal-norepinephrine complexes and that the activation of Na+-K+ ATPase may also be involved (Colburn et al., 1968). The uptake mechanism of the I N E is inhibited by reserpine and also by ouabain. A carriermediated transport dependent on the ionic environment has been postulated. Bogdanski et al. (1968, 1970a, b) have studied the conditions for the uptake of 5-hydroxytryptamine and norepinephrine into INE. The energy for the transport could be derived from the inward movement of Na+ down a concentration gradient. Metabolic poisons, such as 2 ,sdinitrophenol and cyanide, decreased the uptake, probably by reducing the ATP levels. The uptake was virtually abolished in the absence of K+. All these findings suggest a central role for the Na+-K+ ATPase present in the nerve-ending membrane. However, other evidence suggests that the inward directed Na+ concentration gradient and/or the outward directed K+ concentration gradient may not be the only sources of energy for the uptake of [3H]norepinephrine by the I N E (White and Keen, 1970).

262

GEORGINA RODRIGUEZ d e LORES ARNAIZ AND EDUARDO De ROBERTIS

XI. ION PERMEABILITY

A. Sodium

In Section 111, B we mentioned that the nerve-ending membrane has a high content of a Na+-I(+ stimulated Mg2+-dependentATPase (see Rodriguez de Lores Arnaiz el al., 1967). Since this enzyme system appears to be involved in the active transport of Na+ and K+ across cell membranes in general (see Skou, 1965), a similar assumption could be made for the INE. Indeed when Ficoll-isolated nerve endings are put in a medium containing 22Na,a rapid influx is produced which is temperature- and time-dependent. The plateau level reached a t 37" in 20 minutes indicated that an equilibrium between the influx and outflux rates of Na+ was reached. The effect of changing the temperature from 0 to 37°C is evident in the influx, but the reverse cause no change in the total 2zNacontent of the I N E (Fig. 7). The latter process was dependent on the presence of Mgz+ and on the integrity of the nerve-ending membrane. Metabolic inhibitors, such as cyanide and iodoacetate and also ouabain, inhibited the outflux of Na+ but not the influx (Ling and Abdel-Latif, 1968). The addition of K+ produced a release of Na+ in I N E preloaded with 22Na;this suggests the existence of a Na+-K+ exchange transport system in the membrane of the nerve ending, actively able to extrude Na+ and transport K+ inward (Ling and

300 -

0

0 0

Minutes

FIG.7. Effect of temperature changes on Z2Na uptake by isolated nerve endings from rat brain. The arrows indicate changes in temperature. From Ling and Abdel-Latif (1968).

PROPERTIES OF THE ISOLATED NERVE ENDINGS

263

8. Potassium

The ability of INE to accumulate K+ against a concentration gradient has been described (Bradford, 1969). This property is lost in the presence of cyanide and iodoacetate, which prevent ATP and phosphocreatine synthesis. The use of a filtration technique through Millipore filters has facilitated the study of the potassium influx, which is very rapid and reaches a peak in only 3 minutes at 37°C. After incubation of the INE with 10 mM K+, the concentration within the nerve ending increased from 71 mM to 103 mM, indicating a considerable accumulation against a concentration gradient (Escueta and Appel, 1969). Such external conditions are precisely those optimal for Na+-I(+ ATPase activity and for protein synthesis (Appel et al., 1969). The accumulation of potassium within the INE was inhibited by cyanide, 2 ,bdinitrophenol, and ouabain; however, high energy compounds and substances that activate mitochondria1 functions have essentially no effect. Studies on the retention, exchange, and displacement of K+ from the INE indicate that a t least 95% of the intraterminal K+ could be exchanged with the external This exchange declined with decreasing pH. It was suggested that ion exchange might be the sole means by which K+ traverses the plasma membrane (Weinstein and Kuriyama, 1970). Since the available evidence suggests that INE, having intact membranes, are able to extrude Na+ and to absorb K+ against concentrations gradients, it seems possible that they may generate a resting membrane potential. This property, existing in parallel with an active metabolism, could qualify the INE for use in studying the metabolic aspects of transmission. C. Calcium

The importance of calcium in the physiological release of transmitters is well substantiated (Katz and Miledi, 1969). At the arrival of the nerve impulse, Ca ions penetrate the presynaptic region and apparently are instrumental in triggering the release of the transmitter from the synaptic vesicles into the cleft. It has been suggested that four calcium ions are necessary for the release of each quantum of transmitter (Dodge and Rahamimoff, 1967). In slices of cerebra1 cortex, the effect of ouabain on the uptake of calcium was studied. The subcellular fractions thus treated were found to contain more calcium than the controls without ouabain (Stahl and Swanson, 1969). Calcium accumulation was explained as due to an inhibition of the active mechanism involved in the extrusion of calcium. The up-

264

GEORGINA RODRIGUEZ de LORES ARNAIZ AND EDUARDO De ROBERTIS

TABLE IV ATP-

AND

SUBSTRATE-ENERGIZED CALCIUM ACCUMULATION IN SYNAPTOSOMES AND MITOCHONDRIA", b Relative calcium content

Addition (mM)

Succinate (5) Succinate (5) 8-Hydroxybutyrate (5) 8-Hydroxybutyrate (5) ATP (3) .. ATP (3)

Pi

Synaptosomes

Mitochondria-0

(3 mM)

+ +

Mitocondria-W

-

6.0 f 0.5(5) 6.6 f 0.9 (5) 7.0 f 0.8(5) 10.5 f 1.0(8) 0.4 f 0.1(6) 32.2 f 2.1(5) 42.1 f 2.9(5) 16.8 f 0.8(5) 21.1 f 1.2(4) 2.1 f 0.7(4)

+

51.1 f 1.9(11) 63.3 f 3.7(7)

-

+

-

100 f 2.8(10) 100 f 6.2(7) 100 f 4.0(4) 103.5 f 2.5(7) 97.5 f 2.9(3)

-

Data from Lust and Robinson (1970a). The subcellular fractions (approximately 0.7 mg of protein per milliliter) were incubated for 4 minutes a t 30°C in a medium containing 50 mM Tris-HC1 (pH 7.5), 3 mM MgCL, 0.1 mM CaCl2 with tlracer amounts of Wa and the additions listed above. The calcium accumulation was presented in units relative to the W a content of those incubations with 3 mM ATP (the control for the particular fraction). The relationship between synaptosomes, mitochondria-0 (isolated in isotonicity) and mitochondria-W (isolated in hypertonicity) with regard to the rate of ATP-dependent calcium accumulation was 1 :2.5:1.1.Synaptosomal calcium accumulation averaged 46 natoms per milligram of protein for 4 minutes.

take was distributed among the particulate fractions, particularly those containing mitochondria. INE and free mitochondria from rat brain accumulated 46Caby a temperature-sensitive process that requires ATP and Mg2+. Within the nerve ending the structure responsible for the storage of calcium seems to be the mitochondria. This accumulation, although supported by oxidizable substrates and stimulated by organic phosphate, could not be blocked by oligomycin. This suggested that the energy was used without passing into ATP (Table IV) (Lust and Robinson, 1970a). The accumulation of Ca2+ was accompanied by a stoichiometric deposition of phosphate, and the concentration within the mitochondria could reach several hundred times that in the medium. The ef€lux of 45Cawas observed by a further incubation in the absence of ATP, and this outward movement was increased by NaC1,

265

PROPERTIES OF THE ISOLATED NERVE ENDINGS

but not affected by ouabain (Lust and Robinson, 1970b). These findings have been interpreted as indicating that Ca2+enters the nerve ending favored by a n electrochemical gradient and then mitochondria, by incorporating the excess calcium, could regulate the level in the other compartments of the nerve ending. Similar observations have been reported by Diamond and Goldberg (1971). Using &Ca in tracer concentrations, i t was shown that INE take up calcium from a n isotonic saline solution. The initial accumulation occurs rapidly in the first few seconds, and thereafter the entry of Ca2+proceeds linearly for 5-10 minutes. The Ca2+ increases with the concentration of K+ in the medium, but this increase is not observed when the sodium of the saline solution is replaced by lithium (Fig. 8) (Blaustein and Wiesmann, 1970a). The K+-stimulated Ca2+ accumulation is primarily associated with the INE fraction, is temperature dependent, and is reduced by the presence of Mn2+and Cat+ when both ions are in the incubation medium a t equimolar concentration. For Ca2+ uptake the presence of a n intact nerveending membrane is necessary. However, after its accumulation calcium is not in a free form and is relatively insensitive to osmotic shock.

T

a, 1

0

c CL

3

+

N

0

0

O'

5

'

m

a

a

to o

20

50

too

FIG.8. Uptake of Cat+ by isolated nerve endings as a function of the K+ concentration in the incubation medium and the effect of replacing Na+ by Li+. From Blaustein and Wiesmann (1970a).

266

GEORGINA RODRIGUEZ de LORES ARNAIZ AND EDUARDO De ROBERTIS

A marked increase in calcium influx in the I N E was reported when sodium in the medium is replaced by lithium or choline. When the I N E is preloaded with 46Ca,the calcium efflux is reduced when the external sodium is replaced by lithium. Blaustein and Wiesmann (1970b) suggested that a considerable fraction of both Ca2+influx and efflux may be due to a n exchange mechanism between calcium and sodium. D. Anions

The binding of bromide ions by the I N E has been observed. This associatron has been interpreted as due to the presence of ionized cationic components on its surface (Kuriyama, 1970). Only minute amounts of C1- were found associated with the INE, and in a variety of experimental conditions there was virtually no uptake of C1-. Several other anions (I-, Br-, NO;, succinate, pyruvate, and aspartate) appeared to enter the I N E a t physiological pH, while others (acetate, benzoate, formate, SCN-, SO:-, and HCO;) did not. The binding characteristics for anions are probably related to specific selective properties of the membranes (Kuriyama and Roberts, 1971).

XII. CONCLUDING REMARKS

I n the decade that has elapsed since the first isolation of nerve endings from brain, an explosive growth of research on these subcellular particles has taken place. The present review of the literature, carried to the middle of 1971, was not intended to be complete and exhaustive but to give what, in the opinion of the authors, have been the main lines and achievements. The isolation of I N E provided neurobiologists with the possibility of exploring with biochemical and biophysical methods the most important part of the neuron, a unique structure, where transmission of electrochemical signals and probably other fundamental functions of the nervous tissues take place. With the discovery of the synaptic vesicles (De Robertis and Bennett, 1954, 1955) a morphological correlate for the quanta1 release of transmitters (Fatt and Katz, 1952) was established and the nerve ending became recognized as the site of active secretory processes whose activity is mediated by nerve impulses. I n 1964 De Robertis postulated that “nerve endings are essentidly devices differentiated for the secretion of transmitter substances” and that these processes did not markedly differ from the so-called neurohormonal secretion (De Robertis, 1964).

PROPERTIES OF THE ISOLATED NERVE E N D I N G S

267

The work done on the content of active substances and related enzymes in the I N E has confirmed the above postulates and has permitted the recognition of an integrated biochemical machinery involved in this secretory function. Cytochemical studies in relation to the storage of different active substances have led to the localization of transmitters in the I N E and in isolated synaptic vesicles (De Robertis et al., 1963). This is now considered to be an essential criterion in the definition of a chemically mediated synapse. The work on subcellular localization of biogenic amines is now being pursued with success in relation to that of active free amino acids that play a major role in the CNS. Studies on the structural and biochemical organization of the INE have revealed the existence of a t least three subcompartments, i.e., soluble axoplasmic, vesicular, and mitochondrial, that are interrelated within this self-contained unit. This organization may account for the existence of different intraterminal pools of free amino acids or biogenic amines, for the regulation of the absolute rate of reactions of the enzyme systems, for the availability of substrates, and for the permeability of the various membranes limiting such compartments, Although the nerve ending depends on the integrity of the connections with the perikaryon of the cell, through the synthesis and flow of essential proteins, the I N E contains the biochemical machinery able to produce high-energy compounds through glycolysis and oxidative phosphorylation, and to synthesize small molecules and even larger ones, such as lipids, proteins, and polysaccharides. The separation of the nerve ending during cell fractionation leads to the resealing of the nerve-ending membrane and the reformation of a closed structure. Many of the investigations reviewed here demonstrate that the integrity of the limiting membrane is an essential prerequisite for the study of the properties of the INE. Important work is being pursued on the role of the membrane in passive, facilitated, and active transport of molecules and ions. The mechanism of entrance of most of active substances or their precursors into the INE requires the presence of ions and the furnishing of energy-yielding sustrates. Several of these processes seem to be mediated by sterospecific carrier systems. In some cases the energy of transport could be derived from an inward movement of sodium ions down a concentration gradient. In most cases a central role is assigned to the enzyme Na+-K+ ATPase which is built into the structure of the limiting membrane of the nerve ending. Another enzyme which may be essential for the regulatory processes within the nerve ending is adenylcyclase which in our laboratory was shown to be located in the membrane of the I N E with its active sites di-

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GEORGINA RODRIGUEZ de LORES ARNAIZ AND EDUARDO De ROBERTIS

rected to the inner surface of this membrane. According to these observations adenosine 3', 5'-monophospha te might play a presynaptic role which still needs to be uncovered. The readers of this volume will be disappointed with the scarcity of the research regarding ion permeability in INE. These investigations which have been started only the last two years need considerable expansion and provide a n excellent field for the specialists. It is our impression that research on the properties of nerve endings will go for some time in the directions indicated. Other possibilities opened by the I N E are a search for new active substances that may have a transmitter function, the development of methods for the separation of the pre- and postsynaptic membranes, and the study of the mechanism of action of drugs active on the CNS (for a review on this latter point, see Rodriguez de Lores Arnaiz and De Robertis, 1972). We think that the time is now ripe for a breakthrough a t the molecular level, which will come with the application of some of the powerful methods now being employed by molecular biologists. However, in this enterprise we should not lose sight of the main goal-that of tackling the basic mechanisms underlying synaptic transmission. ACKNOWLEDGMENTS The authors wish to thank tjhe following organizations and individuals for permission to use copyrighted material: Elsevier Publishing Co. and Dr. G. G. Lunt for Fig. 1; Pergamon Press for Fig. 2; Elsevier Publishing Co. and I h . H. F. Bradford for Fig. 5 and Dr. G. G. Lunt for Fig. 6; Pergamon Press and I h . C. M. Ling for Fig. 7; Research Institute of National Defence and Dr. M. Blaustein for Fig. 8; Pergamon Press and Dr. H. F. Bradford for Table 111, Wiley (Interscience) and Dr. W. D. Lust for Table I V in this chapter. REFERENCES Abdel-Latif, A. A. (1966). Biochim. Biophys. Acta 121, 403. Abdel-Latif, A. A., and Abood, L. G. (1965). J. Neurochem. 12, 157. Abdel-Latif, A. A., and Smith, J. P. (1970). Biochim. Biophys. Acta 218, 134. Abdel-Latif, A. A., Brody, J., and Ramahi, H. (1967). J . Neurochern. 14, 1133. Abdel-Latif, A. A., Yamaguchi, T., Yamaguchi, M., and Chang, F. (1968). Brain Res. 10, 307. Alberici, M., Rodriguez de Lores Arnaiz, G., and De Robertis, E. (1965). Life Sci. 4, 1951. Albers, R. W., Rodriguez de Lores Arnaiz, G., and De Robertis, E. (1965). Proc. Nat. Acad. Sci. U.S. 53, 557. Appel, S. H., and Parrot, B. L. (1970). J . Neurocheni. 17, 1619. Appel, S. H., Autilio, L., Festoff, B. W., and Escueta, A. V. (1969). J. Biol. Chem. 244, 3166. Austin, L., and Morgan, I. G. (1967). J. Neurochem. 14, 377.

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Autilio, L. A., Appel, S. H., Pettis, P., and Gambetti, P. L. (1968). Biochemistry 7, 2615. Bachelard, H. S. (1966). Biochem. J . 100, 131. Balbzs, R., and Cocks, W. A. (1967). J . Neurochem. 14, 1035. Balbzs, R., Dahl, D., and Harwood, J. R. (1966). J . Neurochem. 13, 897. Baldessarini, R. J., and Vogt, M. (1971). J . Neurochem. 18, 951. Balfour, D. J. K., and Gilbert, J. C. (1971). Biochem. Pharmacol. 20, 1151. Barondes, S. H. (1964). Science 146, 779. Barondes, S. H. (1966). J . Neurochem. 13, 721. Beloff-Chain, A., Cantanzaro, R., Chain, E. B., Masi, I., and Pocchiari, F. (1955). Proc. Roy. SOC.,S o . B 144, 22. Blaustein, M. P., and Wiesmann, W. P. (1970a). In “Drugs and Cholinergic Mechanisms in the CNS” (E. Heilbronn and A. Winter, eds.), pp. 291-307. Research Institute of National Defence, Stockholm. Blaustein, M. P., and Wiesmann, W. P. (1970b). Proe. Nut. Acad. Sci. U.S. 66, 664. Bloom, F. E., and Iversen, L. L. (1971). Nature (London) 229, 628. Bogdanski, I).F., Tissari, A., and Brodie, B. B. (1968). Lije Sci. 7, 419. Bogdanski, D. F., Tissari, A. H., and Brodie, B. B. (1970a). Biochim. Biophys. Acta 219, 189. Bogdanski, D. F., Blaszkowski, T. P., and Tissari, A. H. (1970b). Biochim. Biophys. Acta 211, 521. Bosmann, H. B., and Hemsworth, B. A. (1970). J . Biol. Chem. 245, 363. Bradford, H. F. (1969). J . Neurochem. 16, 675. Bradford, H. F. (1970a). Brain Res. 19, 239. Bradford, H. F. (1970b). In “Drugs and Cholinergic Mechanisms in the CNS” (E. Heilbronn and A. Winter, eds.), pp. 309-321. Research Institute of National Defence, Stockholm. Bradford, H. F., and Thomas, A. J. (1969). J . Neurochem. 16, 1495. Bradford, H. F., Brownlow, E. K., and Gammack, I3. B. (1966). J . Neurochem. 13,1283. Colburn, R. W., Goodwin, F. K., Murphy, D. L., Bunney, W. E., Jr., andDavis, J. M. (1968). Biochem. Pharmacol. 17, 957. Cotman, C. W., and Mahler, H. R. (1967). Arch. Biochem. Biophys. 120, 384. Cotman, C. W., and Taylor, D. A. (1971). Brain Rcs. 29, 366. Cotman, C. W., Mahler, H. R., and Anderson, N. G. (1968a). Biochim. Biophys. Ada 163, 272. Cotman, C. W., Mahler, H. R., and Hugli, T. E. (1968b). Arch. Biochim. Biophys. 126, 821. Davies, W. E. (1970). J . Neurochem. 17, 297. Day, E. G., McMillan, P. N., Mickey, D. D., and Appel, S. H. (1971). Anal. Biochem. 39, 29. Den, H., and Kaufman, B. (1968). Fed. Proc., Fed. Amer. SOC.Exp. Biol. 27, 346. De Robertis, E. (1964). “Histophysiology of Synapses and Neurosecretion.” Pergamon, Oxford. De Robertis, E. (1966). Pharmacol. Rev. 18, 413. De Robertis, E. (1971). Science 171, 963. De Robertis, E., and Bennett, H. S. (1954). Fed. Proc., Fed. Amer. Soc. Exp. Biol. 13,35. De Robertis, E., and Bennett, H. S. (1955). J . Biophys. Biochem. Cytol. 1, 47. De Robertis, E., and Rodriguez de Lores Arnaiz, G. (1969). I n “Handbook of Neurochemistry” (A. Lajtha, ed.), Vol. 2, pp. 365-392. Plenum, New York. De Robertis, E., Pellegrino de Iraldi, A,, Rodriguez de Lores Arnaiz, G., and Salganicoff, L. (1962a). J . Neurochem. 9, 23.

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De Robertis, E., Rodriguez de Lores Arnaiz, G., and Pellegrino de Iraldi, A. (1962b). Nature ( L o n h ) 194,794. De Robertis, E., Rodriguez de Lores Arnaiz, G., Salganicoff,L., Pellegrino de Iraldi, A., and Zieher, L. M. (1963). J. Neurochem. 10, 225. De Robertis, E., Alberici, M., Rodriguez de Lores Arnaiz, G., and Azcurra, J. M. (1966a). Life Sci. 5, 577. De Robertis, E., Lapetina, E. G., Pecci Saavedra, J., and Soto, E. F. (1966b). Life Sci. 5 , 1979. De Robertis, E., Rodriguez de Lores Arnaiz, G., Alberici, M., Butcher, R. W., and Sutherland, E. W. (1967). J. Biol. Chem. 242, 3487. De Robertis, E., Lapetina, E. G., and Wald, F. (1968). Exp. N ~ ~ T o21, Z . 322. Diamond, I., and Fishman, R. A. (1971). Annu. Meet., Amer. Acad. Neurol., New York, April 26-May 1. Diamond, I., and Goldberg, A. L. (1971). J . Neurochem. 18, 1419. Diamond, I., and Kennedy, E. P. (1969). J . Biol. Chem. 244, 3258. Dodge, F. A., and Rahamimoff, R. (1967). J. Physiol. (London) 193, 419. Dowling, J. E., and Boycott, B. B. (1966). Proc. Roy. SOC.,Ser. B 166, 80. Droz, B., and Barondes, S. H. (1969). Science 165, 1131. Eichberg, J., Whittaker, V. P., and Dawson, R. M. (1964). Biochem. J . 92, 91. Escueta, A. V., and Appel, S. H. (1969). Bwchemistry 8, 725. Fatt, P., and Katz, B. (1952). J . Physiol. (London) 117, 109. Feit, H., and Barondes, S. H. (1970). J . Neurochem. 17, 1355. Gfeller, E., Kuhar, M. J., and Snyder, S. H. (1971). Proc. Nut. Acad. Sci. U.S. 68, 155. Goldberg, M. A. (1971). Bruin Res. 27, 319. Gordon, M. W., and Deanin, G. G. (1968). J . Biol. Chem. 16, 4222. Grahame-Smith, D. G., and Parfitt, A. G. (1970). J . Neurochem. 17, 1339. Gray, E. G., and Whittaker, V. P. (1962). J. Anat. 96, 79. Haga, T. (1971). J. Neurochem. 18, 781. Harvey, J. A,, and McIlwain, H. (1969). In “Handbook of Neurochemistry” (A. Lajtha, ed.), Vol. 2, pp. 115-136. Plenum, New York. Hokin, L. E. (1969). In “The Structure and Function of Nervous Tissue” (G. H. Bourne, ed.), Vol. 3, pp. 161-184. Academic Press, New York. Hosie, R. J. A. (1965). Bhchem. J. %, 404. Inouye, A., Kataoka, K., and Shinakawa, Y. (1963). Biochim. Biophys. Acta 71,491. Jones, D. G., and Bradford, H. F. (1971). Bruin Res. 28, 491. Kataoka, K., and De Robertis, E. (1967). J . Pharmacol. E z p . Ther. 156, 114. Katz, B., and Miledi, R. (1969). J. Physiol. (London) 203, 459. Keen, P., and White, T. D. (1970). (1970). J. Neurochem. 17, 565. Keen, P., and White, T. D. (1971). J. Neurochem. 18, 1097. Kornguth, S. E., Anderson, J. W., and Scott, G. (1969). J. Neurochem. 16, 1017. Kornguth, S. E., Flangm, A. L., Siegel, F. L., Geison, R. L., O’Brien, J. F., Lamar, C., Jr., and Scott, G. (1971). J . B i d . Chem. 246, 1177. Kuhar, M.J., Shmkan, E. G., and Snyder, S. H. (1971a). J . Neurochem. 18, 333. Kuhar, M. J., Taylor, K. M., and Snyder, S. H. (1971b). J . Neurochem. 18, 1515. Kuriyama, K. (1970). Life Sci. 9, 1371. Kuriyama, K., and Roberts, E. (1971). Brain Res. 26, 105. Kuriyama, K., Weinstein, H., and Roberts, E. (1969). Brain Res. 16, 479. Kurokawa, M., Sakamoto, T., and Kato, M. (1965). Biachem. J. 97, 833. Lapetina, E. G., Soto, E. F., and De Robertis, E. (1967). Bwchim. Biophys. Acta 135,33.

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Lapetina, E. G., Soto, E. F., and De Robertis, E. (1968). J. Ncurochem. 15, 437. Lapetina, E. G., Rodriguez de Lores Arnaiz, G., and De Robertis, E. (1969a). J . Neurochem. 16, 101. Lapetina, E. G., Rodriguez de Lores Arnaiz, G., and De Robertis, E. (1969b). Biochim. Biophys. Acta 176, 643. Lapetina, E. G., Lunt, G. G., and De Robertis, E. (1970). J. Neurobiol. 1, 295. Ling, C. M., and Abdel-Latif, A. A. (1968). J . Neurochem. 15, 721. Lunt, G. G., and Lapetina, E. G. (1970a). Brain Res. 17, 164. Lunt, G. G., and Lapetina, E. G. (1970b). Brain Res. 18, 451. Lust, W. D., and Robinson, J. D. (1970a). J . Ncurobiol. 1, 303. Lust, W. D., and Robinson, J. D. (1970b). J. Neurobiol. 1, 317. McGeer, P. L., Bagchi, S. P., and McGeer, E. G. (1965). Lijc Sci. 4, 1859. Mahler, H. R., McBride, W., and Moore, W. J. (1970). In “Drugs and cholinergic Mechanisms in the CNS” (E. Heilbronn and A. Winter, eds.), pp. 225-243. Research Institute of National Defence, Stockholm. Mandel, P., and Nussbaum, J. L. (1966). J. Neurochem. 13, 629. Mangan, J. L., and Whittaker, V. P. (1966). Biochem. J. 98, 128. Marchbanks, R. M. (1967). Biochem. J. 104, 148. Marchbanks, R. M. (1968). Biochem. J. 110, 533. Marchbanks, R. M. (1969). Biochcm. Pharmacol. 18, 1763. Maynert, E. W., Levi, R., and De Lorenzo, A. J. (1964). J. Pharmacol. Exp. Ther. 144, 385. Michaelson, I. A., and Whittaker, V. P. (1963). Biochcm. Pharmacol. 12, 203. Morgan, I. G., and Austin, L. (1968). J . Neurochem. 15, 41. Morgan, I. G., and Austin, L. (1969). J . Neurobiol. 1, 155. Nafstad, P. H. J., and Blackstad, T . W. (1966). 2. Zelljorsch. Mikrosk. Anat. 73, 234. Neal, M. J., and Iversen, L. L. (1969). J . Neurochem. 16, 1245. ohman, R. (1971). J. Neurochem. 18, 89. Quastel, J. H. (1969). In “The Structure and Function of Nervous Tissue” (G. H. Bourne, ed.), Vol. 3, pp. 61-107. Academic Press, New York. Rall, W., Shephered, G. M., Reese, T. S., and Brightman, M. W. (1966). Exp. Neurol. 14, 44. Roberts, E. (1968). In “Structure and Function of Inhibitory Neuronal Mechanisms” (C. von Euler, S. Skoglund, and V. Soderberg, eds.), Vol. 10, pp. 401418. Pergamon, Oxford. Rodriguez de Lores Arnaiz, G., and De Robertis, E. (1962). J . Neurochem. 9, 503. Rodriguez de Lores Arnaiz, G., and De Robertis, E. (1964). J. Neurochem. 11, 213. Rodriguez de Lores Arnaiz, G., and De Robertis, E. (1972). In “Pharmacology of the Cell” (S. Dikstein, ed.). Thomas, Springfield, Illinois. Rodriguez de Lores Arnaiz, G., Alberici, M., and De Robertis, E. (1967). J. Neurochem. 14, 215. Rodriguez de Lores Arnaiz, G., Zieher, L. M., and De Robertis, E. (1970). J.Neurochem. 17, 221. Rodriguez de Lores Arnaiz, G., Alberiei de Canal, M., and De Robertis, E. (1971). Brain Res. 31, 179. Ryall, R . W. (1964). J . Neurochem. 11, 131. Ryan, K. J., Kalant, H., and Thomas, E. L. (1971). J. Cell Biol. 49, 235. Sdganicoff, L., and De Robertis, E. (1963). Life Sci. 2, 85. Salganicoff, L., and De Robertis, E. (1965). J . Neurochem. 12, 287.

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Seminario, L. M., Hren, N., and Mmez, C. J. (1964). J. Neurochem. 11, 197. Siege], G. J., and Albers, R. W. (1970). In “Handbook of Neurochemistry” (A. Lajtha, ed.), Vol. 4, pp. 13-44. Plenum, New York. Skou, J. C. (1965). Phyeiol. Rev. 45, 596. Stahl, W. L., and Swanson, P. D. (1969). J. Neurochem. 16, 1553. Tanaka, R., and Abood, L. G. (1963). J. Neurochem. 10, 571. Tanaka, R., and Abood, L. G. (1964). Arch. Biochem. Biophys. 105, 554. Tedeschi, H., and Harris, D. L. (1955). Arch. Biochem. Biophys. 58, 52. Varon, S., Weinstein, H., Kakefuda, T., and Roberts, E. (1965). Biochem. Phurmacol. 14, 1213. von Hungen, K., Mahler, H. R., and Moore, W. J. (1968). J. Bwl. Chem. 243, 1415. Wald, F., Mazzuchelli, A., Lapetina, E., and De Robertis, E. (1968). Exp. Neurol. 21,336. Weinstein, H., and Kuriyama, K. (1970). J . Neurochem. 17, 493. Weinstein, H., Roberts, E., and Kakefuda, T. (1963). Biochem. Pharmacol. 12, 503. White, T. D., and Keen, P. (1970). Riochim. Biophys. Acta 196, 285. Whittaker, V. P. (1966a). Pharmacol. Rev. 18, 401. Whittaker, V. P. (1966b). Ann. N . Y. Acad. Sci. 137, 982. Whittaker, V. P., Michaelson, I. A., and Kirkland, R. J. A. (1964). Biochem. J.90, 293. Wiegant, H. (1967). J. Neurochem. 14, 671. Wofsey, A. R., Kuhar, M. J., and Snyder, S. H. (1971). Proc. Nut. Acad. Sci. U.S. 68, 1102. Zatz, M., and Barondes, S. H . (1971). J . Nmrochem. 18, 1125. Zieher, L. M., and De Robertis, E. (1963). Biochem. Pharmacol. 12, 596. Zieher, L. M., and De Robertis, E. (1964). Congr. Asoc. Latinoamer. Cienc. Fisiol., 6th, 1964 p. 150.

