No title

Current Topics in Membranes and Transport VOLUME 27

The Role of Membranes in Cel Growth and Differentiation

Advisory Board

M . P . Blaustein G . Blobel E. Carafoli J . S . Cook Sir H . L . Kornberg

D . Louvard C . A . Pasternak W . D . Stein W . Stoeckenius K . J . Ullrich

Current Topics in Membranes and Transport Edited by Arnost Kleinzeller Department of Physiology University of Pennsylvania School of Medicine Philadelphia, Pennsylvania

VOLUME 27

The Role of Membranes in Cel Growth and Differentiation Guest Editors

Lazaro J. Mandel

Dale J. Benos

Department of Physiology Duke University Medical Center Durham, North Carolina

Department of Physiology and Biophysics Harvard Medical School Boston, Massachusetts

1986

@) w

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

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COPYRIGHT 0 1986 BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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Contents

Preface, ix Yale Membrane Transport Processes Volumes, xi

DESCRIPTION OF ION TRANSPORT SYSTEMS IN ACTIVATED CELLS

PART 1.

CHAPTER

1.

Mitogens and Ion Fluxes LUIS REUSS, DAN CASSEL, PAUL ROTHENBERG, BRIAN WHITELEY, DAVID MANCUSO, AND LUIS GLASER

I . Introduction and Historical Perspective, 4 II. Ion Transport and pH Regulation in Cells, 5 111. Changes in Cell Membrane Voltage-Role in the Action of Growth Factors, 23 IV. Effect of Mitogens on Ion Fluxes and Intracellular pH, 25 V. Modulation of the Mitogenic Response by Protein Kinase C, 33 VI. Summary and Perspectives, 37 VII. Appendix: Measurement of Intracellular pH, 40 References, 48

CHAPTER

2.

Na+-H+ and Na+-Ca2+ Exchange in Activated Cells MITCHEL L. VILLEREAL

Introduction, 55 Na+-Caz+ Exchange, 57 111. Na+-H+ Exchange, 60 1V. Pharmacological Definition of the Na+-H+ and Na+-Ca2+ Exchange Systems, 78 V. Summary, 81 References, 82 I. II.

vi

CONTENTS

CHAPTER

3.

Chloride-Dependent Cation Cotransport and Cellular Differentiation: A Comparative Approach PETER K. LAUF

I. 11. 111. IV. V. VI

.

Introduction, 90 General Properties, 91 Properties in Blood Cells at Various Stages of Differentiation, 100 Properties in Differentiated Epithelial Cells, 108 Nonepithelial Cells as Models for Cotransport during Differentiation, 1 12 Conclusion, I I5 Note Added in Proof, 116 References, 116

TRIGGERS FOR INCREASED TRANSPORT DURING ACTIVATION

PART 11.

CHAPTER

4.

External Triggers of Ion Transport Systems: Fertilization, Growth, and Hormone Systems JOAN BELL, LORETTA NIELSEN, SARAH SARIBAN-SOHRABY, AND DALE BENOS

I. Introduction, 129 11. Ionic Responses to Fertilization, 132

111. Serum and Growth Factor Activation of Ionic Transport Systems in Cultured Cells and Lymphocytes, 143 IV. Hormonal Stimulation of Ion Transport and Hormone Secretion, 150 V. Concluding Remarks, 155 References. 156

CHAPTER

5.

Early Stimulation of Na+-H+ Antiport, Na+-K+ Pump Activity, and Ca2+ Fluxes in Fibroblast Mitogenesis ENRIQUE ROZENGURT AND STANLEY A. MENDOZA

I. Introduction, 163 11. Ionic Responses Elicited by Growth Factors in Quiescent Cells, 165 111. Protein Kinases and Ion Fluxes, 176 IV. Calcium Fluxes, 180 V. Conclusions and Perspectives, 182 References. 184

vi i

CONTENTS CHAPTER

6.

Volume-Sensitive Ion Fluxes in Amphiuma Red Blood Cells: General Principles Governing Na-H and K-H Exchange Transport and CI-HC03 Exchange Coupling PETER M. CALA

I. 11. 111.

1v. V.

VI. VI1. VIII.

Introduction: The Role of Alkali Metal-H Exchange in Cell Regulatory Processes, 194 Thermodynamic Principles of lon Transport: Electroneutral versus Conductive Alkali Metal Ion Fluxes, 195 Volume-Sensitive Ion Fluxes in Amphiuma Red Blood Cells, 198 CaZ+-DependentAlkali Metal Ion Flux in Amphiuma Red Blood Cells, 202 The Nature of Net Na Flux by Amphiuma Red Blood Cells in Hyperosmotic Media, 205 CI-HC03 Exchange and Its Functional Relationship to Alkali Metal-H Exchange, Alkali Metal-CI Cotransport, and Parallel Alkali Metal and H or Cl Conductance Pathways, 207 Activation and Control of Alkali Metal-H Exchange in Amphiuma Red Blood Cells, 210 Summary, 215 References, 216

PART Ill.

CHAPTER

7.

CONSEQUENCES OF THE ALTERATIONS IN ION TRANSPORT OBSERVED DURING ACTIVATION

lntracellular Ionic Changes and Cell Activation: Regulation of DNA, RNA, and Protein Synthesis KATHl GEERlNG

I. 11.

111. IV. V.

V1.

Introduction, 221 DNA Synthesis, 224 RNA Synthesis, 232 Protein Synthesis, 237 Posttranslational Events Influencing Intracellular Traffic and Cell Surface Expression of Proteins, 247 Conclusions, 249 References, 250

viii CHAPTER

CONTENTS

8.

Energy Metabolism of Cellular Activation, Growth, and Transformation LAZAR0 J . MANDEL

I. Introduction, 261 11. Control of Energy Metabolism in Adult Cells That Maintain a Relatively Constant Metabolic Rate, 264 111. Control of Energy Metabolism of Adult Cells That Can Be Rapidly Activated, 267

IV. Energy Metabolism of Cells in Culture, 272 V. Energy Metabolism of Malignant Cells, 278 References, 286

Index, 293

Preface The biochemical and physiological basis of extracellular signal transduction is an area of research that has burgeoned in recent years. There has been great interest in environmentally activated ion transport systems and the associated intracellular events that may link these transport systems to important processes, such as cellular proliferation, hormone secretion, and initiation of growth. Two such intracellular processes that appear to play prominent connecting roles for these events are inositol phospholipid turnover and oncogene induction. It is hoped that a thorough understanding of the biology of these pathways will provide insight not only to the molecular operation and control of specific ion transport systems but ultimately to the process of tumorigenesis. Our purposes in developing this volume are threefold. First, we think a thorough review of this area is warranted at the present time. Second, we want to address critically the question of whether the increased ion transport resulting from cell activation by growth factors or other stimuli in fact leads to an increased cellular metabolism which in turn stimulates or supports growth. A corollary to this inquiry is the question of whether the expression of ion transport systems caused by growth factors, hormones, cell volume perturbations, and even sperm (the primordial growth factor!) is mediated by common pathways. Third, and perhaps most important, we want to introduce and integrate researchers in the varied disciplines of membrane biophysics, cell biology, metabolism, and cancer and to stimulate thought and further work in areas that have been neglected. This book has been divided into three general parts: description of ion transport changes resulting from cell activation, the triggering mechanisms involved, and, last, the consequences of activation. In the first section are chapters, by Reuss et a / . and by Villereal, providing an overview of the plasma membrane transport systems involved in cell activation. The chapter by Lauf uses the comparative approach to describe a ubiquitous type of cotransport which may be important in cellular activation and differentiation. The first two chapters in the second section by Bell et al. and by Rozengurt and Mendoza detail what is known about the actual initiation of transport and other events subsequently converting a cell from a quiescent to an active state. The chapter by Cala describes in detail the changes in plasma membrane transport which occur in response ix

X

PREFACE

to a specific stimulus, changes in cellular volume. In the third section, the chapter by Geering reviews the effects of alterations in cytoplasmic ionic conditions (e.g., Na, K, H, Ca) on DNA, RNA, and protein synthesis. Finally, the chapter by Mandel describes the alterations in energy metabolism which occur during cellular activation, growth, and transformation. Each chapter synthesizes the relevant results obtained in different cell types and biological systems and highlights current gaps in knowledge. We would like to thank Professor Arnost Kleinzeller for inviting us to develop and edit this book and our colleagues who so graciously submitted excellent contributions to us on time. We hope that this volume will prove of value not only to those studying cell membrane transport, metabolism, and cell transformation, but also to students of cell physiology and biochemistry in general.

LAZARO J . MANDEL DALEJ. BENOS

Yale Membrane Transport Processes Volumes Joseph F. Hoffman (ed.). (1978). “Membrane Transport Processes,” Vol. 1. Raven, New York. Daniel C. Tosteson, Yu. A. Ovchinnikov, and Ramon Latorre (eds.). (1978). “Membrane Transport Processes,” Vol. 2. Raven, New York. Charles F. Stevens and Richard W. Tsien (eds.). (1979). “Membrane Transport Processes,” Vol. 3: Ion Permeation through Membrane Channels. Raven, New York. Emile L. Boulpaep (ed.). (1980). “Cellular Mechanisms of Renal Tubular Ion Transport”: Volume 13 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. William H. Miller (ed.). (1981). “Molecular Mechanisms of Photoreceptor Transduction”: Volume 15 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. Clifford L. Slayman (ed.). (1982). “Electrogenic Ion Pumps”: Volume 16 of Current Topics in Membranes and Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, New York. Joseph F. Hoffman and Bliss Forbush I11 (eds.). (1983). “Structure, Mechanism, and Function of the Na/K Pump”: Volume 19 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. James B. Wade and Simon A. Lewis (eds.). (1984). “Molecular Approaches to Epithelial Transport”: Volume 20 of Current Topics in Membranes and Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, New York. Edward A. Adelberg and Carolyn W. Slayman (eds.). (1985). “Genes and Membranes: Transport Proteins and Receptors”: Volume 23 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, Orlando. Peter S. Aronson and Walter F. Boron (eds.). (1986). ‘“a+-H+ Exchange, Intracellular pH, and Cell Function”: Volume 26 of Current Topics in Membranes and Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, Orlando. xi

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Part I

Description of Ion Transport Systems in Activated Cells

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CURRENT 'TOPICS IN MEMBRANES AND TKANSPOKI', VOLUME 27

Chapter 1 Mitogens and Ion Fluxes LUIS REUSS,* DAN CASSEL,? PAUL ROTHENBERG,? BRIAN WHITELEY,? DAVID MANCUSO,? AND LUIS GLASER? *Department of Cell Biology and Physiology

and tDeparrment of Biological Chemistry Washington University School of Medicine St. Louis, Missouri 63110

Introduction and Historical Perspective .............. Ion Transport and pH Regulation in Cells.. . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Classification of Transport Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Na+ and K + Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. H t Transport: Maintenance and Regulation of lntracellul D. Ca2+Transport: Regulation of lntracellular Ca?' Activity E. Maintenance and Regulation of Cell Volume.. . . . . . . . . . . . . . . . . . . . . . . . . F. Mechanisms of Generation of Cell Membrane Potentials . . . . . . . . . . . . . . . 111. Changes in Cell Membrane Voltage-Role in the Action of Growth Factors . . IV. Effect of Mitogens on Ion Fluxes and lntracellular pH A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Mitogens Increase Na+-H' Excha C. Mechanism of Activation of Na+-H + Exchange.. . . V. Modulation of the Mitogenic Response VI. Summary and Perspectives. . . . . . . . . . . . . VII. Appendix: Measurement of lntracellula A. Intracellular p H Microelectrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Distribution of Amphipatic Molecules in Cells. . . . . . . . . . . . . . . . . . . . . . . . . D. Optical Methods.. . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 11.

4

5 5 6

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13 IS 18

23 25 25 27 32 33 37 40 40 41

41 41 4x

3 Copyright C> 1986 by Academic Press, Inc. All rights of reproduction in any form r e x r v e d .

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LUIS REUSS ET AL.

1.

INTRODUCTION AND HISTORICAL PERSPECTIVE

The past few years have seen the unexpected convergence of two areas of investigation: the first is the study of ionic fluxes across the plasma membrane and the mechanisms that control the intracellular ionic environment, including intracellular pH, and the second is the study of the effects of mitogenic polypeptides on cells and the mechanism by which they stimulate cell growth. This convergence took place when it was discovered in the laboratory of E. Rozengurt that serum addition to quiescent cells in tissue culture increased ion fluxes, notably of sodium and potassium, into the cell (see, for example, Rozengurt and Heppel, 1975, and Smith and Rozengurt, 1978). These early experiments were difficult to interpret since serum is a complex mixture of molecules. More recently, the availability of several pure polypeptides which act as mitogens on cloned cell populations has allowed a detailed reexamination of these observations. Pure mitogenic polypeptides, just as serum, increase ion fluxes in quiescent cells, and as a consequence of these observations a whole new area of investigation has been opened. The convergence of these two fields will benefit investigations in both areas. To ascertain the mechanisms of the effects of mitogens or mitogenic polypeptides, it is important to be able to examine very early events in their action. It is clear that when a mitogenic polypeptide is added to a population of quiescent cells and the biological response assayed is cell division, the effect that is measured is the final result of a progression of events that takes place over a period of 20 to 24 hours. The initial intracellular effect of the interaction of the mitogen with the cells probably occurs within seconds (at the most minutes), and it is the events associated with signal transduction across the membrane, resulting from mitogen binding to its appropriate receptor, that are of particular interest at the present time. The study of the mechanisms by which mitogens activate ion fluxes may provide unique insights into the signal transduction mechanism associated with mitogenic activation of cell growth. Whether any or all of the changes in ionic fluxes observed as a result of mitogen addition to cells are either causal or permissive for the process of cell division, or are perhaps only unrelated consequences of the interaction of mitogenic polypeptides with their cell surface receptors, remains to be established. For the study of the regulation of the ionic composition of cells, the fact that mitogens increase the flux of some ions across the plasma membrane provides a unique tool to gain improved understanding of the mechanisms associated with intracellular homeostasis. In many biological systems, unique insights into regulatory mechanisms have been obtained as a result of the discovery of physiologically important agents that alter these pro-

1. MITOGENS AND ION FLUXES

5

cesses. Fewer insights are obtained by the use of pharmacological agents, which often have complex and unexpected effects. Thus, one can anticipate that further understanding of the mechanisms by which cells control their ionic composition and pH will result from the discovery that defined polypeptides, when added to cells, bind to receptors and alter ion fluxes across the plasma membrane and/or intracellular membranes. II. ION TRANSPORT AND pH REGULATION IN CELLS

In this section we will attempt to provide a basic summary of current knowledge of the mechanisms of transport of Na+, K+, C1-, Ca2+,and H+ by cell membranes and of the mechanisms of maintenance and regulation of cell volume, intracellular pH, and intracellular Ca2+activity. This summary will provide a framework in which we can discuss the action of mitogens in controlling these processes. Whenever pertinent, we will also discuss transport across intracellular membranes and interrelationships between transport of different substrates. Needless to say, the intention of this summary is to provide an overview rather than a comprehensive revision. As references we have chosen mostly review articles that in our opinion provide adequate orientation to readers who are not experts in this field. A. Classification of Transport Protelns

From a didactic point of view, transport proteins can be classified into three groups. 1. Channels (Hille, 1984) are pathways across the membrane which permit downhill electrodiffusional fluxes. The transport pathway in a channel can be exposed to the aqueous phases on both sides of the membrane at the same time. Channels permit transport of a large number of ions per unit time, that is, they have a high turnover number, exhibit varying degrees of ionic selectivity, and are sometimes voltage dependent. 2. Curriers (LeFevre, 1975) bind and translocate one or more solutes by a process thought to involve a conformational change upon binding of the transported substrate(s). As in the case of channels, carrier-mediated transport is energetically downhill, although uphill transport can occurin the case of a carrier that transports more than one solute-if part of the free energy stored in the gradient of one of them is transferred to the other (see below). Even in this case, the overall transport process results in a

6

LUIS REUSS ET AL.

decrease in free energy. Carriers typically exhibit substrate specificity, saturation kinetics, and competitive and noncompetitive inhibition. They translocate ions more slowly than channels. The simplest carriers transport only one solute. This process is commonly called facilitated diffusion or uniport (the carrier being a uniporter). When carriers transport two or more substances in the same direction, the process is called cotransport or symport; when the substrates are transported in opposite directions, the process is called countertransport or antiport. 3. Pumps (Heinz, 1978), which exhibit properties similar to those of carriers, differ in that they can harness metabolic energy, for example, by hydrolysis of ATP, and employ it in energetically uphill transport. Pumpmediated transport, or primary active transport, is thus directly linked to a metabolic energy source. The main ion pumps pertinent to our discussion are the Na+, or Na+-K+, pump, the Ca2+pump, and the H + pumps. All of these pumps are driven by the hydrolysis of ATP. Uphill transport can also occur, in the absence of direct coupling to a metabolic energy source, if a carrier uses the energy stored in the transmembrane chemical or electrochemical gradient of one solute to move another solute against its own unfavorable chemical or electrochemical gradient. This process, or secondary active transport, depends on a preexisting gradient which in turn was established by a primary or other secondary active transport process. Secondary active transport is thus indirectly coupled to a metabolic energy source. One of the main functions of the Na+-K+ pump in animal cells and of the H+ pump in plant cells is to build and maintain large electrochemical gradients favoring influx of Na+ or H+, respectively. Part of the energy stored in these gradients can be used to energize uphill transport of other substances into or out of the cell. Similar mechanisms exist in the bounding membranes of intracellular organelles, such as mitochondria, lysosomes, and secretory vesicles. B. Na+ and K+ Transport

1. Na+ INFLUX

Most cell membranes have an inside-negative voltage whose magnitude is determined by the electrodiffusive permeability coefficients of the permeant ions, their chemical activities on both sides of the membrane, and the current produced directly by charge translocation, such as in the case of electrogenic pumps, as discussed below. In most cells, electrodiffusion is the main determinant of the membrane voltage, with electrogenic

1. MITOGENS AND ION FLUXES

7

pumps playing a smaller role. Generally Kt is the most permeant ion. Hence, frequently the membrane voltage is close to the K+ equilibrium potential. Since K+ ions are more concentrated in the cell than in the extracellular fluid, the cell interior is electrically negative, by values which in most cases lie in the range of 60 to 90 mV. The intracellular Na+ concentration is typically of the order of 10 to 15 mM in cells bathed in an extracellular fluid with 100 to 150 mM Na+. Therefore, both the chemical and the electrical potential differences favor Na+ entry across the membrane. This downhill entry process occurs by several pathways. a . Nu+ channels. Voltage-sensitive Na+ channels are found in excitable cells. They can be blocked by tetrodotoxin and saxitoxin at nanomolar concentrations (Hille, 1984). Voltage-insensitive Na+ channels are found in outer (or luminal) membranes of Na+-transportingepithelia (Lindemann, 1984). They are blocked by the diuretic drug amiloride, with a Ki of the order of 0.4 pM. In a few instances tetrodotoxin-sensitive Naf influx has been demonstrated in nonexcitable cells (Munson et al., 1979; PouyssCgur et al., 1980). Its function remains to be established, since it is not required for the growth of these cells in tissue culture. b. Nu+ carriers. ( 1 ) Cotransport of Na+ with organic solutes has been demonstrated in luminal membranes of intestine and renal proximal tubule epithelial cells for glucose (Sacktor, 1977), and in these and many other cell types for amino acids. Both systems are electrogenic, that is, the net flux, normally inward, results in a flow of positive charge in the same direction. (2) Cotransport of Na+ and anions has also been demonstrated. The best studied examples are Na+-HCO; cotransport in renal tubule cells (Boron and Boulpaep, 1983), and cotransport of NaCl or NaKCI2. Evidence for NaCl cotransport per se (Frizzell et al., 1979) is not as convincing as that for NaKCI2 cotransport, a process clearly demonstrated in many cell types (Palfrey and Rao, 1983). In some cell membranes Na+ is also cotransported with phosphate or with organic anions, such as lactate, citrate, and others. (3) Countertransport of Na+ and H+ (Na+-H+ exchange) has been shown to constitute a major mechanism of maintenance and regulation of intracellular pH in a variety of cell types (Boron, 1983). This process is also essential in Na+ and H+ transport in several epithelia. Its activation may play a role in fertilization (Johnson et al., 1976; see also Bell et al., this volume) and in cell division (see below). Na+-H+ exchange is an electroneutral process which, because of the large inward Na+ chemical gradient, results under most conditions in Na+ influx and H+ efflux. It is also sensitive to amiloride, but with Ki values much higher than those required to inhibit amiloride-sensitive epithelial Na+ channels. (4) Countertransport of Na+ and Ca2+ has been demon-

8

LUIS REUSS ET AL.

strated in excitable and nonexcitable cells, where it appears to play a role in the maintenance of a low intracellular Ca2+activity (see below). 2. Na+ EFFLUX

Under physiologic or near-physiologic conditions the only clearly demonstrated mechanism of net Na+ efflux across the cell membrane is primary active transport by the Na+-K+ pump (Glynn and Karlish, 1975), which appears to operate with the stoichiometry 3 Na+ : 2 K+ : 1 ATP, although it has been suggested that the stoichiometry may change with different experimental conditions. Much progress has been made in recent years in understanding the mechanism of pumping and the regulation of the activity of the pump. Of special interest in the context of our topic are the suggestions of a dependency of the rate of the Na+ pump on intracellular pH (Breitwieser and Russell, 1983) and Ca2+concentration (Brown and Lew, 1983). The main mechanisms of Na+ transport across cell membranes are summarized in Table I. 3. K+ TRANSPORT

Active K+ transport is a. component of the function of the Na+-K+ pump. In most cells, this results in a steady-state intracellular K+ concentration higher than predicted for equilibrium distribution, that is, there is a net driving force for K+ exit from the cells. K+ exit occurs mainly by electrodiffusion through channels, which exhibit diverse properties. Some are voltage sensitive (rapidly or slowly inactivating), and some are activated by increases in intracellular CaZ+(Hille, 1984). K+ channels also exhibit diverse sensitivity to blockers such as aminopyridines, tetraethylammonium (TEA+), Ba2+,and Cs+ (Hille, 1984). The number and properties of K+ channels in different cells are not yet clear. A large amount of information has recently been obtained from patch clamp data (Sakmann and Neher, 1984). The electrodiffusive K+ permeability is also sensitive to intracellular and extracellular pH (Hille, 1984). Two kinds of cotransport mechanisms involving K+ have been described. NaKC12cotransport is a process that exists in a number of cells, including native epithelia, cultured epithelia, red blood cells, nerve cells, Ehrlich ascites cells, and several cell lines in culture (Palfrey and Rao, 1983). In the best studied examples, the transport process is electroneutral and measurements of stoichiometry suggest 1 Na: 1 K: 2 C1. Transport is inhibited by so-called loop diuretics (such as furosemide and bumetanide) and exhibits high ionic specificity. At least in some systems, cotransport is reduced in the absence of ATP. The nucleotide appears to

TABLE I CHARACTERISTICS OF SOMEPLASMA MEMBRANENa' TRANSPORT PATHWAYS Transport mechanism

Physiologic net flux

Dominant driving force"

Na+ channel (voltage sensitive) Na+ channel (voltage insensitive) Na+-H+ exchange

Influx Influx Influx

AFNa+ APNa+ ApNa+

Na+-Cl- and Na+-K+-Cl- cotrans-

Influx

ApNa+

Influx EfAux EfAux

AGNa+ AGHCO; ATP hydrolysis

Modulating factors

Inhibitors

H:(+), Ca:+(+)? cell shrinkage (+)

Tetrodotoxin, saxitoxin Amiloride (K,= 1 p M ) Amiloride (K,= 10 p M )

mitogens (+) Cell shrinkage (+) ATP (+)

Furosemide, bumetanide

Membrane depolarization (+)

cat' (-)

port

Na+-Caz+ exchange Na+-(HCO; )2 cotransport Na+,K+-ATPase a

A p , Chemical potential difference;

ATP (+) HT(-), Caf+(-)?

AG, electrochemical potential difference.