Transport and Discharge of Exportable Proteins in Pancreatic Exocrine Cells: in W r o Studies J . D . JAMIESON The Rockefeller UniuvPrsity New York, New York

I. Introduction. . . . . . . . . . . . . . . . . . . 11. The Secretory Process in Resting Pancreatic Exocrine Cells . . . . . A. General Organizational Features . . . . . . . . . . . B. St,eps in the Secretory Process . . . . . . . . . . . . C. licquirements for Protein Synthesis and Metabolic Energy in the Secretory Pathway . . . . . . . . . . . . . . . 111. Physiological Modulation of t,hc Secretory Process in Pancreatic Exocrine Cells. . . . . . . . . . . . . . . . . . . . . A. Morphological Features of Zymogen Granules Discharge in Vitro . , B. Biochemical Changes Accompanying in Vilro Discharge . . . . IV. Interrelationships of Intercellular Membranes During the Secretory Process References . . . . . . . . . . . . . . . . . . .

273 274 274 279 290 315 317 319 333 336

1. INTRODUCTION

The mammalian pancreatic exocrine cell synthesizes large amounts of digestive enzymes and their zymogens, which it transports, concentrates, and finally stores temporarily in zymogen granules prior to release into the duct system of the gland. For these reasons, this cell type has proved to be a useful system with which to study the initial events involved in the synthesis of exportable proteins and the factors that control and regulate the intracellular transport and discharge of the stored products. The purpose of this article is to review studies done in collaboration with Dr. G. E. Palade on the factors which determine the orderly flow of exportable products from their site of synthesis to their ultimate discharge from the storage granules. Without going into details, I should state that a 273

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number of different transport operations are involved in the intracellular processing of exportable materials and that a11 these are related to the intracellular membrane systems of the cell. Thus while most of the article will be devoted to an assessment of the factors involved in the intracellular transport of the contents for which we have considerable information, it will also be appropriate to consider the dynamic relationships of the membrane containers themselves, although for lack of firm evidence, the discussion will be mainly speculative. Finally since this article is designed mainly to review recent findings concerning the function of the pancreatic exocrine cell, no attempt will be made to cover comprehensively the earlier literature in the field, which has been extensively reviewed by Palade, Siekevitz, and Caro (1962). I n addition several recent reviews have dealt with the biochemistry of the discharge of secretory proteins (Schramm, 1967) and with the relationship between discharge induction and rates of resynthesis of secretory proteins (Webster, ,1969)) and changes in phospholipid (Holtin, 1968) and nucleic acid metabolism (Webster, 1969).

II. THE SECRETORY PROCESS IN RESTING PANCREATIC EXOCRINE CELLS A. General Organizational Features

The general structural features of the exocrine pancreatic cell are diagrammed in Fig. l and illustrated in light and low power electron micrographs in Figs. 2A and 2B. For the sake of brevity, the following description will pertain only to those elements of the cell involved in the synthesis and processing of secretory proteins. As is typical of cells that are highly specialized to synthesize large amounts of exportable proteins, -60% of the volume of the cell is occupied by elements of the rough-surfaced endoplasmic reticulum. These are characteristically arranged in a series of more or less parallel though often convoluted and interconnected flattened saccules or cisternae and which are primarily located a t the basal pole of in width the cell. Each cisterna is bounded by a unit membrane -70 which separates one intracellular compartment (the cisternal cavity) from the surrounding cytoplasmic matrix or cell sap. The outer or cytoplasmic surface of the ER membrane typically is studded with ribosomes which are arranged in whorls, rosettes, or linear arrays comprising the attached polysomes. I n addition to attached polysomes, numerous free polysomes also reside in the cytoplasmic matrix between the ER cisternae.

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FIG.1. Diagram of a pancreatic exocrine cell. The structures involved in the synthesis, transport, and discharge of secretory proteins are indicated and include cisternae of the rough endoplasmic reticulum with attached polysomes (RER) ; transitional elements of the RER (tr); smooth-surfaced vesicles of the Golgi peripheral region (Gv); Golgi cisternae (Gc); condensing vacuoles in the process of maturing to zymogen granules (CV); zymogen granules (Z); and the acinar lumen (L). Numbers indicate the steps in the secretory process discussed in the text.

Again, typical of a resting exocrine cell, the apical cytoplasm is populated with numerous spherical mature storage granules, each bounded by a smooth-surfaced unit membrane (-80-100 A in thickness), and containing material of high electron opacity. The apical plasmalemma of the cell is provided with numerous microvilli which protrude into the duct lumen, the latter being formed by the conjunction of several exocrine cells, each joined laterally to its neighbors by typical junctional elements (zonulae adhaerentes and occludentes, and desmosomes). Located between these two poles of the cell, in the supranuclear region, are the elements of the Golgi complex (Fig. 3). These consist of a number of stacks of laterally placedoflattened saccules bounded by a smooth-surfaced unit membrane -80 A wide and swarms of peripheral located small smooth-surfaced vesicles -45-60 nm in diameter. Centrally located in the complex, and by our definition part of it, are a number of immature zymogen granules or condensing vacuoles. These vacuoles, also bounded by a unit membrane, possess an irregular, scalloped profile and contain material of variable density which, as will be discussed later, reflects the degree of maturation of their content.

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Particular note should be made of those portions of the RER which appear to feed centripetally toward the Golgi peripheral region. Here, as noted in the diagram and seen to advantage in Fig. 3, the E R cisternae are part rough surfaced and part smooth surfaced due to the absence of attached ribosomes. Because of their morphological appearance, these specialized portions of the RER are termed transitional elements. Fre-

FIG.2A. Light micrograph of a portion of a guinea pig pancreatic slice. The solid and dashed lines delineate the exocrine cells comprising an acinus which border on an acinar lumen (L). B, basal portion of a cell containing cisternae of the RER; C, condensing vacuoles in thc Golgi zone; A, cell apex packed with zymogen granules (ZG); m, mitochondria; N, nucleus; n, nucleolus; d, interlobular duct. X 1750.

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FIG.2B. Low magnification electron micrograph t,aken from the same block of tissue as in Fig. 2A. Several exocrine cells are joined at their apices by junctional complexes

(jc and opposed arrows) which delineate an acinar lumen (L). The apical plasmalemma is provided with numerous microvilli (mv). An intercalated duct cell (id) is interposed between two acinar cells. Elements of the rough ER (rer) are basally located in the cells. Golgi vesicles (Cv), cist,ernac (Gc) and several condensing vacuoles in states of increasing maturity are indicated (cv,, cv2, cv3).Numerous zymogen granules (2) populate the cell apices. N, nucleus; n, nucleolus; C, capillary. From Jamieson and Palade, 1967a. Court,esy of t.he Journal o j Cell Biology. X8400.

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FIG.3. Thin section through €he Golgi region of an exocrine cell. Elements of the rough ER (rer) feed into the Golgi peripheral region and terminate in numerous budshaped transitional elements (t). Numerous small, smooth surfaced vericles (GV) populate the Golgi peripheral region and appear to abut on or fuse with (arrows) the condensing vacuole (cv). Note the coated membrane expecially on the proximal side of the condensing vacuole and surrounding some of the Golgi vesicles. GC, Golgi cisternae; a , zymogen granule. From Jamieson arid Palade, 1967a. Courtesy of the Journal of Cell Biology. X26,400.

quently the transitional elements extend toward the Golgi peripheral region in the form of distinct buds. Their unit membranes are usually somewhat thicker than that of the remainder of the RER cisternae and in some instances appear to be provided with a poorly defined fuzzy coat. From the above description it is clear that the exocrine cell contains a number of discrete membrane-bounded compartments. In addition it is clear from the topographical arrangements of these compartments that the

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cell is highly polarized, the synthesizing machinery being located basally, the storage elements apically, with the Golgi complex, which can be regarded as a way station in the processing of the secretory products, occupying a n intermediate posit.ion. While the above description pertains specifically to the exocrine pancreatic cell, it applies, with minor modification, to the majority of cells that temporarily store their secretory products in storage granules including other exocrine as well as endocrine cells. The organization of endocrine cells in general and those of the anterior pituitary in particular have recently been reviewed by Fawcett et al. (1969) and by Farquhar (1971), respect,ively.

B. Steps in the Secretory Process Step 1 is the synthesis of exportable proteins in association with polysomes attached to the outer or cytoplasmic aspect of the cisternae of the RER. While the synthesis of only two of the population of the exportable proteins, a-chymotrypsinogen and a-amylase, has been clearly established to occur in association with attached polysomes (Siekevitz and Palade, 1960; Redman et al., 1966), it can be confidently predicted that this site will also be the synthesis of the remainder since, as will bc seen later, the bulk of all exportable proteins appears to follow the same intracellular transport route and is ultimately sequestered in the content of the zymogen granules. Likewise, in the hepatic parenchymal cell, a specific exportable protein, albumin, has been found to be synthesized in association with attached polysomes (Redman, 1969). The polysomes free in the cell sap apparently are not involved in the synthesis of exportable proteins but produce sedentary, nonexportablc proteins, such as ferritin in the case of the hepatocyte (Redman, 1969). Step 2 effects segregation within, and the movement of secretory proteins through, the ER cisternal space. From their site of synthesis, the secretory proteins eventually gain access to the cisternal space of the RER. This step has recently been explored in detail by Redman, Siekevitz, and Palade (1966), Redman and Sabatini (1966), Blobel and Sabatini (1970), and Sabatini and Blobel (1970), who demonstrated clearly that peptides destined for export are vectorially transferred across the limiting membrane of the RER cisternae during the course of their growth and segregated upon completion of synthesis (either by normal chain termination or artificially with puromycin) in the cisternal cavity. These processes-elongation and discharge-are not dependent on energy (other. than that involved in peptide bond formation and discharge

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J. D. JAMIESON

FIG.4. Model of the ribosome-membrane interaction on the RER. A nascent polypeptide is sliown growing in a channel or groove in the large ribosomal subunit and finally being transferred into the cisternal cavity. Taken from Sabatini and Blobel (1970). Courtesy of the Journal of Cell Biology.

from transfer RNA), are uninfluenced by secretagogues (Redman, 1967), but are governed by structural interrelationships between the ribosome and the cisternal membrane. These features are indicated in the model in Fig. 4, taken from the work of Sabatini and Blobel (1970), which shows the relationship of the large ribosomal subunit to the E R membrane. According to this model, the nascent peptide grows within a channel or groove in the large subunit [which accounts for the resistance of this protected fragment to exogenously added proteases (Sabatini and Blobel, 1970)]and eventually penetrates the ER membrane through an intermittently or continuously patent orifice (Step 2). After segregation in the cisternal space, the secretory proteins percolate through the channels of the RER whence they will ultimately become confined within the zymogen granules a t the cell apex. Here their presence has been reliably determined by the cell fractionation studies of Greene, Hirs and Palade (1963), and of Keller and Cohen (1961), who demonstrated unequivocally that the complement of secretory proteins in zymogen granules is identical to that found in pancreatic juice collected from the gland. It should be mentioned that these results pertain strictly to the bovine pancreas. The cataloguing and analysis of secretory proteins in the juice and granules from other species is as yet incomplete, although for a number of enzymes in other species it has been shown that digestive enzymes are present in high concentrations in zymogen granule fractions. In addition, it should be pointed out that the cell fractionation data noted above give only the average composition of the zymogen granule fraction recovered from the gland and cannot specify whether all granules are alike as to enzyme composition. It remains to be shown whether each granule (or each cell in the gland for that matter) contains all, one, or a limited number of the total complement of secretory proteins produced by the

PROTEINS IN PANCREATIC EXOCRINE CELLS

28 1

gland. Although some immunocytochemical studies (Marshall, 1954; Yasuda and Coons, 1966) have suggested that all exocrine cells are alike in their complement of most digestive enzymes, other studies (Rothman, 1967) have shown that individual secretory proteins may be synthesized or discharged with unique rates. I n the case of most other glandular tissues including the endocrine pancreas, adenohypophysis, adrenal medulla, etc., a single cell type is generally responsible for the production of one exportable product. As seen in the diagram (Fig. l), the intervening steps in the pathway between the R E R and the zymogen granules (steps 2 4 ) involve a number of different types of transport operations each mediated by membranebounded compartments of the cell and a11 concerned with the directed movement of proteins through the cell in the basoapical direction. Over the past 10 years, a number of studies in our and other laboratories have been concerned with defining the route and kinetics of the transport operations over this part of the pathway. According to the EM radioautographic studies of Car0 and Palade (1964) and of van Heyningen (1964), it was evident that labeled secretory proteins become closely associated with elements of the Golgi complex in the course of their passage through the cell, particularly with small smooth-surfaced vesicles located in the periphery of the Golgi region (step 3). Although these studies clearly implicated the small vesicles in this part of the pathway, the results could not be interpreted unambiguously for two reasons. First, owing to the resolution of the radioautographic technique used (-2000 A), it was not possible to ascertain whether the label was associated with the content of the small vesicles of the Golgi or with the surrounding cell sap. complex (whose diameters are -500 i) Second, because the studies were conducted on intact animals, it was not possible to obtain a well defined pulse labeling of secretory proteins which, as will be seen, is required to follow clearly the progress of the wave of labeled proteins through the cell. This is especially important in relation to step 3, which is rapid. To circumvent some of these limitations, we have utilized a n in vitro system of pancreatic slices, the characteristics of which have been published in detail (Jamiesonand Palade, 1967a, b). Briefly, with this system it is possible to pulse-label secretory proteins with radioactive amino acids (e. g., ~-1eucine-3H)for times sufficiently short ( < 3 minutes) to enable us to follow the progress of the wave of labeled proteins through the cell during subsequent incubation in the presence of a large excess of unlabeled amino acids (chase incubation). This provides the necessary time resolution for evaluating the process. I n addition, the slices can be homogenized and

282

J. D. JAMIESON

FIG.5. Electron microscopic radioautogram of a pancreatic exocrine cell pulse labeled for 3 minutes in uitro with leucine-8H. The radioautographic grains mainly (- 86%) mark elements of the rough ER (rer), m, mitochondrion; N, nucleus. From Jamieson and Palade, 1967b.Courtesy of the Journal of Cell Biology. X11,900.

PROTEINS IN PANCREATIC EXOCRINE CELLS

283

subjected to a cell fractionation scheme in which all the subcellular elements involved in the transport scheme can be either isolated or accounted for. This latter procedure thus gives the necessary spatial resolution for defining the roles of subcellular organelles in transport, especially in regard to the partitioning of secretory proteins between the small vesicles of the Golgi periphery and the surrounding cell sap. The cell particulates isolated include rough microsomes derived from fragmented and rehealed portions of the RER, smooth microsomes derived largely from vesicles and cisternae of the Golgi complex (the main source of smooth microsomes in the pancreatic exocrine cell consists of elements of the Golgi complex in contrast to other cell types, such as the hepatic parenchymal cell, where both Golgi and smooth E R elements contribute); a total zymogen granule fraction consisting mainly of mature zymogen granules with a smaller (-5%) population of cosedimenting condensing vacuoles which can be identified in the pellet as in the cell by their morphological characteristics; and a final postmicrosomal supernatant, which represents in part the cytoplasmic matrix with an estimated contribution of -lo’% from proteins stored in the duct system of the gland. By the complementary use of cell fractionation techniques and electron microscopic radioautography applied to the slices at the end of a 3-minute pulse with leucine-3H, and after various times of chase incubation spanning a complete secretory wave, we are now able to reconstruct the intracellular secretory route as described below and illustrated by Figs. 5-9. After initial segregation in the RER cisternae (Fig. 5 ) , the secretory proteins apparently move through this space to the transitional elements of the RER, which, as will be recalled, are the part rough-surfaced, part smooth-surfaced portions of the RER cisternae which abut on and feed into the Golgi peripheral region. Presumably these transitional elements, each surrounding a portion of the ER content, bud off by membrane fusion to become free vesicles now located in, and indistinguishable from, the population of vesicles comprising the Golgi peripheral region. I n some manner the Golgi vesicles move across this region and eventually contribute their content to the condensing vacuoles either by fusion with preexisting empty condensing vacuoles or with each other. The kinetic data show that this ferrying operation is rapid since both cell fractionation and radioautography indicate that the peak of the wave of labeled proteins enters this compartment -10 minutes post-pulse (Figs. 6 and 7). I n addition, the cell fractionation data indicated that at least over this limb of the pathway the secretory proteins are not in transit through the cell free in the cytoplasmic matrix but are associated with the content of the smooth microsomal fraction (Jamieson and Palade, 1967a). Evidently the radio-

2 84

J. D. JAMIESON

FIG.6. Radioautogram of an exocrine cell following 7-minute chase incubation in vitro. The label mainly marks the eIements of the Golgi peripheral region (arrows). A large portion of the labeled proteins have already drained from the ER cisternae (rer). From Jamieson and Palade, 1967b. Courtesy of the Journal of Cell Biology. X13,600.

285

PROTEINS IN PANCREATIC EXOCRINE CELLS

2001

/1I i .:.

LOO

50

pulse

... ... ..

.... .:: :

;:; ..; .:.

:..:. .*.

+ I7

:::

:.......: :.: ... .:.

+ 57

Minutes incubation in chase

FIG.7. Graphic presentation of cell fractionation data taken from Jamieson and Palade (1967a). At the end of the pulse, the majority of the labeled proteins are associated with the rough microsomes (stippled bars) derived from elements of the RER. During chase incubation, the relative specific activity of the smooth microsomes (open bars; fraction derived mainly from the Golgi vesicles) reaches a peak a t 7 minutes, then progressively declines coincident with transfer to condensing vacuoles. Courtesy of the Joumzal of Cell Biology.

autographic grains located over the elements of the Golgi peripheral region including its swarms of smooth vesicles must emanate from labeled proteins within the content of the Golgi vesicles rather than from the surrounding cell sap. In the discussion so far we have assumed that vesicles of'the Golgi complex are responsible for transport of secretory proteins from the RER to condensing vacuoles. This assumption is consistent with the morphological appearance of the Golgi peripheral region after the usual processing procedure (0~01 fixation, Epon embedment) which shows clusters of small smooth-surfaced vesicles located in this region, but the possibility must be considered that transport takes place through tubular connections between the two compartments, such tubules being either labile and subject to artifactual vesiculation or infrequent and difficult to detect without resort to serial sectioning. In the hepatic parenchymal cell, tubular connections between the RER cisternae and saccules of the Golgi complex are readily identifiable by reconstruction of images from serial sections (Claude,

286

J. D. JAMIESON

FIG.8. Radioautogram of an exocrine cell after 37 minutes of chase incubation. Labeled proteins are highly concentrated (- 49%) in condensing vacuoles (cv) at this time with relatively little label over zymogen granules (2). The Golgi peripheral region (arrows) has been drained of labeled proteins in this micrograph. From Jamieson and Palade, 1967b. Courtesy of the Journal of Cell Biology. X 10,400.

PROTEINS IN PANCREATIC EXOCRINE CELLS

2 87

1970), although in this case the nature of the products transported (e.g., lipoprotein droplets among others) differs from those of the exocrine cell. In any event, as will be discussed later, for the pancreatic exocrine cell the two compartments are connected through an energy-requiring lock, and if tubular connections exist they must be functionally discontinuous according to our data. Studies under way using the technique of freeze fracture may help to resolve this question. Finally, the condensing vacuoles are transformed into zymogen granules by the filling and concentration of their content (Fig. 8). According to the radioautographic observations, the condensing vacuoles begin to accumulate large amounts of label -20 minutes post pulse and thereafter become progressively more electron-opaque and more heavily labeled as secretory proteins accumulate within, until they are finally converted into zymogen granules with a typical highly dense content and smooth circular profile (Fig. 9). I n addition to segregation and concentration of secretory proteins, the Golgi complex in the pancreas, as in other secretory cells, appears to mediate a number of modifications of stored products. For example, as shown by the radioautographic studies of Berg and Young (1972) on mouse exocrine pancreatic cells, inorganic sulfate is actively incorporated into a (presumed) macromolecule, beginning a t the level of the vesicles and cisternae of the Golgi complex and finally accumulating within condensing vacuoles and zymogen granules. Further, it can be expected that the elements of the Golgi complex mediate the addition of sugar residues to the polypeptide backbone of at least part of the secreted product since several species of pancreatic RNase (RNase B, C, D> (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

PROTEINS IN PANCREATIC EXOCRINE CELLS

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

PROTEINS IN PANCREATIC EXOCRINE CELLS

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

No. of grains counted

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.

PROTEINS IN PANCREATIC EXOCRINE CELLS

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.

PROTEINS IN PANCREATIC EXOCRINE CELLS

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 <1.5 minutes) and effectively the preceding steps of transport by -95%, was added and incubation was carried out for an additional 60 minutes of chase. This period of chase (total of 80 minutes) is sufficient to allow -62% of the labeled proteins to reach zymogen granules in control, unblocked slices. As seen in Table IV and illustrated in Figs. 12 and 13, during the first 20 minutes of exposure to the drug (20 minute control plus 20 minutes antimycin A), condensing vacuole conversion proceeded without any retardation. After a total of 60 minutes exposure to the drug, however, (20 minutes control plus 60 minutes antimycin A) the conversion efficiency had dropped to -60% of the control level although it is evident that conversion is considerably less affected by antimycin A than the preceding steps of the transport pathway as indicated by the almost complete block of transport in slices which received the drug immedia,tely post pulse. Since the inhibitors effectively block both discharge of storage granules (see later) and delivery to condensing vacuoles from the preceding compartTABLE V ATP CONCENTRATION IN PANCREATIC SLICES EXPOSED TO

METABOLIC INHIBITORS

_ _ _ _ _ ~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

~

Incubation conditions

Control, 60 min 10 mM F-, 60 min 0.01 mM antimycin A, 60 min 10 mM F0.01 mM antimycin A, 10 min 20 min 40 min 60 min

+

ATP (pmoles/mg protein)

Amount

14.8 11.8 1.3

100.0 80.0 8.8

5.9 2.2 1.5 0.8

38.5 14.7 10.1 5.4

(%)

Details concerning incubation conditions and ATP assay are given in Jamieson and Palade (1971a).

301

PROTEINS IN PANCREATIC EXOCRINE CELLS

TABLE VI EFFECTOF ANTIMYCIN A AND FLUORIDE ON THE CONVERSION OF CONDENSING VACUOLES INTO ZYMOGENGRANULEP ~

~

Incubation conditions

Labeled zymogcn granules as percent of sum labeled (zymogen granules condensing vacuoles) No. of labeled structures counted Percent of condensing vacuoles in pellet

20 Min control

80 Min control

16.0

67.2

+

20 hlin control 60 min F75.1

20 Min 20 Min control control 60 min 60 min anti A anti A F-

+

+

61.4

+

57.8

+

214

9.1

689

9.9

225

-

942

7.4

729

6.6

Pancreatic slices were pulse labeled for 4 minutes with leucine-3H and incubated in chase medium for 20 minutes to prelabel condensing vacuoles (see Table IV). Antimycin A (10-5 M ) , NaF (10 mM), or a combination of the two inhibitors was added to the test flasks which were incubated for an additional 60 minutes. After 20 or 80 minutes of incubation, the slices were homogenized, xymogen granule fractions were prepared, and the granule pellets were processed for electron microscopic radioautography as previously described (Jamieson and Palade, 1967b). (1

merits the data also show, as mentioned before, that back diffusion from the condensing vacuoles does not occur. Because chemical analyses indicated that the slices exposed to antimycin A for 1 hour contained about 9% of their initial content of ATP, i t could be presumed that conversion was still energized by ATP produced b y the small but measurable glycolysis of the tissue. To test this possibility, conversion was examined in the presence of antimycin A (0.01 mM) plus N a F a t a concentration (10 mM) sufficient to block glycolysis and further reduce the ATP levels to -5% of the control value (Table V). An experimental protocol similar to that mentioned above for studies on intact cells was employed, but for convenience the results were assessed on radioautograms of zymogen granule pellets isolated from homogenates of the slices. As

302

J. D. JAMIESON

shown earlier, this fraction contains, in addition to zymogen granules, a small but consistent population of condensing vacuoles. As seen in Table VI, a t the end of a 20-minute chase post pulse, labeled zymogen granules accounted for 16% of the sum of labeled zymogen granules plus labeled condensing vacuoles in the pellet, whereas after 80 minutes of chase the comparable figure was -67%. For slices exposed to F- alone for 60 minutes, no depression of conversion was noted, nor did F- enhance the effect of antimycin A. From these results it appears that condensing vacuole conversion does not depend strictly on a continued supply of energy, ruling out the original hypothesis that the process is mediated by endergonic ion pumps located in the condensing vacuole membrane. Additionally, cell fractionation studies have shown that Na+-K+ ATPases are absent from preparations of zymogen granule membranes (Meldolesi et al., 1971b) and other preliminary studies have indicated that 1.0 mM ouabain is without effect on the conversion process or on zymogen granule stability. Finally morphological observations show that condensing vacuoles and zymogen granules do not swell or burst in intact cells deprived of their ATP supply by the experimental conditions employed above. We should also mention that other workers have suggested, based on histochemical evidence, that nucleoside diphosphatase and acid phosphatase activities may be involved in the concentration process in anterior pituitary cells (Smith and Farquhar, 1970) and parotid exocrine cells (Novikoff , 1962) , respectively, but the involvement of these activities in concentration is based solely on their location in elements of the Golgi complex and storage granules. In summary, the evidence to date does not implicate a transport ATPase or comparable enzyme system in the conversion of condensing vacuoles into zymogen granules. What then are the factor (a) involved? One possibility, based on a number of scattered observations, was that secretory proteins might be present in storage granules in osmotically inactive aggregates. To explore this further, the sensitivity of zymogen granule preparations to osmotic shock was determined (Jamieson and Palade, 1971a). To this end, slices were pulse labeled and incubated in chase medium for either 20 or 80 minutes to allow label to reach primarily condensing vacuoles or zymogen granules, respectively. At these times, granule fractions were isolated from homogenates of the gland and then either resuspended in 0.3 M sucrose (the original isolating fluid) or distilled H20. After a brief incubation a t O'C, the suspensions were resedimented and the proportion of radioactive proteins released by the treatment was measured. As seen in Table VII, resuspension in sucrose alone released 11-1775 of the labeled content from both prelabeled condensing vacuoles and zymogen granules. Osmotic shock released a n additional 39% from

303

PROTEINS IN PANCREATIC EXOCRINE CELLS

TABLE VII EFFECTOF RESUSPENDING MEDIAON RELEASEOF LABELEDPROTEINS FROM

ZYMOGENGRANULEFRACTIONW~ Dpm released (%)

Chase incubation time (min)

0.3 M sucrose pH 4.8

Water p H 4.7

0.2 M NaHC03 pH 8.4

0.5% Na deoxycholate pH-7.2

20 80

11.2 f 0 . 6 16.5 f 0 . 1

50.6 f 1 . 4 30.2 f 1 . 1

87.2 f 1 . 3 87.3 f 1 . 2

86.6 89.8

Data from Jamieson and Palade (1971a). "ets of pancreatic slices were pulse labeled for 4 minutes with leucine-SH and incubated in chase media for 20 or 80 minutes. Zymogen granule fractions were isolated by differential centrifugation from homogenates prepared from each set of slices. The pellets were resuspended in the media indicated, and the suspensions were incubated a t 0°C for 5 minutes. After centrifugation of the suspensions at 190,000 g for 20 minutes, the amount of labeled proteins in the supernatant fraction was determined and expressed as the percent of total labeled proteins initially present in the suspension. a

prelabeled condensing vacuoles, but only -14% more from prelabeled zymogen granules. Evidently part of the content or part of the population of condensing vacuoles is somewhat sensitive to osmotic shock while most of the content of labeled proteins in zymogen granules is relatively inert osmotically. Deoxycholate (DOC), which dissolves the limiting membranes of the granules, and mild base treatment, which presumably causes swelling of the granule content and rupture of the limiting membrane, both release most of the content of the supernatant fluid. These experiments thus support the notion that secretory proteins, upon segregation in condensing vacuoles, become progressively aggregated into a form that is relatively inactive osmotically in the zymogen granule. These results confirm the earlier observations of Hokin (1955) and the more recent, studies of Burwen and Rothman (1970) which indicated that the zymogen granules release only 20-25y0 of their content of proteins upon hypotonic treatment, provided the p H of the suspending fluid is within the range of granule stability (-pH 4.5-6.6). The basis of this proposed aggregation is unknown. It may result from ionic or other types of interactions between secretory proteins themselves, or between secretory proteins and divalent cations or binding or matrix substances, etc., such as t,he presumed acid mucopolysaccharides recently reported by Berg and Young (1972) in the granule content. Direct interactions between the

304

J. D. JAMIESON

zymogen granule content and its limiting membrane probably are not important since the surface area of the membrane is such that it could accommodate only a small portion of the content. The presence of filamentous structures in condensing vacuoles and material discharged into the acinar lumina, the formation of dense masses in poorly fixed or partly extracted granules, and the formation of discrete intracisternal granules within the ER cisternae under certain physiological conditions are further evidence that secretory proteins are capable of aggregation under some conditions. Preliminary freeze-fracture studies on unfixed intact cells and zymogen granule fractions have also revealed a characteristic particulate substructure to the cleaved granule content, which may be a further indication of the presence of large aggregates, The formation of complexes between small molecules and large “carriers” resulting in the firm binding of the former have been demonstrated in the case of storage granules of the adrenal medulla (Smith, 1968) and the posterior pituitary (Sachs, 1969). In addition, regularly ordered crystalline or paracrystalline cores have been described within storage granules of (3 cells of pancreatic islets (Fawcett, 1966), eosinophilic leukocytes (Miller et al., 1966), and in hepatic peroxisomes (Baudhuin et al., 1965). Whether in these cases the complexes and the cores of storage granules serve to reduce the osmotic activity of the content or are a secondary manifestation of other factors reducing the osmotic activity remain to be determined. Finally, i t should be reemphasized that transport of secretory proteins from the RER in the luminal direction takes place against a concentration gradient and yet the process is not readily reversible. The formation of aggregates beginning in the condensing vacuoles which may act as a “sink” for incoming secretory proteins may help to explain in part the vectorial nature of the transport operations.

STEP6. DISCHARGE OF THE ZYMOGEN GRANULE CONTENT The final step in the transport operation consists of movement of zymogen granules to the cell apex followed by exocytosis of their content into the acinar lumen (Fig. 14). Although a number of earlier biochemical studies had shown that secretory proteins can be discharged in vitro from gland slices (pancreas and parotid) upon application of appropriate stimuli, and that discharge induct,ion requires energy (Hokin and Hokin, 1962; Schramm, 1967), the results could not be interpreted unambiguously since they were obtained under conditions of ongoing protein synthesis and intracellular transport, both processes requiring energy. In a n attempt to clarify this problem, we have reinvestigated zymogen granule discharge

FIG.14. Zymogen granule discharge at the cell apex. One zymogen granule (z) has closely approached the apical plasmalemma where its membrane will become confluent with the plasmalemma. The end result is the extrusion of the granule content to the duct lumen (L) as seen in the top image where the granule membrane now is continuous with and part of the apical plasmalemma in the region between the arrows. Micrograph by courtesy of Dr. G. E. Palade. X85,000.

306

J. D. JAMIESON

in vitro under conditions independent of protein synthesis and of the preceding steps of intracellular transport (Jamieson and Palade, 1971a). To this end, we have developed a simple radioassay for zymogen discharge based on the radioautographic findings already shown above, which indicated that -65% of the labeled proteins formed during a short pulse labeling with l e ~ c i n e - ~are H transported to and stored within zymogen granules a t the cell apex during an SO-minute chase period, as seen already in Fig. 9. From this site the labeled proteins can be readily discharged to the incubation medium in response either to carbamylcholine, a cholinesterase-resistant analog of acetylcholine, or to pancreozymin, the natural peptidic secretogogue for the exocrine pancreas. As shown in Fig. 15, discharge from prelabeled granules begins without a lag and is sustained a t a linear rate for the duration of the assay, usually 30-60 minutes. In addition to simplicity and high sensitivity in comparison to monitoring of the output of enzyme activity, the radioassay is selective in that it detects only those secretory proteins synthesized during the pulse and transported to granules in the preincubation period. Consequently it is uninfluenced by preformed enzymes and zymogens stored in the duct system of the gland.

5

.-

0

30 0

.-C u) .-C

0

0

0

0

a! c

g

20

'0 a!

-a!

n

0

10

I

4 min 80 min'chose pulse incubation

I

I

+20

+30

1

+ 5 +I0

M in

FIG.15. Kinetics of discharge of labeled proteins from prelabeled zymogen granules. Pancreatic slices were pu1s.e labcled for 4 minutes with l e ~ c i n e - ~ H incubated , post pulse for 80 minutes to label the zymogen granules, a t which time (arrow) the secretagogue carbamylcholine (10-4 M ) was added. Discharge begins without a lag and proceeds a t a linear rate. Controls (0-0) received no stimulant; discharge from them represents spontaneous secretion. 0 - - - 0, Carbamylcholine treated. From Jamieson and Palade, 1971a. Courtesy of the Journal of Cell Biology.

-0

5-

TABLE VIII

v, L

EFFECT OF SECRETAGOGUES ON INTRACELLULAR TRANSPORT OF SECRETORY PROTEINS'

f W

w

z

Chase incubation in 2 mM leucine-'H

2

5

Distribution of radioactivity Microsomal fraction

% Amylase Pulse cIeucine-3H

3 Min 3 Min 3 Min

3 Min

Conditions 0 Min, pulse

37 Min, control 37 Min, 0.1 m M carbamylcholine 37 Min, 10 U/nd pancreozymin

%

ii

Zymogen granule

Postmicrosomal

fraction

supernatant

%

%

in incubation mediumb

Dpm/U amylase

DPm

Dpm/U amylase

DPm

DPdU amylase

DPm

12.0 22.2

8500 4350 6670

32.4 14.8 19.2

206 5100 4280

0.8 12.0 6.7

2730 1550 2400

17.5 11.7 12.0

20.6

6150

15.2

4100

8.6

1480

12.0

a Sets of pancreatic slices were pulse labeled with leucine-3H for 3 minutes, and then incubated in chase media with the indicated additions for 37 minutes. At the end of the pulse, and after 37 minutes of chase incubation, the slices were homogenized and fractionated by differential centrifugation; the radioactivity of proteins in the microsomal and zymogen granule fractions and in the postmicrosomal supernatant was determined. Radioactivity data are expressed as % dpm recovered in the fractions relative to the starting homogenate or as specific radioactivity based on amylase measured in the fractions. The data show relative rather than total changes of radioactivity in cell fractions with time since only the postmicrosomal supernatant Ras completely recovered in our fractionation scheme; recovery of the microsomal and zymogen granule fractions can be estimated to be 30% each. homogenate. Amylase in medium X l[)O/amylase in medium

+

P E 2 r G

-

0

0

u

308

J. D. JAMIESON

The latter constitute up to -10-15% of the total enzyme complement of the gland and are responsible for a t least part of the high resting or unstimulated backgrounds characteristic of enzyme assays for discharge. With this assay we are assuming that the label which appears in the medium derives from the content of zymogen granules. In support of this are cell fractionation and morphological observations which show that with increasing times of stimulation the population of zymogen granules in the cells is progressively decreased and is finally depleted by 3 hours (e.g., Fig. 18D). In addition we are assuming that the secretagogues primarily affect discharge without concurrently accelerating the preceding steps of the transport pathway. To examine this latter assumption the effect of secretagogues on the rate of accumulation of labeled proteins into condensing vacuoles was examined using the fractionation assay described in Table VIII. At the same time, the efficiency of drainage of the RER compartment was assessed by measuring the decrease of total or specific radioactivity .in the microsomal fraction which, according to previous studies (Jamieson and Palade, 1967a) consists primarily of rough microsomes derived from the RER. As indicated in Table VIII, discharge stimulation over a 37-minute chase period accelerates neither the rate of egress of labeled proteins from the RER compartment nor the rate of accumulation of label in the zymogen granule fraction (i.e., primarily into condensing vacuoles recovered in the fraction). Table VIII also shows that discharge stimulation does not lead to the preferential movement of secretory proteins through the cell sap since the amount and specific radioactivity of proteins in the postmicrosomal supernatant (which contains in part the proteins of the cell sap) is not significantly increased by the stimulants. Having thus established the basic premises of the discharge assay, we can return to the question of the metabolic requirements for zymogen granule release. As for the studies on steps 1-4 of the secretory pathway, we first determined whether or not zymogen discharge was tightly coupled to continued protein synthesis. For this purpose, cycloheximide at a concentration which inhibits protein synthesis by >95% was introduced into the assay a t the end of the pulse and was present continuously during both the granule prelabeling period and test period with the secretagogues. A typical experiment, shown in Fig. 16, clearly indicates that the inhibitor was without effect on discharge even though present and active for up to -2.5 hours. Evidently discharge, like the preceding steps of transport, does not require the ongoing synthesis of either secretory proteins or specific proteins, such as carriers and couplers. Nevertheless, such nonexportable proteins may be required but are possibly present in sufficiently large intracellular pools to support discharge over the period of exposure to cycloheximide used here.

309

PROTEINS IN PANCREATIC EXOCRINE CELLS

E .E

I

pulse

incubolion t5X10-4M

Min

FIG.16. Effect of cycloheximide (5 X lo-' M ) on induced discharge. Assay conditions as in Fig. 15. Arrow indicates addition of 10-4 M carbamylcholine. Cycloheximide was added immediat,eiy post-pulse and was present until termination of the assay. At the dose used, protein synthesis was blocked by 98%, yet induced (and spontaneous) discharge was unaffected. Curve a, cycloheximide plus lo-' M carbamylcholine; curve b, control plus carbamylcholine; curve c, cycloheximide ; curve d, control. From Jamieson and Palade, 1971a. Courtesy of the Journal of Cell Biology.

-

Again, since discharge could be uncoupled from protein synthesis, it was possible to examine independently in the uncoupled state the energy requirements for secretagogue action using a number of well known metabolic inhibitors applied to the discharge assay. For each condition tested, the inhibitors were added to the assay a t the time of addition of the secretagogue (i.e., after the 80-minute prelabeling period) and were present for a standard 30-minute test period. In addition cycloheximide was also present in most cases to ensure a uniform degree of inhibition of protein synthesis. The data, sho.wn in Table IX and Fig. 17, indicate that discharge is progressively inhibited by lowering the temperature (QIOof -2), does not depend on glycolysis for an energy supply, but like steps 1-3 of the transport pathway is strictly dependent on a source of respiratory energy, presumably as ATP generated by oxidative phosphorylation. Because inhibition of discharge by respiratory inhibitors is rapid, being completed in -5 minutes (Fig. 17) and since the preceding step in the pathway, the

310

J. D. JAMIESON

maturation of condensing vacuoles, is relatively insensitive to energy deprivation, we conclude that discharge itself is affected by the respiratory blockers used. A number of other compounds which have been implicated in the discharge process were also tested on the assay (Table IX). Dibutyryl adenoTABLE IX EFFECTOF V A H I O UINCUBATION ~ CONDITIONS ON INDUCED DISCHARGE^^^ Chase conditions 80 min with 2 mM leucine-'H Pulse (leucine-3H) 4 4 4 4

Cycloheximide 0.5 mM

Assay conditions 30 min with 2 mM IeucineJH Cycloheximide 0.5 mM

Min Min Min Min

4 Min 4 Min 4 hlin 4 Min 4 Min 4 Min 4 Min 4 Min

+ + + + + +

+ + + + + +

Carbachol 0.1 mM

+ + + + + + + + + + +-

4 Min 4 Min

+

Test conditions

Relative % dpm in medium at 30 min

37" 25" 17" 4"

100.0 45.5 27.3 7.3

10 mM NaF 0.1 mllf I A 95% N2r 5% coz 1 mM D N P 1 mM NaCN

100.0 120.0 70.0 0

-

100.0 17.7

2 mM DCAMP 1 mM theophylline 1 mM ouabain

0 2.3

6.3 117.0

Data from Jamieson and Palade (1971a). Sets of pancreatic slices were pulse-labeled for 4 minutes with ~-leucine-~H, then incubated in chase medium with the indicated additions to prelabel zymogen granules. After this, the secretagogues and test compounds were added to the assays for a further 30 minutes, a t the end of which time the proportion of labeled proteins appearing in the medium was determined and compared to that from stimulated but untreated slices. Data are expressed relative to those maximally obtained with carbachol alone (corrected for resting secretion), which is taken as 100%. The actual proportion of pulse-labeled proteins secrcted usually amounted to 36-40%. IA, iodoacetate; DNP, 2,4-dinitrophenol; DCAMP, dibutyryl cyclic AMP.

31 1

PROTEINS IN PANCREATIC EXOCRINE CELLS

8

1 , 4min 80 min chose pulse incubation

I

I

I

t5

+I0

+ 20

Min

FIG.17. Effect by antimycin A (5 X 10-6M) on induced discharge. Assay as in Fig. 15. Arrow indicates addition of M carbamylcholine. Curve a, carbamylcholine; curve b, control plus antimycin A; curve c, carbamylcholine plus antimycin A. From Jamieson and Paladc, 1971a. Courtesy of the Journal of Cell Biology.

sine-3' ,5'-cyclic phosphate, an active derivative of adenosine 3' ,5'-cyclic phosphate which has been implicated as a common mediator of the secretion response in other tissues (Rasmussen, 1970) produced only a modest response. SimiIarly, the assay was insensitive to theophylline, an inhibitor of the phosphodiesterase which hydrolyzes adenosine-3' ,5'-cyclic phosphate intracellularly. These findings are a t variance to those reported for in vitro preparations of mouse pancreas (Kulka and Sternlicht, 1968) and rat parotid (Babad et al., 1967), but are consistent with the observations of Rasmussen and Tenenhouse [reported in Rasmussen (1970)) that the adenyl cyclase system of the guinea pig pancreas is relatively unresponsive to secretagogues such as used in the present study. Ouabain, a t a dose far in excess of that required to inhibit linked Na+ and I<+transport in other cell types ( <1 mM) was without effect on discharge indicating, as already reported by others (Ridderstap and Bonting, 1969), that a glycoside-inhibitable ATPase is not involved in protein secretion. Finally, bot,h colchicine and vinblastine at levels that interfere with microtubule integrity in other systems and inhibit insulin discharge from /3 cells of the endocrine pancreas (Lacy et al., 1968), inhibit exocrine discharge by only 25-30% (Table X). Both compounds, however, are not without nonspecific cytotoxic effects since our observations indicate that they produce a n inhibition of protein synthesis which, based on our earlier studies, is a sensitive reflection of the metabolic state of slices of exocrine pancreas. If these compounds interfere with an essential aspect of dis-

31 2

1. D. JAMIESON

TABLE X

EFFECT OF COLCHICINE, VINBLASTINE, AND CYTQCHALABIN B ON INDUCED DISCHARGE'

Time

Treatment

Carbachol 10-6 M

+ Colchicine 10-3 M 10-4 M 10-6 M 10-6 M

Carbachol 10-6 M

+ Vinblastine 10-3 M 10-4 M 10-6 M 10- M

Carbachol 10-6 M

+ DMSO 1%

+ cytochalasin B,

Protein Secretion of pulsed proteins synthesis (% of carbachol) (yoof control)

120 Min 120 Min 120 Min 120 Min 120 Min

100 63 87 72 95

100 79 87 98 100

40 Min 40 Min 40 Min 40 Min 40 Min

100 59 75 91 84

100

90 Min 90 Min 90 Min

100 105 120

100 100 81

46

75 122 119

10-4 M

for these experiments, pancreatic slices were pulse labeled for 4 minutes with leucine-", then immediately placed in carbachol-containing media for the indicated times and with the additions noted. Secretory responses are given relative to that obtained with carbachol alone. In parallel experiments, the ability of the gland to incorporate labeled leucine during a continuous incubation was measured. The basis for these assays is given in Fig. 29. DMSO, 1% final concentration, is the vehicle used to solubilize cytochalasin B.

charge then they should be capable of inhibition levels of -95%, i.e., the level reached by the use of metabolic inhibitors. Cytochalasin B, which presumably affects microfilament function and integrity (Wessels et al., 1970), was without influence on induced discharge at doses which rapidly inhibit cellular motility in other systems. Such observations are consistent with the relative paucity of both microtubules and microfilaments in pancreatic exocrine cells. In fact, as Matthews (1970) pointed out, it is not necessary to postulate a structured translational system for /3 cell granule extrusion since, according to Einstein's diffusion law, a particle 200 nm in diameter separated from the cell membrane by 100 A, will reach the membrane in 0.13 msec whereas a particle of the same size will traverse a distance of 5 p in 33 seconds. Calculated times for zymogen granules (1 p in diameter) traversing the same distances are 0.65 msec and -50 seconds, the latter corresponding to the distance from the center of the

PROTEINS IN PANCREATIC EXOCRINE CELLS

313

Golgi region to the apical plasmalemma. Similar calculation for movement of a shuttle vesicle from the transitional elements to the condensing vacuoles (1.5 p distance) predict a time of -1.5 seconds. The calculations are based on a n assumed intracellular viscosity of 0.06 poise and simply describe the possible time course, but give no indication of the probability of the events. The result,s of these studies thus validate the earlier studies of Hokin and Hokin (1962) and of Schramm (1967) and collaborators which indicated that secretory granule discharge requires the continuous production of respiratory energy. The data can be taken to indicate that a second step in the transport pathway operates through an energy-requiring lock or valve. The opening of the first lock described above for steps 1-3 serves to connect two intracellular compartments (the R E R cisternae and the condensing vacuoles), whereas the second lock connects a n intracellular compartment (represented by the zymogen granule) with the extracellular space, i.e., the acinar lumen. I n contrast to the first lock, the opening of the second lock is modulated by secretagogues. The details of the energy-requiring step(s) in the operation of this second lock are, however, still largely unknown, although it is clear that discharge involves movement of the granule to the cell apex and fusion of the granule membrane with that of the apical plasmalemma, foIlowed by exocytosis of the content. Some of the energy requirement may be related to granule translation, although it is likely that part of it may be involved in the reorganization and/or resynthesis of cell membranes which accompanies membrane fusion and fission. I n addition, energy is required for the formation of adenosine-3' ,5'-cyclic phosphate which, as mentioned above, has been proposed as a common mediator for the secretory response in a number of other cell types known to release their stored products by exocytosis No doubt, as the details of the discharge process are unraveled, other enzymatic or energy-requiring steps will be discovered. We should mention that for the pancreas as well as for other cell types which temporarily store their secretion products in storage granules and discharge them by exocytosis, the other essential ingredients for release, in addition to energy, appear to be Ca2f and a n appropriate neural or humoral stimulus (Rasmussen, 1970). Little is known a t present, how or where the stimulus affects the cell except that in the case of polarized cells such as those of the exocrine pancreas the receptor site for the signal is most likely located on the basal plasmalemma. For the future it will be of interest to determine how the primary stimulus is transmitted to the cell apex where discharge actually occurs. A similar sequence of morphological and biochemical steps appears also to pertain for the discharge of stored products from cells of the parotid

314

J. D. JAMIESON

(Amsterdam et al., 1969) adrenal medulla (Schneider et al., 1967; Douglas, 1968), anterior pituitary (Hodges and McShan, 1970), and 0 cells of the endocrine pancreas (Howell et al., 1969a; Lacy, 1970), i.e., cell types with morphologically recognizable storage granules and in which chemical identity between the content of the storage granule and the secreted material has been established a t least in part. Although in the cases mentioned the discharged product eventually reaches the extracellular space (duct lumen or circulation), in others the stored product may be released intracellularly into ingestion vacuoles (e.g., in white-blood cells; Zucker-Franklin and Hirsch, 1964; Cotran and Litt, 1969), or into lysosomes (e.g., in cells of the anterior pituitary in which superfluous secretory granules are disposed of secondary to suppression of secretion; Farquhar, 1971). Finally, although the discharge mechanism discussed above most likely pertains to all cells which possess storage granules, the mechanism of discharge from cells which do not concentrate and temporarily store their secretory products into morphologically recognizable secretory granules remains unclear. Examples of such cell types include plasma cells, fibroblasts, thyroid follicular cells, and hepatocytes. Suggested alternative discharge routes include direct egress from the RER cisternae to the extracellular space (Ross and Benditt, 1965) and passage across the plasmalemma from the cell sap without the intervention of a mechanism involving reversed pinocytosis (Renold, 1970). Discharge of lipoprotein droplets via tubules of the smooth E R has been clearly demonstrated in the case of hepatocytes (Jones et al., 1967; Claude, 1970). Transport through the cell sap has already been considered earlier, and, for the reasons given, the latter mode of discharge, while not proved or disproved, remains speculative. Discharge from the RER directly appears unlikely since, in the cases where it has been suggested (plasma cells and fibroblasts; Ross and Benditt, 1965), the secreted products are glycoproteins, which, as previously mentioned, most likely obtain their polysaccharide moieties in large part at the level of the Golgi complex. This fact must be considered in any alternative mechanism of secretion. A simplified hypothesis, which reconciles the observations to date, is that in those cases where no recognizable storage granules are formed, discharge is mediated by small vesicles which carry the secretory product from the Golgi complex to the plasmalemma where release occurs again by exocytosis. For these cell types, the lack of a concentration phase may be a reflection of continuous (i.e., noncyclic) synthesis and discharge of the product. Alternatively it has been proposed that microvesicle-mediated transport and discharge may provide a second and presumably more rapid means of discharge of exportable

PROTEINS IN PANCREATIC EXOCRINE CELLS

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products such as may occur in stimulated P cells of the endocrine pancreas where mature storage granules may be temporarily bypassed (Renold, 1970). As will be noted later, this form of “mixed” secretion apparently does not occur in hyperstimulated exocrine cells. Because of the absence of morphological criteria to distinguish such putative secretion vesicles from other vesicles b the cell, and because of their small size which precludes tracing their origin and fate by radioautography, support for the hypothesis will most likely come from cell fractionation studies. I n the case of the hepatocyte, the situation is more complex in that the secreted product is apparently given a choice of routes for dischargedirectly from the RER to the extracellular space via tubules of the smooth E R or a more conventional route in which the Golgi complex acts as a way station where secretory vacuoles are formed. Both pathways operating in parallel have been suggested for albumin transport (Ashley and Peters, 1969; Glaumann and Ericsson, 1970) and discharge, although for serum lipoprotein droplets the RER-Golgi pathway is taken (Jones et al., 1967; Claude, 1970).

111. PHYSIOLOGICAL MODULATION OF THE SECRETORY PROCESS IN PANCREATIC EXOCRINE CELLS

The route, timetable, and metabolic requirements for transport and discharge of secretory proteins discussed above were obtained on slices from the pancreases of starved guinea pigs and consequently represent the situation in the resting cell or, a t most, cells stimulated in vitro for relatively short periods of time. Normally, of course, the function of the gland, specifically in relation to its stored products, appears to be cyclic consisting of periods of food deprivation during which granules accumulate in the cell followed by periods of discharge of the granule content in response to secretagogue action triggered by feeding. As noted earlier, the response of the exocrine cell to secretogagues in relation to rates of synthesis of proteins, nucleic acids, and phospholipids has been extensively investigated, although no studies have dealt specifically with the effectsof secretagogues on possible adjustments in the route or timetable of intracellular transport. To investigate this problem, we have taken advantage of the fact that pancreatic slices from starved animals can be progressively depleted of their population of preformed zymogen granules by the in vitro application of either cholinergic agents, such as carbamylcholine, or the natural peptidic secretagogue pancreozymin (Jamieson and Palade, 1971b).

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PROTEINS IN PANCREATIC EXOCRINE CELLS

317

A. Morphological Features of Zymogen Granule Discharge in Vitro

The onset of intensive zymogen granule discharge is evident as early as 30 minutes after application of the stimulant b y the appearance of numerous empty-appearing diverticula and apically situated vacuoles which, by other means, can all be shown to be connected to the acinar lumen (Fig. 18). These diverticula and vacuoles, which result from the insertion of zymogen granule membranes into the apical plasmalemma, coalesce and penetrate the cell deeply as discharge proceeds (1 hour, Fig. 18B). The net result is a marked enlargement of the acinar lumen surface area. By 2 hours of stimulation (Fig. 18C), the depletion of the granule population is evident, though a t this time it is noted that the enlargement of the acinar lumen is less marked than a t 1 hour and has reverted to normal dimensions in many acini. After 3 hours of stimulation, granule depletion is complete (Fig. 18D). Of particular interest is the observation that a t this time the profiles of all the previously enlarged acinar lumina have reverted to normal dimensions despite the fact that the cells continue to synthesize and discharge secretory proteins a t undiminished rates. Cells so depleted of their granules are generally more rounded in outline, although the overall surface area does not appear to be increased despite the massive contribution of membrane to their surface which accompanied discharge. A similar sequence of changes occurs in the parotid exocrine cell stimulated in vivo as reported recently b y Amsterdam et al. (1969). At the electron microscope level, the most striking change noted in the 3 hour-stimulated exocrine cells was a marked hypertrophy of the elements of the Golgi complex (Fig. 19). Whereas in the resting cell the Golgi elements occupy a fairly well circumscribed supranuclear zone, in the hyperstimulated cell they occupy the majority of the apical third of the cell.

FIG. 18. Light micrographs of the in vitro effect of 10-6 M carbamylcholine on pancreatic slices. From Jamieson and Palade, 1971b. Courtesy of the Journal of Cell Biology. All X8500. (A) Thirty-minute exposure to carbamylcholine. Acinar luniina are enlarged in area and are in continuity with diverticula (arrows) arising from symogen granule discharge. (B) Sixty minute carbamylcholine treatment. Luminal profile (L) is still enlarged. Arrow indicates apical diverticulum. (C) Two hours with carbamylcholine. The granule population is markedly depleted in most cells. Acinar lumina (L) have reverted to normal dimensions. Asterisk indicates an arborizing interlobular duct. (D) Three hours of carbamylcholine treatment. Granule discharge is complete, and lumina (L) remain normal in profile.

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PROTEINS IN PANCREATIC EXOCRINE CELLS

31 9

Particularly evident is the increase in number of parallel stacked cisternae which are relatively infrequent in the resting cell. In many instances, the concave, centrally placed cisternae of the stacks contain material of high electron opacity which often accumulates in terminal dilations of these cisternae. In addition the complex contains large numbers of small smoothsurfaced vesicles of uniform (-45 nm) diameter located between characteristic transitional elements of the RER and the outermost cisternae of the stacks, and an abundance of small vesicles of the coated variety which are most often located in the cup-shaped central region defined by the Golgi stacks. Many of the small coated vesicles are filled with dense material similar to that found in the Golgi cisternae. Centrally placed in the Golgi complex and extending to the cell surface are numerous small, often irregularly shaped storage granules possessing a dense content. Some of these granules are bounded by a coated membrane and may represent one extreme in the size range of the small coated vesicles mentioned above. Although most evident after 3 hours of stimulation, an increase in number of Golgi elements and condensation of material in the stacked cisternae was seen as early as 1 hour after onset of stimulation in cells which had not yet been fully depleted of mature granules. 8. Biochemical Changes Accompanying in Vitro Discharge

As expected, the stimulated slices discharge to the medium a substantial amount of their stores of a typical secretory protein, a-amylase. Amylase discharge is init>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.

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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.

PROTEINS IN PANCREATIC EXOCRINE CELLS

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

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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

No. of grains counted

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

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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.

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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.

PROTEINS IN PANCREATIC EXOCRINE CELLS

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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.

PROTEINS IN PANCREATIC EXOCRINE CELLS

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

PROTEINS IN PANCREATIC EXOCRINE CELLS

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.

PROTEINS IN PANCREATIC EXOCRINE CELLS

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).

PROTEINS IN PANCREATIC EXOCRINE CELLS

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

PROTEINS IN PANCREATIC EXOCRINE CELLS

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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Berg, N. B., and Young, R. U’.(1971). J. Cell R i d . 50, 469. Blobel, G., and Sabatini, 1). I). (1970). J. Cell B i d . 45, 130. Burwen, S. J., and Itothman, S. S. (1970). Fed. Proc., Fed. Amer. SOC.Exp. Biol. 29, 1124. Caro, L. G., and Palade, G. E. (1964). J. Cell &ol. 20, 473. Cast)le, J . I)., Jamieson, J. D., and Palade, G. E. (1972). J . Cell B i d . 53, 290. Claude, A. (1970). J. Cell Biol.47, 745. Cotran, R . S., and Litt, M. (1969). J . Exp. Mrd. 129, 1291. Douglas, W. W. (1968). Brit. J . Pharmacol. 34, 451. Ehrenreich, J. H. (1969). Ph.11. Thesis, The Rockefeller University, New York. Farquhar, M. G. (1971). In “Memoirs of the Society for Endocrinology, No. 19,” (H. Heller and K. Lederis, eds.), p. 79. Cambridge Univ. Press, London and New York. Fawcett, 1). W. (1962). Circulation 26, 1105. Fawcett, D. W. (1966). “The Cell.” Saunders, Philadelphia, Pennsylvania. Fanwht, D. W., Long, J. A,, and Jones, A. L. (1969). Recent Progr. Horm. Res. 25,315. Fedorko, M. E., and Hirsch, J . G. (1966). J . Cell Biol.29, 307. Fleischer, B., Fleischer, S., and Oeawa, H. (1969). J . Cell B i d . 43, 59. Glaumann, H., and Ericsson, J. L. E. (1970). J. Ccll Biol.47, 555. Greene, L. J., Hirs, C. H. W., and Palade, G. E. (1963). J. Riol. Chem. 238, 2054. Haddad, A., Smith, M. D., Herskovics, A,, Nadler, N. J., and Leblond, C. P. (1971). J. Cell Biol.49, 856. Herzog, V., and Miller, F. (1970). 2. Zellforsch. Mikrosk.Anat. 107, 403. Hodges, D. It., and McShan, W. H. (1970). Acta Endocrinol. (Copenhagen) 63, 378. Hokin, L. E. (1955). Biochim. Riophys. Acta 18, 379. Hokin, L. E. (1968). Int. Rev. Cylol. 23, 187. Hokin, L. E., and Hokin, M. R. (1962). Exocrine Pancreas, Norm. Abnorm. Funct., Ciba Found. Sump. ( A . V. S. deReuck and M . P. Cameron, eds.), p. 186, Churchill, London. Howell, S. L., Young, D. A., and Lacy, P. E. (1969a). J. Cell Biol. 41, 167. Howell, S. L., Kostianovsky, M., and Lacy, P. E. (1969b). J . Cell Biol. 42, 695. Jamieson, J. ll., and Palade, G. E. (1967a). J. Cell Biol. 34, 577. Jamieson, J. I)., and Palade, G. E. (1967b). J. Cell Biol. 34, 597. Jamieson, J. I)., and Palade, G. E. (1968a). J . Cell Biol. 39, 580. Jamieson, J. I)., and Palade, G. E. (1968b). J . Cell Biol. 39, 589. Jamieson, J. D., and Palade, G. E. (1971a). J. Cell Bid. 48, 503. Jamieson, J . D., and Palade, G. E. (1971b). J. Cell Biol. 50, 135. Jones, A. L., Rudermann, N. B., and Herrera, M. G., (1967). J . Lipid Res. 8, 429. Keller, P. J., and Cohen, E. (1961). J. B i d Chem. 263, 1407. Kern, H. F., and Kern, I). (1969). Virchows Arch., B 4, 54. Kramer, M. F., and Poort,, C. (1968). 2. Zellforsch. Mikrosk. Anat. 86, 475. Kulka, R. G., and Sternlicht, E. (1968). Proc. Nat. Acad. Sci. U.S. 61, 1123. Lacy, P. E. (1970). Diabetcs 19, 895. Lacy, P. E., Howell, S. L., Young, D. A., and Fink, C. J. (1968). Nature (London) 219, 1177. Lin, T. M., and Grossman, M. J. (1956). Amer. J . Physiol. 186, 52. Marshall, J . RI. (1954). E x p . Cell Res. 6, 240. Matthcws, E. K. (1970). Acta Diubet. Lut. 7, Suppl. 1, 83. Meldolesi, J., Jamieson, J. D., and Palade, G. E. (1971a). J. Cell B i d . 49, 130. Meldolesi, J., Jamieson, J. I)., and Palade, G. E. (1971b). J. Cell Biol. 49, 150. Miller, F., deHarven, E., and Palade, G. E. (1966). J . Cell B i d . 21, 349. Morris, A. J., and Dickman, S. R. (1960). J . Biol.Chem. 235, 1404.