Vanadate, benzamyl Disulfonic stilbenes Cardiac steroids

10

LUIS REUSS ET AL.

play a regulatory role since its hydrolysis is not required for transport. NaKC12 cotransport is activated in some cases by osmotic shrinkage of the cells. Increased intracellular levels of cyclic nucleotides have diverse effects on NaKC12 cotransport in different cells: in flounder intestine, cAMP stimulates, whereas cGMP inhibits transport; cAMP has a stimulatory effect in avian red cells and an inhibitory effect in human red cells (Palfrey and Rao, 1983). KCl cotransport is a recently discovered process. It appears to be present in human red cells, in sheep red cells of the LK (low potassium) phenotype (Ellory et al., 1982), and in basolateral membranes of epithelia (Reuss, 1983). There are indications, in some of these systems, that the process is volume sensitive, activated by the sulfhydryl alkylating agent N-ethylmaleimide and inhibited by furosemide. The precise relationship of KCl cotransport and NaKClz cotransport is unclear at present. Whether KCl cotransport is present in other cells is also unknown. C. H+ Transport: Maintenance and Regulation of lntracellular pH

Most cells are subjected to a continuous intracellular acid load by three distinct mechanisms: metabolic production of acid, H+ influx from the extracellular fluid (or its equivalent, OH- efflux), and HCO; efflux. The contributions of the latter two mechanisms are clearly dependent upon the H+ (or OH-) and HCO; permeabilities of the cell membranes. Since under control conditions the intracellular pH is only slightly lower than the extracellular pH and there is a large membrane voltage, with the cell interior negative, the electrochemical gradients favor H+ influx and HCO; efflux. 1. PASSIVETRANSPORT OF H+ EQUIVALENTS

a . Electrodiffusion o f H + and HCO;. H+ permeability ( P H + )is difficult to measure accurately in cells because of the unavoidable presence of HCO;, which makes it difficult to ascribe pH changes to H+ or OHfluxes per se. In artificial lipid membranes, P H +is sizable. However, since the H+ activity in the extracellular fluid is very low, it is likely that net electrodiffusive H+ entry is rather slow and hence capable of producing changes in intracellular pH only over prolonged periods (Roos and Boron, 1981; Boron, 1983). HCO; permeability (PHCOT) is also difficult to prove and quantitate. The best evidence for a HCO; uniport has been obtained by electrophysiologic techniques in mammalian renal proximal tubules (Burckhardt et al., 1984), although there are indications that it might be present in other

1. MITOGENS AND ION FLUXES

11

cells as well. Anions of other weak acids, such as 5,5-dimethyloxazolidine-2,4-dione (DMO), salycylic acid, and short chain fatty acids, are also permeant. Similarly, NH: and other weak acids permeate cell membranes, a feature that provides a convenient technique for acid-loading cells (Boron and DeWeer, 1976). Upon exposure to NH4Cl, NH3 enters the cells rapidly, causing alkalinization; NH: permeates the cell membrane more slowly, causing drift of the intracellular pH in the acid direction. Upon removal of external NHdCI, the intracellular NH3 concentration falls rapidly and dissociation of intracellular NH: causes acidification. b. HCO; Cotransport. An electrogenic, Na+-coupled transport of HCO; , which could consist of Na(HCO3); cotransport, transport of NaCO;, or equivalent processes, has been demonstrated in basolateral membranes of amphibian proximal renal tubules (Boron and Boulpaep, 1983). It is inhibited by disulfonic stilbenes and operates in either direction, according to the direction of the electrochemical gradient. Under control conditions it results in Na+ and HCO; efflux from the cells. OF H + EQUIVALENTS 2. ACTIVETRANSPORT

a . Nu+-H+ Exchange. Na+-H+ exchange, a process discovered by Murer et al. (1976) in renal and intestinal brush border vesicles, has been demonstrated in numerous cell types, including skeletal and cardiac muscle, neurons, erythrocytes, and a variety of epithelial cells. The most detailed analyses of this transporter have been obtained in studies of vesicles obtained from renal and intestinal brush borders (Kinsella and Aronson, 1980; Aronson, 1983). H+ can be transported actively by this process by using energy stored in the Na+ chemical gradient. Na+-H+ exchange is an electroneutral process, independent of the anions present in the system, insensitive to disulfonic stilbenes, and sensitive to amiloride. The Ki for amiloride is about 25 p M in renal brush border vesicles at 1 mM “a+] and pH 7.5 (Kinsella and Aronson, 1981) but far lower in a number of cells in tissue culture (Zhuang et ul., 1984; Vigne et al., 1984a). There is disagreement on the kinetic characteristics of the inhibition of Na+-H+ exchange by amiloride. Kinsella and Aronson, measuring Na+ uptake, found purely competitive inhibition, whereas Ives et al. (1983), measuring H+ extrusion, found mixed inhibition. Lowering intracellular (or intravesicular) pH not only stimulates Na+-H+ exchange by the effect on the driving force but also activates the exchanger (Aronson et al., 1982). CAMPinhibits Na+-H+ exchange in apical membranes of epithelia (Reuss and Petersen, 1985). Insulin and other peptides stimulate Nat-H+ exchange in muscle, causing rises in intracellular pH (Moore, 1981). How-

12

LUIS REUSS ET AL.

ever, this effect of insulin is not observed in fibroblasts (Moolenaar et al., 1983; D. Cassel, unpublished observations). Na+-H+ exchange is a major mechanism of maintenance and regulation of intracellular pH in many cells and plays a role in acid secretion and salt absorption by epithelia (Boron, 1983). b. Cl--HCO; Exchange. First described and best studied in red blood cells (Cabantchik et al., 1978; Knauf, 1979), Cl--HCO; exchange has also been demonstrated in muscle and epithelial cells. It is an electroneutral process, sensitive to disulfonic stilbenes, which operates in an energetically downhill fashion. Since in most cells the C1- gradient inward is dominant, Cl--HCO; exchange causes uphill HCO; extrusion and hence intracellular acidification. Therefore, this transporter may play a role in the recovery of intracellular pH from alkaline loads but not in the maintenance of pHi or in pHi recovery from an acid load (Boron, 1983). c. NaHCOj-HCl Exchange. This complicated antiport appears to be, with some differences between systems, the major mechanism of regulation of intracellular pH in excitable cells. The NaHC03-HCl model, and thermodynamically equivalent ones, extrude 2 H+ equivalents per cycle (e.g., efflux of 1 H+ and influx of 1 HCO;). Na+ and HCO; are required in the extracellular fluid and C1- in the cell. The process is electroneutral, insensitive to amiloride, and sensitive to disulfonic stilbenes. The direction of the net flux depends on the ion gradients (Thomas, 1976, 1977; Boron, 1977, 1983); it may also be present in nonexcitable cells (Rothenberg et al., 1983a). Several factors have stimulatory or inhibitory effects on NaHCO3-HC1 exchange independently of the thermodynamic parameters: lowering intracellular pH activates this transporter, as in the case of Na+-H+ exchange; internal ATP is required in some cases but not in others and is not utilized as an energy source; CAMPstimulates NaHCO3-HC1 exchange in barnacle muscle fibers (Boron, 1983). d. H+ Pump. H+-activated ATPases exist in the membranes of epithelial cells (e.g., parietal cells of the gastric mucosa, mitochondria-rich cells of kidney tubules and the urinary epithelium) and in intracellular organelles such as mitochondria, lysosomes, and chromaffin granules. The gastric H+ pump is an electroneutral Kf ,H+-ATPase(Sachs et al., 1982). The renal H+ pump is electrogenic (Steinmetz and Andersen, 1982), as are also the organelle H+-ATPases and the fungal H+-ATPase. The gastric and renal H+ pumps are sensitive to vanadate, as is the fungal pump, but are insensitive to oligomycin, that is, different from the mitochondria1 H+-ATPase. In experiments in the urinary bladder it has been demonstrated that intracellular acidification stimulates H+ extrusion by insertion of pre-

1. MITOGENS AND ION FLUXES

13

formed, vesicle-contained H+ pumps into the apical membrane (Gluck et al., 1982). There is no compelling evidence for the existence of H+ pumps in the plasmalemma of other cells than those mentioned above. Coated vesicles, which are derived from the plasma membrane, contain an ATPdriven H+ pump, which acidifies the vesicle interior (Stone et al., 1983; Forgac et al., 1983). This pump, if located on the plasma membrane, could serve to extract protons from the cell by an ATP-dependent mechanism, on the assumption that it remains active when present in the latter location. No data are presently available regarding the activity of this pump in the plasma membrane. D. Ca2+Transport: Regulation of lntracellular Ca2+Activlty

The total intracellular Ca2+ concentration varies from about to about low2M in different cell types. Of this total, only about lo-’ M is ionized Caz+in the cytosol, 2 to 5 x lo-’ M is cornplexed with anions or bound to proteins (in particular calmodulin), and the rest is contained in organelles (mitochondria, endoplasmic reticulum). The parameter of most physiologic significance is the cytosolic level of ionized Ca2+,which is regulated by a complex interaction between transport at the cell membrane and sequestration in organelles. 1. Ca2+INFLUX

Because of the large chemical and electrical gradients favoring entry, passive transport mechanisms tend to raise intracellular Ca2+concentration. Ca2+channels have been identified and characterized in excitable cells, where they can be voltage sensitive or voltage insensitive (Tsien, 1983b). Little is known about the precise mechanisms of Ca2+entry in other cells, but it is clear that cell membranes are measurably Ca2+permeable. Recent studies in a number of systems suggest that increase in cytoplasmic Ca2+can be triggered by the conversion of phosphatidylinosito1 to inositol triphosphate in response to activation of surface receptors by neurotransmitters, peptide hormones, and other substances (Nishizuka, 1983a,b). 2. Ca2+BUFFERING A N D SEQUESTRATION

Intracellular Ca2+is partly “buffered” by complexation and binding to cytosolic and membrane proteins and can be also transported into mitochondria and endoplasmic reticulum. Ca2+transport by mitochondria (Scarpa, 1979) is a complicated pro-

14

LUIS REUSS ET AL.

cess. Uptake seems to be electrodiffusive, driven by the large insidenegative voltage across the inner mitochondrial membrane; several pathways for Ca2+ efflux have been demonstrated, not all of them fully understood, which cause intramitochondrial Ca2+ concentration to be much lower than predicted for equilibrium distribution. Mitochondria1 Ca2+uptake and sequestration can be thought of as a high-capacity, lowaffinity process. However, under some conditions it has been shown that mitochondria can reduce the Ca2+ concentration to values as low as 2 x lo-’ M (Becker et al., 1980). Ca2+uptake by sarcoplasmic reticulum membranes (and by analogy by endoplasmic reticulum in non-muscle cells) is an active process mediated by a Mg2+-dependent, Ca2+-activatedATPase (Hasselbach, 1981). Inosito1 triphosphate has been shown to cause Ca2+release from endoplasmic reticulum in several cell types. In contrast with the mitochondrial transport system, the endoplasmic reticulum Ca2+ pump appears to have a lower capacity and a higher affinity, that is, it operates at intracellular Ca2+activities in the physiologic range. The issue of the relative contributions of mitochondria and endoplasmic reticulum to the maintenance of a low intracellular Ca2+activity has not been completely resolved.

3. Caz+ EFFLUX Ultimately, intracellular Ca2+ homeostasis requires extrusion to the extracellular fluid of the Ca2+ that enters continuously across the cell membrane. Intracellular buffering and sequestration can only temporarily maintain intracellular free Ca2+at the low physiologic levels. Two mechanisms of Ca2+ extrusion have been identified in numerous cell types. a. Na+-Ca2+ Exchange. The cell membranes of neurons and other cell types possess a carrier that transports Ca2+in exchange for Na+ (Blaustein and Nelson, 1982; DiPolo and BeaugC, 1983). This process is electrogenic. Coupling ratios of 3 Na+: 1 Ca2+ and 4 Na+: 1 Ca2+ have been proposed. The limit to which the cytosolic ionized Ca2+concentration can be lowered by this transport mechanism can be estimated from the Na+ activities on both sides of the membrane, the membrane voltage, and the Na+ : Ca2+coupling ratio. For Na,f = 100 mM, Na? = 10 mM, V , = -60 mV, and a 3 : 1 stoichiometry, CaT+ would be as low as times the extracellular value. For cells with a lower membrane voltage, a higher intracellular “a+] or both, it is unclear whether this mechanism can account fully for the measured or estimated intracellular free Ca2+level. ATP increases the affinity of the carrier for external Na+, without being hydrolyzed. Even in the presence of ATP, the apparent affinity for Ca2+is

1. MiTOGENS AND ION FLUXES

15

relatively low. In contrast with the kinetic properties of the Ca2+pump (see below), the Na+-Ca2+exchanger can be considered a high-capacity, low-affinity system (DiPolo and Beauge, 1983). 6. Ca2+Pump. A Ca2+pump, that is, a Caz+-activatedATPase, was first demonstrated in red blood cells (Schatzmann, 1983) and is now known to exist in most, if not all, cells. This enzyme requires Mg2+, is activated by calmodulin, and has a half-maximal rate at about M Ca2+, that is, it operates in the physiologic range. In contrast to the Na+-Ca2+exchanger, it is a low-capacity, high-affinity transporter. Intracellular Ca2+ activity is related to a number of cell functions, among them membrane transport events. As mentioned above, Ca2+can activate K+ channels (Lew and Ferreira, 1978) and therefore change the membrane voltage and the rate of ion transport by conductive mechanisms. Ca2+buffering and Ca2+fluxes across the mitochondria1 membrane result in changes in intracellular pH: high intracellular Ca2+levels cause intracellular acidification. Ca2+also has been claimed to activate Na+-H+ exchange, an effect that could be indirect, that is, due to lowering of pHi. Calmodulin, which is activated by Ca2+binding, activates in turn adenylate cyclase and phosphodiesterase, which can result in changes in the intracellular levels of cyclic nucleotides (Rasmussen, 1981). Finally Ca2+ is well known to play a role in membrane fusion, both in artificial systems and in secretory cells. It is possible that membrane recycling, via changes in the number of transporters, is one of the mechanisms by which intracellular Ca2+controls membrane transport processes (Al-Awqati, 1985). The main mechanisms involved in Ca2+transport across the plasma membrane are summarized in Table 11. E. Maintenance and Regulation of Cell Volume

The intracellular fluid contains a high concentration of large anions, to which the cell membrane is impermeable. This results in a large difference in colloid-osmotic pressure between the cytosol and the extracellular fluid, which per se results in entry of permeable ions (and nonelectrolytes) and water. In the absence of a balancing efflux of solute, cells would swell (colloid-osmoticswelling). It is generally accepted that the major mechanism which prevents such swelling is the net efflux of salt which results from the operation of the Na+-K+ pump (Tosteson and Hoffman, 1960). This process, that is, the preservation of cell volume under isosmotic conditions, or, in other words, the prevention of colloid-osmotic swelling, is appropriately referred to as cell volume maintenance (Cala, 1983b). In contrast, cells are also able to return to their control volume after an

TABLE I1 CHARACTERISTICS OF SOMEPLASMA MEMBRANE Ca2+TRANSPORT PATHWAYS

Transport mechanism

Physiologic net flux

Dominating driving force

Modulating factors

~~

~

Caz+channel (voltage sensitive) Ca2+channel (voltage insensitive) "Carrier mediated'

Idux Inilux

mux

A&a+ A&a+ ApCaz+

Na+-Ca2+ exchange Ca2+-ATPase

Efflux Efflux

AFNa+ ATP hydrolysis

a

Inhibitors

Membrane depolarization (+)

Heavy metals, verapamil

Inositol triphosphate from phosphatidylinositol breakdown (neurotransmitters, peptide hormones, etc.) (+) ATP (+) Calmodulin (+)

Divalent cations (competitive)

Ineffective in the absence of ATP; at high C a v the effect is stimulatory (DiPolo and Beaugk, 1983).

Vanadate", benzamyl L.a3+, vanadate, phenothiazines, calmidawlium

1. MITOGENS AND ION FLUXES

17

initial “osmometric” swelling or shrinkage produced by exposure to anisotonic media. The volume recovery under these conditions is referred to as cell volume regulation (Cala, 1983b). Cell volume regulation is discussed in detail in Chapter 6 of this volume. Here, we will briefly summarize current knowledge and speculate on the physiological significance of this process. 1. REGULATORY VOLUMEDECREASE

In many cell types, exposure to a hyposmotic solution results in rapid swelling followed by loss of water, which tends to restore cell volume to control levels. This phase of fluid loss during continued exposure to the hyposmotic medium has been termed regulatory volume decrease (RVD). In Amphiuma red cells, the most extensively studied system (Cala, 1983a), the mechanism of fluid loss during RVD is a net water efflux coupled to a net efflux of KCl which is electroneutral and appears to be due to activation of K+-H+ and Cl--HCO; exchanges. Net solute loss is achieved by efflux of K+ and C1- in exchange for influx of H+and HCO;, which recycle across the membrane as C02 and H20. The coupling between the two exchangers appears to be thermodynamic, that is, attributable to the intracellular pH. The process is sensitive to disulfonic stilbenes, as expected from the role of the anion exchanger, and insensitive to ouabain. In contrast, RVD in Necturus gallbladder epithelial cells, which is also due to KC1 efflux, has been ascribed to an electrogenic KCI cotransport with stoichiometry 3 K : 2 CI (Larson and Spring, 1984). In Necturus intestinal epithelium, cell swelling causes an increase in basolateral membrane electrodiffusive K+ permeability (Lau et al., 1984). 2. REGULATORY VOLUMEINCREASE Initial shrinkage, resulting from exposure of cells to a hyperosmotic medium, is in many cases followed by spontaneous recovery of the control cell volume, due to net influx of salt and coupled entry of water (regulatory volume increase, or RVI). The best studied models of this process are duck and Amphiuma erythrocytes. Salt entry is an electroneutral process, but the species transported and the mechanisms involved differ. In the duck red cell, salt influx involves cotransport of Na, K, and C1, probably with the stoichiometry 1 Na : 1 K : 2 Cl. This pathway, which is inhibited by loop diuretics and insensitive to disulfonic stilbenes, is also activated by catecholamines, in the absence of osmotic perturbations. In the Amphiuma erythrocyte, RVI involves net entry of Na+ and CI- but not of K+. It consists of Na+-H+ and Cl--HCO; exchanges and is,

18

LUIS REUSS ET AL.

therefore, sensitive to amiloride and disulfonic stilbenes (Cala, 1983a,b). Double exchange also appears to be the mechanism of RVI in human lymphocytes (Grinstein et al., 1983a,b). The mechanisms of activation of RVI and RVD have not been established. There are experimental data that suggest, however, the involvement of a calmodulin-dependent intracellular Ca2+effect. In Amphiuma erythrocytes, raising intracellular Ca2+appears to stimulate K+-H+ exchange, an effect that is inhibited by phenothiazines. In the same system, it appears that the effect of Ca2+on Na+-H+ exchange is inhibitory (Cala, 1983b). The preceding discussion clearly indicates that the regulation of cell volume in many cases utilizes mechanisms that are also responsible for other homeostatic mechanisms, such as the regulation of intracellular pH, and for other cell functions, such as transepithelial transport. Only exceptionally are cells exposed to anisotonic media, the preferred experimental perturbation used to study volume regulatory responses. However, cell volume changes can occur by primary alterations of the intracellular solute content in the absence of external osmolality changes, such as upon coupled entry of Na+ and organic solute into epithelial cells of the small intestine or K+ loss by prolonged depolarization of skeletal muscle fibers. It is possible that the physiologic importance of cell volume regulation is to prevent volume changes caused by alterations in solute content, rather than volume changes produced by anisotonic media (Lau et al., 1984). F. Mechanisms of Generatlon of Cell Membrane Potentials

Because of their predominantly lipid composition, cell membranes behave as electrical capacitors. A typical cell membrane may be 7 nm thick and have, under physiologic conditions, a voltage of 70 mV. From these parameters one can calculate that the electrical field (voltage/thickness) is about 100 kV/cm, a very large value indeed. The membrane voltage and the electric field in the membrane exert profound influences on transmembrane transport of permeant ions and carrier-substrate complexes that have a net charge. Furthermore, the orientation and position with respect to the membrane of charged macromolecules is field dependent. A number of cell functions are related to the presence of an electrical potential difference across the plasmalemma or to changes in this potential. In this section we will review the mechanisms of generation of the membrane potential in normal cells and provide general guidelines concerning the interpretation of changes in membrane potential. The literature covering these topics is extensive. For an excellent review, both concise and quantitative, see Schultz (1980).

1. MITOGENS AND ION FLUXES

19

The electrical potential differences across cell membranes are ultimately caused by charge separation resulting from net ion transport. In principle, three distinct mechanisms can account for these net ion fluxes: (1) simple diffusion or facilitated diffusion of ions or of electrically charged carrier-substrate complexes, (2) electrogenic primary active transport, that is, electrogenic pumping, and (3) frictional coupling of an ion flux to a transmembrane water flux induced by hydrostatic or osmotic pressure differences, a phenomenon usually referred to as “streaming potential.” Under most basal and experimental conditions, the first two mechanisms, that is, diffusion potentials and electrogenic pumps, appear to account for the membrane voltages measured in animal cells. 1. MEMBRANEDIFFUSION POTENTIALS

Transmembrane ion fluxes by simple diffusion or by “facilitated diffusion’’ (i.e., mediated by transport proteins) and transmembrane fluxes of electrically charged complexes (e.g., cotransport of 1 Na+ + 2 HCO; or of Na+ and glucose) cause net transfer of electric charge across the membrane, and hence a transmembrane electrical potential difference. The process involved is best understood by first considering a simple case. An artificial membrane (planar bilayer) is exposed at time zero to two aqueous solutions of the same uni-univalent salt at different concentrations ( C , and CZ).The membrane is permeable to only one of the ions. Immediately upon exposure to the solutions, the concentration difference favors net fluxes of both the cation (C+) and the anion (A-) from the side with the high salt concentration to the dilute side. Inasmuch as only one of the ions is permeant, a net flux of that ion will occur, with no flux of the counterion. Since charge is thus transferred across the membrane, a membrane voltage will develop, making the side containing the concentrated solution electrically negative if C+ is the permeant species or electrically positive if A- is the permeant ion. The time course for development of the transmembrane voltage upon “instantaneous” exposure to the two solutions is a single exponential, whose time constant is given by the product of the electrical resistance and the electrical capacitance of the membrane. The electrical potential difference generated across the membrane opposes further translocation of the permeant ion. Therefore, the flux decreases until a membrane voltage is reached at which the chemical potential difference and the electrical potential difference have the same magnitude and the net ion flux is zero, that is, the system is at electrochemical equilibrium. If C+ is the permeant species, equilibrium is given by zV,F

+ RT In([C+I1/[C+l2)= 0

20

LUIS REUSS ET AL.

where z is the valence, Vm is the membrane voltage (VI - Vz),F is the Faraday, [C+]l and [C+]zare the two concentrations, and R and Tare the gas constant and the absolute temperature, respectively. This expression can be rewritten as the more familiar Nernst equation, where Vm is the equilibrium potential of the permeant ion, that is, the voltage at which its net flux is zero:

Vm

RT zF

= - - ln([C+]I/[C+]2)

For a monovalent cation, for example, K+, changing from natural to decimal logarithms, at 37°C V = -61 log([K]I/[K]2)

For a membrane capacitance of 1 pFlcm2,the amount of K+ necessary to transfer in order to bring V, to -61 mV would be about 0.6 X Eq, that is, vanishingly small. If the membrane is permeable to several monovalent ions, for instance, to K+, Na+, and CI-, the voltage generated by electrodiffusion is given by

where P K , P N a , and Pa are the respective electrodiffusive permeability coefficients, which are all assumed to be constant. The above equation is the Goldman-Hodgkin-Katz equation, originally derived assuming a constant electric field in the membrane (Goldman, 1943; Hodgkin and Katz, 1949). Several particular cases of this equation are of interest. (1) If one of the permeability coefficients is much greater than the other two and the concentrations of all three ions are similar, the membrane voltage approaches the equilibrium potential of the permeant ion, given by the Nernst equation. (2) Similarly, if the concentrations of one of the ions are much higher than those of the other two and the permeabilities are similar, the membrane voltage also approaches the equilibrium potential of that ion. (3) If an ion is at electrochemical equilibrium, that is, if its equilibrium potential is equal to Vm, that ion can be-on mathematical reasons alone-eliminated from both numerator and denominator. In this sense, ions distributed at equilibrium do not contribute to the membrane voltage. An expression equivalent to the Goldman-Hodgkin-Katz equation is the following: vm =

EKtK

ENatNa

4- ECltCl

1. MITOGENS AND ION FLUXES

21

where Ei is the equilibrium potential and t i is the ion transference number, that is, the fraction of the membrane current carried by that ion. The transference number can also be defined as a partial ionic conductance ( 4 = gi/g,, where g , is the total membrane conductance). Since the sum of transference numbers of all permeant ions is by definition equal to 1 , it is clear that the ion with the highest transference number, that is, with the highest conductance, makes the largest contribution to V,. Permeability on one hand and conductance and transference number on the other, although related, are different and cannot be interchanged. Permeability is an intrinsic property of the membrane which is in principle independent of the concentration; conductance is a property that depends on both the permeability and the concentration of the ion. In summary, electrodiffusion is a major mechanism of generation of the electrical potential differences observed across membranes. All permeant ions which are not at electrochemical equilibrium contribute to the membrane voltage in proportion to their permeabilities and concentrations on both sides of the membrane.