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Nadler, N. J., Young, B. A.. Leblond, C. P., and Mitmaker, B. (1964). Endocrinology 74, 333. Novikoff, A. B. (1962). Jew. Mem. Hosp. Bull. 7, 70. Omura, T., Siekevite, P., and Palade, G. E. (1967). J. Biol. Chem. 242, 2389. Palade, G. E. (1956). J. Biophys. Riochem. Cytol. 2, 417. Palade, G. E . (1959). I n “Subcellular Particles,” (T. Hayashi, ed.), p, 64. Ronald Press, New York. Palade, G. E. Siekevite, P., and Caro, L. G. (1962). Exocrine Pancreas; Norm. Abnorm. Funct., Ciba Found. Symp., (A. V. S. deIteuck and M. P. Cameron, eds.), p. 23. Churchill, London. Peters, T. (1962). J. Biol. Chem. 237, 1186. Plummer, T. H., and Hirs, C. H. W. (1964). J. Biol. Chem. 239, 2530. Rasmussen, H. (1970). Science 170, 404. Redman, C. M. (1967). J. Biol. Chem. 242, 761. Redman, C. M. (1969). J. Biol. Chem. 244, 4308. Redman, C. M., and Hokin, L. E. (1959). J. Biophys. Riochem. Cytol. 6, 207. Redman, C. M., and Sabatini, D. 1). (1966). PTOC. Nut. Acad. Sci. U.S. 56, 608. Redman, C. M., Siekevite, P., and Palade, G. E. (1966). J. Biol. Chem. 241, 1150. Renold, A. E. (1970). N . Engl. J . Med. 282, 173. Ribet, A., Fedou, R., and Frexinos, J. (1969). Digestion 2, 145. Ridderstap, A. S., and Bonting, S. L. (1969). Pfluegers Arch. 313, 53. Ross, R., and Benditt, E. P. (1965). J . Cell Biol. 27, 83. Rothman, S. S. (1967). Nature (London) 213, 460. Sabatini, D. D., and Blobel, G. (1970). J. Cell Biol. 45, 146. Sachs, H. (1969). Advan. Enzymol. 32, 327. Salnikow, J., Moore, S., and Stein, W. H. (1970). J . Biol. Chem. 245, 5685. Schneider, F. H., Smith, A. D., and Winkler, H. (1967). Brit. J. Pharmacol. Chemother. 31, 94. Schramm, M. (1967). Annu. Rev. Biochem. 36, 307. Siekevite, P. (1970). N . Engl. J . Med. 283, 1035. Siekevite, P., and Palade, G. E. (1960). J . Biophys. Biochem. Cytol. 7, 619. Smith, A. D. (1968). Zn “Interactions of Drugs and Subcellular Components in Animal Cells,” (P. N. Campbell, ed.), p. 239. Churchill, London. Smith, R. E., and Farquhar, M. G. (1970). J . Histochem. Cytochem. 18, 237. van Heyningen, H. E. (1964). Anat. Rec. 148, 488. Webster, P. D. (1969). I n “Exocrine Glands,” (J. Y. Bothelo, F. P. Brooks, W. B. Shelley, eds.), p. 153. Univ. of Pennsylvania Press, Philadelphia. Webster, P. D., and Tyor, M. P. (1967). Amer. J . Physiol. 212, 203. Wessels, N. K., Spooner, B. S., Ash, J. F., Bradley, M. O., Luduena, M. A., Taylor, E., Wren, J. T., and Yamada, K. M. (1970). Science 171, 135. Yasuda, K., and Coons, A. H. (1966). J . Histochem. Cytochem. 14, 303. Zagury, D., Uhr, J. W., Jamieson, J. D., and Palade, G. E. (1970). J . Cell Biol. 46, 52. Zucker-Franklin, D., and Hirsch, J. G. (1964). J . Exp. Med. 120, 569.

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

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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

THE MOVEMENT OF WATER

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

THE MOVEMENT OF WATER

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.

THE MOVEMENT OF WATER

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

THE MOVEMENT OF WATER

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

THE MOVEMENT OF WATER

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

THE MOVEMENT OF WATER

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.

THE MOVEMENT OF WATER

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

THE MOVEMENT OF WATER

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

THE MOVEMENT OF WATER

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

THE MOVEMENT OF WATER

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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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

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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<+ ions out of the cells in an electrogenic fashion, so that

3 70

WILLIAM R. HARVEY AND KARL ZERAHN

the side towards which potassium is transported becomes electrically positive. No specific inhibitor is known, but it is definitely not inhibited by ouabain or acetazolamide.”

STRUCTURE AND FUNCTION IN K PUMPS Despite these similarities which enabled Keynes to classify these Kpumps together, the K-transport in these tissues has differences which we would like to point out. The Malpighian tubules, salivary glands, and labial glands all transport fluid along with potassium, usually as an isotonic solution. However, the midgut does not transport any significant amount of fluid to the lumen. This negligible fluid transport is implied by the finding that the net K-flux to the lumen measured chemically agrees with that expected from the short-circuit current (Nedergaard and Harvey, 1968), and it has now been demonstrated directly (Nedergaard, 1972). Furthermore, the potential difference, PD, across the midgut initially is some 120-150 mV whereas the P D in these fluid secreting tissues is only about 25 mV. The fluid secreting gallbladder (Fig. l ) , which secretes isotonic NaC1, has a P D less than 1 mV (Diamond, 1962). Since the product of these transport systems is so different, one would expect some differences in the transport mechanism. These differences might be expected to show up as differences in the structure of these epithelia. Figure 1 summarizes the main structural features of these insect epithelia. The Malpighian tubules are composed solely of columnar cells having basal infoldings and microvilli on the lumen-side both closely associated with mitochondria. The tubules mainly secrete isotonic K solution, but can in some cases secrete isotonic sodium solution (for review, see Maddrell, 1971). The labial gland, which is a modified salivary gland, is also composed of just columnar cells with basal and apical infoldings both closely associated with mitochondria. The secretion is isotonic KHCO, (Kafatos, 1968). The salivary gland is composed of modified duplex goblet cells with microvilli in an apical cavity. The secretion is isotonic K solution (Oschman and Berridge, 1970; Berridgc and Prince, 1972). The midgut has columnar cells which have basal infoldings associated with mitochondria and apical microvilli free of mitochondria. It also has goblet cells which have mitochondria-filled microvilli in an apical cavity (Section VIII, A). The secretion is K, but as mentioned above the amount of fluid secreted, if there is any, is small. Clearly, although all these systems are secreting potassium, they are not doing it in the same way. Moreover, the structures differ widely, so there is no reason to be convinced that the transport mechanisms are identical. The only structural feature which they all share are spikelike structures

371

TRANSPORT OF ALKALI METALS BY MIDGUT I. Blood

3.

Malpighian tubules Cells

Lumen

2.

L a b i a l glands

5.

Gall Bladder

Cells

Blood

Salivary glands Cells

4.

Lumen

Cells

Blood

Cells

Blood

Midgui

OrnV

Blood

Cells

Lumen

Cells

Blood

Cells

Lumen

FIQ. 1. Summary of main features of K-secreting epithelia from insects. (1) The Malpighian tubules are composed of columnar cells-with basal infoldings and apical microvilli both in close association with mitochondria. Isotonic potassium solution is secreted from blood-side to lumen-side and the P D is low, with the lumen usually positive to the blood-side. (2) The labial gland is also composed of columnar cells only, but there are no microvilli. Instead, there are infoldings of both the apical and basal plasma membranes, which are closely associated with mitochondria in both cases. Isotonic KHCOa is secreted from blood-side to lumen-side and the P D is low, with the lumenside positive. (3) The salivary gland is composed of cells with a complex invagination of the apical surface, which is probably homologous to the goblet cavity of the midgut cells. There are apical projections on the membrane lining the invagination. Isotonic potassium solution is secreted from blood-side to lumen-side and the P D is low with the lumen-side positive to the blood-side. (4) The midgut is composed of columnar cells, which have basal infoldings associated with mitochondria, and apical microvilli, which are free of mitochondria. For approximately every four columnar cells there is a goblet cell which has mitochondria-filled apical projections on the membrane lining the goblet cavity. A solution of potassium, which is far from isotonic, is secreted from blood-side to lumen-side and the P D is more than 125 mV, with the lumen-side positive to the blood-side. ( 5 ) The gall bladder is composed of columnar cells with mitochondria not particularly associated with any membrane. The lateral plasma membrane is convoluted. Isotonic NaCl is absorbed from the lumen to the blood and the P D is virtually zero. Parts 1 and 3 are redrawn from Oschman and Berridge (1971) with permission from Tissue and Cell; Part 2 is redrawn from Kafatos, 1968 with permission from the Journal of Ezperimmtal Biology; Part 4 is redrawn from Anderson and Harvey (1966) with permission from the Journal of Cell Biology and also from Maddrell (1971) with permission from Academic Press; and Part 5 is redrawn from Diamond and Tormey (1966) with permission from Nalure.

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WILLIAM R. HARVEY AND KARL ZERAHN

on the inner leaflet of the unit membrane on the apical surface of some of the cells. These units have been proposed by Gupta and Berridge (1966; see also Berridge and Gupta, 1967; Berridge and Oschman, 1969; Smith, 1969) to be intimately associated with K-transport even to contain the K pumping sites. However, the K-transport mechanisms in these epithelia may be very similar or very different. We simply do not know. Moreover, there are numerous cases in which a tissue without an as-yet-detected change in structure causes its transport role to undergo a dramatic change. The midgut can switch from transporting K to Cs when both ions are available and can even switch to transporting other alkali metal ions when these are the only ones available (Section VI). The Malpighian tubules of Rhodnius can switch from transporting K to C1 in the presence of a diuretic hormone and even change the sign of the potential (Maddrell, 1971 ; Maddrell et al., 1969). In summary one cannot deduce the transport properties of a tissue (especially an insect epithelium) from its structure. Sir Vincent Wigglesworth (1970) has said t h a t . . . cells are full of things that cannot be seen, and that this must be borne in mind when looking at electron micrographs. The problem is whether the stained components are always the most important ones. The real link between structure and function is a t the physicochemical level, as is well shown by the sliding filament theory of muscular contraction. B. The Midgut Transport System

K-transport across the midgut has been studied mainly in the isolated tissue, but a few aspects of its role in the living insect have been useful in deducing its properties.

THEROLEOF

THE

MIDGUTIN VIVO

Harvey and Nedergaard (1964) confirmed the unpublished observations made by Harvey and Zerahn in 1960 that the [K] in the midgut contents is high (208 mM/liter), reflecting the high [K] in the leaf diet, whereas the [K] in the hemolymph is relatively low (27 mM/liter), and that the [Na] was low both in midgut contents (0.7 mM/liter) and in the hemolymph (6 mM/Iiter). More extensive determinations of cation concentrations in vivo were made by Quatrale (1966; see Table 11). Harvey and Nedergaard argued from such data that the midgut must play a n important role in maintaining a low enough [K] in the hemolymph so that nerves and muscles may function (see reviews by Harvey and Haskell, 1966; Treherne, 1967). These same data now allow us to deduce from the competition studies described in Section VI1,A that the only ion actively transported b y the

0

7

TABLE I1 -I

ION CONCENTRATION IN TISSUES FRESHLY ISOLATED FROM MATURE FIFTH INSTAR LARVAEOF Hyalophora cecropia”

E

ul

* 0

Cation concentration (meq/liter of sample water f SE mean)

K

Na

Mg

239 f 1 230 f 18 90.2 f 4.1 22.8 f 1.0 288 f 39

1.13 f 0.14 0.33 f 0.10 0.60 f 0.13 1.98 f 0.32 1.44 f 0.52

151 f 17 18.1 f 2.2 26.9 f 1.2 70.5 f 5.5 170 f 23

Sample

Leaf Midgut contents Midgut tiasueb Hemolymph Fecal pellet

6

5 % H20

Ca 319 38.2 6.86 13.6 312 ~~~

a

3

f 32

f 6.9 f 1.71 f 0.6 f 37

68.9 91.0 81.2 90.5 74.5

~

From Quatrale (1966).

* Samples were washed in 260 mM sucrose solution, and no determination of extracellular

space was made.

f 1.4 f 0.5 f 0.6

f 0.5

f 1.9

N 10 10 7 12 12

4

374

WILLIAM R. HARVEY AND KARL ZERAHN

midgut in vivo is potassium. In summary, the I(-pump in vivo can be considered to be an accessory excretory organ which in plant-eating insects whose diet has a high [K] (see Rladdrell, 1971) relieves the Malpighian tubules and rectum of one of their normal functions, the regulation of blood [K]. The K-transport mechanism is present in Hyalophora cecropia from the earliest developmental stage a t which it has been possible to make measurements (third-instar larvae) until just after the mature fifth-instar larva evacuates the gut contents prior to spinning its cocoon (Haskell et al., 1968). Just after the loss of K-transport the larval midgut epithelium is replaced by an entirely new and different pupal epithelium in a process that has been studied histochemically and autoradiographically b y K. Yamazaki and J. A. Haskell (1969, unpublished results). Since the K-pump is lost before any significant change in epithelial cell structure is observed one cannot account for the pump loss in terms of the loss of the larval epithelium. However, the loss of the K-transport coincides in time with the secretion of ecdysone by the prothoracic gland in the absence of juvenile hormone for the first time in the ontogeny of the silkworm (Burdette, 1962). To test the suggested hypothesis that ecdysone is simply turning off the K-transport mechanism, Harvey and Haskell (1971) exposed the isolated midgut to synthetic ecdysone in the presence and absence of juvenile hormone but could find no effect on the transport mechanism. The loss of the transport mechanism a t a time when the larval midgut epithelium is still intact and virtually unchanged in structure may be a useful control preparation in attempts to isolate chemical components of the transport mechanism. 2. IONTRANSPORT IN THE ISOLATED MIDGUT

The midgut isolated from Hyalophora cecropia (L) or from other lepidopterous larvae will transport potassium actively when it is bathed in solutions containing potassium salts and enough sucrose or other disaccharide such as trehalose to make the solution 260 mOsmM/liter (Harvey and Nedergaard, 1964; see also Nedergaard and Harvey, 1962; Harvey and Nedergaard, 1963; Wood, 1972). The concentration of I< in the bathing solution may be varied from a few to several hundred millimoles/liter and the midgut will still actively transport potassium. There are large increases in the rate of active K-transport as the [I(] is increased from 0 to 20 &/liter and only relatively small increases thereafter. An advantage of using the midgut for studying active ion transport is that it will transport potassium exclusively when K is the main cation in the bathing solutions. The K-transport is not even disturbed very much when Ca or nlg ions are

TRANSPORT OF ALKALI METALS BY MIDGUT

375

added. Neither is it disturbed when several other ions are added in small amounts. The solution can contain as much as 50% of any other alkali metal ion except Rb and the midgut will still transport potassium exclusively. Provided that Ca and Mg are present (see Section VI and VII) even higher proportions of other alkali metal ions will not disturb the exclusiveness of the potassium transport. However, if Mg and Ca are absent and K is omitted from the bathing solution the midgut can transport any one of the other alkali metal ions exclusively provided that the ion is the only cation in the bathing solution. These properties of the midgut make possible a new approach to the study of the active transport mechanism-the competition of ions for the transport mechanism. No other tissue so far tested has the ability to transport exclusively each member of a complete serics of ions. The more familiar frog skin and toad bladder are able to transport Na and to some extent Li. Several other membranes have exchange mechanisms for Na and K so that both are actively transported. Furthermore, there is a wide variety of tissues which can transport Na or I<, but invariably necd to have at least one additional ion (for references, see Keynes, 1969; Ussing, 1960). The midgut does not have this complication; one ion is enough to make the transport system operate and the investigator can choose any ion from the alkali metals. II. METHODS

Most of the studies have been performed on midguts isolated from large (10-20 gm) fifth-instar larvae of Hyalophora cecropia (L). If the larva is simply cut open, the midgut immediately contracts so much that it cannot be worked on. Therefore, it has to be immobilized, and this was first done by simultaneously anesthetizing and cooling the insect under frozen carbon dioxide (Harvey and Nedergaard, 1964). Later J. Bielawski (1966, personal communication) found that cooling alone was sufficient. This was tested later and since 1969 the larvae are simply cooled in crushed ice a t 0°C for about 2 hours. The midsection of the larva is excised and opened to expose the midgut, and the contents are removed. The midgut is then tied across a gap of a section of 5-mm glass tubing, which forms part of a chamber (see Fig. 2 ) , and the rest of the insect is dissected away (see Nedergaard and Harvey, 1968). The midgut is soft, and the transport mechanism in the fragile tissue can withstand only small differences in hydrostatic pressure. The bathing solutions are best equilibrated with oxygen because the consumption of oxygen by the midgut is so high that, in the absence of the tracheal system which is removed during the isolation procedure, an oxygen pressure less than 70% atm. may limit the active transport rate (Haskell

376

WILLIAM R. HARVEY AND KARL ZERAHN

f-Aq

insulated

__

Used solution

n

n

FIG.2. Apparatus for short-circuiting the isolated midgut of Hyalophora cecropia. The midgut is made a sphere by adjusting the volume of fluid in the outer compartment by means of a syringe. This compartment is stirred magnetically. The midgut PD is measured by calomel electrodes which connect via agar bridges to the solutions bathing blood-side (exterior compartment) and lumen-side (interior compartment) of the isolated midgut. To short-circuit the midgut the agar bridge to the lumen-side is placed exactly in the center of the midgut sphere. Then an external potential is applied across the Ag-AgC1 electrodes so that the two coils which form the Ag-AgC1 electrode on the lumen-side become negative. The placement of the midgut as a large sphere between the two symmetrical sections of this central electrode and the two symmetrical sections of a second Ag-AgC1 electrode in the blood-side solution places much of the midgut surface on an equipotential line. Modified from Harvey, Haskell, and Zerahn (1967) and Zerahn (1970) with permission from the Journal of Experimental Biology.

et al., 1965; Wood, 1972). The composition of several sohtions commonly used in midgut work is given in Table 111. In the open chamber the stirring is accomplished by bubbling gas on both sides of the gut. A closed chamber arrangement was developed by Harvey, Haskell, and Zerahn in 1967 for measuring oxygen consumption (Fig. 2). In this chamber the stirring is performed magnetically. The closed chamber has the advantage that the midgut can be arranged as a sphere which will maintain a constant shape

377

TRANSPORT OF ALKALI METALS BY MIDGUT

during the entire experiment as long as one side of the chamber is always closed and its volume kept constant. A leak in the gut will be signaled by the gut sphere collapsing. An additional advantage is that each midgut can be stretched out enough to open the folds in its wall so that diffusion of materials t o and from the gut will be more or less the same in all experiments [see Harvey, Haskell, and Zerahn (1967) and Harvey and Zerahn (1969) for details of this closed chamber or “sphere” method]. The potential difference across the midgut, PD, is measured by connecting the blood-side and the lumen-side solutions through agar-agar bridges filled with these same solutions to calomel cells and thence to a voltmeter. The PD is short-circuited by passing current between Ag, AgCl electrodes. In the sphere technique (Fig. 2) the electrodes of the blood-side (outer) compartment are large and are placed in a large volume of solution far from the midgut to make a uniform electrical field around the tissue. The lumen-side (inner) compartment is small and narrow; consequently, the Ag, AgCl electrodes must be placed symmetrically on both sides of the gap in the tubing and the potential bridges must be centered at the middle of the gap, one in the center of the midgut lumen and the other about 5 mm TABLE I11 COMPOSITION OF SOLUTIONS USEDTO STUDY THE MIDQUT C1

Abbreviation“ 32-K-S 32-N a -S 32-CS-S 100-K 32-K 4-K 32-Na 32-CS 16-K, 16-Na 16-Na, 16-Cs

Alkali metal ion

HCOs

Ca

Mg

Sucrose

(Concentration in millimoles per liter)

32mM K 32mMNa 3 2 m M Cs

50 50 50

2 2 2

5 5

5 5

5

5

166 166 166

100mM K 32 m M K 4mMK 32mMNa 3 2 m M Cs 16mMK 16 mM Na 16mMNa 16 mM Cs

98 30 2 30 30 30

2 2 2 2 2 2

0 0 0 0 0 0

0 0 0 0 0 0

166 166 166 166 166 166

30

2

0

0

166

0 The first number of the abbreviation is the concentration in millimoles per liter of the alkali metal ion and the letter that follows is its symbol. Solutions which contain 5 mM Mg2+and Ca2+ are identified by an “S” in the abbreviation.

378

WILLIAM R. HARVEY AND KARL ZERAHN

away just outside the gut sphere (see Zerahn, 1970, for details). This shortcircuiting arrangement is somewhat empirical because the conductivity of the bathing solutions is low compared to the conductivity of the midgut (see Wood, 1972). However, it can be improved by increasing the ionic concentration in the solutions although no indifferent ion has yet been fully tested. Recently, Wood (1972) has perfected a technique for shortcircuiting the midgut as a flat sheet with obvious advantages for evaluating the accuracy of short-circuiting the tissue. A close agreement between net K-flux and I,, reported in the “sphere” experiments has now been confirmed rigorously in the flat sheet preparation (Section II1,C). Therefore, the current measurement in the sphere cannot be in error by more than 5 1 0 % (we are grateful to Professor Hogben for discussing this problem with us in 1970). The unidirectional fluxes usually have been measured by labeling the ion in the solution on one side of the midgut with a radioactive isotope and removing samples a t appropriate times from the solution on the other side. 111. ACTIVE K-TRANSPORT A. Midgut Potential

The early determinations (Nedergaard and Harvey, 1962) of the midgut potential difference, PD, which were performed in air, always showed the lumen-side to be positive to the blood-side but the magnitude of the PD varied from 40 to 140 mV. With improved methods for isolating the midgut and with oxygenation of the bathing solutions the P D initially exhibited by a typical midgut immediately after isolation has increased over the years. Presently the initial P D of the midgut isolated from a mature fifthinstar larva of H . cecropia and bathed in oxygenated 32-K-S (see Table 111) will vary from more than 150 mV down to about 100 mV, depending rather much on the state of health of the insect and the skill with which the midgut is placed in the chamber. The midgut potential is generated directly by the active K-transport since no counter ion is required (Harvey et al., 1968).

B. Short-Circuii Current When Harvey and Nedergaard (1964) short-circuited the midgut P D in aerated solutions they found short-circuit currents, I,,, ranging from 115 to 1850 MAwith an average of 614 pA for an area of 2.5 cm2.By comparing the I,, with the fluxes measured with 42K, they found that the I,, is carried mainly by the active transport of potassium from blood-side to lumen-side.

TRANSPORT OF ALKALI METALS BY MIDGUT

379

Later work (Harvey et al., 1967) made it clear that the active K-transport depends on the supply of oxygen and the concentration of K as well as the state of the midgut. For midguts with a gross area of about 2 cm2 they found short-circuit currents which ranged from 1000 to as much as 5000 p A . I n oyxgenated solutions they measured a mean current of 2000 p A in 32 mM/litrr K and 2500 pA in 74 mM/liter K. C. Agreement between K-Flux and I,,

Using the open chamber, and sandwiching measurements of fluxes from blood-side to lumen-side between measurements of the fluxes in the opposite direction, Harvey and Nedergaard (1964) found that 83% of the I,, was carried by potassium, but the experimental error in the determination was large. Approximately this same value was confirmed in double-label experiments using 42Kand 86Rb (Nedergaard and Harvey, 1968). Using the closed chamber technique with improved arrangements for short-circuiting Harvey, Haskell, and Zerahn reported that in 32 m M K 89 f 8% (S.E. of the mean; n = 6) of the current was carried by potassium. In 74 m M K solutions the agreement was 92 f 8% (n = 11). They also reported a small current remaining in the absence of potassium but were unable to confirm i t in later unpublished experiments (but see Section VI). Subsequently K. Zerahn (1969, unpublished results) studied the spherical midgut in a closed chamber with further improvements in the arrangements for short-circuiting and found 106 f 1% ( n = 6). Wood (1972) found 99.5 f 1.6% ( n = 20) agreement between I,, and the net Rb-flux across the midgut of Antheraea pernyi perfused as a flat sheet. These data show conclusively that in 32 mM K or higher concentrations and with Ca and Mg present in the bathing solutions, that K (or Rb; see p. 386) carries all the Z6,. These data unequivocally demonstrate thc active transport of potassium by thc midgut and justify the use of the I,, to measure active Ktransport under the conditions just mentioned. Conclusive evidence for active K-transport in midguts with the natural PD is furnished in many experiments (Harvey and Nedergaard, 1964) which show that the flux ratio is very far from that calculated for the passive movement of ions using the flux ratio equation (see Ussing, 1949, 1971).

IV. INFLUENCE OF [K] ON PD AND I,,

Most of the studies of K-transport across the midgut have been performed with the 32 m M K solution (32-K-S, see Table 1x1). Although this

380

WILLIAM R. HARVEY AND KARL ZERAHN

K concentration is close to the value in the hemolymph, which bathes the gut directly in vivo, it is only about one-tenth of the value in the midgut contents (Table 11). Therefore it is an arbitrary value. It does not even yield the highest K-transport rate although it does give almost the maximal PD. A. [K] and PD

Although the influence of [I<] on the PD has been studied extensively, the relationship is still not clear. If a large part of the K is substituted by Na in the standard solution containing 5 mM Ca and Mg, the PD will drop immediately to a lower value and subsequently decrease even more.

t A

01 0

I

I

8

1

I

16 (rnM/liter)

I

1

24

I

I

32

A

FIG.3. Dependence of PD and I,, on [K] in short-term measurements in the absence of calcium and magnesium. The values for P D (squares) and I,. (circles) are both expressed as perccntages of the values in 32 mM K. These values were calculated by dividing by two the sum of the readings in 32 mM K before and after the experimental period. (A) The [K] was changed by replacing an appropriate amount of K by Na. The values (in microamperes) for the I,, in 32 mM K were: a t start, 1820; after 2-K, 1650;

38 1

TRANSPORT OF ALKALI METALS BY MIDGUT

If the [K] is sufficiently low, say, 1 mM, the sign of the P D may even reverse and the lumen-side may become negative with respect to the bloodside (Harvey et al., 1968). This reversal of sign could be explained by a complete cessation of the electrogenic K-pump coupled with a loss of K from the cells to the blood-side but it has not been studied. However, if the [K] is decreased rapidly in a solution with no other cations present there is hardly any change in P D (W. R. Harvey and K. Zerahn, 1970, unpublished results) (see Fig. 3 ) . This result suggests that the decrease in P D found earlier may be attributed just as well to a shunting of the P D by the other ions like Na, Mg, Ca, C1, and HCO, as to a direct effect of [K] on the P D generated by the electrogenic K-pump. Some

.E

I

/

2

0I

8

16

24

4,

32

(mM/liter)

B and after 0-K, 1550. The corresponding values (in millivolts) for the PD in 32 rnM K were: a t start, 109; after 2-K, 98; and after 0-K, 97. (B) The [K] was changed by replacing an appropriate amount of KC1 by a n osmotically equivalent amount of sucrose. The values (in microamperes) for the I,, in 32 mM K were: at start, 1600; and at end, 1480. The corresponding values (in millivolts) for the PD in 32 rnM K were: a t start, 98; and at end, 79. (W. R. Harvey and K. Zerahn, 1970, unpublished results).