2. ELECTROGENIC PUMPS Pumps contribute indirectly to the membrane voltage because they generate and maintain the ion concentration differences responsible for the electrodiffusive mechanisms discussed above. In addition, ion pumps contribute directly to the membrane voltage when they are electrogenic, that is, when their operation results in net transfer of charge across the membrane. This is the case for the Na+-K+ pump, which in many preparations has been shown to transfer 3 Na+ and 2 K+ per cycle, therefore causing a net transfer of charge across the membrane equivalent to onethird of the sodium flux. The H+ pump and the Ca2+ pump are also electrogenic since translocation of the ions indicated is not coupled to cotransport of anions or countertransport of cations. The contribution of electrogenic pumps to the membrane voltage depends on the magnitudes of the pump current and the membrane conductance: where VL is the pump-dependent fraction of V,, i, is the pump current, and g, is the membrane conductance. With few exceptions, accurate measurements of g, and particularly of ip are extremely difficult. Indications of pump electrogenicity in many preparations have been obtained only indirectly, for example, by demonstrating rapid changes in voltage upon pharmacologic inhibition, or upon activation of the pump (use of

22

LUIS REUSS ET AL.

cardiac steroids and intracellular Na+ loading, respectively, in the case of the Na+-K+ pump). In particular cases it is possible to incorporate the contribution of an electrogenic pump into the Goldman-Hodgkin-Katz equation. For instance, Mullins and Noda (1963) showed that if C1- is distributed at equilibrium and the cell is at a steady state (i-e., all net ion fluxes are zero, or, for each ion, influx = efflux), the membrane voltage is given by

where r is the coupling ratio of the pump (JNaIJK). Mullins and Noda calculated that, within the conditions stated, the maximum contribution of the electrogenic Na+-K+ pump to the membrane potential is of the order of 10 mV. Direct measurements in several preparations under control conditions suggest that indeed the Na+-K+ pump contributes directly a few millivolts to the membrane voltage. However, under non-steady-state conditions, for example, during Na+ loading of the cells, the pump current, and hence the direct contribution of the pump to V , , can be substantially larger.

3. MECHANISMS OF CHANGE OF CELLMEMBRANE POTENTIALS The mechanisms by which the cell membrane potential can change follow from the preceding discussion. In general, the experimenter interpreting a change in membrane voltage must consider the following possibilities: (1) alterations in the concentration of one or more permeant ions on one or both sides of the membrane, (2) changes in one or more permeability coefficients, and (3) changes in the current generated by electrogenic pumps. A large number of spontaneous and experimental conditions can result in a change in cell membrane voltage. A few examples are provided for illustration. a . Concentration Changes. (i) V,,, can be changed experimentally by altering the extracellular concentration of one or more permeant ions. If the change in external concentrations is fast, so that no significant changes in intracellular concentrations take place, from the change in V,,, and the change in equilibrium potential the transference number can be estimated (Hodgkin and Horowicz, 1959). Most cell membranes have a high electrodiffusive K+ permeability, and therefore V , is highly dependent on the extracellular Kf concentration. For this reason, it is frequently difficult to rule out the possibility of extracellular changes in K+ concentration at the surface of the membrane itself as a mechanism of

1. MITOGENS AND ION FLUXES

23

change in V,. This is a serious limitation in preparations in which such concentration cannot be controlled, because of the existence of anatomic and/or functional unstirred fluid layers, or monitored with electrophysiologic techniques. (ii) Primary changes in intracellular ion concentrations can also cause changes in V , ; a decrease in intracellular K' concentration produced by direct pharmacologic inhibition of the Na+-K+ pump or of its energy supply, by inhibiting metabolism, will depolarize most cells. Hence, utilization of this result as evidence for pump electrogenicity requires control or measurement of the K+ concentration on both sides of the membrane. b. Permeability Changes. (i) In excitable cells, primary changes in membrane voltage or activation of membrane receptors can result in changes in one or more electrodiffusive permeabilities; if the ion whose permeability is thus changed is not at equilibrium, the membrane voltage will be displaced. (ii) Changes in electrodiffusive permeability can also occur in nonexcitable cells in response to a number of extra- and intracelMar parameters. Among them, the effect of intracellular Ca2+ activity and intracellular pH on PK and the effect of intracellular cyclic AMP levels on a number of ionic permeabilities are typical examples. (iii) A few well-documented cases have been reported in which permeabilities which were not demonstrable under control conditions are induced by specific experimental maneuvers [e.g., increase in PClby cyclic AMP (Petersen and Reuss, 1983)], illustrating the fact that a change in V, cannot be ascribed a priori to preexisting transport processes. c . Changes in Transport Rates of Electrogenic Pumps. The three electrogenic pumps of interest in this discussion, namely, the Na+-K+ pump, the Ca2+pump, and the H+ pump, under normal operating conditions transport positive charges from the cell interior to the extracellular fluid. Therefore, they hyperpolarize the membrane, that is, increase the electrical negativity of the intracellular compartment. Inhibition of any of these pumps will cause cell membrane depolarization. Activation of the pumps will, by itself, hyperpolarize the membrane. Such activation can result from an increase in the concentration of the transported ion(s), from an increase in the number of pump sites, or from an increase in the turnover rate of the pump. 111.

CHANGES IN CELL MEMBRANE VOLTAGE-ROLE ACTION OF GROWTH FACTORS

IN THE

The earliest suggestion of an effect of growth factors on cell membrane ionic permeability was provided by Hulser and Frank (1971), who found

24

LUIS REUSS ET AL.

that exposure of quiescent fibroblasts to serum produced a rapid, large depolarization. Since chemical mitogens stimulate Na+ influx, the observation of cell membrane depolarization suggests that the Na+ influx could be electrodiffusive. This hypothesis has been tested in several cell lines. Moolenaar et al. (1979) observed a two-phase membrane depolarization upon serum stimulation of neuroblastoma cells. Using serum fractions, the slow depolarization was tentatively attributed to the interaction of growth factor-containing fractions with the membrane (Moolenaar et al., 198 1). Although serum also causes membrane depolarization in quiescent fibroblasts, EGF stimulates growth without altering the membrane potential (Moolenaar et al., 1982). In cultured BSC-1 epithelial cells, we found that the average membrane voltages in quiescent or growing cells did not differ and were approximately -48 mV. Addition of serum or mitogenic concentrations of EGF induced a moderate depolarization (5 to 20 mV) of rapid onset, followed by spontaneous repolarization in 5 to 10 minutes (Fig. 1). In BSC-1 cells, the Na+ influx measured by unidirectional tracer uptake techniques was significantly increased shortly after exposure to mitogens and remained elevated for at least 60 minutes, that is, long after the cell membrane had repolarized (Rothenberg et al., 1982). This observation, in conjunction with the lack of effect of EGF on membrane voltage in fibroblasts, indicates that changes in membrane voltage are not a necessary event for mitogen-induced cell growth. Since membrane depo-

5min

FIG.1. Effect of (A) EGF and (B) serum addition on membrane potential of BSC-I cells. Quiescent BSC-1 cells on a plastic dish were impaled with a microelectrode as described (Rothenberg ef al., 1982) to measure membrane voltage. Addition of EGF or fetal calf serum was via a superfusion system. The changes in membrane potential (upward changes = depolarization) start after a delay caused by the dead space of the superfusion system.

1. MITOGENS AND ION FLUXES

25

larization sometimes accompanies the more important activation of electroneutral Na+ influx, mediated by amiloride-sensitive Na+-H+ exchange, an intriguing possibility is that the depolarization is due, at least in part, to an increase in cell membrane Ca2+permeability; the resulting rise in intracellular Ca2+could in turn activate Na+-H+ exchange. However, activation of Na+-H+ exchange can also take place in the absence of extracellular Ca2+ (Rothenberg et al., 1983a). Recent results using quin-2 as an intracellular Ca2+ indicator suggest that following mitogen (serum) addition to fibroblasts, cytoplasmic Ca2+rises due to release from intracellular stores (Moolenaar et al., 1984a; Mix et al., 1984). Using aequorin, which does not strongly chelate Ca2+,McNeil et al. (1985) have shown that the Ca2+rise following mitogen addition is very transient. In Ca2+-containingmedia, this rise is longer-lived than in Ca2+-freemedia, suggesting that in the latter case Ca2+ is lost to the medium. If these observations in two different cell lines can be equated, they suggest that if a rise in cytoplasmic Ca2+is required for activation of Na+-H+ exchange, then a transient rise is sufficient for a prolonged activation. A more conservative interpretation is that a rise in cytoplasmic Ca2+is not required for activation of Na+-H+ exchange. Regardless of the possibility of a role of Ca2+in activating Na+-H+ exchange, the results discussed above indicate that a change in membrane voltage per se is not a necessary event in the transition from quiescence to growth. IV. EFFECT OF MITOGENS ON ION FLUXES AND INTRACELLULAR pH A. lntroductlon

Normal epithelial or fibroblastic cells in tissue culture are arrested in their growth either because they reach a limiting cell density (contact inhibition) or because changes in the medium (removal of mitogens) allow the cells to arrest while remaining viable. Normal cells under these conditions will arrest early in the GI phase of growth (sometimes referred to as G o ) ,while malignant cells may arrest at random when deprived of nutrients (Pardee, 1974; Pardee et al., 1978). At least in culture, contact inhibition of growth is a characteristic only of "normal" cells. The arrested cells can be stimulated to grow either by a crude mixture of components containing mitogenic molecules, such as serum, or preferably by one of several pure mitogenic polypeptides. Changes in ion fluxes, ion contents, and/or intracellular pH are then measured in these cells for appropriate periods of time. From such observations conclusions are drawn about the effect of mitogens on the particular parameters being measured. There are

26

LUIS REUSS ET AL.

several limitations of this general protocol which need to be considered. The first and most obvious one is that the use of relatively crude mixtures of molecules as mitogens, for example, serum, yields inherently ambiguous results inasmuch as the molecules responsible for the effect are not always known and their concentrations may differ in different serum samples. The precise conditions which have led to the arrest of cell growth are also important. Cells arrested for different periods of time or cells arrested by different protocols may have different metabolic characteristics. These variables have often not been controlled or have been impossible to control under the particular experimental conditions used. The metabolically important parameter that needs to be examined in cells is the change of concentration (and/or content) of ions, including hydrogen ion (pH), rather than the ion fluxes. What is often measured, however, is not the new steady-state intracellular concentration or content, but rather the flux. The limiting situation is that a change in ion flux across the membrane may result in no net change in the concentration of the ion, because of compensating activation of other ion transport mechanisms. The distinction between increased flux and increased concentration is often neglected. The major emphasis in this section will be placed on work in our own laboratory focusing on fibroblastic and epithelial cells grown in culture on a solid substratum, that is, either plastic dishes or glass chips. The results obtained by these studies have been made possible by the availability of two pure mitogenic polypeptides, epidermal growth factor (EGF) (for a review see Carpenter and Cohen, 1979) and platelet-derived growth factor (PDGF) (for a review see Heldin and Westermark, 1984). The availability of these pure growth factors plus a rapidly increasing knowledge of the molecular biology of their receptors makes them particularly appropriate for studying the very early steps by which signal transduction occurs across the plasma membrane when these mitogens bind to their receptors. Work in a number of laboratories has established that the receptors for both of these mitogenic polypeptides are protein kinases specific for tyrosine residues. There exists significant homology between PDGF and one of the viral transforming gene products, v-sis, and between the cytoplasmic domain of the epidermal growth factor receptor and the u-erb transforming gene as well as between transforming growth factor a and EGF (for review see Carpenter, 1984; Heldin and Westermark, 1984; Huang et al., 1984). These observations strongly suggest that malignant transformation and the attendant change on growth control can be brought about by the generation in the malignant cell of endogenous growth signals identical to those generated in normal cells as a result of the interaction of the cell with mitogens supplied in the growth medium.

27

1. MITOGENS AND ION FLUXES

B. Mltogens Increase Na+-H+ Exchange and Alter lntracellular pH

Many investigators, following the early observations in the laboratories of Rozengurt and Heppel (1975) and Koch and Leffert (1979), have observed increased flow of ions into cells associated with mitogen stimulation of these cells (Boonstra et al., 1981; Moolenaar et al., 1981; Mummery et al., 1981; Villereal, 1981; Schuldiner and Rozengurt, 1982; Cassel et al., 1983; Moolenaar et al., 1983; Owen and Villereal, 1983; Rothenberg et al., 1983a,b; L’Allemain et al., 1984). The relationship of these ionic fluxes to the mitogenic response has not been clearly established in any system. While much of the early work was carried out with ill-defined mitogens such as serum, recent work has used purified mitogens to demonstrate similar effects. An example of such a response is shown in Fig. 2. The increased sodium influx is electroneutral, that is, it does not necessarily result in a change in membrane potential as discussed in the previous section. In addition, it is sensitive to high concentrations of amiloride (Moolenaar et al., 1982, 1983; Cassel et al., 1983; Rothenberg et al.,

TIME ( m i n )

FIG.2. Effect of mitogens on Na’ influx in NR6 cells. NR6 cells are a derivative of 3T3 cells lacking the E G F receptor. They also adhere well to glass and respond mitogenically to platelet-derived growth factor (PDGF) and fetal calf serum (FCS). Rates of Na’ uptake in cells incubated with 1 m M ouabain to block Na+ extrusion are shown, in the absence of mitogen or as indicated after the addition of PDGF or FCS (for details, see Cassel er d., 1983).

28

LUIS REUSS ET AL.

1983b). The sensitivity to amiloride and the electroneutrality of the Na+ in flux suggest that it may be due to activation of Na+-H+ exchange. In most : > H,+. Since the Na+ gradient (or ApNa+) is cells, N%+ < Na,+ and H greater than the H+ gradient (or ApH+), activation of Na+-H+ exchange will bring sodium into the cells and protons out. A sensitive method for ascertaining the activation of Na+-H+ exchange would be the measurement of intracellular pH by methods which have good temporal resolution and high sensitivity, such as those described in the appendix. The work presented here is centered on the results of such measurements. Activation of Na+-H+ exchange results in an increased influx of Na+ and extrusion of protons; as a consequence pHi rises. The increased Na+ influx results secondarily in an increase in intracellular Ktas Na+ leaves the cell and Kt enters via the Na+,K+-ATPase. Ca2+influx is also stimulated by the addition of mitogens (see, e.g., Sawyer and Cohen, 1981), but activation of the Na+-H+ antiport and of Ca2+influx appear to be independent events in A 431 cells (Rothenberg et al., 1983a) and may be interdependent in other cells where Na+ entry may activate Ca2+ uptake via the Na+-Ca2+ antiport (see, e.g., Smith ef al., 1982). The addition of either EGF or PDGF to appropriately responsive cells results in cytoplasmic alkalinization; an example is shown in Fig. 3. The changes are stereotyped and the same kinetics are observed in all cells

[ i i

0 --J - - - - - - - - _

- --_

'

- -- - I

-_

- - -

,

lomin

FIG.3. Effect of PDGF and FCS on pHi in NR6 cells. pHi was measured using dimethylfluorescein linked to dextran (Cassel et al., 1983). Note the increase in pHi following addition of mitogen and the fact that the rise in pHi occurs only after a lag time.

1. MITOGENS AND ION FLUXES

29

and systems examined to date, that is, upon addition of EGF, PDGF, or serum to responsive cells, there is a short lag (1 to 2 minutes) followed by cytoplasmic alkalinization to a new steady-state value (Moolenaar et al., 1982, 1983; Cassel el al., 1983; Rothenberg et ul., 1983b; see also Table I11 and the references therein). The lag, which has been observed in at least two different laboratories and with different methods of pH, measurement, indicates that the activation of Na+-H+ exchange is unlikely to be the direct consequence of the interaction of mitogen with the receptor. It is much more likely that one or more chemical events, that is, enzymatic reactions, must take place before such activation, even though one cannot rule out with certainty that the stimulation of Na+-H+ exchange is the result of an unusually slow conformational change. The fact that a new steady-state pHi is reached suggests that the cells compensate for this change in ion fluxes. The precise mechanism by which the new steady state is controlled has not yet been determined. Note that all the experiments described in this section, unless otherwise indicated, are carried out in the nominal absence of bicarbonate. Additional pH regulatory mechanisms come into play when bicarbonate is present; for example, a Na+-dependent CI--HCO; exchange system appears to be present in A 431 cells (Rothenberg et al., 1983a). The lag represents a real set of events and not a methodological artifact. If the function of the Na+-H+ exchanger is prevented either by allowing the cell to interact with the mitogen in the absence of external Na+ or in the presence of amiloride, and then Na+ is restored or the inhibitor is removed, the lag is abolished and, in fact, an overshoot occurs. These results indicate that full activation of Na+-H+ exchanger has taken place (Fig. 4) under conditions in which it cannot function, suggesting the operation of control mechanisms of pHi and/or ion content or concentration. Table 111 shows a partial list of measurements of intracellular pH. These methods have been used to determine whether addition of mitogens to cells, and the consequent activation of Na+-H+ exchange (see Section V), results in a change (alkalinization) of pHi. It is comforting that the same results have been obtained by different methods in similar if not identical cell types. Since the errors inherent in each of these methods and the technical problems associated with them are likely to be different, they reinforce the basic conclusion that mitogen addition to some cells results in an alteration of pH, as a consequence of activation of Na+-H' exchange. Mitogens also alter the rate of other ionic fluxes into cells. For example, Ca2+entry has been shown to be increased as a result of the addition of mitogens to cells (Sawyer and Cohen, 1981). In addition, data implicating the release of calcium from intracellular stores into the cytoplasm, based

30

LUIS REUSS ET AL.

-0.1L

M

FIG.4. Effect of amiloride on PDGF-induced alkalinization. NR6 cells were incubated with PDGF or FCS as in Fig. 3 , but in the presence of amiloride. Under these conditions no alkalinization is observed. Upon removal of amiloride there is a rapid alkalinization with no lag and pHi overshoots (compare Fig. 3 ) . After about 10 minutes, pHi returns to a value similar to that seen in Fig. 3 (for details, see Cassel et a/., 1983).

on experiments with the calcium-sensitive fluorescent dye quin-2, have been reported (Moolenaar et al., 1984a; Mix et al., 1984). These experiments suggest that calcium entry may precede the exit of protons from the cells, but a causal relationship between the two events has not been proven. Mitogen activation of Ca2+and Na+ influx are totally independent events (Rothenberg et al., 1983a) (for discussion, see Section 111). While Na+ influx into cells is usually measured in the presence of an inhibitor of Na+,K+-ATPase,for example, ouabain, in order to prevent the exit of sodium from the cell and its exchange for potassium, under physiological conditions the ATPase is active so that the increased Na+ entry will be followed by active Na+ extrusion in exchange for K+. We are then faced with the possibility that an increase in intracellular K+, an increase in intracellular Na+, an increase in intracellular pH, or independently the increase in intracellular Ca2+ may all, singly or together, be responsible for some of the initial events which drive the cells through the cell cycle. It is fair to say that at the present time no causal relationship of

TABLE 111 CHANGES IN INTRACELLULAR pH INDUCED BY MITOGENS" Cell line A 431 Human epidermoid carcinoma

NR6 Mouse fibroblasts

Human foreskin fibroblasts Chinese hamster ovary cells 3T3 Mouse fibroblasts

Mitogen

Method

APHi

EGF Serum PMA PDGF Serum PMA EGF Serum Thrombin

0.2 0.2 0.1 0.15 0.15 0.15 0.1 0.1 0.2

E E E E E E

Phorboldibutyrate PDGF Serum

0.08 0.18 0. I8

DMO

c

C + microelectrodes Benzoic acid

References Rothenberg et al. (1983b) Whiteley el a / . (1984) Cassel er 01. (1983) Whiteley et a / . (1985) Moolenaar el al. (1983) Paris and Pouyssegur (1984); L'Allemain er al. (1984b) Schuldiner and Rozengurt (1982); Bums and Rozengurt (1983)

a The table lists the magnitude of ApHi observed upon addition of mitogen to cells. Except for foreskin fibroblasts, all measurements are at 37°C. Methods indicated by letters refer to Fig. 9. The maximum steady-state change in pH is indicated rather than the maximum initial change where an overshoot may take place. PMA, phorbol 12-mynstate 13-acetate.

32

LUIS REUSS ET AL.

any of these events to cell division can be established with certainty. Inhibition of Na+-H+ exchange by amiloride or amiloride analogs, which inhibits cell growth, cannot be interpreted to indicate that Na+-H+ exchange is specific for cell growth, since amiloride as well as its more potent analogs all have some toxicity (see, e.g., Zhuang et al., 1984; L’Allemain et al., 1984a), that is, they inhibit other metabolic processes so that long-term experiments using amiloride to inhibit cell growth are not easily interpretable (L’Allemain et al., 1984a’b). Similarly, incubation of cells in a medium containing low Na+ generally blocks cell growth and growth of cells in a medium containing low Na+ is more sensitive to inhibition by amiloride (or amiloride analogs) since amiloride is competitive with Na+, These observations cannot be used to correlate activation of Na+-H+ exchange with cell growth, because exposure of cells to low Na+ (50 mM) is likely to have pleotropic effects on cell metabolism. Recently, a mutant has been isolated which appears to lack the Na+H+ exchange and yet can grow, provided that the extracellular medium is maintained at an alkaline pH in the presence of bicarbonate ions (Pouys6ggur et al., 1984). These observations suggest that an alkaline intracellular pH is permissive for cell growth but is not by itself adequate to drive the cells through the cell cycle, since these cells still require mitogen to grow even when bicarbonate buffers are used to raise pHi. The precise role of changes in intracellular concentrations of K+ and Ca2+,either transiently or permanently in response to mitogens, remains to be determined. The changes in pHi observed upon addition of mitogens to cells are small, -0.2 pH units, and the metabolic reactions that are affected by these small changes in pH are not known. It is clear, however, that both simple enzymatic reactions, for example, phosphofructokinase (Trivedi and Danforth, 1966; Pettigrew and Frieden, 1979), and complex enzymatic pathways such as gluconeogenesis (Kashiwagura et al., 1984) can be affected in an all or none manner by pH changes of this order of magnitude. C. Mechanism of Activation of Na+-H+ Exchange

It is tempting to speculate that activation of Na+-H+ exchange by mitogens is a consequence of the activation of the known enzymatic activity of the mitogen receptor, namely, the tyrosine-specific protein kinase activity. No direct evidence for this idea is presently available, but a number of indirect observations agree with this hypothesis. The first is the observation that vanadate (Cassel et al., 1984), a known inhibitor of tyrosine phosphate phosphatase activity (Swarup et al., 1982), also stimu-

1. MITOGENS AND ION FLUXES

33

lates Na+-H+ exchange and has also been shown under certain conditions to be mitogenic (Carpenter, 1981). The simplest, but certainly not the only interpretation of the experiments using this relatively nonspecific inhibitor, is that vanadate acts by blocking the dephosphorylation of protein or proteins containing phosphotyrosine, and therefore has the same metabolic consequences as activation of protein kinases specific for tyrosine phosphorylation. The kinetics of activation of Na+-H+ exchange by vanadate are identical to those observed upon addition of mitogens to cells. Thus the activation of Na+-H+ exchange by vanadate is in a temporal sense no closer to the Na+-H+ exchange molecule than that by activation by the mitogen receptor. The molecular mechanisms of the activation of Na+-H+ exchange are not known. In addition to stimulation by mitogens and by low pHi in some cells, two mechanisms of activation of Na+-H+ exchange have been described which appear to be more proximal to the transporter than the activation by mitogens. First is the activation by high intracellular sodium. Na+-H+ exchange is activated in fibroblastic or epithelial cells in culture when intracellular Na+ is elevated either by addition of ouabain or by incubation of the cells in a K+-free medium, without significant changes in pHi. The second mechanism of activation of Na+-H+ exchange in these cells resembles that seen in Amphiuma erythrocytes (Cala, 1983a,b) and human lymphocytes (Grinstein et al., 1982, 1983a,b) as a part of the volume regulatory response. When A431 or NR6 cells are exposed to a hypertonic medium, Na+-Ht exchange is activated, even though in cells examined to date this does not result in an increase in cell volume (Cassel et al., 1985) (see also Section 11). This activation appears to be more proximal to the Na+-H' exchanger than the mitogen-dependent activation. Mitogens do not activate Na+-H+ exchange indirectly by changing cell volume. No volume changes are observed upon addition of mitogens to A431 cells when measured with 3-O-methyl-~-glucose(Cassel et al., 1985) over a 30-minute period. The most compelling evidence to suggest that the tyrosine kinase activity of the mitogen receptor is involved in the activation of Na+-H+ exchange arises from experiments in the regulation of the mitogenic response by protein kinase C, as discussed in the next section. V.

MODULATION OF THE MITOGENIC RESPONSE BY PROTEIN KINASE C

Phorbol esters are biological active molecules which in a variety of systems have been shown to act as tumor promoters (Mastro, 1983) and

34

LUIS REUSS ET AL.

when added to cells in tissue culture under some conditions influence cell growth either by acting as mitogens or by potentiating the mitogenic effect of other compounds but in some cases also induce cell differentiation and inhibit cell growth. A major advance in our understanding of how phorbol esters may exert these effects is the discovery that they interact with protein kinase C because they are structural analogs of diacylglycerol (for review, see Nishizuka, 1983a). Protein kinase C exists as a soluble and inactive molecule in the cell. Upon addition of diacylglycerols and negatively charged phospholipids and with appropriate calcium concentrations, protein kinase C binds to the plasma membrane, is activated, and phosphorylates various proteins on threonine or serine residues. Activation of protein kinase C is presumed to be the major cellular effect of phorbol esters. During studies of phorbol esters as potential activators of Na+-H+ exchange, we noted that activation of this antiport by mitogens such as EGF was, in fact, blocked by phorbol esters (Fig. 5 ) (Whiteley et al., 1984). Only biologically active phorbol esters, that is, those that can interact with protein kinase C, show this activity. For example, phorbol diacetate, which is not a tumor promoter, does not alter the mitogenic response of the Na+-H+ exchanger. A possible explanation for these results comes from the observation that protein kinase C phosphorylates the EGF receptor on threonine residues (Cochet et al., 1984; Iwashita and Fox, 1984; Davis and Czech, 1984; Hunter et al., 1984; Friedman et al., 1984). This modified form of the EGF receptor has diminished tyrosine-specific protein kinase activity. Thus, the blockage by phorbol esters of the mitogen-dependent activation of Na+-H+ exchange is most readily interpreted as a consequence of the diminished tyrosine-specific protein kinase activity of the EGF receptor. The apparent discrepancy between these observations and the observation that phorbol esters also act as mitogenic compounds or potentiate mitogenic action is documented by the fact that at higher concentrations and in the presence of extracellular calcium, phorbol esters can mimic the action of mitogens by activating Na+-H+ exchange. Observations in other laboratories have shown that phorbol esters can increase the cellular content of tyrosine phosphate residues (Gilmore and Martin, 1983; Bishop et al., 1983; Cooper et al., 1984; Grunberger et al., 19841, that is, that activation of protein kinase C can indirectly cause the activation of one or more as yet undetermined tyrosine kinases or the inhibition of tyrosine specific phosphatases, thus potentially mimicking the effect of mitogenic polypeptides and therefore the activation of Na+-H+ exchange. Phorbol esters, and by inference activation of protein kinase C, may

1. MITOGENS AND ION FLUXES

35

FIG.5 . Effect of phorbol myristic acetate (PMA) on the activation of Na'lH' exchange of A 431 cells. In each panel A-E, the activation of Na+-H+ exchange measured by cytoplasmic alkalinization using dimethylfluorescein dextran is shown, either in control cells or M PMA for 30 minutes. Note that PMA abolishes the in cells preincubated with cytoplasmic alkalinization due to addition of EGF, serum, and vanadate, the first two being potential activators of tyrosine-specific kinases and vanadate being an inhibitor of tyrosine phosphate phosphatases. PMA has no effect on the alkalinization induced by exposure of cells to hypertonic medium (rnannitol).

also act as negative modulators of the activity of other cell surface receptors, similar to the down regulation of 0-adrenergic receptor by activation of protein kinase C (Sibley et al., 1984; Kelleher et al., 1984) and the inhibition of the glucagon-stimulated adenylcyclase (Heyworth et al., 1984). It is tempting to generalize from these observations to all cellsurface hormone or mitogen receptors, but such conclusions may be premature. Figure 6 presents a hypothetical diagram of how these effects may take place. We suggest that the major action of protein kinase C may be as a negative control of the mitogenic response. Preliminary observations in at least two laboratories have suggested that oncogenes that have tyrosine kinase activity also phosphorylate phosphatidylinositol to di- and triphosphoinositol and that they can also phosphorylate diacylglycerol to phos-

36

LUIS REUSS ET AL.

in

out

tza=EGF

pty; phosphotyrosine

yh,rphorphothreonine €18

phorbol esters or diacylglycerol

FIG.6. Hypothetical diagram for modulation of EGF-dependent alkalinization by PMA. Reaction 1 is the binding of EGF to its receptor. It results in a conformational change on the cytoplasmic side of the receptor, thereby activating the tyrosine kinase activity of the receptor. Reaction 2 is the hypothetical phosphorylation of a control molecule by the EGF receptor, which results in activation of Na+-H+ exchange. The regulatory protein binds protons better when phosphorylated, and protonation of one or more residues results in dissociation of the protein from the Na+-H+ exchanger (see Fig. 8). Reaction 3 involves a tyrosine phosphate phosphatase, which is inhibited by vanadate and effectively reverses the effect of the kinase. Addition of vanadate would be expected to mimic the action of mitogenic polypeptides (see, e.g., Cassel er al., 1984). Reaction 4 is the hydrolysis of tyrosine phosphate on this protein by a phosphatase (inhibitable by vanadate); as a result the pK, of the protein would shift, protons would dissociate, and it would again be able to bind to the Na+-H+ exchange protein. Na+, which enters the cell as a result of activation of Na+-H+ exchange, will exit via the Na+,K+-ATPase,thereby raising intracellular K+levels. Reaction 5 is the binding of PMA (an analog of diacylglycerol), which binds to protein kinase C. As a result of the binding of PMA, protein kinase C binds to the cytoplasmic membrane and is now active and can phosphorylate the EGF receptor on a threonine residue (reaction 6). This last reaction inactivates the tyrosine kinase activity of the EGF receptor and thereby defines a potential feedback loop for the response of the cell to mitogens since mitogens are presumed to increase the level of diacylglycerol in the cell.

1. MITOGENS AND ION FLUXES

37

phatidic acid (Sugimoto et al., 1984; Macara et a / . , 1984). This is apparently a paradoxical effect, inasmuch as the first reaction, the generation of triphosphoinositol, is currently believed to result in a release of Ca2+from intracellular stores (Berridge, 1984); the resulting rise in cytosolic Ca2+ should active protein kinase C. Diacylglycerol is also an activator of protein kinase C, and thus high Ca2+and diacylglycerol should act synergistically. One possible interpretation of these observations is that the purpose of this dual kinase activity is to prevent activation of protein kinase C and therefore to prevent an inhibition of the mitogenic response. The major effect of these two kinase activities would be to raise cytoplasmic Ca2+without activating protein kinase C, thereby allowing some of the mitogenic effects to take place. This is contrary to the view (Berridge, 1984) that mitogens act by activation of protein kinase C. Only detailed analysis of the cellular content of these various ligands will provide an appropriate test of these hypotheses. VI.

SUMMARY AND PERSPECTIVES

Many major questions remain open regarding the regulation of ion fluxes by mitogens and the role that such ion fluxes have on the mitogenic response. Inhibitors are powerful experimental tools in the study of membrane transport processes. However, two kinds of problems are frequently faced when employing these agents. The first is related to lack of specificity of some of these agents. Examples particularly relevant to this topic are the inhibitory effects of amiloride on the Na+-K+ pump (Soltoff and Mandel, 1983) and protein synthesis (Lubin et al., 1982; Fehlman et al., 1981) and the effects of loop diuretics on anion exchange (Brazy and Gunn, 1976). Disulfonic stilbenes, in addition, inhibit not only anion exchange but also C1-independent Na-HC03 cotransport (Boron and Boulpaep, 1983) and HCO; transport (Burckhardt et ul., 1984). The second problem related to the use of transport inhibitors is the lack of consideration of secondary effects. Exposure of cells to cardiac glycosides is a good example: ouabain treatment not only inhibits the Na+,K+-ATPase but in most cells will eventually cause alterations in Na+ and K+ contents, membrane voltage, intracellular pH, intracellular Ca2+activity, cell volume, and probably other parameters. Some of these effects may, by themselves, cause activation or inactivation of specific transport processes. Careful experimental design and adequate controls are required in such studies. Genetic manipulation of cells in culture, that is, selection of mutants defective in one or more transporters involved in the mitogenic response,

38

LUIS REUSS ET AL.

is a new and exciting avenue that will be explored with increasing success. The multiplicity of mechanisms of ion transport makes difficult the design of useful experiments to be applied to a particular cell type unless the transport pathways of that cell under control conditions are well known. Extrapolations are frequently not justified, that is, the presence of a transporter of given properties in a cell does not indicate its presence or the same characteristics in another cell. Multiple mechanisms of transport for a particular ion complicate the issue further. For example, stimulation of K+ efflux, in the absence of primary changes in chemical or electrical gradients, can be the result of an increased electrodiffusive K+ permeability, stimulation of K+-H+ exchange, increased KCl cotransport, or increased NaKC12 cotransport. Dissection of these possibilities is a must. The conclusion of, for example, an “increased K+ permeability” does not constitute an explanation. Transport regulation varies not only from cell to cell, as illustrated by the widely diverse effects of elevations of intracellular CAMP levels, but also under different transporting conditions. Seemingly minor changes in ionic composition of the incubation media can result in profound changes in transport. Mitogen-cell interactions are highly complex and subject to both positive and negative regulation. The understanding of these systems in detail will require the ability to generate in uitro systems in which, by the tried and true biochemical process of taking things apart and putting them together again, it will be possible to ascertain which components are involved and the molecular basis of their interaction. Figure 7 illustrates, for example, the fact that it is now possible to obtain membrane vesicles which show Na+-H+ exchange activity from cells which are mitogen responsive. It is comforting that the characteristics of Na+-H+ exchange seen in this preparation are similar to those observed in well-characterized vesicles, such as those obtained from kidney cortex (Aronson et al., 1982, 1983; see also Vigne et al., 1984b). The most interesting property, which is illustrated in Fig. 7, is that Na+-H+ exchange in these vesicles, as in the renal tubule vesicles, is activated allosterically by low intravesicular pH. The apparent midpoint of this activation is at pH 6.4 to 6.6, which by comparison to data obtained in whole cells suggests that the Na+-H+ exchanger in these vesicles is in an inactive form. One of the mechanisms, perhaps not the only one, by which activation of Na+-H+ exchange may take place is by shifting its pHi sensitivity as a result of mitogen action (Paris and Pouyssegur, 1984). Many of the observations presented in this chapter are summarized in Figs. 6 and 8. The reader should be cautioned that this is a simplified

39

1. MITOGENS AND ION FLUXES

\ pH, 5%- KR-64Z-M$K87- 71pn.119 14 763 787 807 832

\

pH GRADIENT

\

-Al

1 1

I

I

I

I

I

pH.5.96- 6.18- 6.42- 6.65- 6.87- 71pHb 7.19 74 7.63 7.07 8.07 8.32 pH GRADIENT

FIG.7 . Na+-H+ exchange in plasma membrane vesicles from A 431 cells. Plasma membrane vesicles from A 431 cells were prepared as described. These vesicles take up *?Na+in response to a pH gradient (interior acid), and this pH-dependent uptake is amiloride sensitive. The figures illustrate that activation of Na+-H' exchange requires protonation of a regulatory group(s), with apparent pK around 6.5. In this experiment the magnitude of the proton gradient is constant but the absolute pH is varied on both sides of the vesicle membrane. Na+-H+ exchange in the presence of a constant proton gradient is maximally active when the pH in the vesicle interior is in the range where this group is protonated. 0 , Na+ uptake in vesicles; 0 , uptake in the presence of amiloride. The insert shows net Na+ uptake after subtraction of the amiloride-sensitive component. This behavior is characteristic of the Na+-H+ exchanger, which has not been activated by mitogen stimulation (Paris and Pouyssegurt, 1984). (For experimental details, see Mancuso and Glaser. 1985.)

view, and that its purpose is not to define the various steps in molecular terms but simply to present a graphic summary of the various results and hypotheses described in this chapter. We note that positive and negative control mechanisms exist at the level of the mitogen in this system, and that multiple controls are available to control Na+-H+ exchange. The task for the future is to try to define in real terms the components presented in this diagram. While some of them are by now reasonably well understood, such as the mitogen receptor and protein kinase C , others are still only understood at the level of activities which can be measured in the laboratory; their molecular basis remain to be elucidated.

40

LUIS REUSS ET AL. IN PH 7.0 l2OmMNa'

pH 6.0 IOmMNa'

IN

OUT

pH 7.0

pH 7.0

Na'

OUT

Na'

FIG.8. Summary diagram of the activation of Na+-H+ exchange. This diagram serves primarily as a visual summary of the experiments detailed in the text and should not be interpreted literally. It illustrates, using the symbols in Fig. 6, the activation of Na+-H+ exchange by high intracellular Na+, low intracellular pH, or (not shown) phosphorylation (Fig. 6) of a regulatory protein that can interact with protons or Na+ and whose affinity for protons is modulated by phosphorylation.

VII.

APPENDIX: MEASUREMENT OF INTRACELLULAR pH

There are a number of methods that have been used to measure intracellular pH. Since many of these have been described in detail elsewhere (Roos and Boron, 1981), in this section we will focus only on newer developments in the measurement of intracellular pH, comparing the advantages and disadvantages of these methods as applied to cells in culture. A. lntracellular pH Microelectrodes

This method has the advantage of an absolute determination of pHi. Only recently has it been applied with considerable difficulty to cells such as fibroblasts. Its use requires experimental tools and skills available in only a few laboratories (Moolenaar et al., 1984b).

1. MITOGENS AND ION FLUXES

41

B. NMR

This method makes use of the pH-dependent shift of the resonance of inorganic phosphate to assess pHi. At the present time, it is relatively insensitive. Therefore, the measurements are obtained by averaging signals over a significant period of time and using cell densities comparable to those observed in tissues or organs (see, e.g., Prichard er al., 1983). C. Distribution of Amphipatic Molecules in Cells

This method takes advantage of the fact that organic molecules which are sufficiently amphipatic to cross the plasma membrane and which have appropriate ionization constants, that is, either weak organic acids or weak organic bases, distribute across the membrane reflecting the ratio of intracellular pH to extracellular pH. The time resolution of this method is usually of the order of minutes and the intracellular distribution of the molecules is not always known with certainty. Therefore, the calculated pHi may reflect a composite of the pH values in various intracellular compartments. Radioactively labeled 5,5-dimethyloxazolidine-2,4-dione (DMO) has been frequently used, as has benzoic acid. This method is technically simple but has the disadvantage of being destructive, that is, the cells have to be separated from the medium and their radioactivity analyzed separately for each measurement. D. Optical Methods

These methods allow continuous monitoring of pH, with excellent temporal resolution, ideally without altering cellular metabolism, but they also suffer from some ambiguity in the location of the dye within the cell. The sensitivity is greater if fluorescence, rather than absorbance, is the method of signal detection. Introduction of dye into the cell should as far as possible be via a unidirectional process whereby the probe is trapped within the cell. This can be achieved in two ways: first, by using dye derivatives which are highly hydrophobic (i.e., rapidly permeant) but inside the cell are hydrolyzed usually by esterases; the hydrolysis products, which are ionized, are less hydrophobic and hence less permeant. In the extreme, the dye would be permanently trapped in the cytoplasm. This technique, originally developed by Thomas et al. (1979), has proven useful in a number of cells. The applicability of any dye to any given cell, however, must be investigated. The structures of some of these recently developed pH-

42

LUIS REUSS ET AL.

sensitive dyes are shown in Fig. 9. The most useful ones appear to be those recently synthesized by Tsien and co-workers (Tsien, 1983a). Before any dye can be used for the determination of pHi in a given cell, the following parameters must be considered. 1 . LEAKAGE RATE

If the dye leaks from the cells rapidly, for example, fluorescein derivatives in certain fibroblasts (Rothenberg et al., 1983a), it becomes necessary to remove external dye continuously by changing the medium. The leakage rate appears to be temperature dependent, at least in some cells (Moolenaar ef al., 1983). Working at 25°C rather than at 37°C may slow down the leakage rate, but the extrapolation of observed changes in pHi to physiological conditions at 37°C is not necessarily valid.

C

A c

H

,

-

L

o c =- o ~

~

-

II

c

H

, HOIC

7 \

CO,H

CO,H

/

7 \

0

II

COiH

2CH,-COH

CO,H

BEECF

corboxyfluorescein diocetate

bis(corboxy ethy1)carboxyfluorescein

D COtH C02H

no 4-methylumbelliferone CH’: COzH CO,H

Quin I

8

- [ bis(ethoxycarbony1methyl)ornmo]6methoxy-Z-[trcns-Z [bis (ethoxycarbonyl methyl)omino] styr y I] qui no Ii ne

E H

O

e

6’””

dimethyl fluorescein

FIG. 9. Examples of fluorescent dyes that have been used to measure cytoplasmic pH. Listed below is the pKa of each dye and selected references which describe their use in the measurement of pH,. Note that compound A becomes a pH probe only after the acetyl groups have been removed by cytoplasmic esterases. The pKa listed is for the hydrolysis product. Compound E is used covalently linked to dextran and the pKa is for that compound. (A) pK, = 6.1 (Thomas er a / . , 1979); (B) pK, = 6.97 (Tsien, 1983a); (C) pK, = 7.3 (Rogers e t a / . , 1983); (D) pK, = 7.8 (Gerson and Kiefer, 1982); (E) pK, = 6.75 (Rothenberg ef al., 1983b).

43

1. MITOGENS AND ION FLUXES

2. CELLULAR LOCATION OF

THE

DYE

It is assumed that the dye in the cell remains free, that is, it is not bound to intracellular structures. To the extent that the dye is bound to intracelMar components, its ionization constant and optical properties may change, and hence the calculated pHi could be substantially incorrect. For example, in A 431 cells, carboxymethylfluorescein has been found to bind to the cell nucleus (Rothenberg et al., 1983a).Shortly after introduction of the dye into the cell, only a small fraction of the dye is bound to the nucleus, but as the free dye leaks out of the cell an increasingly larger fraction of the intracellular dye is bound. The bound dye has an unknown pK,, and therefore pHi could not be determined in these cells using this dye. In all cases it is desirable to confirm the cytoplasmic localization of the dye at least by examining the cells by fluorescence microscopy. Two apparently unrelated observations have been combined recently to develop a new method for the measurement of cytoplasmic pH. The pH of endocytic vesicles has been measured by coupling fluorescein to dextran (Okhuma and Poole, 1978). If cells are incubated in a medium containing fluorescein linked to dextran, these molecules will be taken up nonspecifically from the medium by fluid pinocytosis. The dye coupled to dextran remains in the endocytic vesicles since the dextran will not cross the vesicle membrane and the fluorescein serves as a reporter group for intravesicular pH. The same principle could be applied to the measurement of pHi (i.e., cytoplasmic pH) if a mechanism for introducing macromolecules into cells could be devised. Microinjection, while feasible, is tedious and only allows measurement on a limited number of cells using quantitative microscopy. Okada and Rechsteiner (1982) have developed a method for introducing macromolecules into the cytoplasm by osmotic shock which can be used to introduce fluorescent dyes coupled to dextran into the cytoplasm. This system, shown in outline in Fig. 10, has been used to introduce fluorescein or its derivatives into the cytoplasm. Fluoresceindextran is not suitable for the measurement of pHi because its pK, is too low (6.1), but dimethylfluorescein, as well as other dyes of higher pK,, can be used to measure pH,. The method illustrated in Fig. 10, whereby a fluorescent dye linked to dextran is introduced into cells by an osmotic shock method, has several advantages. First, it has the excellent temporal resolution associated with optical measurements. Second, it has the high sensitivity associated with fluorescent measurements and can therefore be used on a small number of cells growing as a monolayer in the area illuminated by the beam of a conventional fluorimeter and can be potentially adapted as a microscopic method to examine the fluorescence and therefore pHi of single cells.

. . .. . . . . . . FIG. 10. Introduction of fluorescein-dextran to cells. The method is based on that of Okada and Rechsteiner (1982). Cells are allowed to take up macromolecules by fluid pinocytosis from the growth medium under hypertonic conditions for a short period of time (5-10 minutes). Subsequent exposure of cells to a hypotonic medium results in rupture of endocytic vesicles and release of their contents into the cytoplasm. The cells remain viable and recover from this treatment. Dyes linked to dextran are permanently trapped in the cytoplasm, and if they have an appropriate pK., as, for example, does dimethylfluorescein, they can be used to measure cytoplasmic pH as diagrammed in the bottom of the figure (for details, see Rothenberg ef al., 1983a,b).

1. MITOGENS AND ION FLUXES

45

Third and most importantly, this method obviates the difficulties associated with dye leakage from the cells. Cells loaded with dextran derivatives appear to be morphologically normal-they divide and retain the dye for prolonged periods of time. Although the method has so far only been used for relatively short periods of time-hours, not days-there is no a priori reason to assume that it cannot be used to follow pHi over periods of days. The method can be limited by the capacity of other cell types to withstand the initial conditions required to introduce the probe. Alternative methods for introducing macromolecules into cells being developed for other purposes in various laboratories may solve this problem (see, e.g., McNeil et al., 1984). The second limitation is that a small but potentially significant fraction of the dye may be located in lysosomes or other acidic vesicles. If the pH of these vesicles is below 5.5, their contribution to cellular fluorescence is negligible. However, if their pH becomes more alkaline, for example, during calibration, the intravesicular dye may contribute to the observed fluorescence and introduce some uncertainty into the pHi measurements. Since this fraction is very difficult to estimate with precision, it seems likely that measurements of intracellular pH by this method are much more precise as relative measurements rather than as absolute measurements. For any given experimental perturbation, for example, addition of mitogen to cells, it is important to ascertain directly whether intravesicular pH has been altered. This can be done by introducing fluorescein-dextran into endocytic vesicles, including lysosomes, to monitor intravesicular pH. Fluorescein has an isosbestic point so that the pH of the fluoresceincontaining solution can be determined by the ratio of fluorescence after excitation at two different wavelengths. Dyes with more appropriate p&’s for measurement of pHi do not have an isosbestic point, and fluorescence intensity is measured in order to quantitate pH. This introduces a small uncertainty in the measurement of intracellular pH, which can in principle be resolved by the use of two dyes, one pH sensitive and the other one pH insensitive, introduced into the cells simultaneously. The calibration of the optical signal involves the use of either ionophores to equilibrate internal and external pH (Thomas et al., 1979) or of full activation of Na+-H+ exchange for the same purpose. The former method is extremely versatile, but it has the disadvantage that it also allows equilibration across internal vesicles. In contrast, activation of Na+-H+ exchange by raising cytoplasmic Na+ will result in equilibration of cytoplasmic and external pH, and therefore it appears to yield a better calibration of the optical signal. To raise intracellular Na+, cells are

A

B

1 - 11

I

I

w

-1 IA

3. V

3.01

1

5

10

15 20 25 TIME (minutes)

" 3

FIG. Changes in intracellular I: in cells equilibrz :d with extracellular Na+. A 431 cells were fully equilibrated with extracellular Na+ by incubation in the presence of I mM ouabain for 2 hours. Under these conditions Na+-H+ exchange is fully activated. The cells had previously been loaded with fluorescein-dextran (pK, 6.1), as illustrated in Fig. 2. Decrease in fluorescein ratio denotes acidification (for details, see Rothenberg et al., 1983a). Reduction of external Na' in steps results in rapid acidification (panel A) which is reversible and inhibited by amiloride, as shown in panel B. Note in panel A that if cells remain in low Na+ for a prolonged period, there is a slow alkalinization, which presumably reflects passive proton transport across the cytoplasmic membrane.

47

1. MITOGENS AND ION FLUXES

a;2F":GF Acetate

6.8 I

-

Control

66 6.5-

6.4-

I

1

I

MINUTES

FIG. 12. Acidification of A 431 cells by a permeant anion. In the experiment shown, cytoplasmic pH was measured in A 431 cells with dimethylfluorescein-dextran. Where indicated, the medium was replaced with a medium containing 25 mM sodium acetate under isotonic conditions. CH3COOH enters rapidly and acidifies the cell. This is followed by recovery to the original pH. Both EGF and vanadate enhance the rate of recovery and amiloride inhibits the recovery (data not shown), as is discussed in Section IV (Cassel ef al., 1984).

treated with ouabain or incubated in K+-free medium. Both procedures inhibit the Na+,K+-ATPase. Figure 11 illustrates the effects of altering external Na+ concentration ("a+],) on pH, in cells with high intracellular Na+. Amiloride is used to show that the changes in pHi under these conditions are due to Na+-H+ exchange (see below). When "a+], is decreased, the cells become more acid. When "a+], is raised to the control value, the reverse pHi change takes place. Both pHi changes are amiloride sensitive (Rothenberg et al., 1983a). Figure 12 shows a classical acid-loading experiment. When cells are exposed to a medium containing 25 mM acetate, pHi falls because of the influx of undissociated acetic acid, which enters the cell rapidly and dissociates, releasing protons. This initial acidification is followed by a recovery phase. This is a classical experiment in cellular acid-base balance (Roos and Boron, 1981) which is reproduced here to illustrate that pHi measurements using dimethylfluorescein linked to dextran yield predictable results under well-defined conditions. Results obtained by the use of this dye in studying the action of mitogens in intracellular pH are described in Section V. ACKNOWLEDGMENTS

Work in the authors' laboratories has been supported by Grants GM 18405 and AM 19580 from the National Institutes of Health as well as by a grant from Monsanto Chemical Company. P. R. was supported by Grant GM 02016, B. W. by Grant GM 07067, and D. M. by Grant CA 091 18.