382

WILLIAM R. HARVEY AND KARL ZERAHN

insight is provided by a consideration of the effects of I( concentration on the short-circuit current. B. [K] and I,,

Nedergaard and Harvey (1968) studied the dependence of I,, on [K] by replacing K with Na in the standard Ca and Mg containing solution. In the curve constructed from mean values the current drops with decreasing [K] and can be extrapolated to zero for zero [K]. However, it is obvious that the results are uncertain because the decreases in current were reversed less than 50% when the original [K] was restored. Therefore we tried to repeat the experiments and found that by changing the solutions rapidly (reading the I., a t 2.5 minutes after changing the solution) the decreases in I,, were completely reversible (W. R. Harvey and I(. Zerahn, 1970, TABLE IV AGREEMENT BETWEEN K-FLUXFROM BLOOD-SIDE TO LUMEN-SIDE OF ISOLATED MJDQUT A N D I., I N 4 M M K SOLUTION"

May 15 22

June 11A June 11B

13 22 23 27 24 33 32

10 18 17 21 18 26 23

130 122 135 128 133 127 139

K-$uz from lumen-side to blood-side May 28 June 10A June 10B

1.0 1.0 1.6

-

-

-

a (W. R. Harvey and K. Zerahn, 1970, unpublished results). Mean influx = 25 Mean flux = 23.8 Mean efflux = 1 . 2 Mean I., = 19

Mean flux ~X 100 = 125y0 Mean I.,

TRANSPORT OF ALKALI METALS BY MIDGUT

383

unpublished results) (Fig. 3A). At [I<] of 24 mM and 16 mM the drop in current agreed with that reported earlier, but a t [IC] of 4 m M and 2 mM there was no agreement with the previous work. The I,, was much higher than previously reported, and we now know that it is because of a Na current (see Section V1,C). Therefore, we measured the dependence of I,, on [Ii] when no cations other than Ii were present. The results are shown in Fig. 3B. It is clear that the I,, decreases with decreasing [I<]. RIoreover, the entire current was carried by I< in 32, 24, 16, and 8 mM K as demonstratrd by agreement between I,, and IC-flux in these solutions. However, the I,, values at 4 mM I< and below are uncertain due to the difficulty of short-circuiting the midgut in solutions with such low conductivity. Howrvrr, the unidirectional Ii-fluxes wrre measured in 4 mM IT (Table IV), and the net flux was found to be about 125% of the I,,. Although this agreement b r t w e n I,, and nrt flux in 4 mM is far from perfect i t does indicate that the I s , in 4 mM Ii still is a fair approximation of the active K-flux. Short-circuiting experiments in solutions with [I<] less than 4 mM were considered to be too inaccurate to be worthwhile. However, we can say with certainty that thr I<-flux at P D = 0 does drop with decreasing [K] and should be close to zero at zero Ii concentration. To summarize, in short-term experiments in solutions in which IC is the only cation present the P D is virtually unaffected whereas the I,, (K-transport rate) decreases drastically when the [I<] is decreased (Fig. 3 R ) . C. Interpretation of [K], PD, and I,, Relationships

Koefoed-Johnsen and Ussing (1958) found in the frog skin that a 10-fold change in “a] on the outside yields a 60 mV change in PD. We have just seen that the results are not a t all the same when the [K] is decreased in the solutions bathing the midgut. When Na replaces K with Ca and Mg present, Na-transport and its associated electrogenic potential is inhibited by the Ca (see Section VI1,D). However, in the experiments in which this effect is clear the inhibition of the I,, is not reversed when the higher [K] is restored so that irreversible damage to the tissue contributes to the result. With Ca and Mg absent the P D does not drop very much when K is substituted by Na, presumably because the shunting is less and the P D from the Na pumping replaces the P D from the K pumping. However, with K the only cation present, as the [K] is decreased the I,, decreases, signaling a decrease in pumping rate by the electrogenic K-pump. Nevertheless, the P D is not affected until the I., has become virtually zero. The K-transport mechanism is able to build up the full P D even with a low [K] and slow pumping rate. This result should be no more surprising than the

384

WILLIAM R. HARVEY AND KARL ZERAHN

observation that a small dry cell which generates but a small current is able to build up as large a potential as a large dry cell. Whether or not the midgut does build up a large PD depends on conditions such as the degree of shunting by other ions and the generation of potentials by the transport of other ions.

V. COUPLING OF K-TRANSPORT TO METABOLISM A. Ratio of K-Transport to O2 Uptake

It has been found in several epithelia, such as the frog skin, toad bladder, and kidney, that the Na-transport triggers the oxygen consumption and that there is a constant ratio between active Na-transport and the extra oxygen consumption correlated with this transport. Nothing was known about the energy needed for K-transport, so it was investigated by Harvey et al. (1967). Surprisingly enough, the results showed that the I<-transport does not trigger the oxygen consumption. This result was unexpected since the K-transport depends closely on the oxygen consumption. The midgut consumes a great deal of oxygen, some 300 keq ( = 1700 p1) per gram of wet weight per hour. The same amount of oxygen is consumed whether the K-transport rate is high in the short-circuited gut, is diminished by the natural PD of about 100 mV, or is diminished further by imposing an additional 60 mV. Similarly, the oxygen consumption remained unchanged when the K-transport is abolished by substituting the I< in the blood-side solution by Na (in the presence of Ca and Mg; see Section VI1,D) or enhanced by increasing the [K] to 73.5 mM. In 32 mM K solution 1.23 peq K was transported per peq of total oxygen consumed whereas in 73.5 mM K the value was found to be 2.0. In fact, since the K-transport can be changed from zero to large numbers with no change in oxygen uptake, the K : O ratio will range from 0.0 to about 2.0. This lack of any direct coupling between K-transport and oxygen consumption means that, when no transport is taking place, either the transport mechanism is idling and the energy is lost as heat or the energy is then geared to some other endergonic process. Although the oxygen consumption is independent of the K-transport the opposite is not the case. The inhibition of K-transport by anoxia is complete but highly reversible (Haskell et al., 1965; see also Haskell and Clemons, 1963). With constant potassium concentration the degree of transport depends almost directly on the rate of oxygen consumption. The oxygen consumption of the isolated midgut depends in turn on the oxygen pressure in the bathing solu-

385

TRANSPORT OF ALKALI METALS BY MIDGUT

TABLE V DEPENDENCE OF ACTIVE K-TRANSPORT ON TEMPERATURE^ Total Temper- oxygen conature sumption ("C) (wdhr) 25 15 0

K-flux (peq/hr)

56 36

68 44

K:O 1.23 1.22

From Harvey et al. (1967).

tion but the dependence is complex depending on such factors as the thickness of the tissue (Harvey et al., 1967). B. Dependence on Temperature

The active K-transport by the midgut as expected depends on the temperature. Mean values for the midgut a t 25" and 15°C in 32 m M K are given in Table V. The ratio of the K-flux rates a t 25":15" was 1.55. This ratio of the flux rates at the two temperatures is exactly the same as the ratio of the rates of oxygen consumption a t these two temperatures, a result which is to be expected from the close dependence of flux on oxygen consumption. The ratio for Na-transport by the frog skin at these same two temperatures is 1.66 (Zerahn, 1956). C. ATPare

The energy for active Na-transport is derived from a Mg-dependent ATPase which is activated by Na and K ions (Skou, 1957). To examine the prospect that the energy for active K-transport may also be derived from ATP, Turbeck et al. (1968) isolated an ATPase from the midgut. The enzyme's activity was stimulated by bicarbonate and chloride, but not by potassium. Therefore, this ATPase may have no direct role in active K-transport and appears to be a mitochondria1 enzyme. However, mitochondria are likely to be important in supplying energy for the K-transport so that mitochondrial ATPase(s) may be involved directly or indirectly in the transport mechanism. The enzymology undcrlying the transport remains an unsolved and challenging problem (see Turbeck and Foder, 1970).

386

WILLIAM R. HARVEY AND KARL ZERAHN

VI. TRANSPORT OF OTHER ALKALI METAL IONS AND OTHER SUBSTANCES A. Rubidium

Harvey and Nedergaard (1964) showed that neither sodium nor lithium will change the P D or the I,, when added in the same amount as potassium. When they substituted all the K by Na in the presence of Ca and Mg the P D disappeared almost completely, and they concluded that sodium is not transported by the midgut under these conditions (but see Section IV). However, all the potassium could be substituted by rubidium without any change in the P D for more than an hour (Harvey et al., 1968). These workers concluded that rubidium is actively transported by the midgut almost as well as potassium. In midguts isolated from Antheraea pernyi Wood (1972) found that the net Rb-flux accounted completely for the I,, (see Section II1,C).

B. Cesium Cesium is chemically very similar to potassium and rubidium. So cesium was also tested to see whether it is act(ive1y transported by the midgut (Zerahn, 1970). However, the midgut proved to be very unpredictable when bathed with solutions containing Cs in the presence of Ca and Mg; some preparations would maintain a PD and an I., for longer than 2 hours whereas others would have no P D left after a few minutes. The cesium flux from blood-side to lumen-side in the short-circuited midgut was found in the mean to be 11.8 peq/hour and from the lumen-side to the blood-side to be 0.8 peqlhour. The net flux of 11.0 peqlhour agreed well with the short-circuit current of 10.8 peqlhour. These data prove without any doubt that cesium is actively transported undcr these conditions. However, even in the best experiments thc I,, in cesium after about 10 minutes is only about a quarter of that previously exhibited in potassium. C. Sodium

As just mentioned, midguts bathed in cesium solutions which also contain Ca and Mg very often show a very low P D after 5 minutes. Harvey and Nedergaard had shown in 1964 that sodium is not actively transported by t,he midgut in solutions containing Ca and Mg. If the Ca and Mg are omitted from the solutions, however, we obtain quite a different result. When the solution bathing the midgut is changed from 32-K to 32-Na (neither of which contain Ca or Mg; see Table 111) the P D and I., are maintained for more than an hour, the initial current in 32-Na being about

TRANSPORT OF ALKALI METALS BY MIDGUT

3 87

half of its value in the 32-K solution (Harvey and Zerahn, 1971). The sodium flux from blood-side to lumen-side was found in the mean of 10 values to be 22.5 peq/hour and from the lumen-side to the blood-side to be 1.2 peqlhour. The net flux of 21.3 agreed well with the short-circuit current of 22.9 peqlhour. The flux ratio was about 20. These data prove without any doubt that sodium is actively transported by the midgut under these conditions. In general, the active sodium transport mechanism in living membranes is inhibited by ouabain. In contrast the electrogenic transport mechanism for potassium in the midgut is not affected even by high concentrations of ouabain (Haskell et al., 1965) and neither is the active sodium transport (Harvey and Zerahn, 1971). 0. Lithium

Lithium has no usable radioactive isotope, and therefore was not investigated with the same rigor as sodium. However, when the midgut is bathed in solutions containing lithium as the only cation, it will maintain a potential difference that can be short-circuited and yield an I,, that is about 50% of the K-current and in other respects is almost identical with the Na-current. It can be considered quite certain that lithium, like sodium, is transported actively by the midgut (Harvey and Zerahn, 1971). E. Ammonium

Nedergaard and Harvey (1968) found that ammonium is not transported actively in the standard solution containing Ca and Mg. The I,, dropped to zero when all the potassium was substituted by ammonium and did not return when the potassium was restored. Ammonia did not poison the transport mechanism because with 16 mM NH4, 16 m M K in the solution (and therefore almost as much NH, as in 32 m M NH, solution) the drop in active transport rate was limited as deduced from the I.,. The amount of ammonia could be reduced by decreasing the pH. However, once again the main determinant is calcium. When this divalent cation is absent, ammonium does get transported actively (Zerahn, 1971). F. Hydrogen Ions

Harvey and Nedergaard (1964) suggested that hydrogen ion is exchanged for potassium in the active K-transport mechanism and this prospect is also discussed positively by Haskell et al. (1965). Moreover, it has becn cited in papers published by others. However, the pH in the lumenside solution can be changed from about 6 to about 9 without changes in the short-circuit current. This indicates that K-transport is independent of H+. Now that it has been established that there is a competition between K

300

WILLIAM R. HARVEY AND KARL ZERAHN

and the other members of the alkali metal series, a competition between potassium and hydrogen ions can be considered. However, hydrogen ions a t the usual concentration in the bathing solutions of 1 nanomole/liter to 1 micromole/litcr would have difficulty in taking part in the active transport process. Even if the hydrogen ion has as high an affinity for the pump as does the most favored alkali metal ion, potassium, Hf in the bulk solution would still be of no importance in the transport process. At a p H of 6 (nearly the lowest pH a t which the midgut can function), the affinity of the mechanism for H+ would have to be more than lo4 times higher than that for potassium for competition to occur. G. Amino Acid Absorption

Recently, Ncdergaard (1972) reported that the midgut will actively transport the nonmetabolizable a-aminoisobutyric acid from lumen-side to blood-side, i.e., in a dircction opposite to the alkali metal ion transport. This amino acid transport was diminished but by no means abolished when the midgut was short-circuited. Therefore, the dependency of the amino acid absorption on K secretion is not a simple one and is quite different from the absorption of amino acids in the intestine of rabbits (Schultz and Zalusky, 1965). Vll. COMPETITION BETWEEN ALKALI METAL IONS

The interpretation of data for competition of transport across epithelia depends to some extent on the route which the transport takes. If the transport is between cells or via some other non-mixing route, then a knowledge of the transport rate and concentration ratio in the solution from which transport occurs is all that is required. However, if the ions actually enter the cells and the pumping sites are competed for by intracellular ions, then obviously the information required is the transport ratio compared to the concentration ratio of the ions within the cells (J. L. Wood and W. R. Harvey, in preparation). Precise information about concentrations of ions within epithelial cells is difficult if not impossible to obtain with present techniques. Therefore we will rcstrict our analysis to a consideration of the transport ratio compared to the ion ratio in the blood-side compartment, i.e., competition for the overall transport mechanism, and to a consideration of the uptake of competing ions compared to the transport rate of those ions. A. Competition for Metal Ions

Overall

Transport Mechanism between Alkali

The competition between alkali metal ions for the overall transport mechanism is different when Ca and Mg are present than when they are

389

TRANSPORT OF ALKALI METALS BY MIDGUT

absent. Zerahn (1970) showed that in the presence of Ca and Mg the competition between cesium and potassium is anomalous. Thus when Cs and K are present in the same concentration (both a t 16 mM), only K is transported. However, when the [K] is 10% or 20% of the total only Cs is transported. These results mean that the overall transport mechanism changes its affinity and prefers whichever ion is available in the highest concentration. However, in the absence of Ca and Mg no such anomalous competition is seen. In Ca- and Mg-free solutions, when N a and K are present in the same concentration (both a t 16 mM), again only K is transported. However, when the K concentration is 10% or 20% of the total 32 m M of alkali ion present, then both Na and K are transported (Zerahn, 1971a). These results mean that with Ca and Mg absent the mechanism does not seem to have changed its affinity as the concentration ratio between the ions is changed. It is the calcium rather than the magnesium which is important (see Section VI1,D). In order to compare the affinity of the overall transport mechanism for the alkali metal ions we have to know (1) whether or not calcium is present, (2) the concentration of the ions to be compared, and (3) the ratio of those ions in the compartment before the pump. The competition was measured by comparing transport rates in solutions containing 16 mM/liter of each of two alkali metal ions, for example in 16-Na, 16-Cs (see Table VI). The measurements were made on short-circuited midguts in the absence of Ca and Rlg. The flux of one of the ions was measured with a radioisotope and compared with the flux of the other ion or with the I,, minus the measured flux of the first ion, This latter method makes the reasonable but untested assumption that the I,, is approximately equal to the sum of the net flux of the two ions and that the effluxes are small. Since the concentration ratios are 1:1 the ratios of the flux of each ion from blood-side to lumen-side gives the competition ratio directIy. This ratio is not very accurately known for Na: K because the Na flux is small, but the inaccuracy TABLE VI

RATIOOF FLUXES OF ALKALI METALIONSFROM BLOOD-SIDE TO LUMEN-SIDE WHENTHE CONCENTRATION RATIO IS 1 A N D THE CONCENTRATION OF EACH IONIS 16 M W . ~ Li

Na

K

Rb

CS

NH,

1.00 -

1.00 0.05

-

1.0

1.0

1.6 0.05

0.25

K. Zerahn (1972, unpublished results). Na was compared to I,i and Cs whereas K was compared to Cs, Rb, Na, and NH,. a

390

WILLIAM R. HARVEY AND KARL ZERAHN

wiIl not change the sequence. These competition ratios are shown in Table VI and they yield the competition sequence K > Rb >> NH, > Cs > Na = Li (Zerahn, 1971a), which is Sequence IV in the analysis given b y Diamond and Wright (1969). This sequence reflects neither the sequence of increasing radii of the unhydrated ions from crystal data nor the sequence of hydrated ions. However, it is a sequence that Diamond and Wright reported is often found for the active transport of these ions. B. Uptake of Sodium by the Midgut

Zerahn (1970) showed that when the midgut is bathed in the 32-Cs-S solution (in which Cs is actively transported exclusively) the midgut takes up Cs and loses potassium. After it was learned that Na is actively transported exclusively in 32-Na1 Zerahn (1971a) studied the Na-K exchange and found a similar uptake of Na and loss of K from the midgut tissue. The midgut was first isolated in a chamber and maintained in 32-K for from 15 to 30 minutes, and then washed and maintained in 32-Na for periods of 5, 10, 20, or 25 minutes. Then the midgut was removed from the chamber, dried gently on a tissue paper, weighed, and homogenized in 0.3 M perchloric acid. The ion content was determined with a flame spectrophotometer. The extracellular space was determined by measuring the content of sucrose (Zerahn, 1971a), and the ion concentrations in the tissue were corrected for extracellular space. It can be seen (Table VII) that after 5 minutes the Na content of the midgut is very close to its maximum value, that short-circuiting the midgut seems to have but little influence on its Na contents, and that after about 25 minutes the [K] has dropped to 20 mM. These data reflect a n uptake of Na in exchange for tissue K because the [Na] of the midgut tissue freshly excised from the larva is but 0.6 m M (Quatrale, 1966). This fast exchange TABLE VII CONCENTRATION OF K AND N A IN MIDGUT CELLSWHENTNE MIDQUTIS BATHED IN 3 2 - N ~ ~ - ~ Time (minutes)

[Nal (mM)

[KI (mM)

pAmp

mV

5 10 10 25

109 110 100 100

54 35 45 20

800 700 0 800

0 0 80 0

a

K. Zerahn (1972, unpublished results).

* Results are corrected for extracellular space.

391

TRANSPORT OF ALKALI METALS BY MIDGUT

TABLE VIII CONCENTRATION OF K, NA, A N D Cs I N MIDGUTCELLS, WHENTHE MIDGUTIs BATHEDI N 1 6 - N ~ ,16-K, OR IN 16-C~,16-K0,b Timein minutes

“a] (mM) ~

10 10 10 20 30 a

[K] (mkf)

[Cs] (mM)

IAmp

mV

1600 -

0 130 83 86 94

~

44 34

-

-

29 34 38

-

-

95 93 103 107 99

-

K. Zerahn (1972, unpublished results). Results are corrected for extracellular space.

of external Na with tissue K is in agreement with the fast exchange of labeled K with unlabeled tissue K found in the earlier work by Harvey and Zerahn (1969). C. Competition Sequence for Uptake of Alkali Ions

The kinetics of Na uptake were also examined when the midgut was maintained in a chamber with the 16-K, 16-Na (Ca- and Mg-free) solution in which just active K-transport is observed. The results were corrected for extracellular Na and K. Similarly the kinetics of Cs uptake in 16-K, 16-CS solutions were examined. The results are reported in Table VIII. The amount of Na taken up is not very much affected by short-circuiting the midgut and has reached nearly its maximal level after 10 minutes. The same results were observed with Cs under these conditions. The concentration of Cs increases rapidly at first but does not increase much more from 10 to 20 minutes. The concentration of K hardly decreases. These results show that there is quite a large exchange between external Na or Cs and tissue K in bathing solutions in which only active K-transport is observed. A complete sequence for the uptake of alkali metal ions was made by comparing the uptake of these ions in solutions containing two members each at 16 mM. Table IX shows the molarity of each alkali metal ion in the midgut cells. Clearly the ions do not equilibrate to a n equal concentration. The cells prefer K, but Rb is almost as favored. Na and Li are much less favored, but the rather astonishing finding is that Cs is the last in the sequence which takes the following form: K > Rb >> Na > Li > Cs. This sequence is different from the one given for the overall active trans-

392

WILLIAM R. HARVEY AND KARL ZERAHN

TABLE IX

CONCENTRATION AND CONCENTRATION RATIOOF ION 1 AND 2 IN MIDQUT CELLSWHENTHE MIDQUT Is BATHED WITH 50% ION1 AND 50% ION2 WITH A TOTAL CONCENTRATION OF 32 M M " ! ~ Ion 1 ( m M )

Ion 2 (mM)

Na Na Rb

Li Cs Na Na

73 82 67 K 94 Isotopes

K

Ratio 2:l

53 40 30 39

Rb

0.73 0.49 0.45 0.41 1.1

K. Zerahn (1972, unpublished results). Results are corrected for extracellular space.

port in that there is an inversion between Na > 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).

TRANSPORT OF ALKALI METALS BY MIDGUT

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

WILLIAM R. HARVEY AND KARL ZERAHN

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.

TRANSPORT OF ALKALI METALS BY MIDGUT

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

WILLIAM R. HARVEY AND KARL ZERAHN

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.

TRANSPORT OF ALKALI METALS BY MIDGUT

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

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WILLIAM R. HARVEY AND KARL ZERAHN

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

TRANSPORT OF ALKALI METALS BY MIDGUT

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.

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WILLIAM R. HARVEY AND KARL ZERAHN

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

TRANSPORT OF ALKALI METALS BY MIDGUT

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

WILLIAM R. HARVEY AND KARL ZERAHN

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.

TRANSPORT OF ALKALI METALS BY MIDGUT

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

WILLIAM R. HARVEY AND KARL ZERAHN

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

TRANSPORT OF ALKALI METALS BY MIDGUT

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

WILLIAM R. HARVEY AND KARL ZERAHN

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

TRANSPORT OF ALKALI METALS BY MIDGUT

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<] From Section VIII, I, 4, we see that in 2 mM K solutions the pool size is small, amounting to about 0.5 peq of K (Table X). Harvey and Zerahn showed that, aftcr 5 minutes, when they reported that the isotope steady state had been reached (within 20%), the midgut cells were labeled, but 8% by the 42Kbeing transported through the midgut. Is this small size of the measured pool valid? This small pool size is the same as that measured from the flux, and the flux is of the small size that one expects a t the low K concentration and is stable. These two facts-that the lag time in low K still has the same small value as in high K and that it corresponds to a small pool-are consistent with the conclusion that the pool is a transported pool. Moreover, the low labeling of the midgut is to be expected

408

WILLIAM R. HARVEY AND KARL ZERAHN

when the transport is by model 4 (goblet cell route) or by model 5 (nonmixing route). Is there any evidence that favors the view that the small pool in low K is a transport pool? Harvey and Wood argued that the transport pool was represented by the exchangeable fraction of K, which they suggested was in the cytoplasmic matrix. They reported that isolated midgut pieces not mounted in a chamber became labeled with 42Kto a very low level even after 60 minutes of incubation. They further reported that the total K (uncorrected for extracellular spaces) decreased to the low value of about 30 mM/liter after an hour. These data seem to show that the size of the exchangeable fraction of K which was large in high [K] is small in low [K], and thereby account for the small transport pool in low K. Confirmation of these results is needed from measurements on midguts mounted in chambers where the viability and transport rates can be evaluated. The low cellular [K] implied by these results presents an osmotic problem. The ground cytoplasm must have ions to maintain the osmotic pressure of the transporting cell system. If this role is taken over by ions other than K, then the [K] in the mitochondria and nucleus would have to be high and no proof is given that these organelles will not exchange K with that in the cytoplasmic matrix. However, perhaps the greatest problem with a transport pool is in supplying proof that a cellular pool which is large enough to yield a 3-minute lag time with a flux of 120 peq/hour could become small enough to yield a 3-minute lag time with a flux of 5 peq/hour. How can the cellular pool size change so much when tissue [K] changes so little?

K.

Summary of Transport Route

On present evidence it is not possible to reject model 3 (variable cell number), model 4 (goblet cell route), or model 6 (variable [K] in cytoplasmic matrix), all of which are mixing models implying a transport pool in the cells. On the other hand, the kinetic evidence is easily accommodated by model 5 , the non-mixing model implying a ' transported pool in the extracellular space on the lumen-side of the midgut. The main conclusion from these kinetic studies is that, far from being able to assume that transport across epithelia always goes through cells, as in the traditional view, one must consider the question of the route to be unanswered in both the frog skin and the midgut, two tissues in which it has been studied extemively. Therefore, although it is generally assumed that a mixing route is followed, there is no basis for assuming either a mixing route or a nonmixing route in epithelial tissues in which the route has not been studied.

TRANSPORT OF ALKALI METALS BY MIDGUT

409

ACKNOWLEDGMENTS We thank Dr. John L. Wood for helpful discussions. Some of the hitherto unpublished research described here, as well as the writing of this article, was supported in part by a research grant (A1 09503) from the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service. REFERENCES Andersen, B., and Zerahn, K. (1963). Acta Physiol. Smnd. 59, 319. Anderson, E., and Harvey, W. R. (1966). J . Cell. Biol. 31, 107. Barry, P. H., and Hope, A. B. (1969). Biophys. J. 9, 700. Berridge, M. J., and Prince, W. T. (1972). Aduan. Insect Physiol. (in press). Berridge, M. J., and Gupta, B. L. (1967). J . Cell. Sci. 2, 89. Berridge, M. J., and Oschman, J. L. (1969). Tissue Cell 1, 247. Burdette, W. J. (1962). Science 135, 432. Cereijido, M., and Rotunno, C. A. (1968). J . Gen. Physiol. 51, 280s. Dahlstrom, A. (1971). Phil. Trans. Roy. SOC.London, Ser. B 261,325. De Robertie, E. D. P., Nowinski, W. W., and Saes, F. A. (1970). Cell Biology, Saunders, New York. Diamond, J. M. (1962). J. Physiol. (London) 161, 474. Diamond, J. M., and Tormey, J. M. (1966). Nature 210, 817. Diamond, J. M., and Wright, E. M. (1969). Annu. Rev. Physiol. 31, 581. Gupta, B. L., and Berridge, M. J. (1966). J. Cell Biol. 29, 376. Harvey, W. R. (1968). J . Cell Biol. 39,59A. Harvey, W. R., and Haskell, J. A. (1966). Aduan. Insect Physiol. 3, 133. Harvey, W. R., and Haskell, J. A. (1971). Endocrind. E x p . 5, 47. Harvey, W. R., and Nedergaard, S. (1963). J. Cell Biol. 19,32A. Harvey, W. R., and Nedergaard, S. (1964). Proc. Nat. Amd. Sci. U.S.51,757. Harvey, W. R., and Wood, J. L. (1972). Ini. Conf. Biol. Membranes, Gargnuno, Ituly, June, 1971 (in press). Harvey, W. R., and Zerahn, K. (1969). J . Exp. Biol. 50,297. Harvey, W. R., and Zerahn, K. (1971). J.Exp.Biol. 54, 269. Harvey, W. R., Haskell, J. A., and Zerahn, K. (1967). J. E x p . Biol. 46, 235. Harvey, W. R., Haskell, J. A,, and Nedergaard, S. (1968). J . Exp. Biol. 48, 1. Haskell, J. A., and Clemons, R. D. (1963). J . Cell Biol. 19, 77A. Haskell, J. A,, and Clemons, R. D., and Harvey, W. R. (1965). J. Cell. Comp. Physiol. 65,45. Haskell, J. A., Harvey, W. R., and Clark, R. M. (1968). J. E x p . Biol. 48,25. Hodgkin, A. L., and Keynes, R. D. (1955). J. Physiol. (London) 128,28. Hoshiko, T., and Ussing, H. H. (1960). Actu Physiol. Smnd. 49, 74. Johnstone, B. M. (1967). Curr. Top. Bioenerg. 2,335. Kafatos, F. C. (1968). J. Exp. Biol. 48,435. Keynes, R. D. (1954). Proc. Roy. SOC.,SeT. B 142, 359. Keynes, R. D. (1969). Quart. Rev. Biophys. 2, 177. Koefoed-Johnsen, V., and Ussing, H. H. (1958). Acta Physiol. Scand. 42, 298. Lassen, U. V. (1971). First European Biophysics Congress, Baden near Vienna, Austria. Loewenstein, W. R. (1966). Ann. N . Y . Acad. Sci. 137, 441. Maddrell, S. H. P. (1971). Aduan. Insect Physiol. 8, 199.

410

WILLIAM R. HARVEY AND KARL ZERAHN

Maddrell, 8.H. P., Pilcher, D. E. M., and Gardiner, B. 0. C. (1969). Nature (London) 222,784. Nedergaard, 8. (1972). J. Exp. BWl.56, 167. Nedergaard, S.,and Harvey, W. R. (1962). 2nd Annu. Meet., Amer. SOC.Cell B i d . p. 131. Nedergaard, S., and Harvey, W. R. (1968). J. Exp. B i d . 48, 13. Oschman, J. L., and Berridge, M. J. (1970). Tissue Cell 2,281. Oschman, J. L., and Berridge, M. J. (1971). Fed. Proc. Amer. SOC.Exp. B i d . 30,49. Quatrale, R. P. (1966). Ph.D. Thesis, University of Massachusetts, Amherst. Ramsay, J. A. (1953). J . Exp. B i d . 30,358. Schulte, S . G., and Zalusky, R. (1965). Nature (London) 205,292. Skou, J. C. (1957). Biochim. Biophys. Acta 23,394. Smith, D. S. (1969). Tissue Cell 1,443. Smith, D. S. (1970). In “Insect Ultrastructure” (A. C. Neville, ed.), p. 14. Blackwell, Oxford. Solomon, A. K. (1964). In “Transcellular Membrane Potentials and Ionic Fluxes” (F. M. Snell and W. K. Noell, eds.), p. 47). Gordon S. Breach, New York. Templeton, J. R. (1964). Comp. Biochem. Physio2.11,223. Treherne, J. E. (1967). In “Insects and Physiology” (J. W. L. Beament and J. E. Treherne, eds.), p. 175. Oliver, Boyd, Edinburgh and London. Turbeck, B. O., and Foder, B. (1970). Biochim. Biophys. Acta 212, 139. Turbeck, B. O., Nedergaard, S., and Kruse, H. (1968). Biochim. Biophys. Actu 163,354. Ussing, H. H. (1949). Acta Physiol. Scand. 19, 43. Ussing, H. H. (1960). In “Handbuch der experimentellen Pharmakologie,” (0. Eichler and A. Farah, eds.), Vol. 13, p. 1. Springer-Verlag, Berlin and New York. Ussing, H. H. (1971). PhysioE. Veg. 9, 1. Ussing, H. H., and Zerahn, K. (1951). Acta PhysioZ. Scand. 23, 110. Wigglesworth, V. B. (1970). Cited in Smith (1970). Wood, J. L. (1972). Ph.D. Thesis, Cambridge University, Cambridge, England. Wood, J. L., Farrand, P. S., and Harvey, W. R. (1969). J . Exp. Biol. 50,169. Zerahn, K. (1956). Acta Physiol. Scund. 36,300. Zerahn, K. (1969). Acta Physiol. Scand. 77,272. Zerahn, K. (1970). J. Exp. B i d . 53, 641. Zerahn, K. (1971a). Phil. Trans. Roy. SOC.London, Ser. B 2662,315. Zerahn, K. (1971b). Proc. Int. Union Physiol. Sci. 8. This bibliography contains all references known to us of work published on the ion transport system in the silkworm midgut up to March, 1972.

AUTHOR INDEX Numbers in italics refer to the pages on which the complete references are listed.

A Abdel-Latif, A. A., 243, 246, 249, 256, 257, 262, 268, 271 Abood, L. G., 11, 81, 246, 249, 253, 268, 272 Abrams, A,, 118, 176 Abramson, M. B., 102, 175 Acheson, G. H., 35, 77 A d a m , M. J., 172, 174, 175 Adam, R. D., 168,171, 172,174,176,190 Adelman, W. J., Jr., 200, 204, 205, 206, 207, 208, 209, 210, 214, 221, 222, 224, 228, 229, 230, 231, 232, 233, 234, 236 Adrian, R. H., 10, 73, 145, 176 Aftergood, L., 173, 176 Ager, M. E., 10, 12, 15, 49, 82 Agosthi, B., 196 Ahmed, K., 9, 11, 14, 20, 29, 32, 33, 36, 39, 73, 77 Ajmone Marsan, C., 217, 223, 234 Akera, T., 40, 41, 73, 134, 176 Alberici de Canal, M., 243, 244, 246, 247, 248, 250, 262, 268, 270, 271 Albers, R. W., 9, 11, 20, 22, 25, 26, 27, 28, 31, 34, 36, 37, 38, 40, 44, 55, 73, 76, 81, 136, 190,246, 272 Albright, C. D., 12, 13, 36, 40, 80 Albu, E., 162, 189 Albuquerque, E. X., 161, 164,176 Alexander, D. R., 32, 73 Alh-Slater, R. B., 173, 175 Allard, A. A., 36, 81 Allen, J. C . , 15, 27, 38, 39, 40, 41, 52, 58, 65, 73, 81, 132, 133, 134, 176, 191 Almendares, J., 91, 183 Almendares, J. A., 60, 63, 64, 73, 78 Aloisi, M., 85, 138, 151, 155, 157, 159, 161, 163, 176, 188 Alvarado, F., 71, 73

Alving, B. O., 10, 74 Amsterdam, A., 314, 317, 335, 336 Andersen, B., 340,344, 359, 365, 395, 396, 404,409 Anderson, E., 371, 394, 401, 409 Anderson. J. W., 251,270 Anderson, L. D., 15, 78 Anderson, N. G., 243, 248, 269 Anderason-Cedergren, E., 157, 159, 176, 186, 188 Andreoli, T., 355, 366 Angelini, C., 137, 140, 169, 188 Appel, S. H., 71, 75, 243, 254, 258, 259, 263, 268, 269, 270 Arcos, J. C., 135, 176 Argus, M. F., 135, 176 Armitage, J. L., 134, 171, 190 Ash, J. F., 312, 338 Ashley, C. A., 289, 315, 336 Ashley, C. C., 84, 107, 114, 176, 190 Askari, A., 11, 34, 35, 73, 78 Atwood, H. L., 196 Auditore, J. V., 36, 39, 73, 134, 176 Ausiello, D. A., 60, 63, 7 8 Austin, L., 248, 258, 259, 268, 271 Autilio, L. A., 254, 258, 259, 263, 268, 269 Avi-dor, Y., 76 Awad, M. X., 10, 79 Axelsson, J., 167, 176 Azcurra, J. M., 244, 247, 270 Azzi, A., 141, 178 Azzone, G. F., 20,73,80, 159,188

6 Babad, H., 311, 336 Bachelard, H. S., 258, 269 Bachmann, E., 128, 177 Bader, H., 22, 25, 30, 31, 33, 34, 73, 74 Baer, R. D., 172, 173, 194 41 1

412 Bar, U., 136, 176 Bagchi, S. P., 246, 871 Bainton, D. F., 289, 336 Baird, G. D., 123, 176 Bajuss, E., 135, 160, 163, 176, 185 Baker, P. F., 10, 12, 13, 15, 38,41, 73, 131, 132, 176 Bal&zs,R., 248, 255, 869 Baldessarini, R. J., 243, 869 Balfour, D. J . K., 254, 869 Balser, H., 90, 176 Bangham, A. D., 119, 120, 176, 184 Bar, R. S., 6, 73 Barany, K., 137, 165, 176 Barany, M., 137, 160, 165, 176 Barker, S. B., 5, 81 Barlogie, B., 93, 116, 117, 176 Barnett, R. E., 19, 38, 41, 73 Barondes, S. H., 248, 258,869, 870, 878 Barry, P. H., 201,833, 356, 365,409 Baskin, L. S., 21, 73 Baskin, R. J., 88, 101, 107, 123, 124, 139, 146, 152, 153, 157, irs, 179 Baudhuin, P., 155, 179, 304, 336 Bauer, C., 176 Baylor, D. A., 200, 201,211, 213,214,220, 221, 833, 834 Bdolah, A., 311, 336 Beamer, D. W., 6, 73 Beatty, C. H., 136, 176 Beaty, C., 50, 76 Beaufay, H., 304,336 Beckett, S. B., 167, 177 Begin, N., 50, 73 Behnke, O., 147, 153, 190 Beitner, R., 157, 184 BelofF-Chain, A., 255, 869 Bendall. J. R... 84., 90., 176 Benditt, E. P., 314, 338 Bendler, E., 167, 177 Benedetti, E. L., 122, 176 Bennett, A. L., 164, 167, 186,193 Bennett, H. S., 238, 266, 869 Benson, E. S., 126, 142, 146, 188, 198 Ben-Zvi, R., 311, 336 Berg, H. C., 149, 176 Berg, N. B., 287, 303, 337 Bergman, R. A., 171, 179 Berliner, R. W., 341, 366

AUTHOR INDEX

Berridge, M. J., 369, 370, 371, 372, 393, 409, 410 Berry, J., 164, 177 Bertaud, W. S., 86, 142, 143, 149, 150, 176, 189 Bertrand, H . A., 164, 190, 195, 196 Beach, H . R., Jr., 15, 73, 131, 132, 133, 134, 176, 195 Beuren, A,, 130, 187 Beusch, R., 29, 36, 81 Bhatnagar, G. M., 196 Bianchi, C . P., 107, 114, 115, 140, 176,177 Bihler, I. P., 44, 62, 63, 66, 67, 74 Bing, R. J., 130, 135, 177, 187 Bird, J . W . C., 170, 189 Birks, R. I., 142, 147, 150, 151, 153, 154, 155, 177 Bishop, D. W., 171, 180 Bittner, J., 50, 51, 74 Blackstad, T. W., 238, 871 Blanchaer, M. C., 135, 136, 168, 176, 177, 183, 194 Blank, N., 71, 74 Blasie, J. K., 109, 186 Blaszkowski, T. P., 261, 869 Blaurock, A. E., 108, 112, 194 Blaustein, M. P., 12, 15,73, 131, 132, 176, 265,266,869 Blinks, J . R., 154, 177 Blobel, G., 279, 280, 337, 338 Bloch, R., 341, 366 Blok, M. C., 169,177 Blomberg, F., 135, 177 Blond, D. M., 49, 88 Blondin, G. A., 95, 177 Bloom, F. E., 260, 869 Blostein, R., 22, 74 Bocek, C., 151, 177 Bocek, R. M., 136, 176 Bogdanski, D. F., 69, 70, 74, 88, 261, 269 Bohr, G., 16, 79 Bond, G. H., 30, 74 Bonting, S. L., 311, 338 Bos, J. C., 11, 75 Bosman, A. R., 135,185 Bosmann. H. B... 257.. 258.. 869 Botelho, k. Y., 167, 177 Bowen, W. J., 134, 192 Boycott, B. B., 238,870 Bozler, E., 154, 177

413

AUTHOR INDEX

Bradford, H. F., 36,81, 246,253, 254, 255, 256, 263, 269, $YO Bradley, M . O., 312, 338 Brady, A. J., 132, 186 Brand, L., 14, Y6 Brandt, P. W., 142, 145, lY7 Branton, D., 11, Y4, 108, 109, 111, 121, lY9,193 Braunwald, E., 130, 135, lY9, 191 Bresnick, E., 2, 43, 44, 48, 50, 74 Bressler, R., 124, 132, 180 Brierley, G. P., 128, 177 Briggs, A. H., 172, 174, 177 Briggs, F. N., 123, 129, 132, 134, 177, 181, 182, 184, 196 Brightman, M. W., 238.2Yl Brink, A. J., 135, 136, 186, 188 Brinley, F . J., Jr., 215, 218, 220, 223, 233, 234 Briskey, E. J., 146, 152, 153, 182 Britten, J. S., 71, Y 4 Brodie, B. B., 69, 70, 74, 261, 269 Brodkey, J. S., 167, 186 Brodsky, W. A., 22, 23, 29, 36, 24, 74, 81 Brody, I . A., 162, 167, 170, 171, 172, 177 Brody, J., 246, 268 Brody, T. M., 40,41, 73, 134, 176 Brooke, M. H., 160, 180 Broom, A. H., 31, 73 Brown, G. L., 171, l7Y Brown, W. C., 165, 182 Brownlow, E. K., 246, 969 Brust, M., 167, 168, 173, lY7, 190 Bryant, R. E., 130, 177 Buccino, R. A., 130, 191 Bucher, N. L. R., 174, 1YY Bucher, T., 155, 179 Buller, A. J., 137, 160, 165, 167, 177, 187 Bunch, W., 164, 177 Bungenberg de Jong, H. G., 2, 74 Bunney, W. E., Jr., 261, 269 Burdette, W. J., 374, 409 Bure;, J., 215,233 Bureiov4, O., 215, 233 Burg, M., 341, 366 Burns, T. W., 171, 172, 173, lY7, 194 Burwen, S. J., 303, 33Y Bum, J., 160, l Y Y Buse, M. G., 160, 177 Butcher, R. W., 247, 248, 2YO

Butler, J., 167, 169, 1Y8 Butler, K. W., 111, 177 Butow, R. A., 196

C Cahill, M. A., 151, 184 Cahn, R. D., 167, 184 Cain, J., 109, 186 Caldwell, K. A., 161, 170, 192, 196 Caldwell, P. C., 19, 20, 36, 38, 74, 140, 178 Campbell, M. J., 166, 178 Cantanzaro, R., 255,269 Carafoli, E., 127, 128, 138, 140, 141, 178, 179, 186, 189 Carnay, L., 110, 192 Caro, L. G., 274, 281,337,338 Carpenter, D. P., 10, 74 Carpenter, P. C., 169, 181 Carr, C . W., 178 Carson, V., 133, 188, 196 Carsten, M . E., 88, 122, 123, 124, 128, 132, 133, 178, 196 Carvalho, A. P., 87, 109, 153, 178 Caspersson, T., 157, 178 Cass, A., 347, 366 Cassens, R. G., 146, 152, 153, 182 Castle, J. D., 289, 337 Caughey, J. E., 172, 178 Caulfield, J. B., 168, 178 Cereijido, M., 395, 409 Chain, E. B., 255, 269 Challoner, D. R., 128, 178 Chamberlain, C., 104, 193 Chance, B., 96, 107, 109, 110, 111, 127, 141, 178, 187 Chang, F., 256, 257, 268 Chang, K. Y., lY8 Changeux, J . P., 13, 14, 74, 79, 80 Chapman, D., 12, 74, 121,178 Charalampous, F . C., 2, 50, 51, 74 Chargaff, E., 74 Charlton, J. P., 111, 119, 183 Charnock, J. S., 19, 22, 25, 31, 74, 79, 147, 189 Chatfield, P. O., 167, 188 Chen, Y., 14, Y6 Chevallier, J., 196 Chiarandini, D. J., 10, 74 Chidsey, C. A., 124, 125, 129, 130, 135, 178, 183, 192

AUTHOR INDEX

414 Chignell, C. F., 27, 31, 32, 37, 74, 103, 178 Chimoskey, J. E., 132, 133, 178 Chinoy, D. A., 69, 79 Chipperfield, D., 133, 188, 196 Choi, S. J., 132, 186 Christensen, H. N., 45, 46, 47, 56, 57, 76, 76, 79, 80, 81, 82

Civan, M. M., 346, 366 Clark, A. J., 131, 179 Clark, R. M., 374, 459 Claude, A., 285, 314, 315, SS7 Clausen, J., 11, 76 Clausen, T., 66, 76 Cleland, K. W., 127, 141, 178, 191 Clemons, R. D., 375, 384, 387, 409 Close, R. I., 136, 137, 160, 165, 167, 176, 178,196

Cobb, L. A., 136, 191 Cockrell, R. S., 20, 76, 77, 93, 178 Cocks, W. A., 248, 269 Coggeshall, R. E., 210, 233 Cohen, A,, 109,178 Cohen, B. R., 122, 184 Cohen, E., 280,337 Colburn, R. W., 261, 269 Coleman, A. L., 50, 80 Coleman, J., 10, 77 Coleman, P., 221, 235 Coleman, R., 112, 178 Colfer, H. F., 216, 2S3 Connelly, C. M., 13, 15, 73 Connolly, R., 196 Conrad, J. T., 16, 178 Conti, F., 110, 178 Conway, E. J., 9, 76 Cook, J. W., 124, 132, 180 Cooke, I. M., 10, 81 Coons, A. H., 281, 338 Cooper, S., 165, 178 Copenhaver, J. H., 11, 76 Corfford, 0.B., 61, 79 Cori, C. F., 43, 76, 157, 184 Cori, G. T., 43, 75 Cornwell, D. G., 6, 73 Cosmos, E., 167, 169, 178 Costantin, L. L., 128, 141, 152, 153, 1'79 Cotlier, E., 50, 76 Cotman, C. W., 243, 248, 258, 269 Cotran, R. S., 314, 337 Coursaget, J., 160, 190

Covell, J. W., 130, 179 Craig, R. J., 130, 182 Crane, R. K., 44, 49, 59, 60, 71, 73, 74, 75 Criddle, R. S., 102, 105, 184 Crowell, J., 218, 836 Csaky, T. Z., 44, 46, 60, 76 Cuervo, L. A., 205, 233 Cunningham, G. G., 171,180 Curran, P. F., 44, 53, 54, 60, 76, 81 Currie, J., 166, 186 Czok, R., 155,179

D Dahl, D., 255, 269 Dahlstrom, A., 402, 409 Daile, P., 133, 188 Dainty, J., 347, 355, 365 Dale, H. E., 171, 172, 173, 177, 194 Dallner, G., 155, 179 Daly, I., 131, 179 Danielli, J. F., 8, 11, 76 Datta, D. K., 134, I79 Davey, D. F., 151, 154, 155,177 Davies, W. E., 248, 269 Davis, A. H., 60, 63, 78 Davis, D. G., 195 Davis, J . M., 261, 269 Davis, R., 147, 159, 179 Davson, H., 8, 11, 75 Dawson, I). M., 167,179, 183 Dawson, R. M., 249, 270 Dawson, R. M. C., 120,182 Dawson, R. M. G., 29, 30, 76 Day, E. G., 243, 269 Dayani, K., 80 Deamer, D. W., 88, 101, 107, 109, 121, 123, 124, 139, 146, 152, 153, 176, i r 9 Deanin, G. G., 270 De Bernard, L., 120, 179 deDuve, C., 155, 179, 304,336 deHarven, E., 304, 337 De Lorenzo, A. J., 244, 271 DeLuca, H. F., 88, 179 De Meis, L., 88, 89, 179, 196 Demel, R. A., 120, 179 Den, H., 249, 269 Denny-Brown, D., 171, 176 De Robertis, E., 238, 239, 241, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,

415

AUTHOR INDEX

255, 262, 266, 267, 268, 269, 270, 271, 272, 403, 409 Deeai, I. D., 136,161,170,187,192,196 DeSalles, L., 133, 159, 189 Desmedt, J. E., 133, 179 Devine, C. E.. 197 Dhalla, N. S., 130, 133, 134, 179, 187, 199,197 Diamond, I., 254, 259, 265, 270 Diamond, J., M., 36, 38, 76, 161, 179, 370, 371, 390, 409 Dickman, S. R., 289,337 Diggs, C. H., 171, 179 Dkon, M., 13, 22, 76 Dodge, F. A., 263, 270 Domonkos, J., 136, 137, 179, 182 Donley, J. R.. 88, 89, 91, 92, 96, 97, 98, 99, 100, 110, 113, 118, 119, 130, 140, 169, 170,179, 187 Dorow, W., 173, 184 Douglas, W. W., 335, 337 Dowling, J. E., 238, 270 Drachman, D. B., 163, 167, 180 Drahota, Z., 141, 179 Dransfeld, M., 133, 179 Dresel, P. E., 66, 74 Dreyfus, J. C., 160, 190 Droz, B., 258, 270 Dubowitz, V., 165, 179 Dugas, H., 111, 177 Duggan, P. F., 85, 93, 106, 109, 110, 111, 116, 117, 118, 119, 155, 169, 179 Duke, J. A., 111,187 Dunham, E. T., 20, 36, 39, 76 Dunham, P. B., 226, 23s Dunkle, L. M., 171, 179 Durbin, R. P., 341, 366 Dutta, S., 134, 179 Dydynska, M., 10, 76, 154, 179 Dyro, F. M., 221,233

Eccles, R. M., 137, 165, 167, 177, 180 Eddy, A. A., 49, 76 Edelman, I. S., 346, 366 Edelman, P. M., 134, 190 Edstrom, L., 136, 180 Edwards, C., 164, 177 Ehrenreich, J. H., 335, 337 Eichberg, J., 29, 30, 76, 249, 270 Eisenberg, B., 142, 144, 180 Eisenberg, R. S., 142, 144, 145, 149, 151, 180, 181 Eisenman, G., 17, 76, 120, 180 Elbrink, J., 66, 74 Elfvin, L. G., 92, 151, 152, 153, 182 Elison, C., 128, 141, 180 Ellis, R. A., 5, 76 Ellis, S., 128, 180 Ellory, J. C., 93, 186 Ells, H. A., 90, 180 Emmelot, P., 11, 76, 122, 176 Endo, M., 84, 85, 87, 116, 125, 132, 142, 155, 179, 180 Engel, A. G., 92, 136, 142, 171,180,193 Engel, W. K., 160, 161, 171,180,183,184 Engelman, D. M., 6, 76, 108, 112, 122, 192, 194 Engstrom, A., 180 Engstrom, G. W., 128, 179 Entman, M. L., 15, 58, 81, 124, 132, 133, 159, 180 Epstein, F. H., 4, 14, 15, 76, 77 Epstein, S., 170, 194 Epstein, S. E., 133, 159, 180 Ericsson, J. L. E., 315, 337 Ernst, S. A., 5, 76 Ernster, L., 155, 179 Escueta, A. V., 254, 259, 263,268, 270 Essex, H. E., 216, 233 Est, M., 134, 189 Eyzaguirre, C., 171, 174, 180 Ezerman, E. B., 142, 162, 180

E Eavenson, E., 46, 82 Ebashi, F., 87, 122, 179, 188 Ebashi, S., 84, 85, 86, 87, 122, 123, 125, 151, 152, 167, 168, 179, 180, 183, 188, 192 Eberstein, A,, 167, 173, 180, 181 Eccles, J. C., 137, 165, 167, 177, 178, 180

F Fahimi, H. D., 85, 155,180 Fahn, S., 22, 25, 26, 31, 36, 76 Fahrenbach, W. H., 149, 180 Fairhurst, A. S., 128, 141, 162, 163, 180, 183 Falk, G., 145, 180

416 Fanburg, B. L., 122, 123, 124, 125, 127, 1417, 163, 167, 168, 180 Farmer, R. E. L., 363, 366 Farquhar, M. G., 279, 289, 297, 302, 314, 336, 537, 338 Farrand, P. S., ,393,401, 402, 4 l O Fasman, G. D., 43, Y6 Fatt, P., 145, 147, 180, 266, 2YO Faulkner, P., 90, 180 Fawcett, D. W., 85, 123, 126, 128, 157, 159, 181, 210, 233, 279, 304, 333, 335, 337 Fedelesova, M., 134, 192 Fedorko, M. E., 289, 33Y Fedou, R., 333, 338 Feinstein, M. B., 164, 181 Feit, H., 248, 2YO Feldberg, W., 216, 233 Feldman, D., 132, 134, 192 Feng, T. P., 160, 181 Fenichel, I. R., 355, 365 Fenn, W. O., 144,181 Fenster, L. J., 11, 75 Feretos, R., 85, 87, 88, 93, 113, 115, 116, 117, 125, 132, 147, 159, 18Y Fernald, G. P., 60, Y5 Fertziger, A. P., 220, 224, 225, 226, 233 Festoff, B. W., 71, 76, 254, 269, 263, 268 Fex, S., 165, 181 Fiehn, W., 85, 90, 99, 100, 111, 113, 118, 170, 172, 174, 176, 181, 182, 191, 195 Fifkova, E., 216, 235 Filmet, D., 14, Y8 Finean, J. B., 96, 97, 107, 112, 114, 152, lY8, 181 Finegold, M. J., 174, 192 Fink, C. J., 311, 33Y Finke, E. H., 154, 190 Finkel, R. M., 122, 123, 124, 125, 168, 180 Finkelstein, A., 19, Y6, 347, 366 Fischer, H., 45, Y 5 Fisher, E., 160, 181 Fisher, J. R., 14, 81 Fishman, R. A., 254,270 Fishman, S. N., 19, 82 Fitzpatrick, K., 168, 181 Flangas, A. L., 243,ZYO Fleckenstein, A., 131, 181 Fleischer, B., 287, 337

AUTHOR INDEX

Fleischer, S., 96, 98, 99, 100, 18Y, 196, 287, 33Y Flitney, F. W., 196 Foder, B., 385, 4 l O Folk, B. P., 171, 174, 185 Ford, L. E., 116, 132, 181 Formby, B., 11, Y6 Forssmann, W. G., 123, 126, 128, 142, 144, 146, 154, 155, 181 Forster, R.. E., 340, 366 Fox, A. C., 135, 181 Fox, M., 50, Y 5 Frank, G. B., 181 Frank, €I., 341, 365 Frankenhaeuser, B., 200, 201, 202, 203, 204, 210, 211, 217, 228,233 Franki, N., 348, 349, 353, 357, 358, 366

Franzini-Armstrong, C., 86, 128, 136, 137, 141, 142, 149, 150, 151, 152, 153, 154, 160, 161, 170, lY9, 181, 189 Fratantoni, J. C., 11, 73 Frazier, H., 346, 365 Frexinos, J., 333, 338 Freygang, W. H., Jr., 147, 189, 216, 233 Frieden, C., 14, Y 5 Frizzell, R. A,, 53, 55, 75 Froberg, S. O., 173, 181 Frumento, A. S., 10, Y 5 Fuchs, F., 123, 134, 140, 181 Fujino, M., 144, 181 Fujita, M., 10, 11, 16, 28, 33, Y6, Y9 Fyhn, A., 127, 18s

0 Gage, P. W., 144, 145, 149, 151, 180,181 Galsworthy, P. R., 22, 30, 31, YY, 95, 184 Gambetti, P. L., 258, 269 Gamble, R. L., 141, lY9 Gammack, D. B., 246, 269 Gan-Elepano, M., 195 Gardiner, B. 0. C., 372, 410 Gardos, G., 10, Y6 Garrahan, P. J., 10, 15, 17, 19, 20, 34, 36, Y6, 80 Gasser, H. S., 200, 202, 233 Gauthier, G. F., 136, 160, 161, 165, 181, 188

Geison, R. L., 243, BY0

417

AUTHOR INDEX

Gentile, D. E., 22, 36, 81 Gerber, C. J., 22, 77, 171, 177 Geren, B. B., 200, 233 Gergely, J., 92, 109, 122, 123, 127, 132, 133, 134, 137, 138, 139, 146, 147, 152, 153, 162, 165, 167, 168, 169, 172, 174, 178, 180, 181, 183, 187, 190, 191, 192, 1.96 Gerlach, V., 16, 76 Gertler, M. M., 135, 181, 189 Gertz, E. W., 123, 129, 132, 133, 134, 177, 181, 184 Gfeller, E., 246, 270

Gibbs, E. L., 217, 233 Gibbs, F. A,, 217, 233 Gibbs, R., 22, 36, 76 Gilbert, D. I,., 144, 181, 225, 226, 234 Gilbert, J. C., 254, 869 Ginsburg, B. Z., 347,366 Girardier, L., 123, 126, 128, 142, 144, 145, 146, 154, 155, 177, 181 Glaser, G. H., 167, 178, 219, 226, 236 Glaumann, H., 315, 337 Glick, G., 132, 133, 134, 176 Glick, M. C., 101, 194 Glossman, H., 10, 76 Gluckman, M. I., 68, 82 Glynn, I. M., 3, 9, 10, 11, 12, 15, 17, 19, 20, 29, 30, 35, 36, 38, 39, 53, 54, 55, 76, 76, 93, 134, 181, 185 Godinee, M. H., 169, 181 Godri, I., 162, 189 Goldberg, A. L., 160, 181, 265, 270 Goldberg, M. A., 258, 270 Goldman, D. E., 201, 213, 233 Goldring, S., 223, 234 Goldspink, G., 137, 186 Goldstein, D. A., 189 Goldstein, M. A., 147, 153, 189 Goldstone, L., 168, 187 Gollan, F., 130, 181 G&mez,C. J., 249,272 Goodgold, J., 173, 180, 181 Goodman, J., 89, 114, 125,183 Goodner, C. J., 136, 191 Goodwin, F. K., 261, 269 Gordon, M. W., 270 Gorlin, R., 130, 182 Gorman, A. L. F., 10, 79 Gorter, E., 6, 76

Goswami, S., 134, 279 Gotterer, G. S., 45, 80 Gough, W., 196 Graff, G. L. A,, 164,181 Grafstein, B., 215, 216, 223, 233 Graham, R. C., 142, 155, 182 Grahame-Smith, D. G., 261, 870 Granit, R., 216, 223, 233 Grantham, J., 341, 366 Gray, E. G., 243, 244, 252, 270 Gray, G. M., 149, 186 Greaser, M. L., 146, 152, 153, 182 Greef, K., 133, 179 Green, A. L., 12, 13, 76 Green, D. E., 128, 177 Green, J. D., 217, 218, 223, 224, 233,234 Green, R., 167, 182 Greene, L. J., 280, 337 Greenfield, N., 43, 76 Greengard, P., 202, 226, 233 Greenwaalt, J. W., 128, 186 Grendel, F., 6, 76 Griffin, C. C., 14, 76 Griffith, 0. H., 167,182 Grodecker, H., 46, 78 Grossman, M. J., 289,337 Grossman, R. G., 211, 233 Gruener, N., 76 Grundfest, H., 10, 79, 142, 145, 177 Gudbjarnason, S., 135, 177 Gupta, B. L., 372, 393, 409 Guth, L., 136, 161, 164, 165, 167, 182,190 Gutmann, E., 136, 137, 140, 160, 162, 164, 165, 167, 170, 182, 196

H Haber, J. E., 14, 76 Haddad, A., 287, 337 Hadek, R., 142, 186 Haga, T., 260, 270 Hagopian, M., 151, 154, 182 Hagyard, C. J., 165, 186 Hait, G., 57, 78 Hajdu, S.,132, I82 Hhjek, I., 160, 170, 182 Hajjar, J. J., 53, 54, 75 Hall, T., 128, 141, 152, 153, 289 Halpin, R. A., 85, 88, 89,91,92, 95,96, 97, 98, 99, 100, 101, 103, 104, 105, 106,

418 109, 110, 113, 118, 119, 130, 140, 169, 170, 187 Hamburger, H., 131, 184 Hamer, J., 133, 182, 191 Hamilton, I. R., 133, 179 Hampton, T., 211, 233 Hanai, T., 347, 366 Handler, J. S., 363, 366 Hanikova, M., 160, 182 Hansen, J. L., 180 Hansen, O., 37, 76 Hanzlikova, V., 136, 137, 139, 190 Harary, I., 128, 182 Hare, D., 46, 76 Harigaya, S., 38, 81, 87, 124, 125, 134, 135, 137, 138, 167, 168,182,185,191 Harkness, S. H., 344, 366 Harris, D. L., 251, 272 Harris, E. J., 10, 20, 75, 76, 93, 154, 160, 178, 182 Harris, J., 166, 182 Harris, R. A., 15, 27, 40, 73, 78, 134, 176 Harrison, L. I., 57, 76 Hartzog, H. G., 60, 75 Harvey, A. M., 171, 177 Harvey, E. N., 8, 11, 76 Harvey, J. A., 238, 870 Harvey, W. R., 369, 370, 371, 372, 374, 375, 376, 377, 378, 379, 380, 381, 382, 384, 38.5, 386, 387, 389, 391, 392, 393, 394, 395, 396, 397, 398, 401, 402, 403, 404, 407, 409, 410 Harwood, J. R., 255, 269 Haskell, J. A., 372, 375, 376, 377, 378, 379, 381, 384, 385, 386, 387, 409 Hasselbach, W., 84, 85, 86, 87, 88, 90, 92, 93, 99, 100, 108, 111, 113, 115, 116, 118, 122, 124, 125, 141, 147, 151, 152, 153, 170, 172, 174, 176, 181, 182, 186, 191, 196, 196, 197 Hatchett, S. L., 128, 141, 152, 153, 189 Haugaard, E. S., 128, 18% Haugaard, N., 127, 128, 182, 183 Hauser, H., 120, 182 Hauss, W. H., 16, 76 Haydon, D. A., 347, 366 Hays, R. M., 341, 342, 343, 344, 345, 346, 348, 349, 350, 351, 353, 354, 357, 358, 360, 361, 366 Heald, P. J., 29, 76