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Reuss, L. (1983). Basolateral KCI co-transport in a NaC1-absorbing epithelium. Nature (London) 304,723-726. Reuss, L., and Petersen, K.-U. (1985). Cyclic AMP inhibits Na+/H+exchange at the apical membrane of Necfurus gallbladder epithelium. J . Gen. Physiol., 85, 409-429. Rogers, J., Hesketh, T. R., Smith, G. A., and Metcalfe, J. C. (1983). lntracellular pH of stimulated thymocytes measured with a new fluorescent indicator. J. Eiol. Chem. 258, 5994-5997. Roos, A., and Boron, W. F. (1981). Intracellular pH. Physiol. Rev. 61, 296-434. Rothenberg, P., Reuss, L., and Glaser, L. (1982). Serum and epidermal growth factor transiently depolarize quiescent BSC-I epithelial cells. Proc. Narl. Acad. Sci. U . S . A . 79, 7783-7787. Rothenberg, P., Glaser, L., Schlesinger, P., and Cassel, D. (1983a). Epidermal growth factor stimulates amiloride sensitive Na’ uptake in A431 cells. J . Eiol. Chem. 258, 4883-4889. Rothenberg, P., Glaser, L., Schlesinger, P., and Cassel, D. (1983b). Activation of Na’/H+ exchange by epidermal growth factor elevates intracellular pH in A431 cells. J . Eiol. Chem. 258, 12644-12653. Rozengurt, E., and Heppel, L. A. (1975). Serum rapidly stimulates ouabain-sensitive 86Rb’ influx in quiescent 3T3 cells. Proc. Natl. Acad. Sci. U . S . A . 72, 4492-4495. Sachs, G., Faller, L. D., and Rabon, E. (1982). Proton/hydroxyl transport in gastric and intestinal epithelia. J . Membr. Eiol. 64, 123-135. Sacktor, B. (1977). Transport in membrane vesicles isolated from mammalian kidney and intestine. In “Current Topics in Bioenergetics” (R. Sanadi, ed.), pp. 39-82. Academic Press, New York. Sakmann, B., and Neher, E. (1984). Patch clamp techniques for studying ionic channels in excitable membranes. Annu. Rev. Physiol. 46, 455-472. Sawyer, S. T., and Cohen, S. (1981). Enhancement of calcium uptake and phosphatidylinositol turnover by epidermal growth factor in A431 cells. Biochemistry 20, 6280-6286. Scarpa, A. (1979). Transport across mitochondria1 membranes. Membr. Transp. Eiol. 11, 263-355. Schatzmann, H. J. (1983). The red cell calcium pump. Annu. Rev. Physiol. 45, 303-312. Schuldiner, S., and Rozengurt, E. (1982). Na+/H+ antiport in Swiss 3T3 cells. Mitogenic stimulation leads to cytoplasmic alkalinization. Proc. N a f l .Acad. Sci. U . S . A .79,77787782. Schultz, S. G. (1980). “Basic Principles of Membrane Transport.” Cambridge Univ. Press, London and New York. Schwarz, W., and Passow, H. (1983). Ca2+-activated K’ channels in erythrocytes and excitable cells. Annu. Rev. Physiol. 45, 359-374. Sibley, D. R., Nambi, P., Peters, J. R., and Lefkowitz, R. J. (1984). Phorbol esters promote 0-adrenergic receptor phosphorylation and adenylate cyclase desensitization in chick erythrocytes. Eiochem. Eiophys. Res. Commun. 121, 973-979. Smith, J. B., and Rozengurt, E . (1978). Serum stimulates the N a + , K +pump in quiescent fibroblasts by increasing Na+ entry. Proc. Natl. Acad. Sci. U . S . A . 75, 5560-5564. Smith, R. L., Macara, I. G., Levinson, R., Hausman, D., and Cantley. L. (1982). Evidence that a Na+/Ca2+antiport system regulates murine erythroleukemia cell differentiation. J. Biol. Chem. 257, 773-780. Soltoff, S. P., and Mandel, L. J. (1983). Amiloride directly inhibits the Na,K-ATPase activity of rabbit kidney proximal tubules. Science 220, 957-959. Steinmetz, P. R., and Andersen, 0. S. (1982). Electrogenic proton transport in epithelial membranes. J . Membr. Eiol. 65, 155-174.

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Stone, D. K., Xie, X.-S., and Racker, E. F. (1983). An ATP-driven proton pump in calthrincoated vesicles. J . Biol. Chem. 258,4059-4062. Sugimoto, Y . , Whitman, M., Cantley, L. C., and Erikson, R. L. (1984). Evidence that the Rous sarcoma virus transforming gene product phosphorylates phosphatidylinositol and diacylglycerol. Proc. Natl. Acad. Sci. U . S . A . 81, 2117-2121. Swarup, G., Cohen, S., and Garbers, D. L. (1982). Inhibition of membrane phosphotyrosylprotein phosphatase activity by vanadate. Biochem. Biophys. Res. Commun. 107, 1104-1 109. Thomas, J. A., Buchsbaum, R. W., Zimniak, A., and Racker, E. (1979). Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18, 2210-2216. Thomas, R. C. (1976). Ionic mechanism of the H+ pump in a snail neuron. Nature (London) 262, 54-55. Thomas, R. C. (1977). The role of bicarbonate, chloride and sodium ions in the regulation of intracellular pH in snail neurons. J . Physiol. (London) 273, 317-338. Tosteson, D. C., and Hoffman, J. F. (I%O). Regulation of cell volume by active cation transport in high and low potassium sheep red cells. J . Gen. Physiol. 44, 169-194. Trivedi, B., and Danforth, W. H. (1966). Effect of pH on the kinetics of muscle phosphofructokinase. J. Biol. Chem. 241, 41 10-41 14. Tsien, R. Y. (1983a). Intracellular measurements of ion activities. Annu. Rev. Biophys. Bioeng. U,91-116. Tsien, R. W.(1983b). Calcium channels in excitable cell membranes. Annu. Rev. Physiol. 45, 341-358. Vigne, P., Frelin, C., Cragoe, E. J., Jr., and Lazdunski, M. (1984a). Structure-activity relationships of amiloride and certain of its analogues in relation to the blockade of the Na+/H+exchange system. Mol. Pharmacol. 25, 131-136. Vigne, P., Frelin, C., and Lazdunski, M. (1984b). The Na+-dependent regulation of the internal pH in chick skeletal muscle cells. The role of the Na+/H+exchange system and its dependence on internal pH. EMBO J . 3, 1865-1870. Villereal, M. L. (1981). Sodium fluxes in human fibroblasts: Kinetics of serum-dependent and serum-independent pathways. J. Cell. Physiol. 108, 251-259. Whiteley, B., Cassel, D., Zhuang, Y.X., and Glaser, L. (1984). Tumor promoter phorbol12-myristate 13-acetate inhibits mitogen stimulated Na+/H+exchange in human epidermoid carcinoma A431 cells. J . Cell Biol. 99. 1162-1166. Whiteley, B., Deuel, T. F., and Gleser, L. (1985). Modulation of the activity of the platelet derived growth factor receptor by phorbol myristate acetate. Biochem. Biophys. Res. Commun. U9, 854-861. Zhuang, Y.X.,Cragoe, E. J., Jr., Shaikewitz, T., Glaser, L., and Cassel, D. (1984). Characterization of potent Na+/H+exchange inhibitors from the amiloride series in A431 cells. Biochemistry 23,4481-4488.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 27

Chapter 2

Na+-H+ and Na+-Ca2+ Exchange in Activated Cells MITCHEL L . VILLEREAL Department of Pharmacological and Physiological Sciences The University of Chicago Chicago, Illinois 60637

1. Introduction. .. ... 11. Na+-Ca2+ Exchange

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

A. General Properties of the Na+-Ca2+ Exchanger in Non B. Na+-Ca2+ Exchange in Activated or Differentiating Cell 111. Na+-H+ Exchange ..................................... A. Role of Na+-H+ Exchange in the Regulation of Intracel Nonactivated Cells.. ................................................ B. General Properties of the Na+-H+ Exchanger in Nonactivated Cells . . . . . C. Na+-H+ Exchange in Activated Cells.. ............................... D. Na+-H+ Exchange in Differentiating Cells E. Mechanism for Stimulation of Na+-H+ Ex IV . Pharmacological Definition of the Na+-H+ and Na+-Ca2+ Exchange Systems . A. Amiloride Inhibition of the Na+ Channel in Tight Epithelia Cells.. . . . . . . . B. Amiloride Inhibition of Na+-H+ Exchange . . . . . . . . . . . . . . . . . . C. Amiloride Inhibition of Na+-Ca2+ Exchange. . . . . . . . . . . . . . . . . D. Generalities Concerning the Pharmacological Interaction of Amiloride Analogs with Na+ Transport Systems.. ............................... E. Nonspecific Effects of Amiloride ..................................... V. Summary :. . . . . . .

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Since the discovery that Na+ ions are not in electrochemical equilib-

rium across the plasma membrane of cells, the importance of a Na+ ion gradient across the plasma membrane has been extensively investigated. 55 Copyright $0 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.

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The first physiological role of the Na+ ion gradient to be generally appreciated was its involvement in the regulation of plasma membrane potential and membrane excitability. As the Na+ electrochemical potential energy gradient across the plasma membrane provides a constant driving force for the movement of positive ions into the cell, an increase in Na+ conductance and hence an influx of Na+ ions can cause a dramatic membrane depolarization. Subsequently, it was suggested that one purpose of the Na+-K+ pump was to maintain Na+ in disequilibrium so that the energy stored in its concentration gradient could be utilized to modify the membrane potential in excitable cells. However, only with the demonstration of Na+-sugar and Na+-amino acid cotransport in the early 1960s (Crane et al. 1961; Christensen et al., 1962) did the extent of the utilization of the energy stored in the Na+ electrochemical gradient by a diverse group of transport processes begin to be appreciated fully. These studies demonstrated that Na+ and certain organic substrates interact with a single membrane transport protein, which results in Na+ and the organic substrate crossing the plasma membrane together (cotransport). Because of the kinetics of these transport systems the energy stored in the Na+ electrochemical gradient can drive organic substrates across the plasma membrane, against their concentration gradient, so that these substrates are accumulated to intracellular concentrations which far exceed what would be their normal equilibrium value. Thus, these cotransport systems utilize the energy stored in the Na+ electrochemical gradient to perform an important cellular function, namely, the maintenance of high intracellular levels of amino acids and, in some cases, sugars in the cell. Recently, it has become apparent that there are other membrane transport systems which utilize the energy of the Na+ electrochemical gradient to move ions in a direction which is opposite to the direction that they would move in response to their electrochemical gradients and which is also counter to the direction in which Na+ moves on these transporters. Thus, these transporters are referred to as countertransport systems and will be the topic of discussion in this article. In particular, two specific countertransport systems will be considered, namely, the Na+-Ca2+ and the Na+-H+ exchange systems. The evidence for the existence of these two systems will be discussed and then the involvement of these two systems in maintaining and perhaps modifying cytosolic Ca2+activity and intracellular pH in activated cells will be discussed. For the purpose of this discussion, an activated cell will be considered to be any cell in which an external stimulus can elicit a physiological response, be it growth factor stimulation of fibroblasts or the activation of sea urchin eggs by fertilization.

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II. Na+-Ca2+ EXCHANGE A. General Properties of the Na+-Ca2+ Exchanger in Nonactlvated Cells

Mammalian cells maintain an intracellular free Ca2+concentration of 10-8-10-7 M in the face of an external Ca2+concentration of lop3M. At present there are a number of recognized mechanisms by which the cells maintain such a low Ca2+activity. Calcium is actively extruded from the cell across the plasma membrane and is also pumped into intracellular Ca2+storage sites. The two major transport systems for pumping Ca2' are the Ca2+-ATPaseand the Na+-Ca2+ exchange system. The latter system utilizes the energy stored in the Na+ electrochemical energy gradient to pump Ca2+out of the cell against its electrochemical potential gradient. It is this Ca2+extrusion mechanism which will be discussed in this section. The existence of a Na+-Ca2+ exchanger was first postulated by Reuter and Seitz (1967, 1968) based upon studies in cardiac muscle. They found that 15Ca2+efflux from guinea pig atria was stimulated by the addition of Na+ to a Na+-free medium. This observation was consistent with the classic trans-stimulation phenomenon seen in facilitated transport systems (for a review, see Stein, 1967). Previous studies had already demonstrated that the addition of extracellular Naf would inhibit Ca2+ influx (Wilbrandt and Koller, 1948; Luttgau and Niedegerke, 1958), but these results had been previously explained on the basis of competition for fixed charge groups at the membrane surface. Reuter and Seitz proposed that Na+ and Ca2+interact with a common transport protein which normally carries two Na+ ions into the cell in exchange for a single Ca2+ ion. In later studies in the squid axon, Baker et al. (1969) and Blaustein and Hodgkin (1969) provided evidence which supported the concept of a Na+-Ca2+ exchanger but offered the suggestion that the coupling ratio was three Na+ ions to one Ca2+ ion. Although the exact stoichiometry may still be in question in some tissues, the existence of Na+-Ca2+ exchange has been extensively documented in heart and other tissues (for a review, see Sulakhe and St. Louis, 1980). Because Na+ ions can be transported into the cell down their electrochemical gradient in exchange for Ca2+,the energy in the Na+ gradient can be utilized to drive Ca2+out of the cell. One can calculate the theoretical limit to the extent that this system could lower the intracellular Ca2+ concentration. If one assumes that the counterexchange of Na+ for Ca2+ is electroneutral, then only the energy in the Na+ concentration gradient is available for the work of extruding Ca2+ from the cell. Under these

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circumstances the steady-state Ca2+activity would be given by [Cali = [Cal,([Nailn/[Na,l") where n is the number of Na+ ions transported with each Ca2+ion. Of course, this theoretical Ca2+activity would be higher if there were substantial leakage of Ca2+by other transport mechanisms so that the Ca2+ extrusion were partially shunted. This theoretical value of Ca2+activity would also be modified if there were other Ca2+extrusion mechanisms at work. Now if the mechanism is electrogenic, the entire energy stored in the Na+ electrochemical energy gradient is available for the extrusion of Caz+.Thus, the theoretical equation for the steady-state concentration of intracellular Ca2+would become exp[(n - 2)FV,,,/RT] [Cali = [Ca]o([Nqln/[Nao]n) Based on these equations, internal and external Na+ concentrations of 15 mM and 150 mM, respectively, and a membrane potential of -75 mV, one can calculate theoretical values for [Cali of 20 p M (for the electroneutral case) and 100 nM (for the electrogenic case). Thus, it is clear that a coupling ratio of 3 Na+ to 1 Ca+ allows the system to extrude more Ca2+ than would be possible for an electroneutral Na+-Ca2+exchanger. Also, based on these equations it is easy to see that small changes in the internal Na+ concentration will be reflected in large changes in the intracellular Ca2+activity. In studies by Sheu and Fozzard (1982), it was shown that increasing the activity of Na+ from 8.5 mM to 30 mM led to a rise in the intracellular Ca2+activity from 51 to 320 nM, as measured by Na+- and Ca*+-specificmicroelectrodes. Thus, these studies support the concept that Na+-Ca2+exchangers can control intracellular Ca2+activity as well as attesting to the sensitivity of the Ca2+levels to modifications of intracellular Na+. B. Na+-Ca2+ Exchange In Activated or Differentiating Cells

Although there is not extensive information available on the involvement of Na+-Ca2+exchange in activated or differentiating cells, there are several interesting examples of involvement. 1. FRIENDERYTHROLEUKEMIC CELLS

A useful cell system for investigating the differentiation process is the murine erythroleukemia cell system. These cells will grow indefinitely in cell culture but can terminally differentiate in response to a number of external agents, the most effective of which is dimethyl sulfoxide. Recent

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studies demonstrated that dimethyl sulfoxide and other differentiating agents inhibit the Na+,K+-ATPase in this cell system (Mager and Bernstein, 1978), suggesting that there may be an ionic factor involved in the differentiation signal. This is further supported by the observation that ouabain is also an effective differentiating agent in this cell system (Bernstein et al., 1976). Since both dimethyl sulfoxide- and ouabain-induced pump inhibition should lead to a rise in Na+ concentration inside the cell, investigators began to ask whether this could be an important signal in the differentiation process. From our earlier discussion of the properties of the Na+-Ca2+ exchanger, it is clear that a modest rise in [Naf]i can lead to a substantial increase in the intracellular Ca2+concentration in cells where the internal Ca2+activity is regulated by a Na+-Ca2+ exchanger. Thus, recent investigations have probed the involvement of Na+-Ca2+ exchange in the differentiation process of murine erythroleukemia cells. In a recent report by Smith et al. (1982), the existence of Na+-Ca2+ exchange in this cell system was demonstrated by the observations that external Na+ would inhibit an influx of Ca2+ into uninduced cells. In addition, this group demonstrated a stimulation of Ca2+efflux following the addition of Na+ to the external medium. As discussed earlier, these observations are consistent with the presence of a Na+-Ca2+ exchanger. It was also observed that the addition of amiloride would completely block the Na+ stimulated efflux of Ca2+from the erythroleukemia cells. As discussed in more detail below (Section IV), amiloride has recently been shown to inhibit Na+-Ca2+ exchange in several cell systems. Previous data from Levenson et al. (1980) had demonstrated that the addition of amiloride to dimethyl sulfoxide-induced cells would block their differentiation. The observation that amiloride inhibits the Na+-Ca2+ exchanger at the same concentrations that it inhibits differentiation suggests that the block of differentiation may be via its effect on this transport pathway. Although, as discussed in more detail in Section IV, there are clearly nontransport effects of amiloride which one must always take into account when interpreting amiloride effects on long-term processes such as cell growth and differentiation, the effects on the erythroleukemic cells occur at doses below those required for these nonspecific effects. 2. PANCREATIC p CELLS Glucose is the major physiological stimulant for the release of insulin from pancreatic p cells. Much work has been done toward identifying the mechanism by which the entry and metabolism of glucose leads to the release of insulin. Certainly, because this is a protein secretion phenomenon, the involvement of Ca2+ has been speculated. Thus, it became of interest to determine whether the intracellular Ca2+activity in /i' cells is at

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least partially regulated by a Na+-Ca2+ exchanger and whether modifications of its activity could be involved in the release of insulin. Work by Herchuelz et al. (1980) has provided evidence for the existence of a Na+-Ca2+ exchanger in rat pancreatic islets. Studies of 45Ca2+efflux from this tissue demonstrated that the removal of external Na+ from the assay medium in the absence of external Ca2+leads to a marked reduction of Ca2+efflux. It was also observed that the addition of glucose significantly reduced the efflux of Ca2+in the presence of external Na+, suggesting that glucose may modify the activity of a Na+-Ca2+ exchanger. Although the mechanism for the glucose-induced effects on the Na+-Ca2+ exchanger are not clear, these data are suggestive that the Na+-Ca2+ exchanger may be modulated in an important fashion during the glucose induction of insulin secretion. 3. PARATHYROID HORMONE AND BONERESORPTION

The effect of parathyroid hormone on bone resorption is thought to be mediated by its actions on the plasma membrane. Although the process by which this hormone causes release of bone Ca2+is not clear, recent studies suggest that its actions could involve the regulation of the activity of a Na+-Ca2+ exchanger in the plasma membrane (Krieger and Tashjian, 1980). It was demonstrated that several agents which modify ion transport across the plasma membrane inhibit parathyroid hormone-stimulated bone resorption. These agents include ouabain, veratridine, and monensin, all of which would be expected to elevate intracellular Na+ concentration. As mentioned above, an increase in intracellular Na+ concentration should inhibit Ca2+extrusion via the Na+-Ca2+ exchanger. In further support of the idea that Na+-Ca2+ exchange may be important in the resorption process was the observation that the parathyroid hormone stimulation was inhibited by the removal of external Na+. It is clear that there is not extensive information available on the involvement of Na+-Ca2+ exchange in the processes of activation and differentiation of cells and that the evidence available is only of a suggestive nature. However, the evidence is sufficiently intriguing that this will undoubtedly be an active area of investigation in coming years. 111.

Na+-H+ EXCHANGE

A. Role of Na+-H+ Exchange in the Regulatlon of lntracellular pH In Nonactivated Cells

In the early years of membrane transport physiology, it was thought that protons were in electrochemical equilibrium across the plasma mem-

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brane. This hypothesis arose because many of those early studies were performed in human erythrocytes, where protons do appear to be in equilibrium. With the introduction of techniques for the measurement of intracellular pH and membrane potential, it became apparent that protons in most cell systems are not in electrochemical equilibrium. Clearly, the intracellular proton concentration is much lower than would be predicted based on the external proton concentration and the membrane potential. If protons were passively distributed, then based on an external pH of 7.4 and a membrane potential of -60 mV, the intracellular pH at equilibrium would be approximately 6.4. Since the measured intracellular pH is on the order of 7.1 in most cells, there must be some active mechanism for extruding protons against their electrochemical gradient. There are three major extrusion mechanisms that occur in various cell types: (1) a Na+-dependent, CI--HCO; exchange system, (2) a proton ATPase. and (3) a Na+-H+ exchange system. This section will deal with only the Na+-H+ exchange system. For a complete review of the other mechanisms for regulating intracellular pH, see the excellent review by Roos and Boron ( 1981). The initial existence of a Na+-H+ exchange system was suggested by Mitchell and Moyle (1969, 1967) and Brierley et al. (1968), who demonstrated that in mammalian mitochondria Na+ was exchanged with protons in an electroneutral process. Similar systems were also reported in Escherichia coli (West and Mitchell, 1974) and Streptococcus faecalis (Harold and Pappineau, 1972). The first description of Na+-H+ exchange in mammalian plasma membranes was by Murer et al. (1976), who demonstrated that Na+-H+ exchange could be observed in isolated intestinal and renal brush border membrane vesicles. In these experiments proton transport was measured by recording extravesicular pH. Addition of an inwardly directed Na+ gradient caused an acidification of the extravesicular medium due to proton extrusion from vesicles. In addition, they observed that an outwardly directed proton gradient stimulated uptake of Na+ into the vesicular space. In subsequent studies, Aickin and Thomas (1977) showed that the recovery of the intracellular pH of mouse soleus muscle following an acid load appeared to be mediated by a Na+-H+ exchange system. Introduction of an acid load in the absence of extracellular Na+ prevented the normal recovery of the intracellular pH back to its control level. These workers also demonstrated that the recovery from an acid load could be blocked by the diuretic amiloride. Since those initial observations were made, the existence of Na+-H+ exchange systems has been postulated in fibroblasts, neurons, cultured kidney cells (Smith and Rosengurt, 1978a; Rindler et al., 1979; Moolenaar et al., 1981a), and many other cell types.