AUTHOR INDEX

Hechter, O., 154, 188 Heimberg, K. W., 108, 182 Heiner, L., 136, 137, 179, 182 Heine, E., 23, 46, 50, 51, 74, 76, 78 Helman, S., 341,365 Helmreich, E., 157, 184 Hempling, H. G . , 46, 76 Hems, D. A., 22, 25, 30, 76, 80 Hemsworth, B. A., 257, 258, 269 Henderson, A. H., 130, 182 Hendler, R. W., 6, 12, 76 Herpol, J. E., 167, 194 Herrera, M. G., 314, 315, 337 Herskovics, A., 287, 337 Hers, R., 84, 86, 87, 93, 113, 115, 116, 122, 124, 125, 127, 128, 140, 141, 145, 164, 193, 194 Herzog, V., 289, 337 Hess, A., 200, 233 Hess, M. L., 129, 132, 133, 181, 182, 184, 191, 196 Hexum, T . , 15, 20, 21, 76 Higashino, S., 151, 177 Hilberg, C., 22, 29, 81 Hill, D. K., 142, 1.54, 155, 159, 182, 183 Hill, T. L., 14, 76 Himes, R. H., 15, 20, 21, 76 Hirs, C. H. W., 280, 287, 337, 338 Hirsch, J. G . , 289, 314, 337, 338 Hirschhorn, N., 44, 76 Hirschowits, B. I.,35, 80 Hoagland, C. L., 160, 183 Hodges, D. R., 314,337 Hodgkin, A. L., 10, 20, 36, 38, 74, 76, 131, 132, 145, 176, 183, 200, 201, 202, 203, 204, 206, 209, 210, 211, 212, 213, 217, 222, 224, 228, 229, 230, 232, 233, 234, 369,409 Hoekstra, W. G., 146, 152, 153,186 Hoeschen, R. J., 67, 76, 130, 188 Hofer, M., 20, 77 Hoffman, J. F., 9, 12, 23, 35, 36, 38, 76, 77 Hogan, E. L., 167, 183 Hogancamp, C. E., 130, 187 Hogenhuis, L. A. H., 160, 183 Hokin, L. E., 9, 11, 20, 22, 29, 30, 31, 36, 77, 79, 95, 184, 249, 270, 274, 289, 303, 304, 313, 337, 338 Hokin, M. R., 9, 11, 20, 29, 77, 304, 313, 337

419

AUTHOR lNDEX

Holland, D. L., 163, 167, 183 Hollands, M., 66, Y 4 Hollenberg, P. F., 46, 82 Holliday, T. A., 170, 195 Homburger, F., 135, 188 Hoogeveen, J. T., 10, YY Hope, A. B., 201, 233, 356, 365, 409 Horn, R. S., 127, 128, 182, 183 Horowicz, P., 22, Y Y , 145, 183 Horowicz, P. J., 141, 183 Horowitz, S. B., 355, 366 Hoshiko, T., 395, 409 Hosie, R. J. A., 246, 270 Howler, F. E., 10, Y7 House, C. R., 347, 366 Howell, J. N., 141, 144, 152, 153, 162, 165,

Ishikawa, H., 142, 162, 180, 183 Israel, Y., 34, 77 Itg, Y., 130, 183 Iversen, L. L., 244, 260, 269, 2Y1 Izumi, F., 33, 79, 82

J

Jackson, J. H., 216,234 Jacob, F., 13, 79 Jacobson, B. E., 135, 136, 168, 183, 194 Jacquez, J. A., 47, 49, Y7 Jarnieson, J. D., 277, 278, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303,306, 308, 309, 310, 180, 183, 189 311, 315, 317, 319, 320, 321, 322, 323, Howell, S. L., 289, 311, 314, 33Y 324, 325, 326, 327, 328, 329, 330, 334, Hoyle, G., 149, 151, 183 335, 337, 338 Hren, N., 249, 27.2 Jampol, L. M., 4, Y7 Hsu, Q . S., 168, 185, 196 Jardetzky, O., 17, 19, 77 Huang, C., 111, 119, 183 Jarnefelt, J., 12, 14, Y7 Hudson, A. J., 164, 169, 181, 186 Jarrett, L., 154, 190 Hughes, B. P., 140, 169, 185, 188 Jasper, D., 142, 183 Hugli, T. E., 248, 269 Jean, D. H., 25, 30,31, 75 Hull, R. M., 95, 120, i Y Y , 183 Jeanrenaud, B., 66, 74, 78 Hulsmans, H. A. M., 123, 183 Jenden, D. J., 128, 141, 144, 152, 153, 162, Hurley, M. R., 25, 26, Y6 163, 180, 183, 189 Husson, F., 122,186 Jensen, J., 23, 24, 79 Huxley, A. F., 123, 142, 144, 154, 183, 200, Jensen, W. N., 6, 82 201, 203, 204, 206, 209, 210, 212, 213, Jewett, P. H., 126, 183 222, 224, 229, 230, 232, 233, 234 Ji, T. H., 42, 82, 112, 183, 193 Huxley, H. E., 149, 153, 155, 185 Jirmanova, I., 165, 181 Jobsis, F. F., 84, 107, 115, 184 Johns, R. J., 171,179 I Johnson, D., 128, 186 Johnson, E. A., 126, 147, 155, 183, 184, 191 Ikemoto, N., 92, 123, 146, 152, 153, 167, Johnson, H. L., 61, 79 168, 169, 183, 192, 196 Johnson, 5.M., 119, 120, 184 Iles, G. H., 106, 107, 186 Johnstone, B. M., 154, 192, 369, 409 Imai, K., 123, 146, 147, 183 Johnstone, R. M., 48, 77 Imai, S., 122, 188 Inesi, G., 88, 89, 91, 93, 94, 103, 107, 108, Jolly, P. C., 10, 36, 80 111, 114, 122, 123, 125, 183, 184, 186, Jones, A. L., 279, 314, 315, 333, 33Y Jones, D. G., 254, 2YO 195, 196 Jorgensen, P. L., 10, Y7 Inouye, A., 244, 2YO Judah, J. D., 9, 11, 14, 20, 29, 33, 36, 39, Inturrisi, C. E., 26, 34, 35, 77 Y 3 , YY Inui, Y., 46, 82 Juliano, R., 10, 7Y Isaacson, A., 140, 183 Junge-Hulsing, G., 16, 76 Ishida, H., 32, 73

420

AUTHOR INDEX

K Kafatos, F. C., 370, 409 Kahlenberg, A., 30, 77, 95, 184 Kahlke, W., 173, 184 Kahn, J. B., Jr., 35, 77 Kaji, S., 219, 234 Xakefuda, T., 244, 260, 272 Kalant, H., 243, 271 Kalant, N., 157, 184 Kaldor, G., 168, 183, 196 Kallsen, G., 164, 277 Kalrnan, C. F., 61, 79 Kanazawa, T., 22, 28, 30, 33, 77, 79, 93, 184, 196, 197 Kandel, E. R., 215,218,220,223,233,234 Kaplan, D. M., 102, 105, f84 Kaplan, N. O., 167, 184 Kar, N. C., 167, 189 Karahashi, Y., 223, 234 Karnovsky, M. J., 85, 142, 154, 155, 180, 182, 184, 189 Karpati, G., 160, 161, 180,184 Karpatkin, S., 85, 157, 159, 184 Kataoka, K., 244, 245, 270 Katchalsky, A., 16, 77, 347, 351, 366 Kato, M., 243, 246, 270 Kato, T., 160, 161, 185 Katz, A. M., 89, 122, 123, 124, 125, 134, 138, 184,189,190 Katz, B., l E , 161,184, 201, 203, 213, 234, 263, 266, 270 Katsrnan, R., 102, 175 Kaufrnan, B., 249, 269 Kaufrnann, R. L., 131, 284 Kavanau, J. L., 16, 77 Kanabori, I., 136, 191 Kawada, J., 5, 81 Kawarnura, H., 11, 79 Kedem, O., 341, 351, 366 Keen, P., 251, 252, 261,270, ,972 Keenan, M. J., 136, 188 Keith, A., 108, 111, 193 Keller, P. J., 280, 337 Kelly, D. E., 149, 150, 151, 154, 155, 184 Kennedy, B. I,., 134,184 Kennedy, E. P., 259,270 Kepner, G. R., 10, 11, 77 Kerkut, G. A., 10, 78 Kern, D., 333, 337 Kern, H. F., 333, 337

Kernan, R. P., 10, 78, 164, 184 Keynes, R. D., 10, 12, 15, 36, 38, 73, 74, 76, 78, 201, 234, 369, 375, 402, 409 Khandelwal, R. L., 133, 279 Kimelberg, H. K., 119, 184 Kimizuka, H., 120, 284 Kinsolving, C. R., 12, 13, 80 Kipnis, D. M., 50, 79 Kirkland, R. J. A., 243, 244, 272 Kirsten, E., 137, 184 Xirsten, R., 137, f84 Kirton, C. B., 15, 58, 81 Kittel, C., 14, 74 Klachko, 1).M., 172, 173, 194 Kleinzeller, A., 60, 63, 64, 65, 73, 78 Kleitke, B., 131, 194 Klicpera, M., 160, 182 Kline, M. H., 36, 79 Knotkova, A., 64, 78 Koefoed-Johnsen, V., 36, 38, 78, 340, 342, 366, 383, 409 Koelle, G . B., 147, 159, 179 Koketsu, K., 120, 184 Kolinska, J., 64, 78 Komnick, H., 127, 184 Kornnick, U., 127, 184 Kono, T., 61, 70 Korn, E. D., 6, 12, 78 Kornberg, R. I)., 108, 109, 111, 121, 284 Kornguth, S. E., 243, 251, 270 Koshland, I).E., Jr., 9, 14, 42, 76, 78, 94, 184 Kostianovsky, M., 289,337 Kouvelas, E. D., 169, 184 KOvac6, I,., 163, 167, 292 Koval, G. J., 22, 23, 26, 27, 28, 31, 34, 36, 37, 38, 40, 7'3, 76, 81 Kover, A., 163, 167, 292 Koyal, D., 34, 35, 73, 78 Kozak, W., 167, 180 Kraft, G. H., 15, 58, 82 Kramer, M. F., 320, 337 Kramer, R., 22, 29, 31, 36, 82 Kreisberg, R. A., 67, 78 Krivacic, J., 42, 81, 112, 193 Xiiv&nek,J., 215, 233 Krolenko, S. A., 144, 184 Krompecher, S., 136, 184 Kromphardt, H., 46, 78 Kruckenberg, P., 159, 193

421

AUTHOR INDEX

Kruse, H., 385, 410 Kuffler, s. 164,184, 200,210, 220,934 Xuhar, M. J., 244, 246, ,270, 272 Kuhn, E., 172,173,174,175,177,184,191 Kujalova, V., 68, 79 Kulka, R. G., 311, 557 Kume, S., 24, 25, 26, 29, 80 Kunau, R. T., Jr., 5, 78 Kuriyama, K., 260, 263, 266, 270, 272 Kurtz, J. B., 196 Kushmerick, M. J., 120, 184 Kypson, J., 57, 78

w.,

1 Lacy, P.E., 289,311,314,337 Laczko, J., 136, 184 Ladanyi, P., 136, 184 Ladinsky, H., 129, 186 Lagutchev, S. S., 135, 187 Lain, R. F., 129, 132, 133, 181, 184 Lamar, C., Jr., 243, 270 Lambert, E. H., 171, 180 Landau, B. R., 184 Landau, W. M., 216, 23.3 Landgraf, W. C., 91, 107, 108, 111, 114, 18.3, 184 Landin, E., 346, 366 Langer, G. A., 15, 41, 78, 126, 127, 131, 132, 141, 185 Langley, J. N., 160, 161, 186 Langley, P. L.,172, 173, 177, 194 Langmuir, I., 109, 185 Lant, A. F., 20, 78 Lapetina, E. G., 240, 241, 244, 249, 250, 257, 270, 271 272, Lardy, H. A., 128, 186 Lark, P. C., 12, 36, 78 Larsen, F. S., 40, 41, 73, 78, 134, 175 Laseter, A. H., 134, 190 Lassen, U. V., 395, 409 Laszlo, M. B., 136, 184 Latzkovits, I,., 136, 179 Laughter, A. H., 22, 26, 27, 37, 38, 78, 81, 134,191 Lavin, B. E., 22, 25, 29, 80 Leaf, A., 341, 342, 343, 344, 345, 346, 348, 351, 360, 361, 366 Leblond, C. P., 287, 289, 3.38 Lee, C. p., 109, 111, 141, 178

Lee, J. Y., 141, 178 Lee, K. S., 117, 125, 126, 129, 132, 134, 135, 186, 191 Lee, N. H., 128, 182 LeFevre, P. G., 61, 71, 78 Legato, M. J., 126, 127, 186 Lehninger, A. L., 96, 127, 128, 140, 141, 179, 186 Lenard, J., 11, 42, 78 Lengyel, L., 43, 82 Lenman, J. A. R., 161, 167,186 Leo, B., 87, 109, 153, 178 Leslie, R. B., 21, 73 Lesslauer, W., 109, 186 Lester, G., 154, 188 Letarte, J., 66, 78 Letchworth, P. E., 12, 36, 78 Levai, G., 136, 184 Levey, G. S., 133, 159, 180 Levi, R., 244, 271 Levin, R., 128, 183 Levine, Y. K., 108, 112, 186 Levy, H. M., 79 Lew, V. L., 19, 20, 55, 76, 93, 186 Lewis, D. M., 137, 160, 165, 167, 177, 186 J. 15, 78 Lewis, P. R., 201, 2.34 Leyton, R. A., 123, 126, 186 N* s . ~ 349p .366 Lieberman, M., 126, 184 Lilienthal, J. L., Jr., 171, 174, 180 Lin, C. H., 169, 186 Lin, T. M., 289, 337 Lindenmayer, G. E., 14, 15, 26, 27, 36, 37, 38, 39, 40, 41, 42, 43, 73, 78, 79, 134, 135,186,188,191 Lindower, J. O., 134, 179 Ling, C. M., 262, 271 Ling, G. N., 9, 16, 78, 79 Lipicky, R. J., 225, 226, 234 Liprnsnn, F., 84, 86, 94, 151, 152, 157,180, 186, 190 Litt, M., 314, 5.37 Liuzzi, E.y 226* 2.3.3 Lochner, A., 135, 136, 186, 188 Locker, R. H., 165, 185 Loeb, s.9 350, 36'6 Loewenstein, W. R., 151, 177, 186, 194, 368,409 Loewi, O., 131, 186 Lichtensteini

422

AUTHOR INDEX

Long, J. A., 279, 333, 887 Lorand, L., 90, 133, 159, 186,189 Lotapeich, W. D., 71, 79 Lu, D.-L., 160, 181 Luckenbach, W., 186 Lucy, J. A., 11, 79,122, 186 Luduena, M. A., 312, 388 Luff, A. R.,137, 186,196 Luft, J. H., 142, 147, 154, 155, 186 Lukacs, M., 170, 194 Lullman, H., 164,186,186 Lunt, G. G., 240, 249, 250, 257, 271 Lust, W. D., 264, 271 Luthi, U., 20, 76 Luttgau, H. C., 131, 186 Lutz, F., 10, 76 Luzzati, V., 122, 186

M McBride, W., 243, 271 McCallister, L. P., 142, 186 McCollester, D. L., 134, 186 McComas, A. J., 166, 167, 168, 178,186 McConnell, H. M., 107, 108, 109, 111, 121, 184,186 McDonald, R. M., 140, 187 Macey, R. I., 10, 11, 77,363, 866 McFarland, B. G., 107, 108, 111, 186 McFarland, B. H., 89, 93, 94, 103, 114, 188,186,196 McGeer, E. G., 246,271 McGeer, P. L., 246, 871 McIlwain, H., 25, 36, 81,238, 270 McInnes, I., 133, 188,196 Mclntyre, A. R., 164, 167, 186,198 McIsaac, R. J., 161, 164, 176 McLain, L. R., 4, 82 McLean, A. E. M., 14, 29, 33, 77 MacLennan, D. H., 103, 106, 107, 109, 110, 186,198 McMaster, J., 160, 177 McMillan, P. N.,243, 269 McMurray, W. C., 128, 186 McNamara, D. B.,134, 192,197 McNutt, N. S., 126, 181 McShan, W. H., 314, 337 Maddox, C. E. R., 137, 165, 186 Maddrell, 5. H. P., 370,371,372,374, 409 410 Maddy, A. H., 149, 186

Maeda, H., 10, 11.79 Maffly, R. H., 346, 366 Mahler, H. R.,243, 248, 269,27i,272 Maizel, J. V., 103, 191 Makinose, M., 84,85,86,87,88,89,90,91, 92, 93, 94, 111, 113, 115, 116, 117, 124, 176,182,186 Malamed, S.,354, 866 Malhotra, S. K., 218, 236 Manchester, K. L., 160, 169, 182,184 Mandel, P., 249, 271 Mangan, J. L., 244, 271 Manil, J., 12, 15, 38, 41, 78 Mann, W. S., 136, 186 Marchbanks, R. M., 252, 259, 260, 271 Margreth, A., 85, 138, 142, 151, 155, 157, 159, 161, 163, 176,186,188,190 Marinetti, G. V., 149, 186 Maring, E., 89, 93, 94, 114, 188 Marks, B. H., 134, lr9 Marmor, M. F., 10, 79 Marsh, B. B.,84, 90, 186 Marsh, J. B., 101, 194 Marshall, J. K., 71, 78 Marshall, J. M., 281, 837 Marshall, W. H., 215, 220, 223, 283 Martin, A. W., 159, 191 Martinez-Palomo, A., 147, 186 Martonosi, A., 84,85,87,88,89,90,91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113, 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 130, 132, 139, 140, 147, 151, 152, 155, 157, 159, 167, 168, 169, 170, 173, 179,180,181,186, 187,189,191,193 Martt, J. M., 171, 172, 173, 194 Maruyawa, N., 219, 284 Masi, I., 255, 269 Masoro, E. J., 95, 105, 140, 187,194,196, 196 Masotti, L., 112, 193 Massari, S., 20, 78 Matsudaira, H., 10, 11, 79 Matsui, H., 10, 22, 27, 36, 37, 38, 41, 79, 81 Matsumoto, H., 217, 223, 834 Matthews, E. K., 312, 887 MattingIy, P., 134, 1.92 Matsui, H., 134, 187,191

423

AUTHOR INDEX

Mauro, A., 161, 187 Maxwell, D. S., 217, 218, 223, 233 Maynert, E. W., 244, 271 Mazzuchelli, A., 251, 272 Mead, J. F., 196 Medzihradsky, F., 36, 79 Meerson, F. Z., 135,187 Meisler, J., 133, 169, 189 Meissner, G., 96, 98, 99, 100, 187, 196 Mela, L., 96, 141, 178, 187 Meldolesi, J., 302, 335, 337 Mellow, A., 136, 187 Menon, T., 133, 192 Menozzi, P., 154, 188 Meretsky, D., 123, 193 Merrillees, N. C. R., 126, 188 Merritt, C. R., 12, 13, 80 Merritt, H. H., 225, 234 Michaelson, I. A., 243, 244, 271, 272 Michal, G., 130, 187 Mickey, D. D., 243, 269 Middleton, H. W., 19, 79 Migala, A. J., 85, 111, 113,182, 195 Miledi, R., 159, 160, 161, 164, 167, 179, 187, 263, 270 Milhorat, A. T., 168, 170, 187, 194 Miller, F., 289, 304, 337 Mitchell, P., 33, 79 Mitrnaker, B., 289, 338 Mizuno, N., 10, 11, 16, 22, 30, 33, 76, 79 Molino, C. M., 109, 187 Moll, D., 163, 167, 180 Molnar, J., 90, 185 Mommaerts, W. F. H. M., 88, 104, 112, 123, 124, 128, 133, 137, 147, 154, 160, 165, 177, 178, 187, 191, 19s Monod, J., 13, 14, 79 Moore, S., 287, 338 Moore, W. J., 243, 248, 271, 272 Morales, M., 134,192 Morgan, H. E., 61,79 Morgan, I. G., 248, 258, 259, 268, 271 Morgan, T., 341, 366 Morris, A. J., 289, 337 Morrow, A. G., 135,178 Mosinger, B., 68, 79 Mossawy, S. J., 167, 168, 186 Mota, A. M., 109, 178 Mrozek, K., 166, 167, 186 Mueller, P., 121, 187

Muir, J. R., 130, 187 Mullens, L. J., 10, 79 Muller, P., 144, 187 Munsat, T. L., 167, 189 Murata, F., 136, 188 Murer, E., 128, 177 Murphy, A. J., 89, 93, 94, 111, 114, 183, 187 Murphy, D. L., 261, 269 Murray, L., 36, 39, 73, 134, 176 Muscatello, U., 138, 157, 159, 161, 163, 176, 186, 187, 188 Myrianthopoulos, N. C., 172, 178

N Nadler, N. J., 287, 289, 338 Nafstad, P. H . J., 238,271 Nagai, K., 33, 34, 42, 79, 82, 134, 188 Nagai, T., 90,92, 137, 139, 140, 141, 162, 167, 188,198, 194 Nagano, K., 10, 11, 16, 22, 28, 30, 33, 76, 79, 134, 188 Nageotte, J., 200, 234 Nakagawa, Y., 34, 82 Nakahara, T., 120, 184 Nakajima, S., 10, 79 Nakamaru, Y., 196 Nakamura, A., 92, 146, 152, 153, 183 Nakao, M., 10, 11, 16, 22, 28, 30, 33, 76, 79, 134, 188 Nakao, T., 10, 11, 16, 22, 28, 30, 33, 76, 79, 134, 188 Nakazawa, Y., 99, 193 Narahashi, T., 202, 205, 234 Nayler, W. G., 126, 133, 188, 196 Neal, M . J., 244, 271 Nechay, B. R., 69,79 Nedergaard, S., 369, 370, 372, 374, 375, 378, 379, 381, 382, 385, 386, 387, 388, 409,410 Needham, D. M., 136,188 Neets, K. E., 9, 14, 42, 78 Negendank, W., 16, 79 Nelson, D. A., 126, 142, 146, 188 Nelson, I. A., 130, 181 Nelson, P. G., 160, 180 Nemethy, G., 14, 78 Neufeld, A. H., 79 Newman, D. L., 165, 179

424

AUTHOR INDEX

Nicholls, J. G., 161, 164, 188, 200, 201, 210, 211, 213, 214, 220, 221, 233, 234 Niedergerke, R., 126, 131,186, 188 Nixon, C. W., 135, 188 Norby, J. G., 23, 24, 79 Norman, D. P., 154, 188 Norris, F. H., 167, 188 North, S. R., 197 Norton, Pi. T., 102, 176 Novikoff, A. B., 302, 338 Nowinski, W. W., 403, 409 Nussbaum, J. L., 249, 271 Nystrom, B., 136, 180

0 Oakley, J., 223, 224, 234 Obara, S., 10, 79 O’Brien, J. F., 243, 270 O’Connor, M. J., 84, 107, 115, 184 Ohman, R., 249, 271 Oertelis, S. J., 142, 146, 191 Ogata, T., 136, 188 Ogawa, Y., 87, 116, 132, 136, 137, 138, 139, 140, 167, 168, 180, 182, 188 Ohad, I., 314, 317, 335, 336 Ohashi, T., 34, 82 Ohki, S., 109, 121, 188 Ohnishi, T., 11, 79, 87, 188, 196 Ohtsuki, I., 84, 180 Okimoto, K., 167, 168, 192 Okinaka, S., 167, 168, 192 Olson, R. E., 130, 135, 169, 181, 187, 188 Omura, T., 123, 146, 147, 185, 336, 338 O ’ N ~ IR. , M., 130, 177 Onsager, L., 120, 188 Opie, L. H., 135, 188 Opit, L. J., 19, 31, 74, 79, 147, 189 Orcutt, B., 24, 25, 26, 29, 80 Orkand, R. K., 200, 210, 220, 234 Orloff, J., 341, 363, 566, 366 Orteza, M., 130, 187 Oschman, J. L., 369, 370j 371, 372, 409, 41 0 Otsuka, M., 122, 188 Overby, J. L., 135, 176 Overton, E., 6, 79 Owens, K., 137, 140, 169, 188 Oxender, D., 47, 79 Ozawa, H., 287, 337

P Padieu, P., 160, 188 Padykula, H. A., 136, 160, 161, 165, 181, 188 Paganelli, C. V., 341, 366 Pagano, R., 121, 188 Page, E., 10, 79 Page, S., 142, 145, 146, 154, 155,183, 188 Palade, G. E., 123, 189, 274, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 306, 308, 309, 310, 311, 315, 317, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 334, 335, 336, 337, 338

Palatine, I. M., 45, 76 Palmer, R. F., 132, 188 Palti, Y., 200, 204, 205, 206, 207, 208, 209, 210, 214, 221, 222, 224, 228, 229, 230, 231, 232, 233, 234 Panet, R., 103, 188, 196 Papa, S., 109, 111, 178 Papahadjopoulos, D., 109, 119, 120, 121, 184,188 Pappenheimer, J. R., 340, 366 Parfitt, A. G., 261, 270 Park, C. R., 61, 79 Park, R. B., 11, 74 Parker, C. J., Jr., 90, 188 Parrish, J. E., 50, 79 Parrot, B. L., 254, 268 Patlak, C. S., 46, 76 Patriarca, P., 127, 138, 140, 141, 161, 164, 169, 178, 187, 189, 195 Peachey, L. D., 84, 86, 142, 144, 145, 147, 149, 151, 152, 153, 154, 175, 189 Pearee, G. W., 168, 189 Pearson, C. M., 167, 171, 176, 189 Pease, D. C . , 141, 152, 153, 189 Pecci Saavedra, J., 241, 250, 270 Pellegrino, C., 136, 137, 160, 161, 170,189 Pellegrino de Iraldi, A., 238,243,244,245, 246, 252, 255, 267, 269, iro Pennington, R. J., 159, 168, 181, 189 Perlman, P., 135, 177 Perry, S. V., 123, 137, 163, 165, 167, 1’76, 185, 186 Peter, J. B., 88, 125, 134, 167, 168, 172, 174, 175, 189, 194, 196

425

AUTHOR INDEX

Peters, T., 289,315,336, 338 Quigley, J. P., 45,80 Quintana, N.,147, 153,180 Peterson, R. D.,136, 1Y6 Pethica, B. A., 120, 179 Petsche, H.,217, 223,233 Pettis, P., 258,269 R Pfisterer, H., 173, 184 Raabe, G., 131,194 Phillips, C.G.,216,223, 233 Rabinowitz, M.,133, 159,189 Philpott, C.W.,147, 153, 189 Radda, G.K.,109, 111, 178 Pick, U.,196 Radu, A., 162,189 Pierce, L. E.,6,82 Radu, H.,162,189 Pierobon, S.,136, 137, 139,290 Rahamimoff , R. , 263, 270 Pilcher, D.E.M., 372,410 Ran, T.W.,133,192 Plaut, G.W.E., 135, 189 Rall, W.,238, d71 Plummer, T.H., 287,338 Ramahi, H.,246,268 Pocchiari, F.,255, ,869 Ramsay, J. A.,369,410 Poche, R., 131, 189 Ramsey, V. W.,160, 181 Podleski, T.R., 14,74 Ranck, J. B., Jr., 220,224, 225, 233 Podolsky, R. J., 116, 120, 128, 132, 141, Rang, H.P., 10, 15,80,221,234 152,153, 179, 181,184, 189 Ranvier, L.A., 136, 165,189 Polascik, M. A., 122, 123, 124,184 Rao, S. N.,34,35,73,78 Pollack, M.S.,170,189 Rapoport, S. I., 19, 80, 147, 189 Pollen, D.A.,215, 224, 234 Rasmussen, H.,311, 313, 338 Pool, P. E.,135, 178, 191 Rayner, M.D.,136,189 Poort, C.,320, 337 Rayns, D.G.,86, 142, 143, 149, 150,176, Porter, K.R., 86,123, 142,149,181,189 189 Portius, H.J., 14, 35, 79,80, 134,189 Rebeis, J., 168, 172, 174, 190 Posey, V.A.,132,188 Reckard, T.,137, 165, 176 Post, R. L.,9,10,11, 12, 13,20,21,22,24, Redman, C.M., 279,280, 289,338 25, 26, 27, 29, 30, 36, 38, 40, 61,73, Reed, G.E.,135,181 74, 79, 80, 81 Reedy, M.K.,196 Potter, D. O., 200,210, 220, 234 Reese, T.S., 238, 871 Potter, H.A.,31,74, 147,189 Rega, A. F.,34,80 Pouchan, M. I., 34, 80 Reich, E.,111, 193 Pragay, D.,139, 181 Reid, D.,154,188 Pressman, B. C.,20, 76, 77, 93, 178 Reiss, I.,84,86,87,93,113,115, 116,122, Preston, A. J., 363,366 124, 125, 127, 128, 141,193, 194 Prewitt, M.A.,165,189 Rendi, R., 10, 22, 77, 80 Priestland, R. N.,15, 20,45,78,80 Renold, A. E.,66,78,314, 315, 338 Prince, D.A.,221, 234 Repke, D.I., 122, 123, 124, 125, 138, 184, Prince, W.T.,370,409 189 Pruett, J. K.,134,189 Repke, K.,14,35,65,79,80,131,134,189 Pshennikova, M.G.,135, 187 Repke, R. H.,15, 59,81 Pucell, A. G.,88,89,90,91,92,95,96,97, Reuben, J. P., 142, 145,177 98, 99, 100, 101, 104, 105, 109, 110, Revel, J. P., 85, 128, 142, 149, 154, 157, 118, 170,187,189 159, 181, 189 Putnam, T. J., 225, 234 Ribet, A.,333, 338

0 Quastel, J. H., 43,80,255, 271 Quatrale, R. P.,372, 373, 390, 410

Richardson, K.C.,154, 190 Ridderstap, A. S.,311,338 Rideout, D.F.,167, 182 Ridgway, E. B., 84, 107, 114, 176, 190

426

AUTHOR INDEX

Riggs, c., 32, 73 Riggs, T. R., 45, 76, 80 Riklis, E., 43, 80 Ring, K., 46, 78 Ringer, S., 5, 80 Ritchie, J. M., 10, 15, 80, 200, 201, 221, 934