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B. General Propertles of the Na+-H+ Exchanger In Nonactivated Cells

Since many of the kinetic properties of the Na+-H+ exchange system were first described in nonactivated cells, it is useful to spend some time discussing these properties, This will provide some baseline information with which to compare the kinetic properties for transporters in those activated cell systems where the kinetics have been well studied. First, in most cases the Na+-H+ transport system appears to be electroneutral. Studies in both renal and intestinal vesicle systems indicate that the transport of Na+ is not affected by manipulations which would induce alterations in the membrane potential (Murer et a]., 1976). This is supported by similar studies in cultured kidney cells (Rindler et al., 1979). The Na+-H+ exchange system is inhibited by the Na+ transport inhibitor amiloride. This inhibition occurs at significantly higher amiloride concentrations than is seen for inhibition of the Na+ channel in tight epithelial cells (see Benos, 1982). In most studies of the Na+-H+ exchanger, the inhibition by amiloride has been shown to be competitive with Na+. Kinsella and Aronson (1981) showed that the inhibition of Na+ influx into renal microvillus membrane vesicles is independent of the time of exposure to the inhibitor and that the inhibitory effects were rapidly reversed by washing away the amiloride, suggesting that the drug inhibits Na+ influx by acting at a readily accessible external site. By varying the amiloride concentration at a Na+ concentration of 1 mM, they were able to show that the inhibition data were consistent with the presence of a single amiloride inhibitory site which has an apparent Ki of 25 p M . In another study, where external Na+ was varied and Na+ transport measured in the presence of varying concentrations of amiloride, the resulting Lineweaver-Burke plot indicated that amiloride changed only the apparent Na+ affinity and not the V,,, of the transport system, indicating that the inhibition was purely competitive. The true Ki (in a Na+-free medium) for the inhibition was estimated to be 30 p M . A similar analysis in chick skeletal muscle gave a comparable result (Vigne et al., 1982). In contrast, however, Ives et al. (1983) found that for the Na+-H+ exchanger in renal microvillus membranes the inhibition of Na+ influx by amiloride was of the mixed type. Current evidence indicates that the simplest description of the Na+-H+ exchanger is that it has a single cation transport site which alternates between being accessible to the two sides of the membrane, although it may also have an internal proton site which serves as a modifier site. The cation transport site can bind and transport Na+, H + , Li+, and NH: ions. Competition studies in the renal microvillus membrane show that external

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protons, Li+ ions, and NH: ions all competitively inhibit the uptake of Na+ via the amiloride-sensitive system (Kinsella and Aronson, 1981b). Similar results are seen in cultured kidney cells (Rindler et al., 1979). Efflux of Na+ from the kidney vesicles is stimulated by external Na+ and NH: but inhibited by external Li+.The Ki's for inhibition by Li+ and NH: ions were 1.9 and 4.3 mM, respectively, as compared to the K1/?for Na+ transport of 6 mM. Evidence for an internal proton modifier site is provided by the studies of Aronson et al. (1982). An asymmetry in the transport system is suggested by the observation that while external protons appear to interact at only a single site, internal protons appear to interact at both a transport site and an activator site. Evidence for an internal activator site is threefold: (1) the influx of Na+ shows a response to changes in internal proton concentration which is greater than can be explained by a simple interaction at a single site (while one could postulate a 2 : 1 H+-Na+ coupling ratio, this is contrary to a number of experimental observations); (2) elevation of intracellular proton concentration can stimulate Na+-Na+ exchange, which should only be inhibited if protons are binding to a single internal site; (3) elevation of the intracellular proton concentration stimulates the efflux of Na+ from vesicles, which again is contrary to predictions based on a single site of action. C. Na+-H+ Exchange in Activated Cells 1. SEAURCHIN EGGS

The initial observation that an external stimulus could activate Na+-H+ exchange was made by Johnson et al. (1976) when they saw a stimulation of Na+ influx and proton efflux in sea urchin eggs activated by sperm. Activation of sea urchin eggs is a two-stage process. The first stage includes the exocytosis of cortical granules, which occurs in the first 60 seconds after insemination and appears to be dependent on a rise in intracellular free Ca2+concentration. The second phase, which begins approximately 5 minutes after insemination, involves synthesis of protein and DNA. This second phase is dependent on the presence of external Na+ and thus could theoretically be triggered by the increased Na+ influx or the resulting extrusion of protons which occurs subsequent to activation of Na+-H+ exchange. The evidence for the activation of Na+-H+ exchange in this system comes from the measurement of acid extrusion into the extracellular medium and from the measurement of Na+ influx and intracellular pH. Initial experiments by Johnson et al. (1976) dealt with eggs which had been stimulated with sperm and then transferred to a

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Na+-free medium. The effect of Na+ addition on acid extrusion was measured by monitoring the extracellular environment of an egg suspension with a pH electrode. The addition of Na+ to choline-arrested eggs produced an acidification of the extracellular medium. The rate of H+ efflux was found to be dependent on extracellular Na+ concentration in a linear fashion. Li+ was found to stimulate H+ efflux, although less effectively than Na+. The Na+-induced efflux of protons was found to be inhibited by the addition of M amiloride. Measurement of 22Na+influx demonstrated that fertilization induced an increase of Na+ influx within 1 minute and that this stimulated Na+ flux could be blocked by the Na+ transport inhibitor amiloride. In addition to amiloride blocking the proton efflux, the acid extrusion was blocked by an elevation of the external proton concentration. Since the proton efflux rates were quite high after fertilization, the investigators measured the effect of activating this proton extrusion system on the intracellular pH level. They estimated intracellular pH by homogenizing eggs in an unbuffered medium and measuring the pH of the resulting homogenate. Intracellular pH was found to rise from 6.48 in unfertilized eggs to 6.76 in fertilized eggs. However, it should be noted that Cuthbert and Cuthbert (1978) found no amiloride inhibition of the fertilization-induced acid release in eggs from a different species of sea urchin.

FIBROBLASTS 2. CULTURED Smith and Rozengurt (1978a) demonstrated that the addition of serum to quiescent 3T3 mouse fibroblasts would stimulate influx of Na+ as measured by either 22Na+or by net Na+ influx. In a subsequent study (Smith and Rozengurt, 1978b), these authors demonstrated that serum would also stimulate influx of Li+ in 3T3 cells and that the serum-stimulated Li+ flux could be inhibited by amiloride. Studies in human fibroblasts (Villereal, 1981a) demonstrated that amiloride would inhibit Na+ influx in serum-stimulated cells, but amiloride had no significant effect on basal Na+ influx in serum-deprived cells, suggesting that at least in some cell systems the amiloride-sensitive pathway is basally inactive and is turned on in response to growth factors. The Na+ influx in response to mitogen stimulation appears to be mediated by an electroneutral Na+-H+ exchange system. Pouyssegur et al. (1982) showed that in Chinese hamster fibroblasts the addition of thrombin produces an activation of Na+-H+ exchange. In these studies an amiloride-sensitive Na+ influx was observed as well as a Na+-dependent H+ extrusion in the presence of mitogens. The Na+-dependent H+ extrusion was inhibited by amiloride at concentrations which inhibited the

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mitogen-induced influx of Na+. In a subsequent study from Pouyssegur’s laboratory (Paris and Pouyssegur, 1983), it was demonstrated that in addition to an inwardly directed Na+ gradient driving H+ efflux, an outwardly directed Li+ gradient could drive the uptake of protons, thus demonstrating the reversibility of this exchange system. In those studies, the initial rate of amiloride-sensitive H+ uptake by Li+-loadedcells was measured at a number of external pH’s, and the H+ uptake appeared to saturate with increasing H+ concentration. An apparent K , of 1.6 x was observed. A coupling ratio of 1.3 : 1 for H+-Lit exchange was measured, suggesting a stoichiometry of 1 : 1 , which would imply an electroneutral exchange of ions via this transporter. The electroneutrality of this mitogen-stimulated Na+ influx pathway is further supported by the direct electrophysiological studies of Moolenaar et al. (1982). Work from Pouyssegur’s laboratory served to characterize further the kinetics of the Na+-H+ exchange system in fibroblasts. The Na+ dependency of H + uptake was measured at two different external pH’s in stimulated Chinese hamster lung fibroblasts (Paris and Pouyssegur, 1983). The apparent Km’s for Na+ stimulation of H+ extrusion were 13 mM and 60 mM at pH 7.4 and 6.8, respectively. External Na+ ions inhibited the H+ uptake into Li+-loaded cells in a competitive fashion, with a Ki which compared favorably to the K, for Na+ stimulation of H + efflux. Although the simplest explanation would appear to be that H+ and Nat ions share a common binding site, this appears not to be the case, because amiloride behaves as a competitive inhibitor of Na+-stimulated H+ efflux and as a noncompetitive inhibitor of H+ influx in Li+-loaded cells, suggesting two distinct and mutually exclusive binding sites for Na+ and H+. Since H+ extrusion could be demonstrated via the Na+-H+ exchanger, several investigators sought to determine the effect of mitogen-induced activation of this system on intracellular pH. Work by Schuldiner and Rozengurt (1982), based on intracellular pH measurements utilizing the weak acid 5,5-dimethyloxazolidine-2,4-dione (DMO), demonstrated that in cultured mouse fibroblasts (3T3) the addition of Na+ to Na+-depleted cells would induce an alkalinization of the intracellular pH. The Na+dependent alkalinization of the intracellular pH could be blocked by the addition of amiloride. Addition of mitogens to quiescent 3T3 cells produced an alkalinization of the intracellular compartment from a pH of 7.21 +- 0.07 to 7.36 5 0.09. Although the use of DMO allowed Schuldiner and Rozengurt to demonstrate that internal pH rises in response to mitogenic stimulation, it did not allow a continuous monitoring of the pH following mitogenic stimulation. Subsequent studies utilizing fluorescence techniques for continuous monitoring of intracellular pH demonstrated the time course of the change in intracellular pH following the

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mitogenic stimulation of cultured cells. In a study in the epidermoid carcinoma cell line A431, Rothenberg et al. (1983) loaded cells with fluorescein-labeled dextran as a probe of the cytoplasmic pH. They were able to demonstrate that EGF stimulates an influx of Na+ into A431 cells and that there exists a Na+-H+ exchange system in these cells. Although they were not able to show an intracellular alkalinization in response to EGF using fluorescein-labeled dextran, because the pK of fluorescein was too low to see a rise in pH above the basal level, they were able in a subsequent study (Cassel et al., 1983) to demonstrate growth factor-induced alkalinization in NR-6 cells (mouse fibroblasts) using dimethylfluoresceinlabeled dextran. At an external pH of 7.18, mitogens induced a rise in pH of 0.1 to 0.14 pH units. A subsequent report utilizing a similar fluorescent technique showed that the addition of mitogens to human fibroblasts (HF cells) would stimulate an intracellular alkalinization (Moolenaar et al., 1983). The fluorescence indicator utilized by Moolenaar et al. was 2’,7’bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The advantage of this compound over that utilized by Cassel et al. (1983) is that BCECF enters the cell by uptake of its membrane-permeable ester, which is subsequently cleaved by intracellular esterases to generate the pH-sensitive probe, whereas the fluoresceinated dextran is loaded by endocytosis and freed from endocytotic vesicles by osmotic shock. Utilizing BCECF, Moolenaar et al. (1983) demonstrated that the resting pHi of HF cells in a HC0;-free, Hepes buffered medium at pH 7.4 is 7.05 f 0.02 (n = 8). Following stimulation of cells with fetal calf serum, the intracellular pH rose by approximately 0.2 pH units. This mitogen-induced rise in intracellular pH is completely blocked by amiloride. Similar results have been observed in another strain of cultured human fibroblasts (Muldoon et al., 1985). 3. HEPATOCYTES While work was being done in the fibroblast system on the mitogen stimulation of Na+-H+ exchange, parallel work was in progress on the effects of mitogens on this transport system in cultured hepatocytes. The initial studies in this cell system were reported by Koch and Leffert (1979), who, based on the evidence for Na+ involvement in sea urchin egg activation, began to investigate possible involvement of Na+ flux in the activation of hepatocytes. The addition of growth factors to primary cultures of hepatocytes stimulated the influx of **Na+.The addition of amiloride to these mitogen-stimulated cultures dramatically inhibited the influx of Na+. Subsequent studies showed that the addition of amiloride to hepatocyte cultures blocked the stimulation of mitotic activity, although later studies by this laboratory (Leffert et al., 1982) and by Lubin et al.

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(1982) demonstrated that the effects on cell growth were probably the result of nonspecific effects due to amiloride inhibition of protein synthesis. In contrast to some of the fibroblast systems described above, amiloride inhibits a significant portion of the basal Na+ influx in cultured hepatocytes. 4. PLATELETS

The addition of various stimuli (e.g., thrombin or ADP) to platelets causes a sequence of events to occur which progresses from a response at the plasma membrane to execution of a shape change, secretion, and aggregation of the platelets. Studies by Horne and Simons (1978, 1979) suggested that an influx of Na+ may be important in this process because the thrombin-induced depolarization of the platelet membrane potential, which is temporally correlated with the aggregation, is blocked by the addition of amiloride. A subsequent study in the platelet system, in which ADP was shown to stimulate Na+ influx, lends supports to the possible involvement of Na+ influx in the aggregation phenomenon (Sandler et af., 1980). The ADP-stimulated Na+ influx was also shown to be blocked by amiloride. It appears that the Na+ influx stimulated in the platelets is via a Na+-H+ exchange system since studies by Horne et al. (1981) indicate that stimulation of platelets with thrombin will induce an alkalinization similar to that seen in mitogen-stimulated fibroblasts. Utilizing fluorescent methods similar to those described above for fibroblasts, Simons et af. (1982) demonstrated that intracellular pH increases by 0.2 to 0.3 pH units upon stimulation of platelets with a maximal dose of thrombin. The addition of M amiloride dramatically inhibits the thrombin-induced change in intracellular pH, suggesting that it is mediated by a thrombinactivated Na+-H + exchanger. 5 . NEUTROPHILS

Chemotactic factors such as formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) stimulate a number of responses in polymorphonuclear leukocytes (or neurophils), including chemotaxis, aggregation, respiratory bursts, and, in the presence of cytochalasin B, secretion of enzymes. Although the biochemical events following stimulation of neutrophils with chemotactic factors is not fully understood, there is considerable information concerning the stimulation of ionic events, which have been postulated to have an important role in the activation process. The early chemotactic factor-induced changes in ion permeability lead to alterations in intracellular ionic concentrations as well as in membrane potential (Naccache et al., 1977; Korchak and Weissman, 1981). Early reports from

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Naccache et al. (1977) indicated that the addition of f-Met-Leu-Phe stimulated a large and rapid increase in Na+ influx into rabbit neutrophils. Subsequent studies by Simchowitz and Spilberg (1979) in human neutrophils supported these findings. It now appears likely that the stimulated Na+ influx is mediated by a Na+-H+ exchange system that is activated by chemotactic factors. Molski et al. (1980) demonstrated that the addition of f-Met-Leu-Phe to rabbit neutrophils would stimulate a biphasic change in the intracellular pH. Utilizing the distributive pH probe DMO, this group demonstrated a rapid decrease in intracellular pH followed by a dramatic rise in pH to a level above the resting pH values. It appears that the delayed rise in intracellular pH is mediated by a Na+-H+ exchanger since the rise in pH is blocked by amiloride (Sha’afi et al., 1982), which had been shown to block the f-Met-Leu-Phe-inducedNa+ influx (Sha’afi et al., 1981). Subsequent studies in human neutrophils utilizing the pH-sensitive fluorescent probe BCECF (Grinstein and Furuya, 1984) substantiated the biphasic nature of the f-Met-Leu-Phe-stimulated change in intracellular pH. These authors showed that amiloride would block the slower rise in pH and that the alkalinization was dependent on the presence of Na+ in the external medium. These observations strongly support the notion that the alkalinization is the result of activation of a Na+-H+ exchanger. Grinstein and Furuya (1984) proceeded to demonstrate that the Na+-H+ exchanger in the human neutrophils also could be activated by acidifying the intracellular compartment. These authors explained the activation of Na+-H+ exchange by an acid load on the basis of observations by Aronson et al. (1982) that there appears to be an intracellular proton modifier site on the Na+-H+ exchange system in renal brush border membranes. In the neutrophil system, Grinstein and Furuya (1984) demonstrated that the rate of change of pHi following an acid load is higher at a given pH in the presence of f-Met-Leu-Phe than in its absence, suggesting that the chemotactic factor induces a shift in the affinity of protons for the internal modifier site, thereby leading to activation of Na+-H+ exchange at resting pH values.

6. LYMPHOCYTES

The intracellular pH of lymphocytes has been measured by a number of laboratories utilizing the techniques of weak acid distribution (Deutsch et al., 1979), nuclear magnetic resonance (Rink et al., 1982; Deutsch et al., 1982), and fluorescent pH indicators (Rink et al., 1982; Gerson and Kiefer, 1982). A comparison of the intracellular pH to the calculated equilibrium value, based upon available membrane potential measurement (Deutsch et d . , 1979; Grinstein et d . , 1982), shows that there must

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be a pH-regulating process present in lymphocytes. The presence of such a regulatory device is further evidenced by the observation that for a change in external pH from 6.9 to 7.3, the lymphocyte is able to maintain a constant pHi (Deutsch et al., 1982). Recent evidence suggests that the pH-regulating device may be a Na+-H+ exchange system. Recent papers provide strong evidence for the existence of a Na+-H+ exchange system in rat thymic lymphocytes (Grinstein et al., 1984) and human peripheral blood mononuclear cells (Grinstein et al., 1983). In the study of human cells it was reported that those cells undergo a regulatory volume increase which can be blocked by the removal of external Na+ or by the addition of amiloride, suggesting that, as observed by Siebens and Kregenow (1978) for Amphiuma red cells, a Na+-H+ exchange system may be involved in the regulation of cell volume. In studies utilizing pH-sensitive fluorescent probes to monitor intracellular pH, Grinstein et al. (1984) demonstrated that acid loading these cells stimulated an amiloride-sensitive, Na+-dependent pH recovery system which restored the pH, to its original level. They also demonstrated an intracellular alkalinization which accompanied the volume regulatory event. In the study on rat thymic lymphocytes, Grinstein et al. (1984) demonstrated that intracellular acidification activated a pH recovery system that was Na+ dependent and amiloride sensitive. At normal levels of Na: and pHi, the system was not operative but could be readily activated by intracellular acidification. By measuring the rate of change of the intracellular pH and the cellular buffering capacity, these authors calculated the rate of H+ extrusion at various concentrations of extracellular Na+. In acid-loaded cells the initial H+ efflux rate was proportional to the external Na+ concentration. Kinetic analysis of these data demonstrated that the data followed Michaelis-Menton-type kinetics, with the K1/2 for Na+ stimulation of H+ efflux occurring at 59 mM. The apparent Na+-H+ exchange was inhibited by lowering external pH, and the inhibition was not of a purely competitive nature. In contrast, the inhibition of the H+ efflux by amiloride is strictly due to competition between amiloride and extracellular Na+ ions. The stoichiometry of the Na+-H+ exchange system was measured in the rat lymphocyte system and was found to be approximately 1 : 1 , suggesting that the exchange is an electroneutral process. However, there is an amiloridesensitive hyperpolarization which occurs upon acid loading and which is argued by the authors to be the result of activation of the electrogenic Na+-K+ pump. Although lymphocytes clearly have a Na+-H+ exchanger which can be activated either by cell acidification or by cell shrinkage, it is not clear whether stimulation of cells with mitogens will activate this system. Reports from Gerson et al. (1982) indicate that there is a mitogen-induced alkalinization which could be the result of stimulation of this Na+-H+

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exchanger. However, the rise in intracellular pH is extremely slow in comparison to the activation of this system in fibroblasts. The first shift in intracellular pH reported by this group was from 7.18 to 7.35 and reached its peak 6 to 8 hours after stimulation of cells with Con A. A second alkalinization begins 12 hours after stimulation and coincides with a rise in DNA synthesis. There is agreement in two other reports (Deutsch et al., 1984; Rink et al., 1982) that little change in intracellular pH occurs within 1 hour of stimulation of lymphocytes with mitogens. Thus, there appears to be a distinct difference between the way the Na+-H+ exchange system responds to mitogens in lymphocytes versus fibroblasts. 7. CULTURED NEURALCELLS A number of studies on Na+-H+ exchange have been performed in cultured neuroblastoma and glioma cells. Moolenaar et al. (1981a) demonstrated that the addition of serum to mouse neuroblastoma cells (NlE115) in culture would stimulate an influx of Na+ which could be blocked by the addition of amiloride. In a subsequent publication Moolenaar et al. (1981b) provided evidence that the serum-stimulated influx of Na+ was mediated by a Na+-H+ exchange system. External medium pH was monitored as a measure of the extrusion rate of internal protons. The addition of Na+ and Lit to cells rapidly stimulated the extrusion of protons, while the addition of choline, K+,or Ca2+had no effect on the rate of extrusion. The Na+-induced extrusion of H+ was blocked by amiloride. Studies based on the weak acid uptake method indicated that intracellular pH rises following the introduction of Na+ into the external environment. These authors also demonstrated that the influx of Na+ was stimulated by the acidification of the intracellular environment, which is consistent with intracellular protons stimulating the turnover of a Na+-H+ exchanger. Other studies in the NGlO8-15 neuroblastoma-glioma cell line indicate that serum stimulates Na+ influx in this cell system via an amiloridesensitive pathway (O’Donnell and Villereal, 1982). In contrast to the case in most fibroblasts, the amiloride-sensitive Na+ pathway in NGlO8-15 cells has significant basal activity in the absence of mitogens. Measurements of Na+ influx in response to peptide factors have also been performed in rat pheochromocytoma cells (PC-12) by Boonstra et al. (1983). These authors demonstrated that the addition of nerve growth factor (NGF) and EGF to PC-12 cells would stimulate an influx of Na+. The peptide-stimulated Na+ influx is blocked by the presence of amiloride. Stimulation of PC-12 cells did not effect the membrane potential of these cells, suggesting that the Na+ influx occurs by an electroneutral

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process. The neutrality of this Na+ influx was further supported by the observation that the addition of Na+ to cells induced a rapid efflux of protons which was inhibited by amiloride, suggesting that the Na+ influx occurred by an exchange of Na+ for protons. Conversely, elevating the internal proton concentration through the addition of weak acids was found to stimulate the influx of Na+, as expected for a Na+-H+ exchanger. Benos and Saperstein (1983) have recently demonstrated that an amiloride-sensitive Na+ influx pathway can be activated in neuroblastoma (NIE and NB2A) and glioma (C,) cells by the addition of serum. There were several unique properties which were demonstrated for the activation of Na+ influx in C6 glioma cells in these studies. First, the activation of Na+ influx by serum occurred only in cells which had been serum deprived for at least 4 hours. The authors’ suggestion that transporters were being synthesized during this time of serum deprivation was supported by the observation that if cycloheximide was added during this time period, there would be no subsequent stimulation of Na+ influx when serum was added back to the system. In addition, the effects of serum deprivation on the proposed synthesis of transporters could be mimicked by preincubation of cells in dibutyryl CAMP. Second, the Na+ dependency of the amiloride-sensitive flux was linear up to a Na+ concentration of 140 mM, which is in contrast to most studies, which show that the transporter is saturated with Na+ at concentrations well below 140 mM. However, it should be pointed out that there are several other studies (Villereal, 1981b; Johnson et al., 1976) in which a saturation of the system is not seen over a Na+ concentration range which normally saturates the Na+-H+ exchanger in other cell systems.

8. CULTURED SMOOTH MUSCLECELLS Two recent studies in cultured smooth muscle cells have provided evidence for a Na+-H+ exchange system which can be activated by receptor stimulation. Brock et al. (1982) demonstrated that in primary cultures of smooth muscle from rat thoracic aorta angiotensin stimulates a threefold increase in Na+ influx. The activated Na+ influx has been demonstrated to be mediated by an amiloride-sensitive transport system (Smith and Brock, 1983). In another study utilizing the cultured smooth muscle cell line A10, Owen (1984) demonstrated that the addition of platelet-derived growth factor (PDGF) or fetal bovine serum would dramatically stimulate Na+ influx. The Na+ flux stimulated by both agents was inhibited by amiloride.

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D. Na+-H+ Exchange in Differentiating Cells

In recent years data have become available concerning the possible importance of the Na+-H+ exchanger in cells which undergo differentiation. 1. Dictyostelium

There have been several recent studies which support the contention that the Na+-H+ exchanger may be important in the differentiation of Dictyosteiium discoideum amoebae. Martin and Rothman (1980) demonstrated that removal of Na+ from the external medium slowed the differentiation of Dictyostelium in response to starvation. In addition, Gross et al. (1983) demonstrated that treatment of Dicfyosteliumwith NH&l modifies the spore versus stalk proportioning of differentiating Dictyostelium cells. These observations provided a basis for investigating the role of pH and the possible involvement of Na+-H+ exchange in differentiation. In a subsequent study, Jamieson et al. (1984) utilized fluorescent techniques to monitor intracellular pH in differentiating Dicfyostelium. This group observed that at approximately 2 hours into the starvation-induced differentiation process the intracellular pH underwent a dramatic alkalinization from a pH value of 6.2 to 7.1. The cells then returned to their normal pH value of 6.2. The alkalinization can be blocked either by removing Na+ from the external medium or by the addition of amiloride, suggesting that the alkalinization is mediated by a Na+-Hf exchange system which is activated during the differentiation process. The addition of amiloride to cells blocked the differentiation process. Although amiloride is known to have nonspecific effects on protein synthesis and other intracellular events (see Section IV), the block of differentiation may be independent of these nonspecific effects because amiloride only blocks differentiation if added prior to the time of the normal alkalinization and has no effect if added after the alkalinization has occurred. 2. PRE-BLYMPHOCYTE CELLLINE

Differentiation of the pre-B lymphocyte cell tumor line 70Z/3 can be induced by lipopolysaccharide (LPS), a polyclonal B cell mitogen. This cell line behaves as a pre-B lymphocyte, but stimulation with LPS appears to induce its differentiation to a later stage in B cell maturation. Rosoff and Cantley (1983, 1984) have demonstrated that treatment of these cells with LPS stimulates an influx of Na+ which is amiloride sensitive. The addition of LPS also induces a rise in intracellular pH from 7.0 to 7.2 which can be blocked by the addition of amiloride. The LPS-

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induced alkalinization was also found to be dependent on the presence of external Na+. These data suggest that LPS activates a Na+-H+ exchange system in 70Z/3 cells. E. Mechanlsm for Stlmulation of Na+-H+ Exchange in Activated Cells

A number of cell systems were considered above in which the addition of an external agent led to the stimulation of a Na+-H+ exchanger. The investigation of the mechanism by which binding of agents to receptors at the cell surface leads to the activation of Na+-H+ exchange has been an area of intense interest over the past few years. In this section some of the evidence for certain activation sequences in several cell systems will be discussed. The choice of systems discussed was based upon the availability of evidence in those systems and upon a desire to point out that there may be different mechanisms which control the Na+-H+ exchange pathway in different cell systems. Two possible mechanisms for activation of the Na+-H+ exchanger will be considered, one of which involves Ca2+as the second messenger and the other of which involves diacylglycerol as the second messenger, although it will be pointed out that these two mechanisms need not be considered to be mutually exclusive. 1. INVOLVEMENT OF Ca2+IN

THE

ACTIVATION OF Na+-H+ EXCHANGE

The discussion of the involvement of Ca2+in the activation of Na+-H+ exchange will be organized around evidence from my laboratory on the involvement of Ca2+in the mitogen activation of Na+-H+ exchange in cultured human fibroblasts (HSWP cells), with reference to other systems where applicable. If Ca2+acts as a second messenger in the activation of Na+-H+ exchange in response to mitogens, then there are four criteria which must be met: (1) an artificially induced rise in intracellular Ca2+ should lead to activation of Na+-H+ exchange; (2) mitogens should induce a rise in intracellular Ca2+activity; (3) any agent which can block the mitogen-induced rise in cellular Ca2+ should block the activation of Na+-H+ exchange; (4) there should be some demonstrable mechanism by which a rise in cellular Ca2+could activate the Na+-H+ exchanger. Evidence will be presented that these criteria have been met in HSWP cells. Introduction of the Ca2+ionophore A23 187 (or ionomyocin; Villereal, unpublished observations) to HSWP cells in the absence of mitogens leads to a dramatic stimulation of Na' influx which can be inhibited by amiloride (Villereal, 1981a). In addition to an increased Na+ influx, the addition of A23 187 also induces an intracellular alkalinization which is

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Na+ dependent and amiloride sensitive (Muldoon et al., 1985). For short time periods and moderate doses of A23187, the alkalinization is strictly dependent on the presence of external Ca2+,although incubation with A23187 for longer time periods and at higher doses can mobilize intracellular Ca2+,thereby activating the Na+-H+ exchange system. Stimulation of Na+ influx by an amiloride-sensitive transport system using A23 187 has also been seen in sea urchin eggs (Johnson et al., 1976), cultured kidney cells (Taub and Saier, 1979), 3T3 cells (Owen and Villereal, 1985), cultured smooth muscle cells (Owen, 1984), and WI-38 cells (Owen and Villereal, 1985). Thus, the first criterion has been met. In recent studies our laboratory has demonstrated that the addition of mitogens to fibroblasts induces a rise in intracellular Ca2+ activity as monitored by the Ca2+-sensitivefluorescent probe quin-2 (Mix et al., 1984). Subsequent studies (Moolenaar et al., 1984a; Morris et al., 1984) have confirmed this observation in fibroblasts and have quantitated the charige in Ca2+activity. In resting cells the basal Ca2+activity appears to be approximately 150 nM and upon stimulation with mitogens rises to approximately 300 nM, as measured by quin-2. However, in a recent report, utilizing aequorin to monitor intracellular Ca2+activity, it was observed that the intracellular Ca2+activity rose to 1 pM upon mitogen stimulation (McNeill et al., 1984). These authors suggested that quin-2 might be buffering the Ca2+released from intracellular stores so that the transient rise in Ca2+activity is somewhat suppressed in the presence of the high doses of quin-2 needed to monitor intracelluar Ca2+ activity. Regardless of the question of the magnitude of the rise in Ca2+activity, it is clear that mitogens do elevate the free Ca2+concentration. Thus, the second criterion is satisfied. In our studies of Ca2+mobilization, we found that the previously described intracellular Ca2+ antagonist TMB-8 would block the mitogeninduced rise in intracellular Ca2+activity (Mix et al., 1984) and appeared to do so by blocking the mobilization of intracellular Ca2+(Owen and Villereal, 1983a). Studies of the effect of TMB-8 on the mitogen activation of Na+ influx in HSWP cells demonstrated that blocking the rise of intracellular Ca2+would also block the mitogen-induced activation, but not the A23 187-induced activation, of the Na+-H+ exchange system (Owen and Villereal, 1982a). Thus, the rise in intracellular Ca2+ appears to be a necessary event for activation of Na+ influx in HSWP cells. Previous studies from our laboratory suggested the involvement of a Ca2+-dependentregulatory protein in the activation of the Na+-H+ exchanger in HSWP cells (Owen and Villereal, 1982a,b). We found that a series of six psychoactive agents and two naphthalene sulfonamides, which were known inhibitors of calmodulin, inhibited the activation of

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Na+ influx in HSWP cells in a potency order which agreed well with their potency for inhibiting calmodulin-mediated events. At that time, these data were interpreted as implicating calmodulin in the activation process. 2. POSSIBLEINVOLVEMENT OF PROTEIN KINASEC OF Na+-H+ EXCHANGE

I N THE

ACTIVATION

Recently there have been several reports which have provided evidence for the involvement of protein kinase C in the activation of Na+-H+ exchange in different cell types. Dicker and Rozengurt (1981) demonstrated that the tumor promoter TPA would stimulate Na+ influx in 3T3 cells, although at the time it was not known that the primary site of action for this agent is protein kinase C. However, with this realization, based upon the work of Nishizuka’s laboratory (1984), an intense interest in protein kinase C and the Na+-H+ exchanger has developed. Protein kinase C involvement has been suggested based upon effects of TPA in 3T3 cells (Dicker and Rozengurt, 1981), lymphocytes (Rosoff and Cantley, 1984), neuroblastoma cells (Moolenaar et al., 1984b), HeLa cells (Moolenaar et al., 1984b), glial cells (Saperstein and Benos, 1984), and A 431 cells (Whitely et al., 1984). Our laboratory first became interested in protein kinase C when recent studies indicated that the calmodulin antagonists that we utilized for our previous studies also could inhibit protein kinase C. Thus, we initiated studies in HSWP cells to test for the involvement of protein kinase C in the activation of Na+-H+ exchange. The initial studies sought to determine whether TPA alone would activate Na+-H+ exchange in HSWP cells in the absence of mitogens. We found that over a range of TPA doses of 1 to 1000 ng/ml, TPA alone would not stimulate Na+ influx in HSWP cells (Vicentini and Villereal, 1985). The effects of TPA were tested in low-density versus high-density cells, in acutely serum-deprived versus chronically serum-deprived cells, in cells preincubated with TPA for time periods from 1 to 30 minutes, and under all combinations of these assay conditions. Under no circumstances did TPA alone stimulate Na+ influx in HSWP cells. We next sought to determine whether TPA would affect Na+ influx in the presence of a small dose of A23187, to elevate the intracellular Ca2+ activity. TPA was found to synergize with A23187 in the activation of Na+ influx in HSWP cells. At this point it was of interest to determine whether TPA would also synergize with mitogens in the stimulation of Na+-H+ exchange. To our surprise not only did this compound not synergize with mitogens but it dramatically inhibited the mitogen-induced stimulation of Na+-H+ exchange (Vicentini and Villereal, 1985). This last observation turned out to be consistent with a recent observation in A 431 cells that

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doses of TPA which have no effect on Na+-H+ exchange when given alone will dramatically inhibit the EGF activation of Na+-H+ exchange (Whiteley et al., 1984). Although the above effects of TPA on Na+ influx observed in HSWP were also observed in another human foreskin strain (Jackson fibroblasts), there are cell lines which behaved differently in studies also conducted in our laboratory. For example, when a 3T3 cell culture is treated with TPA alone, there is a dramatic activation of the Na+-H+ exchanger (Vincentini and Villereal, 1985). In light of these conflicting observations in different cell types, care must be taken in interpreting results from any one cell type in a global fashion. Clearly in the HSWP cell and the Jackson fibroblast strains addition of TPA is not a sufficient signal to activate the Na+-H+ exchange system. It does appear that TPA is working in HSWP cells since it can synergize with A23 187 and it does inhibit the mitogen-induced stimulation of Na+ flux. Thus, activation of protein kinase C in the absence of a rise in Ca2+activity does not appear to be a universal mechanism for stimulating Na+-H+ exchange. Several other cautionary notes should be provided. Although TPA effects on Na+ influx have been reported in several recent studies, at least some of the cells required doses of TPA which are uncomfortably high for action via protein kinase C. The K d for TPA binding to isolated protein kinase C has been reported to be in the 1-1044 range (Nishizuka, 1984). This estimate agrees well with the doses of TPA needed in HSWP cells to inhibit the mitogen-induced stimulation of Na+ influx, where half-maximal inhibition occurs at approximately 2 nM. If one compares this to the doses of 20 to 300 nM utilized to stimulate maximally Na+ influx in the absence of other agents (Moolenaar et al., 1984b; Dicker and Rozengurt, 1981; Rosoff and Cantley, 1984), it is important to ask whether TPAinduced effects at these concentrations are mediated via protein kinase C. Second, caution must be exercised in the interpretation of pharmacological activation studies as proof of the physiological mechanism for activation of the Na+-H+ exchanger. It is now well known that a number of mitogens stimulate the breakdown of 4‘,5’-phosphatidylinositolbisphosphate by phospholipase C to release inositol trisphosphate and diacylglycerol. There is very good evidence indicating that the release of inositol trisphosphate leads to the mobilization of intracellular Ca2+ and that diacylglycerol is the physiological equivalent of TPA for the activation of protein kinase C. Thus, the stimulation of cells by mitogens results in both a rise in Ca2+activity and a rise in diacylglycerol activity, both of which can stimulate the Ca2+-dependentprotein kinase C. Thus, one could view the synergism between A23187 and TPA in HSWP cells either as the

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interaction of Ca2+and TPA at protein kinase C or the dual action of a Ca2+-dependent calmodulin-mediated and a protein kinase C-mediated regulation of the Na+-H+ exchanger. One could envision two separate phosphorylations by separate kinases as regulating the Na+-H+ exchanger in a similar manner to the regulation of phospholamban (Davis et al., 1983) and myosin light chains (Naka et al., 1984) by multiple phosphorylations. Regardless of the point at which Ca2+and TPA synergize in HSWP cells, it should be remembered that Ca2+and diacylglycerol are thought to both interact at protein kinase C and that both a rise in Ca2+ and a rise in diacylglycerol occur in response to the physiological stimulus. Since the proposed action of diacylglycerol is to increase the Cat+ affinity of protein kinase C, it is possible that at pharmacological doses of TPA one can activate protein kinase C in the absence of a rise in intracellular Ca2+,or that in the presence of pharmacological doses of A23187 protein kinase C can be activated in the absence of a rise in diacylglycerol concentration. However, neither of these pharmacological observations excludes the possibility that under physiological conditions a rise in both diacylglycerol and Ca2+concentration may be necessary to activate protein kinase C. OF PHOSPHOLIPASE ACTIVITY IN 3. INVOLVEMENT Na+-H+ EXCHANGE

THE

ACTIVATION OF

In discussing the effects of the phorbol ester TPA, mention was made of mitogens stimulating phospholipase C activity to release diacylglycerol and inositol trisphosphate. For several years our laboratory has been studying the possible involvement of phospholipase activity in the mitogen activation of Na+-H+ exchange. The involvement of phospholipases was first suspected based on the observation that bradykinin, a peptide known to stimulate phospholipase activity, would stimulate Na+ influx and DNA synthesis in HSWP cells (Owen and Villereal, 1983b) and that melittin, a known activator of phospholipases, would stimulate Na+ influx into 3T3 cells (Rozengurt et al., 1981). In brief, our recent work has demonstrated that inhibitors of phospholipase activity will block the mitogen-induced increase of Na+-H+ exchange in HSWP cells (Vicentini et al., 1984) at concentrations comparable to those required for the inhibition of phospholipase activity. In addition, melittin will stimulate an amiloride-sensitive increase in Na+ influx, suggesting that it is stimulating the Na+-H+ exchanger. This is in contrast to the hypothesis put forward in the paper on 3T3 cells, which suggested that melittin was creating nonspecific leaks in the plasma membrane through which Na+ would enter (Rozengurt et d., 1981). Stronger support for the activation of Na+-H+ ex-

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change by melittin is provided by the observation that this agent stimulates an intracellular alkalinization which is dependent of the presence of external Na+ and is blocked by the presence of amiloride (Muldoon et al., 1985). Subsequent studies from our laboratory indicated that the phospholipase activity stimulated by mitogens probably is involved in the mobilization of intracellular Ca2+,and therefore has its effect on the Na+-H+ exchanger via a Ca2+-dependent activation sequence. We have shown that melittin will stimulate a rise in intracellular Ca2+activity which is the result of intracellular mobilization (Mix et al., 1984). In addition, the mitogen-induced rise in intracellular Ca2+can be blocked by phospholipase inhibitors (Muldoon et al., 1985). A rise in inositol trisphosphate concentration in response to mitogens has been demonstrated in HSWP cells (Vicentini and Villereal, 1984; Jamieson and Villereal, 1985), and this compound has been demonstrated to release Ca2+ from intracellular stores (Muldoon and Villereal, 1989, thereby supporting the contention that phospholipase activation is important in the mobilization of intracellular Ca2+and the subsequent Ca2+-dependentactivation of Na+-H+ exchange. IV. PHARMACOLOGICAL DEFINITION OF THE Na+-H+ AND Na+-Ca2+ EXCHANGE SYSTEMS

In our discussions of the properties of the Na+-H+ and Na+-Ca2+ exchangers it was mentioned that both of these transport systems can be inhibited by the Na+ transport inhibitor amiloride. At this point it would be useful to go back and clarify the pharmacological effects of amiloride on various Na+ transport systems and see if some general conclusions about the relative potencies of amiloride and its analogs for these Na+ transport systems can be outlined. It might also be of value to list the nonspecific effects of these agents which have been reported to date so that future users of these compounds may proceed with caution in the interpretation of their results. A. Amllorlde lnhlbltion of the Na+ Channel In Tight Epithelia Cells

The first Na+ transport system that was reported as being inhibited by the diuretic amiloride was the Na+ channel in tight epithelia such as frog skin and toad bladder (Eigler and Keifer, 1967; Ehrlich and Crabbe, 1968; Bentley, 1968). There is good evidence, based on noise analysis techniques (Lindemann and Van Driessche, 1977), that this Na+ pathway is

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indeed a channel. The Ki of amiloride for this Na+ channel is about

M.In general, substitutions on amiloride at the 5 NH2 position or removal of the 6 C1 group render the resulting amiloride analog less effective than amiloride for inhibition of the Na+ channel in frog skin (see Benos, 1982, for discussion and references). However, substitution at the guanidinium group enhances the Ki of this compound to about lop9 M. One such compound, benzamil, has been successfully utilized in binding studies to quantitate the number of Na+ channels in frog skin (Cuthbert and Edwardson, 1979). B. Amiloride Inhibition of Na+-H+ Exchange

In comparison to the low doses of amiloride required to inhibit the Na+ channel in tight epithelia, much larger doses of amiloride are required to block the Na+-H+ exchanger. The effective range of Ki for amiloride inhibition of Na+-H+ exchange in a variety of tissues is 3 pM to 1 mM, depending upon the external Na+ concentration. Clearly, with such low affinity amiloride is of little use as a probe to identify or quantitate the Na+-H+ exchange systems. Thus, several years ago a number of laboratories began screening analogs of amiloride, with the hope of obtaining an inhibitor with high enough affinity to be useful as a probe for identifying the Na+-H+ exchanger. The initial studies in our laboratory involved the use of amiloride analogs which were substituted on the guanidinium group, because substitutions at other locations seemed to inactivate amiloride as an inhibitor of the Na+ channel in frog skin. While early studies from our laboratory indicated that in HSWP cells benzamil was a much more potent inhibitor of Na+ influx than was amiloride (O’Donnell and Villereal, 1982), it was clear that this was not universally true for all cell systems. For HSWP cells, where the Ki for amiloride is near I mM, benzamil, with a Ki of 15 p M , is a much more effective inhibitor of Na+H+ exchange. However, in cells where the Ki for amiloride is already in the 3-100.pM range, benzamil showed little improvement or in some cases was less effective than was amiloride (Benos and Saperstein, 1983; L’Allemain et al., 1984; O’Donnell and Villereal, 1982; Villereal, unpublished observations). In a subsequent screen of analogs substituted at other positions, it became apparent that modifications which reduced the inhibition of Na+ channels actually enhanced the effects of these compounds on the Na+-H+ exchanger. Substitutions on the 5 NH2 group of amiloride generated compounds such as ethylisopropyl amiloride and methylisopropyl amiloride, whose Ki’s for inhibition of Na+-H+ are in the range of 10 to 100 nM (L’Allemain et al., 1984; O’Donnell et al., 1984;

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Vigne et al., 1983). Thus, compounds could be identified which exhibit a degree of specificity for inhibition of the Na+-H+ exchanger in’comparison to the Na+ channel in tight epithelia. C. Amiioride inhibition of Na+-Ca2+ Exchange

As discussed in the section on erythroleukemia cell differentiation, evidence exists that amiloride can inhibit Na+-Ca2+ exchange (Levenson et al., 1980). As is the case for Na+-H+ exchange, there is a considerable range of Ki’s for inhibition of Na+-Ca2+ exchange. In the murine erythroleukemia cells a concentration of 40 p M was sufficient to totally block the Na+-Ca2+ exchanger (Smith et al., 1982), while the Ki’s for amiloride inhibition in heart mitochondria and sarcolemma vesicles were 200-350 p M (Jurkewitz et al., 1983; Sordahl et al., 1984) and 1000 p M (Siegl et al., 1984), respectively. The low value for the erythroleukemic cells could have resulted from the preincubation of these cells with amiloride prior to measurement of Ca2+fluxes. In the mitochondria1preparation, a screen of amiloride analogs identified benzamil (with a Ki of 167 p M ) as a more effective inhibitor of Na+-Caz+ exchange than amiloride (Jurkowitz et al., 1983). In the cardiac membrane vesicles (Siegl et al., 1984) the benzamil analog is approximately 10-fold more potent than amiloride, and 3,4-dichlorobenzamil is another 10-fold more potent than benzamil (Ki = 17 p M ) . D. Generalities Concerning the Pharmacological interaction of Amiloride Analogs with Na+ Transport Systems

Based on the early results with amiloride analogs, it is probably premature to try to generalize too much concerning any specificity acquired from a given substitution. However, in terms of differentiating between the Na+-H+ exchanger and the Na+ channel, the results do appear to be dramatic enough to mention at this time. A large number of early studies of the Na+ channel indicated that substitutions at the 5 NH2 group would reduce the effectiveness of the compound as an inhibitor of the Na+ channel. Since substitution at this location results in a dramatic improvement of inhibition of the Na+-H+ exchanger, it seems clear that 5 NHzsubstituted compounds show a high degree of specificity for the Na+-H+ exchanger over the Na+ channel. Specificity for the Na+-Ca2+ exchanger is less well defined. The substitutions which have been reported to give a higher affinity for the Na+-Ca2+ exchanger are those occurring at the

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guanidinium group, where substitutions also increase the affinity for the Na+ channel. However, even the best affinity reported for the Na+-Caz+ exchanger is still well below that seen for benzamil inhibition of the Na+ channel. The analogs substituted at the guanidinium group have been suggested to distinguish between the Na+-Ca2+ and the Na+-H+ exchange systems. However, I would suggest that this is not the case, since in HSWP cells, NG108-15 cells (O’Donnell and Villereal, 1982), and L(TK-) cells benzamil will inhibit Na+-H+ exchange with Ki’s in the 1050-pM range and in Chinese hamster lung fibroblast (L’Allemain et al., 1984) with a Ki of 80 pM.These doses for inhibition of Na+-H+ exchange are not dramatically different from the doses of benzamil which inhibit Na+-Ca2+ exchange. E. Nonspecific Effects of Amiloride

As mentioned earlier in the discussion, amiloride has been demonstrated to block protein synthesis in a cell-free extract (Leffert et al., 1982; Lubin et al., 1982). In addition, other studies demonstrated that amiloride would inhibit mitochondria1 processes (Taub and Saier, I98 1) and Na+,K+-ATPase activity in renal proximal tubules (Soltoff and Mandel, 1983). The most recent addition to the list of nonspecific events inhibited by amiloride is provided by a report that amiloride inhibits protein kinase C activity (Besterman et d., 1985). Given the fact that amiloride readily enters cells, these nonspecific effects must be taken into account when interpreting the effects of amiloride or its analogs on biological processes such as cell growth and differentiation. V.

SUMMARY

As discussed above, there are a number of cell systems in which Na+H+ exchange or Na+-Ca2+ exchange are modified by the addition of external agents. Thus, it appears that the modification of either intracellular pH or intracellular free Ca2+concentration, via alterations in the activity of these exchangers, may be an important signaling process. In this regard, several possible mechanisms were suggested for the mitogeninduced activation of the Na+-H+ exchanger. In the future it is likely that attention will be focused on the identification and isolation of the membrane proteins responsible for mediating Na+-H+ and Na+-Ca2+ exchange. With these proteins in hand it will be possible to determine whether the exchangers are biochemically modified (e.g., phosphory-

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lated) in response to external stimuli. This would allow a much clearer understanding of the regulation of these transport functions. Clearly, this will be a fertile area for future investigations.

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Sheu, S. S., and Fozzard, H. A. (1982). Transmembrane Na+ and Ca2+ electrochemical gradients in cardiac muscle and their relationship to force development. J. Gen. Physiol. 80, 325-351. Siebens, A., and Kregenow, F. M. (1978). Analysis of amiloride-sensitive volume regulation in Amphiuma red cells. Fed. Proc., Fed. A m . Soc. Exp. Biol. 39, 379. Siegl, P., Cragoe, E., Jr., Trumble, M. J., and Kaczorowski, G. J . (1984). Inhibition of Na+/ Ca2+exchange in membrane vesicle and papillary muscle preparations from guinea pig heart by analogs of amiloride. Proc. Nut/. Acud. Sci. U . S . A . 81, 3238-3242. Simchowitz, L., and Spilberg, I. ( 1979). Chemotactic factor-induced generation of superoxide radicals by human neutrophils: Evidence for the role of sodium J. Irnmunol. 123, 2428-2435. Simons, E. R., Schwartz, D. B., and Norman, N. E. (1982). Stimulus response coupling in human platelets: Thrombin-induced changes in pH,. I n “Intracellular pH: Its Measurement, Regulation and Utilization in Cellular Functions” (R.Nuccetelli ed.), pp. 463482. Liss, New York. Smith, J. B., and Brock, T. A. (1983). Analysis of angiotensin-stimulated sodium transport in cultured smooth muscle cells from rat aorta. J. Cell. Physiol. 114, 284-290. Smith, J. B., and Rozengurt, E. (1978a). Serum stimulates the Na+/K+pump in quiescent fibroblasts by increasing Na+ entry. Proc. N u t / . Acud. Sci. U.S.A. 75, 55605564.

Smith, J. B., and Rozengurt, E. (1978b). Lithium transport by fibroblastic mouse cells: Characterization and stimulation by serum and growth factors in quiescent cultures. J . Cell. Physiol. 97, 441-450. Smith, R. L., Macara, I. G., Levenson, R., Housman, D., and Cantley, L . (1982). Evidence that a Na+lCa2+antiport system regulates murine erythroleukemia cell differentiation. J. Biol. Chem. 257, 773-780. Soltoff, S. P., and Mandel, L . J. (1983). Amiloride directly inhibits the Na,K-ATPase activity of rabbit kidney proximal tubules. Science 220, 952-954. Sordahl, L. A., LaBelle, E. F., and Rex, K. A. (1984). Amiloride and diltiazem inhibition of microsomal and mitochondria1 Na+ and Ca2+ transport. A m . J. Physiol. 246, C172(2176. Stein, W. D. (1967). The coupling of active transport and facilitated diffusion. I n “The Movement of Molecules across Cell Membranes” (W. D. Stein, ed.), pp. 177-206. Academic Press, New York. Sulakhe, P. V., and St. Louis, P. J. (1980). Passive and active calcium fluxes across plasma membranes. Prog. Biophys. Mol. B i d . 35, 135-195. Taub, M., and Saier, M. H., Jr. (1979). Regulation of 22Na+transport by calcium in an established kidney epithelial cell line. J. B i d . Chem. 254, 11440-1 1444. Taub, M., and Saier, M. (1981). Amiloride-resistant Madin-Darby canine kidney (MDCK) cells exhibit decreased cation transport. J. Cell. Physiol. 106, 191-199. Vicentini, L. M., and Villereal, M. L. (1984). Serum, bradykinin and vasopressin stimulate release of inositol phosphates from human fibroblasts. Biochem. Biophys. Res. Commun. l23, 663-670. Vicentini, L. M., and Villereal, M. L. (1985). Activation of Na+/H+exchange in cultured fibroblasts: Synergism and antagonism between phorbol ester, CaZ+ionophore and growth factors. Proc. Nurl. Acud. Sci. U . S . A . 82, 8053-8056. Vicentini, L. M., Miller, R. J., and Villereal, M.L. (1984). Evidence for a role of phospholipase activity in the serum stimulation of Na+ influx in human fibroblasts. J. Biol. Chem. 259, 6912-6919.