Robbhs. P. W., 157. 190 Roberts,. E., 244, 260, 266, 870, 271, 272 Robertson, J. D., 8, 11,80,200, 202, 234 Robinson, C. V., 346, 366 Robinson, J. D., 14, 15, 21, 34, 35, 72, 80, 264, 271 Robinson, J. W. L., 65, 80 Robu, R., 162, 189 Roddy, P. M., 22, 36, 76 Rodnight, R., 22, 25, 29, 30, 32, 73, '76, 80 Rodriguez de Lor- Arnair, G., 238, 239, 241, 243, 244, 245, 246, 247, 248, 249, 250,252,255, 260,262,267, 968,269, 9'70,2'71 Roe, R. D., 167, 168, 190 Rojas, E., 120, 190 Romanul, F. C . A,, 136, 165, 167, 179,183, 190 Roodyn, D. B., 155, 190 Rose, J. D., 35, 80 Rosenberg, I. H., 44,50, 76, 80 Rosenberg, L., 50, 76, 80 Rosenberg, T., 44, 61, 82 Rosenbluth, J.., 85, 86, 157, 158, 159, 190 Rosenheck, K., 42, 81, 112, 157, 190 Rosenthal, A. S., 21, 22, 24, 25, 26, 36, 38, 74, 80 Rosenthal, S. L., 134, 190 Ross, J., Jr., 130, 179 Ross, R., 314, 338 Rossi, C. S., 127, 128, 140, 141, 178, 179, 186 Rossi, E., 20, 80 Rossi, F., 164, 169, 196 Rostgaard, J., 147, 153, 190 Roth, S. I., 163, 167, 180 Rothman, S. S., 281, 303, 337, 338 Rothstein, A., 10, 77 Rotunno, C. A., 395, 409 Rowell, L. B., 140, 187 Rubin, B. B., 89, 122, 124, 184, 190 Rubin, M., 14, 80 Rubio, R., 144, 190

Rudermann, N. B., 314, 315, 337 Rudin, D. O., 121, 187 Ruscak, M., 49, 82 Ryall, R. W., 244, 271 Ryan, K. J., 243, 271

5 Sabatini, D. D., 279, 280, 337, 338 Sachs, G., 35, 80 Sachs, H., 304, 338 Sachs, J. R., 15, 80 Saez, F. A., 403, 409 Saito, M., 22, 28, 77 Sakai, F., 341, 366 Sakamoto, T., 11, 81, 243, 246, 270 Salafsky, B., 136, 165, 166, 186, 189, 190 Salganicoff, L., 238, 243, 244, 245, 246, 252, 255,267, 269, 870, 271 Salnikow, J., 287, 338 Saltin, A., 25, 81 Samaha, F. J., 134, 136, 137, 138, 162, 167, 168, 172, 174, 182, 190 Samson, F. F., Jr., 15, 20, 21, '76 Sandblom, J. P., 120, 180 Sandlin, R., 110, 192 Sandow, A., 84, 140, 142, 144, 162, 164, 167, 168, 180, 182, 183, 190 Sanslone, W. R., 164, 190, 196 Sardesai, V. M., 135, 176 Sarkar, S., 165, 192 Sastry, P. S., 22, 30, 31, 77 Sata, M., 10, 82 Sato, R., 123, 146, 147, 183 Sawa, M., 219, 234 Sawant, P. L., 136, 187 Sawh, P. C . , 66, 67, 7' .4 Scarpa, A., 196 Schade, J. P., 219, 236 Schafer, J. A., 47, 49, 77, 80, 355, 366 Schapira, F., 160,190 Schapira, G., 160,190 Schatzmann, H. J., 11, 12, 36, 80, 81, 126, 190 Schein, M., 135, 192 Schellnack, K., 169, 190 Scherphof, G. L., 169, 17'7 Schiaffino, S., 136, 137, 139, 142, 157, 161, 190 Schild, R. F., 142, 144, 154, 189

AUTHOR INDEX

Schmitt, F. O., 200, 233 Schneider, A. S., 42, 81,112, 157, 190 Schneider, F. H.,338 Schneider, H.,111, 177 Schneider, M. J. T., 42, 81,112, 157,190 Scholefield, P., 50, 73 Scholefield, P. G.,36, 39, 48, 73,79 Scholz, A. F.,139, 181 Schoner, W., 22, 29, 31, 36, 81 Schorn, A., 133, 179 Schramm, M., 274, 304, 311, 313, 314, 317, 335, 356, 338 Schrodt, G. R., 142, 149, 193 Schroeder, J. M.,168, 172, 174, 190 Schulman, J. H., 120, 191 Schultz, S., 60, 81 Schultz, S. G., 46, 50, 53, 55, 76,81,388, 410 Schuke, W., 132, 134, 147, 190,194 Schutta, H. S., 171, 190 Schwarts, A., 2, 14, 15, 22, 26, 27, 36, 37, 38, 39, 40, 41, 42, 43, 44, 48, 50, 52, 58, 68, 73,74,78,79,81,82,87, 124, 125, 132, 133, 134, 135, 137, 138, 176, 182,186, 187,188, 190,191, 196, 196 Schwartz, I. L., 134, 190 Schwartz, T. L., 214, 834 Scott, G., 243, 251, 270 Seelig, J., 196 Seeman, P., 106, 107, 186 segal, S., 50,76 Seidel, J. C.,137, 139, 165, 181,191,192 Seiler, D.,172, 173, 174,191,197 Selby, C. C.,126, 181 Sehger, Z., 103, 109, 178, 188,196 Semente, G.,134,186 Seminario, L. M., 249,972 Sen, A. K., 9, 10, 11, 12, 20, 21, 22, 24, 25, 26, 27, 29, 30, 33, 34, 36, 37, 38, 40, 73,80,81,82 Senft, J. P., 10, 81 Seraydarian, K., 124, 137, 147, 154, 160, 165, 177,187,191 Serena, S. D., 15, 78,131, 132, 141, 186 Settembrini, P., 190 Seubert, W.,22, 31, 81 Shafiq, S. A., 168,187 Shah, D. O., 120, 191 Shamoo, A. E., 22, 23, 24, 29, 36, 74,81, 95, 191

427 Shanes, A. M., 201,234 Shankley, K. H., 154,198 Shapiro, A. L., 103,191 Shaskan, E. G.,246, 270 Shaw, F. H.,154, 192 Shaw, M. L., 107,182 Shaw, T. I., 12, 15, 20, 36, 38, 73,74 Sheff, M. F., 155, 194 Shelburne, J. W.,132, 134, 19% Shepherd, G. M.,238, $71 Sherman, J. H., 47, 77 Sherwood, S. L.,216, 233 Shibko, S., 161, 170, 192,196 Shimamoto, T.,217, 233 Shmakawa, Y., 244, 670 Shinebourne, E. A., 133, 18.9, 191, 194 Shirachi, D.Y.,36, 81 Shon, R., 15, 59, 81 Shonfeld, W., 15, 59, 81 Short, F. A., 136, 191 Shyr, C. I., 111, 119, 183 Sica, R. E. P., 166, 178,186 Sidbury, J. B., Jr., 171, 177 Siegel, F. L., 243, 870 Siegel, G. J., 26, 27, 28, 37, 38, 40, 73,81, 246, 878 Siekevitz, P., 274, 279, 335, 336, 338 Simon, S. E.,154, 198 S i p s o n , F. O.,86,142,143, 146,149,150, 176, 189, 191 Sims, E. A. H., 184 Singer, B., 354, 366 Singer, I., 115, 198 Singer, S. J., 11, 42, 78 Sjodm, R. A., 141, 191 Skelton, C. L.,133, 180 Skou, J . C.,9, 10, 11, 12, 13, 21, 22, 25, 28, 29, 36, Y7,81,131, 134, 191,246, 262, 972, 385, 410 Slater, C. R., 159, 160, 161, 187 Slater, E. C.,127, 128, 136, 141, 178,188, 191 Slayman, C. W., 10, 29,30,73, 76 Smith, A. D.,304, 338 Smith, D. S., 141, 142, 191,372, 410 Smith, I. C. P., 111, 177 Smith, J. P., 249, 268 Smith, M. D.,287, 337 Smith, R. E.,302,338 Smulders, A. P., 356, 366

428 Snyders, S. H., 244,246, 270, 272 Sobel, B.E., 135,191 Soberman, R.,357,358,366 Sokolove, P. G.,10, 81 Sola, 0.M.,159,191 Solomon, A. K.,341,366,366,398,410 Solomonson, L.P.,10,77 Sorners, J. E.,171, 172, 173,194 Somlyo, A. P.,197 Sornlyo, A. V., 197 Sommer, J. R.,126, 147, 153, 155, 183, 191 Sonnenblick, E. H.,123, 126, 130, 132, 134, 135, 179,182, 186,191,192 Sordahl, L. A., 135, 186 Soto, E.F.,241,244,249,250,270,871 Spach, M.S.,147,191 Spann, J. F.,Jr., 130, 135,191 Spencer, W.A., 218,234 Sperelakis, N.,144, 190 Spiegler, P.,104,193 Spiro, D.,126,151,154,182,186,194 Spooner, B. S.,312, 338 Squires, R.F.,14, 15, 21,81 Sreter, F. A., 87, 88, 92, 123, 137, 138, 139,140, 146,152, 153, 162, 163,165, 167, 168, 169,181,185,191,198,196 Stahl, W.L.,25,81,263,878 Staley, N. A., 126, 192 Stam, A. C.,Jr., 126, 132, 134, 191, 192 Standish, M. M.,119, 176 Stefani, E.,10,74 Stein, O.,159,192 Stein, W.,175, 184, 192 Stein, W.H.,287, 338 Stein, Y.,159, 198 Steinbach, H.B.,154,192 Steinberg, D.,128,178 Steinhardt, R.A.,12,15, 73,131, 132,176 Sternlicht, E.,311,337 Stewart, D.M.,160, 192 Stillman, I. M.,225, 226, 234 Stoeckenius, W.,122,192 Stone, A. J., 19,81 Stoner, C. D.,103, 192 Storm, S. R., 10, 79 Stowring, L.,111, 134,187,192 Straub, R.W.,183,200,202,226,233, 234 Strickland, K. P., 11, 81, 164, 169, 181, 186

AUTHOR INDEX

Struthers, J. J., 132,186 Stryer, L.,107,110,111, 193,194 Stuckey, J. H.,129, 186 Sugita, H.,87, 137, 138, 139, 167, 168, 188,192 Sugita, M.,363,366 Suko,J., 124, 125,129, 130,183,199,197 Sulakhe, P.V.,133,179,192,197 Sutherland, E.W.,133,198,247,248,870 Suzuki, K.,144,181 Svennerholm, L.,101,192 Swanson, P.D., 25, 36,81,263,972 Sweeny, J. R.,14,81 Swift, M.R.,174, 192 Sypert, G.W., 223, 224, 234 Syro\S, I., 160, 170, 182 Szabolcs, M.,163,167,192 Szekeres, L.,135, 192

T Takagi, A., 197 Takahashi, H.,92,188 Takahashi, K.,10,79 Takauji, M.,90, 92, 137, 139, 140, 141, 167, 188,192, 194 Takayama, K.,103,192 Tanaka, M.,116,132,180 Tanaka, R.,11, 81, 246,253,2'78 Taniguchi, M.,140,192 Tappel, A. L.,123,136,161, 170,187,192, 196 Tasaki, I., 110,178 Tashima, Y.,10,16,22,28,30,33,76,79, 134,188 Tasker, P., 154, 192 Taylor, C.B.,12,13, 44,76,81 TBylor, D.A., 258, 269 Taylor, E.,312,338 Taylor, J. W.,141, 183 Taylor, K. M.,244, 246, 970 Taylor, R. E.,144, 183 Taylor, R. E.,Jr., 5, 81 Tedeschi, H., 251,978 Templeton, J. E.,372,410 Teorell, T., 347,366 Terribile, V., 164, 169, 196 Terris, J., 47, 77 Thale, M., 44, 76 Thau, G.,341, 366

429

AUTHOR INDEX

The, R., 88, 186 Themann, H., 16, 76 Thesleff, S., 161, 164, 167, 176, 176, 192 Thier, S., 50, 76 Thiery, J., 14, 74 Thomas, A. J., 255, 269 Thomas, E. L., 56,81,243, 271 Thomas, R. C., 10, 78, 81 Thomas, W. A., 130, 177 Thompson, J. E., 112,178 Thompson, J. W., 172, 174, 176 Thompson, M. M., 137, 139, 181, 191 Thompson, T . E., 6,82, 111, 119, 121,183, 188 Thorell, B., 157, 178 Tice, L. W., 92, 136, 142, 153, 180, 195 Ting, B. T., 133, 179 Tissari, A. H., 70, 82, 261, 269 Titus, E., 22, 26, 27, 31, 32, 34, 35, 36, 37, 74, 76, 77, 103, 178 Tobias, J. M., 120,190 Tobin, T., 24, 25, 26, 27, 29, 37, 40,80,81, 82 Tonomura, Y., 22, 28, 77, 89, 90, 91, 93, 94, 114,184,194,196,197 Tormey, J. McD., 356, 366, 371, 409 Tosteson, D. C., 12, 77 Tourtellotte, M. E., 108, 111, 193 Tower, D. B., 216, 217, 219, 224, 254 Trachtenberg, M. C., 215, 224 Traut, R. R., 157, 190 Treherne, J. E., 372, 410 Trevor, A. J., 36, 81 Triester, S., 68, 82, 133, 194 Trumpower, B. L., 169, 181 Tung, Y., 14, 74 Turbeck, B. O., 385, 410 Tuttle, L. C., 94, 185 Tyor, M. P., 320, 338 Tzagoloff, A., 103, 192

U Uchida, K., 123, 195 Uda, Y., 99, 193 Uejo, H., 120, 184 Uhr, J. W., 287, 289, 358 Ulbrecht, G., 159, 193 Ulbrecht, M., 90, 195 Upshaw, J. E., 122, 123, 124, 184

Urry, D. W., 42, 82, 96, 112, 183, 195 Ussing, H. H., 340, 342, 344, 359, 565, 566, 369, 375, 379, 383, 395, 398, 409,410

V Breemen, V. L., 168, 195 Deenen, L. L. M., 120, 179 den Bergh, S. G., 135, 193 der Kloot, W. G., 117, 123, 126, 132, 144, 159, 193 Vanderkooi, J., 87, 107, 108, 109, 110, 111, 116, 119, 120, 193 Van Der Meulen, J. P., 165, 190 Van Harreveld, A., 216, 218, 219, 253, 256 Van Heyningen, H. E., 281, 338 Van Wazer, J. R., 32, 82 Varon, S., 260, 272 Vegh, K., 104, 193 Verhaag, D. A., 175, 189 Vidaver, G. A., 46,89 Villani, G., 161, 189 Villegas, J., 208, 235 Vincenzi, F. F., 126, 190 Vinuela, E., 103, 191 Vogel, J. H. K., 124, 125, 129, 130, 192 Vogt, M., 243, 269 Volkenstein, M. V., 14, 19, 81 Volpe, A., 137, 165, 176 von der Decken A., 159, 188 von Euler, C., 217, 223, 234 von Hungen, K., 248, I72

Van Van Van Van

W Waggoner, A. S., 107, 109, 110, 111, 178, 18.2, 193 Waggoner, D. M., 141, 185 Waku, K., 99,193 Wald, F., 250, 251, 270, 272 Walker, J. L., Jr., 120, 180 Walker, L. N., 80 Walker, S. M., 142, 149, 193 Wallach, D. F. H., 11, 42, 8.2 Walster, G., 140, 178 Wang, J. H., 346, 566, Ward, A. A., Jr., 223, 224, 954 Ward, D. C., 111, 195 Ware, F., Jr., 164, 193

430 Warren, C. M., 41, 82 Warren, L., 101, 193, 194 Watanabe, A., 110, 192 Watanabe, S., 89, 114, 122, 123, 125,183 Watkins, J. C., 119, 120, 176, 188 Watson, P. K., 165,181 Wattiaux, R., 155, 179 Webb, E. C., 13,22,76 Weber, A., 84, 86, 87, 88, 90,93, 113, 114, 115, 116, 122, 124, 125, 127, 128, 133, 140, 141, 145, 164, 193, 194 Webster, H. L., 123, 194 Webster, P. D., 274, 320, 338 Weinbach, E. C., 135,178 Weinstein, D. B., 101,194 Weinstein, H., 244, 260, 263, 970,272 Weinstock, I. M., 170,194 Weissmann, G., 119, 176 Welt, L. G., 15, 80 Weeaels, N. K., 312, 338 Westerman, N. P., 6,82 Westley, J., 22, 82 Wheeler, K. P., 11, 13, 46, 82 White, R. J., 133, 191, 194 White, T. D., 251, 252, 261, 270, 272 Whiteside, L., 211, 233 Whittaker, V. P., 243, 244, 249, 252, 970, m1, m2 Whittam, R., 10, 11, 12, 13, 15, 20, 36, 38, 45, 49, 76, 77, 78, 80, 88 Widdas, W. F., 61, 8%' Wiegant, H., 249, 972 Wieme, R. J., 167, 194 Wiener, J., 151, 194 Wiessmann, W. P., 266, 269 Wigglesworth, V. B., 372, 410 Wilbrandt, W., 43, 44,61, 82 Wilkes, A. B., 25,30, 31,73 Wilkie, D. R.,154, 155,179, 183 Wilkins, M. H. F., 108, 112, 186, 194 Williams, J. T., 123, 194 Williamson, J. R., 67, 78 Willis, J. S., 41, 49, 73, 82 Willmer, E. N., 118, 194 Wilson, P., 166, 182 Winegrad, S., 107, 114, 126, 128, 132, 146, 152, 161, 194, 196, 197 Wmer, N., 171, 172, 173,194 Winicur, S., 122, 193 Winkler, H., 338

AUTHOR INDEX

Wirth, H., 131, 184 Wirth, W., 16, 76 Wirtr, K. W. A., 169, 177, 194 Wofsey, A. R., 246, ,972 Wolcott, L., 171, 172, 173, 194 Wolf, H. U., 126, 194 Wolff, H. H., 142,194 Wollenberger, A., 132, 134, 142, 147, 190, 194 Wong, P. T. S., 106,109,110,186 Wood, J. L., 374, 375, 378, 379, 386, 389, 392, 397, 401, 402, 403, 404, 410 Woodbury, D. M., 226, 836 Woodbury, J. W., 132, 194 Woods, E. F., 134, 189 Worsfold, M., 88, 125, 134, 159, 167, 168, 172, 174, 189, 194 Wren, J. T., 312, 338 Wright, E. B., 221,236 Wright, E. M., 356, 366, 389, 409 Wright, J. H., Jr., 61, 79 Wrogemann, K., 135, 136, 168, 177, 183, 194 Wu, C., 135, 177 Wyman, J., 14, 79

Y Yamada, K. M., 312, 338 Yamada, S., 93, 95, 184, 194, 196, 197 Yamaguchi, M., 256, 257, 268 Yamaguchi, T., 144,181,256,257, 268 Yamaji, K., 167, 168, 190 Yamamoto, T., 89, 90,91, 93, 94, 95, 114, 137, 139, 140, 141, 167, 192, 194, 196, 197 Yamanouchi, I., 87, 179 Yamaaaki, T., 202, 234 Yamauchi, A., 120, 184 Yasuda, K., 281, 338 Yates, J. C.,133, 192 Yguerabide, J., 107, 111, 194 Yip, C. C., 106, 107, 186 Yoda, A., 22, 30, 31, 77, 94, 194, Yonemura, K., 10, 8.8 Yoshida, A., 34, 8.8 Yoshida, H., 33, 79 Young, B. A., 289,338 Young, D. A., 311, 314, 337 Young, R. W., 287, 303, 337

431

AUTHOR INDEX

Yu, B. P., 95, 105, 164, 187, 190, 194, 196, 196 Yu, D. H., 68,82, 132, 133, 134,186,194

Z Zacks, S. I., 155, 19.4 Zadunaisky, J. A., 142, 194 Zagury, D., 287, 289,338 Zahler, P. H., 11, 42, 82 Zak, R., 160,195 Zaletayeva, T. A., 135, 187 Zalkin, H., 123, 161, 170, 192, 196 Zalusky, R., 46, 50, 81, 388, 410 Zatti, M., 164, 169, 196

Zatz, M., 248, 27.2 Zaugg, W. S., 4, 82 Zebe, E., 141, 196 ZelenA, J., 160, 161, 167, 182, 187, 196 Zerahn, K., 369, 376, 377, 378, 379, 384, 385,386,387, 389,390, 391,392, 395, 396, 397, 398, 401, 404, 407,409, 410 Zhukow, Y. K., 137, 196 Zieher, L. M., 238, 244, 245,246,260, 267, 270, 271, 272 Zierler, K. L., 171, 174, 180 Zilversmit, D. B., 169,194 Zucker-Franklin, D., 314, 338 Zuckermann, E. C., 219, 226, 236

SUBJECT INDEX A Alkali ions, transport by silkworm midgut, 367-410 Amines, biogenic in nerve endings, 245 role in membrane transport,, 69-70 Amino acids absorption by silkworm midgut, 388 metabolism in isolated nerve endings, 255-256 uptake related to protein synthesis, 258-259 transport of, role of Na+, K+-ATPase system in, 43-59 p-Aminohippuric acid, transport of, ouabain-sensitive, 69 Ammonium ions, transport by silkworm midgut, 387 8-Anilino-1-naphthalene sulfonate (ANS), in structural studies on sarcoplasmic reticulum membrane, 108-111 Anions, accumulation by isolat,ed nerve endings, 266 ATPase, sodium, potassium and in membrane transport system, 1-82, 134135 Axons giant, of squid, potassium ion changes in, 229-231 potassium ion accumulation related to activity of, 201-203 model for, 226-229

B Bladder, water diffusion studies on cell layers of, 346-353 Brain potassium accumulation related to 432

activity of, 215-220 significance of, 224-226

C Calcium accumulation by isolated nerve endings, 266 effects on ion transport by silkworm midgut, 392 regulation in sarcoplasm, in cardiac muscle, 122-136 release from sarcoplasmic reticulum, and sodium activation, 14-15 uptake by microsomes in cardiac m u 5 cle, 138 Calcium transport conformational probes of, 107-112 ANS, 108-111 circular dichroism, 112 EPR, 111 x-ray diffraction, 112 energy sources for, 8&90 mechanism of, 86-112 phospholipids in, 96-102 phosphoprotein intermediate in, 90-96 Cardiac glycosides antilipolytic effects of, 68-69 effect on Ca uptake of cardiac sarcoplasmic reticulum, 131-134 effects on membrane-linked functions, 68-69 Cell membrane, role in Na+, K+, -ATPase transport system, 5-8 Cerebral cortex, spreading depression of, related to potassium accumulation, 215-216 Cesium, transport by silkworm midgut, 386 Choline, uptake by isolated nerve endings, 259-260

433

SUBJECT INDEX

Circular dichroism, in studies of sarcoplasmic reticulum, 112

D 2,4-D, myotonia induced by, sarcoplasmic reticulum in, 174-175 Denervation of muscle, sarcoplasmic reticulum in, 159-166 Diasocholesterol, myotonia induced by, saroplasmic reticulum in, 172-174

E Electron paramagnetic resonance (EPR), in studies of sarcoplasmic reticulum, 111 Energy sources for calcium transport, 88-90 transduction of, by sodium pump, mechanism of, 20-23 Enzymes, in nerve endings, 244-246 Epileptiform seizures, potassium accumulation in neural membranes and, 216220 Epithelia, vasopressin-sensitive, see Vasopressin-sensitive epithelia Excitation-contraction coupling, in heart muscle, role of mitochondria in, 126-128 Exercise, muscle contracture in, sarcoplasmic reticulum role, 170

GABA, uptake by isolated nerve endings, 260 Glycolysis, in nerve endings, 252-253 Goblet cell model, for K transport by sikworm midgut, 401-402

Heart failure of mitochondria in, 135-136 sarcoplasmic reticulum role in, 129131

sarcoplasmic reticulum function in, 122-1 26 Hexose monophosphate pathway, in isolated nerve endings, 254 Hydrogen ions, transport by silkworm midgut, 388

I Immunology, of isolated nerve endings, 250-251 Insulin-sensitive systems, sugar transport in, 66-67 Intestine, sugar transport in tissues of, 62-66

K Kidney, sugar transport in tissues of, 62-67

1 Lipids, in nerve endings, 249-250 Lithium, transport by silkworm midgut,

387

M McArdle's disease, sarcoplasmic reticulum in, 171 Membrane transport, system, Na+, K+ATPase in, 1-82 Membranes, of nerves, see Neural membranes Mitochondria in heart failure, 135-136 role in regulation of excitation-contraction coupling in heart muscle, 126-128 Muscle diseases of, sarcoplasmic reticulum role in, 159-175 of heart, sarcoplasmic Cae+ regulation in, 122-136 red skeletal type, sarcoplasmic reticulum in, 136-141 transverse tabular system of, 141-151 Muscular dystrophy, sarcoplasmic reticulum in, 166-170

SUBJECT INDEX

434 Myotonia, sarcoplasmic reticulum in, 171174

N Nerve endings active substances in, 244-246 amino acid metabolism in, 255-256 uptake related to protein synthesis, 258-259 chemical composition of, 244-250 choline uptake by, 259-260 enzymes in, 244-246 phosphohydrolases, 246-248 GABA uptake by, 260 high-energy compound synthesis, 252254 in glycolysis, 252-253 in H M P pathway, 254 in respiration, 253-254 immunological properties of, 250-251 ion permeability of, 262-266 to anions, 266 to calcium, 263-266 to potassium, 263 to sodium, 262 isolation of, 239-244 lipids in, 249-250 norepinephrine uptake by, 261 osmotic properties of, 251-252 properties of, 237-272 proteins and ribonucleic acid in, 248 membranes of, isolation, 243-244 tryptophan uptake by, 260-261 uptake mechanisms related to transmitter function of, 259-261 Neural membranes ionic flow across, periaxonal and perineyronal spaces in, 199-235 reconstruction of action potential of, 231-232 Neurons, potassium accumulation related to activity of, 210-215 Norepinephrine, uptake by isolated nerve endings, 261

0 Osmotic properties, of isolated nerve endings, 251-252

P Pancreatic exocrine cells intracellular membrane interrelationships in, 333-336 physiologrical modulation of secretory process in, 315-333 protein transport and discharge by, 273-338 resting type, secretory process in, 274315 organizational features, 274-279 protein synthesis and metabolic energy in, 290-315 steps in, 279-289 zymogen granule formation and discharge by, 297-315,319-333 Paralysis, hypokalemic periodic type, sarcoplasmic reticulum in, 170-171 Perioaxonal space, ion accumulation in, model for, 226-229 Phlorizin, effects on membranes, 70-72 Phosphohydrolases, cation-stimulated, in nerve endings, 246-248 Phosphoprotein, intermediate in calcium transport, 90-96 Potassium accumulation external to neural membrane, 201-220 axonal activity and, 201-203 brain activity and, 215-220 significance of, 223-226 membrane currents during voltage clamping, 203-210 neuronal activity and, 210-215 significance related to axon and neuron behavior, 220-223 accumulation by isolated nerve endings, 263 active transport of across silkworm midgut, 367-410 coupling to metabolism, 384-385 midgut potential, 378 short-circuit potential, 378-379 transepithelial pumps for, 369-372 structure and function in, 370-372 Potassium phosphatase, in sodium pump mechanism, 33-35 Protein(s) in isolated nerve endings, 248

435

SUBJECT INDEX

transport and discharge of, from pancreatic exocrine cells, 273-338

R Respiration, in nerve endings, 253-254 Ribonucleic acid, in nerve endings, 248 Rubidium, transport by silkworm midgut, 386

S Sarcoplasmic reticulum calcium release from, 114-119 carrier-mediated efflux, 116-1 17 by passive permeability, 117-119 in calcium transport, 86-112 function of, biochemical and clinical aspects of, 83-197 regulation of, 112-122 model membranes of, calcium permeability of, 119-122 in muscle disease, 159-175 protein composition of membrane of, 102-107 in red skeletal muscles, 136-141 regulation of Ca*+ in, in cardiac muscle, 122-136 cardiac glycoside role, 131-134 mitochondria1 role, 126-128 in regulation of Ca uptake, 112-114 role in heart failure, 129-131 tubules of, content of, 151-159 Silkworm midgut alkali ion transport by, 367410 competition among, 388-392 kinetic studies of, 395396,398403 lag time in, 396-397 route of, 393408 amino acid absorption by, 388 ammonium ion transport by, 387 calcium effects on ion competition in, 392 cesium transport by, 386 hydrogen ion transport by, 388 lithium transport by, 387 microelectrode potential profiles of, 393-395 potassium active transport across, 367410 methods for, 375-378

pool location, 406408 role of in vivo, 372-374 rubidium transport by, 386 sodium transport by, 386 uptake of, 390-391 structure of, 393 Sodium accumulation by isolated nerve endings, 263 transport by silkworm midgut, 386-387 Sodium, potassium-ATPase membrane transport system, 1-82 cell membrane and, 5-8 phlorizin effects on, 70-72 physiological aspects of enzyme system in, 43-67 in amino acid transport, 43-59 in sugar transport, 59-62 complications of, 62-67 role in biogenic amine transport, 69-70 sodium pump mechanism in, 9 4 3 Sodium pump cardiac glucoside inhibition in, 3 5 4 3 energy transduction by, 20-33 mechanism of 9 4 3 monovalent cation activation and transport, 9-20 potassium phosphatase and, 33-35 sulfhydryl groups and enzyme activity in, 43 Squid giant axon, potassium changes upon voltage clamping of, 229-231 Sugar transport in insulin-sensitive systems, 66-67 in intestine and kidney tissue, 62 66 role of Na+, K+-ATPase in, 59-62 Steroid myopathy, sarcoplasmic reticulum in, 175

T Transverse tabular system, structure and function of, 141-151 Triad, structure and function of, 149-151 Tryptophan, uptake by isolated nerve endings, 260-261

V Vasopressin-sensitive epithelia, movement across, 339-366

water

436

SUBJECT INDEX

W

X

Water movement, across vasopressinsensitive epithelia, 339-366 activation energy for, 345-346, 357-359 duaI barrier hypothesis of, 344-345 pore enlargement hypothesis of, 340-346 solvent drag effect, 359-364 unstirred layer effects, 361-364 true diffusion rate across luminal membrane, 346-357 epithelial cell effects, 355 supporting layer effect, 350-352 sweeping away effect, 355-356 unstirred layer effect, 347-349

X-ray diffraction, in studies of sarcoplasmic reticulum, 112

Z Zymogen granules, in pancreatic exocrine cells discharge of, 304-315 biochemical features of, 319-333 morphological features, 317-319 formation of, 297-304