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Vigne, P., Frelin, C., and Lazdunski, M. (1982). The amiloride-sensitive Na+/H+exchange system in skeletal muscle cells in culture. J. Biol. Chem. 257, 9394-9400. Vigne, P., Frelin, C., Cragoe, E. J., and Lazdunski, M. (1983). Ethylisopropyl-amiloride: A new and highly potent derivative of amiloride for the inhibition of the Na+/H+exchange system in various cell types. Biochem. Biophys. Res. Commun. 116, 86-90. Villereal, M. L. (1981a). Sodium fluxes in human fibroblasts: Effect of serum, Ca and amiloride. J. Cell. Physiol. 107, 359-369. Villereal, M. L. (1981b). Sodium fluxes in human fibroblasts: Kinetics of serum dependent and serum-indeDendent pathways. J. Cell. Physiol. 108, 251-259. West, I. C., and Mitchel, P. (1974). Protonhodium ion antiport in Escherichia coli. Biochem. J . 144,87-90. Whiteley, B.,Cassel, D., Zhuang, Y.,and Glaser, L. (1984). Tumor promoter phorbol 12myristate 13-acetate inhibits mitogen-stimulated Na+/H+ exchange in human epidermoid carcinoma A431 cells. J . Cell Biol. 99, 1162-1166. Wilbrandt, W., and Koller, H. (1948). Die Calciumwirkung am Froschherzen als Funktion des Ionengleichgewichts zwischen Zellmembran und Umgebung. Helv. Physiol. Pharmacol. Acta 6 , 208-221.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 21

Chapter 3

Chlo ride-Dependent Cation Cotransport and Cellular Differentiation: A Comparative Approach PETER K . LAUF’ Department of Physiology Duke University Medical Center Durham, North Carolina 27710

I. Introduction. . . 11.

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97 98 99 100 111. 100 A. General Aspects.. . . . . . . . . . . . . . . . . . B. Volume-Stimulated K-CI Cotransport . . . . . . . . . . . . 103 104 . . . . . . . . . . . . . . . . 105 108 IV . V. Nonepithelial Cells as Models for Cotransport during Differentiation . . . . . . . . . 112 115 VI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 Note Added in Proof . . . . . . . . . . . . 116 References . . . . . . . . .

F. Inhibitors

Present address: Department of Physiology and Biophysics. Wright State University, School of Medicine, Dayton, Ohio 45401-0927. 89 Copyright 0 1986 by Academic Press. Inc All nghts of reproduction in any form reserved

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1.

INTRODUCTION

Monovalent cation fluxes tightly coupled to the presence of chloride constitute a major portion of ouabain-resistant (OR) “leak” cation transport in a variety of biological membranes. Depending on the participating ions, we speak of Na-Cl, K-Cl, or Na-K-2CI transport or cotransport, or use the terms C1-dependent Na and K or Na-K transport. It is the aim of this contribution to describe briefly the properties and to review the occurrence of CI-dependent cation transport in differentiated cells or cell lines and to attempt to describe their changes during cellular differentiation and development. It will become apparent that the first part of this task is less difficult than the latter because a large body of information has been accumulated from studies of C1-dependent cation fluxes in physiologically and biochemically well-defined epithelial and nonepithelial cells. Nevertheless, at the time of writing of this article, no exhaustive review on C1-dependent cation cotransport has been published. A series of subtopical reviews, however, has dealt with aspects of the theme discussed here (26,28,39,47,55,73,90,111,126-129,134, 147,159). In contrast, reports on C1-dependent cation transport in differentiating cells are scarce and reviews are absent because, not only is there a problem in obtaining sufficient quantities of cells, but also few really differentiating cell lines, mostly from hemopoietic tissues, are available. To remedy this shortcoming, a vertical approach was chosen in which C1-dependent cation transport will be compared between less differentiated and more differentiated cell types of the same ontogenetic (but not necessarily species) origin. The nucleated red cells of fish or birds and the reticulocytes and enucleate mature red cells of mammals provide examples. Another measure for changes throughout differentiation is the response of C1-dependent cation transport during cell activation commissioned by a variety of stimuli such as culture media, hormonal, and ionic effectors, which are used to gain insight into the role of other passive and active membrane transport systems such as the Na-H exchanger (see Chapters 2, 5 , and 6, this volume) or the Na-K pump in differentiation. While there is considerable progress in our understanding of the purported role of these other membrane transport processes for cellular differentiation, the role, if any, of the C1-dependent cation transporters still needs to be established for many of the differentiated cells in which they have been found. This problem is compounded by the fact that the physicochemical basis of tightly coupled cation-anion transport has barely been addressed.

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II. GENERAL PROPERTIES A. Thermodynamic Considerations

In the description of the biophysical properties of Cl-dependent cation transport, only those transport modes in which the cation and chloride gradients are thermodynamically directly coupled will be considered and not those modes where cation-C1 cotransport is the result of a Cl-sensitive cation-proton antiport coupled to a C1/HCO3exchanger (28). Equation (1) defines the driving forces for C1-coupled Na-K or Na-K fluxes in a general form:

where R and T have the usual meanings and the exponents m ,n , p are not only dependent on whether Na-Cl, K-CI, or Na-K-2Cl cotransport is considered but also include variations in the individual stoichiometries. This equation emphasizes the coupling of the chemical gradients of the individual ions, that is, the fact that any participating ion may be driven by another “driver” ion outwardly or inwardly and uphill against its own chemical gradient as long as the ionic product is greater at the inside (i) or outside (0)of the membrane. The membrane potential term is omitted in Eq. (1). When the net driving force is not equal to zero, that is, becomes negative or positive, net efflux or net influx by cotransport ensues, leading to an electroneutral outward or inward shift of salt and water, isosmotic with the transmembraneous fluid milieu. Hence CI-dependent cation transport performs “isosmotic intracellular regulation” by up or down regulation of the number of effective osmotic particles within the cell, a process resulting in cell swelling or shrinkage, when no other dissipative forces are at work. For human (33) and duck (65,151) red cells, Cl-dependent cation net fluxes are close to zero under physiologic conditions, that is, the Na-K pump is maintaining the normal ion gradients across the membrane. Thus, lowering the extracellular K concentration, [K],, leads to a net outward Na-K-2Cl transport (34,49,76). Although hydrolysis of ATP and hence direct energy dependence of C1-dependent cation transport on ATP has been ruled out (54), there is clear evidence for requirement of intact metabolism and perhaps ATP beyond that necessary for gradient maintenance by the Na-K pump (see Section 11,G).

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B. Anisosmotic Activation of Cotransport

When there is no net flux through the C1-dependent cation transport system(s) and with the basal leak or “ground permeability” balanced by the activity of the Na-K pump, the cell is at steady-state volume. The importance of the pump-leak relationship was established some 25 years ago by Tosteson and Hoffman (161), and its approach is still valid today although details of the “passive” membrane permeability have changed considerably. In relation to C1-dependent cation transport, the constant cell volume V , has been defined by Geck et al. (56) to be not far from a threshold volume V , in order to explain the fact that C1-dependent cation fluxes are activated when V , deviates by a critical value from V, by swelling or shrinking. Thus it has become evident that the predominance of the individual cation-C1 cotransport mode, that is, K-Cl, Na-Cl, or Na-K2C1 cotransport, is determined by the ratio VcIVt.When V , > V , , K-Cl cotransport is activated, and when V , < V , , the Na-C1 or Na-K-2Cl cotransport system is activated. As can be seen from Figs. 1 and 2, which detail the two boundary conditions, water moves first into or out of the cell when its chemical activity is increased or decreased in the medium, the so-called “osmotic phase” (87,90). The volume regulatory phase, setting in as soon as V , =

K’CI‘

FIG.1. K-CI cotransport effects regulatory volume decrease. In this diagram the model step 1) which is cell is placed into a hyposmotic medium resulting in rapid water entry (H20, accompanied by cell swelling from the original volume, V O ,to the volume of a sphere ( V , , maximum volume, step 21, called the osmotic phase (90). The mechanism [step 3, “volume stat” (93)] by which ouabain-resistant K-C1 eftlux (step 4) is elevated to cause RVD (step 5) remains to be elucidated. [From P. K. Lauf (1985), Molecular Physiology, 8, 215-234.1

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No+(:K: 2) C I-

FIG.2. Na-CI or Na-K-2CI cotransport effects regulatory volume increase. Here the model cell is placed into a hyperosmotic medium, causing the cell water to leave (step I ) together with volume reduction from V, to V - , a smaller than normal cell volume. Following or during this osmotic phase an unknown mechanism (step 3) activates inward salt transport (step 4), leading to a gradual return of the cell volume to V,.

V , and becoming evident only after the osmotic phase, entails regulatory volume decrease (RVD) (see Fig. 1 ) and regulatory volume increase (RVI) (see Fig. 2) accompanied by net K-CI loss in the former and Na-CI or Na-K-2CI gain in the latter case. The rate at which restoration of the original V , is achieved is different between various cell types studied, being much faster in Ehrlich ascites tumor cells (73) and duck erythrocytes (87) than in fish red cells (96) and very slow in enucleate red cells (33,37,38). One of the central questions concerns the mechanism that senses deviation from and return to V,, which Kregenow coined the “volume stat” (87). What are the identities of the molecules which detect volume deviation and/or implement a counterregulatory response, that is, K-CI efflux to cause RVD and Na-K-2Cl influx for RVI? C. Activation by Hormones

Table I lists the major modes and triggers for C1-dependent cation transport and the cell types in which it occurs. It is clear that activation of C1dependent cation transport has been extensively studied in many cell types exposed to anisosmotic media. However, under isosmotic conditions certain pharmacologic effectors such as hormones have been shown to activate exclusively the RVI cotransporters, perhaps by altering the threshold settings of the hypothetical volume stat or altering the “membrane memory” for cell volume.

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TABLE I CHLORIDE-DEPENDENT CATION TRANSPORT I N DIFFERENTIATED CELLS: MAJORMODES,TRIGGERS, A N D CELLTYPES A. K-CI cotransport (Tables I1 and V, Fig. I ) I. Hyposmotic 1. Cells of the hemopoietic system (19,20,37-39,87,90,92,96,110,111,133) 2. Epithelial cells (32,57,158) 11. Chemical modification (SH reagents) 1. Cells of the hemopoietic system (81,97-107) 2. Epithelial cells (Ehrlich cell) (86) 111. Ionophores and bivalent cations I . Cells of the hernopoietic system (50,103,107) B. Na-CI or Na-K-2CI cotransport (Tables 111, IV, and VI, Fig. 2) I. Hyperosmotic shrinkage 1. Cells of the hemopoietic system (34,88-92,111,120,121,137,l49,151,162) 2. Epithelial cell lines, including Ehrlich cells (54-56,73-75) 3. Fibroblasts (5) 11. Pharmacologic effectors I . Cells of the hemopoietic system (6,12,lS,19,20,51,65-67,88-92,111,112,120, I26-129,137,l40,14 1,150,162) 2. Epithelial cells (4,24,25,4l,62,64,7l,113,116,136,153,l55,156,l65,167) 3. Fibroblasts (123) 4. Smooth muscle (78-80,122)

Most of the studies were performed with P-adrenergic agonists such as catecholamines, which activate the adenylate cyclase (109). Thus norepinephrine, isoproterenol, etc., as well as CAMP, have been shown to activate Na-K-2Cl cotransport in isosmotic media in a variety of cell systems (see Table I). This process was paralleled by phosphorylation of high-molecular-weight proteins (63,140). Attempts failed, however, to correlate CAMP levels and Na-K-2Cl cotransport activity induced by catecholamines (88,91,127,128,137)with the phosphorylation of high-molecular-weight membrane protein. Nevertheless, there seems to be sufficient consensus in the literature on the striking similarity between Na-K2C1 cotransport augmented by hypertonicity and by catecholamines to propose that these two stimuli initiate events converging into a common (distal) process which activates cotransport (88,91,150,162).The similarities entail (1) the sensitivity of Na-K-2Cl or Na-Cl cotransport to the inhibitory action of loop diuretics, (2) the K0.5 of 4-10 mM for activation by external KO,and (3) the pH dependence. Less understood is the mechanism by which deactivation of catecholamine or hypertonicity-induced Na-K-2Cl cotransport occurs. In turkey red cells the time-dependent deactivation of Na-K-2Cl cotransport was different after these two

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35

methods of activation (162). The inactivation of cotransport in duck red cells stimulated by norepinephrine was several times faster than the one turned on by hypertonicity (150). D. Alterations by Chemical Modifications

Since the discovery of C1-dependent cation transport is rather recent, there are no data available on chemical modification of the Na-K transport system operational during RVI. However, as early as 1957 Tosteson and Johnson (160), while working on the metabolic basis of membrane transport in duck red cells, discovered that N-ethylmaleimide (NEM) selectively increased the K permeability in these cells. More than 20 years later it became apparent that NEM stimulated CI-sensitive K fluxes (98,104,106). At first it could not be decided whether NEM stimulated the same transport moiety which also responded to cell swelling with increased flux rates (36). Furthermore, in all cells where there is K-CI transport activity, whether or not associated with RVD, NEM stimulates this transport system by reacting with SH groups, most probably on the cytoplasmic side of the membrane (102). The effect has been found in reticulocytes of sheep (99) and piglets (108) and in the mature red cells of humans (104,105,166), pigs (108), and low-K sheep (106,110). Chemical modification by NEM fixes the K-CI transporter in a conformation permissive for maximum transport rates observed in untreated but swollen cells (103). Hence shrinkage of the cells subsequent to treatment with NEM in iso- or hyposmotic media does not reduce the K-Cl flux as compared to controls. This explains the reported volume insensitivity of the NEM-stimulated flux (102). The situation, however, is far more complex since the NEM-stimulated flux is also refractory to further stimulation when cells are transferred from hyperosmotic to hyposmotic solutions (103). The strongest evidence for the fact that both NEM and cell swelling affect the same transport molecule comes from an immunological observation: anti-Ll , an antibody prepared in high-sheep against low-K sheep red cells (97), reduces both NEM- as well as swelling-stimulated KCI transport in low-K red cells (102,110). How can an NEM-alkylated SH group activate K-CI flux? Recent experiments have shown that the Ca ionophore A23187 in the presence of ethylene glycol tetracetic acid (EGTA) was able to activate volume-dependent K-CI fluxes but had only little or no effect on NEM-treated cells (103,107), which maximally stimulated K-CI fluxes. This finding suggests that the activation of the K-CI transporter by A23187 and EGTA may be under the control of SH groups. On the other hand, increasing cellular

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concentrations of divalent metal cations leads to inhibition of K-Cl cotransport involving chemical groups probably different from SH groups necessary for activation (107). E. Kinetlcs and ionic Interdependence

Aside from the thermodynamic aspects (see Section II,A), there are kinetic considerations of C1-dependent cation cotransport. In fact, the kinetic dependence of the fluxes of one cation on the presence of the other was first recognized for ouabain-resistant (OR) Na-K fluxes in human red cells, where OR K influx was reduced by replacement of Na, with choline (16). However, the applications of specific inhibitors, such as furosemide (166), or C1 replacement by non-C1 anions of the Hofmeister series (36,54) was necessary to define the tight relationship between the monovalent cations and Cl anions in the electroneutral transport process. Although attempts have been made to understand mechanistically the Cl-dependence, presently available data do not permit any conclusion about its nature. When in ouabain-poisoned cells, Na or K influxes are measured in C1 media with varying [Na], or [K],; a relationship between cation influxes, iJy$, and the external cation concentration, [cat],, is obtained as described by Eq. (2), from which the half-maximum activation constant, Kgi , of the hyperbolic saturation component can be calculated:

The second term on the right-hand side of Eq. (2) defines the linear relationship between “ground permeability” and [cat], , where ‘k;$ is the pseudo-first-order rate constant for ouabain-resistant cation influx, usually determined in non-C1 media or in C1 media in the presence of loop diuretics. This approach has been widely used to determine K6ai for epithelial and nonepithelial cells alike. In general, for coupled Na-K cotransport the K8aj values are 4-10 mM for [K],, -20 mM for “a],, and >15 mM for [K], for C1-dependent K fluxes. The K6f values obtained, of course, apply to coupled Na-K cotransport as well as to C1-dependent K-K or Na-Na exchange pathways which may be operational modes of or distinct from the C1-dependent coupled net transport (33,67). Hence KFi values may be close to or even higher than [cat],, at which thermodynamically no net transport occurs. Furthermore, evidence is mounting that the mutual interdependence of cations is kinetically much more com-

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plex, requiring extrapolation maneuvers in some epithelial cell lines ( 139,147). In contrast to the mostly hyperbolic cation activation curves of C1dependent cation transport, the CI activation curves reported vary from sigmoidal(36) or hyperbolic (102) or simply linear (30) to parabolic shapes (102). These curves display not necessarily true site heterogeneity but also experimental problems. As in most of the studies, lyotropic anions of the Hofmeister series, such as NO3, SCN, or I, were used to replace C1. The work with anions, such as methylsulfate, which are claimed to be milder than their inorganic chaotropic counterparts is not yet developed enough to define unequivocally a Kf,\for any system studied. Nevertheless, any experiment in which CI is replaced by lyotropic anions (except Br, which in some cases seems to substitute fully for CI) yields an impressive diminution of OR cation fluxes when Cl-dependent cation cotransport is present. F. Inhibitors

Before the now classic loop diuretics were used experimentally on a broader scale, work with related compounds such as ethacrynic acid revealed the presence of ethacrynic acid-sensitive Na fluxes (pump 11, ref. 72), which much later were identified as being due to loop-diuretic-sensitive, C1-dependent Na-K cotransport (168). In fact, the first explanation of the diuretic effect of furosemide was its inhibition of an active CI transport system (26), a notion superseded later by the brilliant work of Greger’s group (58-62) showing that it was the reabsorptive Na-K-2CI cotransport in the renal cortical thick ascending limb that was inhibited by loop diuretics. The chemistry of loop diuretics in relation to its inhibition of cotransport pathways has been addressed by Schlatter et al. (148) and by Palfrey et al. (126,127), and these papers and their references should be consulted for further pharmacological details. In spite of the wide use of loop diuretics for identifying cotransport pathways as being structurally separate from other transporters such as the Na-K pump or the Ca-activated K channel (82), a few caveats are appropriate. First, although much evidence attests to the fact that the inhibition of cation cotransport by loop diuretics approximately equals that caused by total CI replacement with NO3 or other anions, no systematic study has been done to deal directly with this problem. Second, the inhibition of Na-K-2CI cotransport is usually effected by furosemide or bumetanide concentrations much lower (49,58-62,77,123,126,127,162)

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than those necessary for abolishing K-Cl cotransport (101,104). In fact, when the furosemide concentration exceeds M, inhibition of other transport systems important for the steady-state equilibration of living cells may occur (23). Moreover, Aull (10,ll) has reported that the K-Cl stoichiometry of furosemide-sensitive K-K exchange in Ehrlich cells changes as a function of the drug concentration. Third, the mechanism of the molecular action of these compounds is far from understood. It seems that K and/or Na may augment (46,101,126) and C1 (in some cases) reduce the inhibitory potency of furosemide or bumetamide (66), and a possible metabolic dependence of the loop diuretic effects (2) needs to be considered. It is also not clear whether the target site for the action of loop diuretics is on the external or cytoplasmic side of the putative Na-K-2CI or K-Cl carrier, since loop diuretics may be permeable. G. Role of Cellular Metabolism

That a part of OR cation flux is dependent upon metabolism was suspected early from work on metabolically depleted human red cells (16,72), a fact leading to the coining of the term “pump 11” (72) in analogy to the ATP-driven Na-K pump (pump I). Subsequent work showed that only the Na-dependent K fluxes and not the ground leak were reduced upon metabolic depletion (16). It is now clear that Cl-dependent, furosemidesensitive cation cotransport is metabolically dependent in a variety of cells and that the cellular ATP levels have to be lowered into the micromolar range to affect Na-K cotransport (2,31,113,124). Restoration of metabolism (as measured by cellular ATP levels) after reversible metabolic depletion (by starvation or 2-deoxy-~-glucose)is followed by full recovery of Na-K cotransport (2,124). The exact mechanism by which metabolism affects Na-K cotransport is unknown. The metabolic dependence certainly cannot be simply explained by assuming depletion of substrate ATP required for membrane phosphorylation, because in perfused squid giant axons ATP as well as ADP reversibly supports C1dependent Na-K inward cotransport (142,144). Future work will have to decide how metabolism or ATP influence Na-K-2Cl cotransport. The formation of phosphoinositol-oligophosphates(18) and their role in passive membrane permeability has just begun to be evaluated (40). Of even greater mystery is the well-documented metabolic dependence of thiol-stimulated K-Cl fluxes in certain red cells (100,102,105). The basal K-Cl fluxes which respond to cell swelling were found to be metabolically independent. The time courses of ATP depletion and disappear-

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ance of the NEM-stimulated K-CI fluxes are different, suggesting that factors additional to ATP may control these fluxes (105).

H. Functional Aspects In epithelial cells an intracellular C1 activity above equilibrium indicates the presence of coupled, electroneutral, cation-CI-cotransport mediating solute (re-)absorption or secretion. In the case of NaCl- absorption, as in the thick ascending limb of certain mammals (58-61), the early distal tubule of the amphiuma nephron ( I 18,119), or the intestinal epithelium (47,64,113), the coupled Na-K-2CI or Na-CI cotransporter is on the apical or luminal side, Na leaves the cell uphill through the Na-K pump on the basolateral membrane, and K and CI ions traverse the membrane via specific channels on the apical and basolateral membrane, respectively. In some epithelia, for example, in the Necturus gall bladder, a special symport at the basolateral membrane mediates K-CI exit (95,135). Secreting epithelia possess coupled transport pathways at the basolateral side, together with the Na-K pump (41,71,153) and a barium-inhibited K channel. The intricacies of the detailed arrangement of coupled Cl-dependent ion transport parallel or sequential with other transport mechanisms have been discussed by others (26,42,47,57,157). The striking polarity of epithelial cells with respect to their cotransportdetermined functions is lost upon isolation and suspension of epithelial cells. Although the suspended cell is not carrying out vectorial transcellular solute flow, it has, dependent on its origin, maintained its Cl-dependent Na-K, Na, or K transport capability commensurate with the activity found in the original tissue. When epithelial cells establish a cell colony, they regain their polarity with respect to C1-mediated cation transport (70,138) and transepithelial solute and water flow may occur again. One of the questions frequently raised is whether volume-responsive cation-Cl cotransport indeed serves as a volume regulator in the cell suspended in uiuo or whether one should consider, at least for red cells, the system to be of vestigial nature. For invertebrate red cells of the bivalve Noetia ponderosa (7,155), it seems that RVD through K fluxes makes sense to cope with hyposmotic stress due to salinity changes. In these cells, as well as in the red cells of teleosts (48) and in the axons of Carcinus (83), K fluxes are accompanied by amino acids and/or CI (20,27,96), and it is unclear at present whether one or two transport systems need to be considered. In cells of organisms evolutionarily higher and removed from tidal salinity changes, C1-dependent cation trans-

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porters can take on the role of isosmotic salt transporters, or may function in conjunction with activation of other transport systems involved, for example, in substrate transport “a-dependent amino acid or glucose transport (13,93)] or in pH equilibration by Na-H antiport. However, the exact relationships have not been worked out as yet. 111.

PROPERTIES IN BLOOD CELLS AT VARIOUS STAGES OF DIFFERENTIATION

A. General Aspects One would think that the hemopoietic tissue would be most suitable for studying the role of C1-dependent cation cotransport during differentiation. However, among the available cell lines only one is useful, namely, the HL-60 promyelocyte, which may be induced by dimethylsulfoxide to differentiate into a polymorphonuclear leukocyte or by phorbol esters into a macrophage (53). Unfortunately, the cells differentiating from the HL-60 cell line lack the C1-dependent cation transporter (53) and, in contrast to cells from the erythroid line, mature polymorphonuclear cells have no furosemide-sensitive monovalent ion transport, although this drug interferes with their electroneutral Cl-Cl exchange (154). In light of this dilemma, the investigator who wants to assess the presence of such transporters in differentiating hemopoietic cells has to turn to a comparative approach, assembling bits and pieces of information from transport data gathered in hemopoietic cells naturally arrested at various points of differentiation but unfortunately studied in several species of birds, mammals, and fish. In order to facilitate this task, data available on Cl-dependent K as well as on C1-dependent Na or Na-K cotransport have been assembled in two separate tables (Tables I1 and 111) and discussed separately. One can assume that Na-independent and Na-dependent K fluxes are carried out by molecular systems which are functionally and perhaps structurally as different from each other as they are from the Na-K pump (82). Normal erythrocyte formation consists of a sequence of some three mitotic divisions, starting with the omnipotential stem cell, followed by the proerythroblast, and the early and late nucleated erythroid cells, the latter maturing without further division into the nucleated normoblast, the enucleated reticulocyte, and finally the mature erythrocyte. Within about 4 days the terminally differentiated, mature red cell is produced with a much reduced cell volume as compared to the erythroblast and containing mainly hemoglobin and the glycolytic enzymes. Only very recently have

APPARENTLY NI-INDEPENDENT

TABLE I1 K-CI TRANSPORT: PROPERTIES IN ERYTHROCYTES AT VARIOUS

STAGES OF

DIFFERENTIATION"

Inhibitors

Cell type/species differentiation (references) Nucleated red ceUs Muscovy duck (87.90.92) Rkin duck (111,160)

Ions

Ko.5 ( m W for [KI,

Stimulus activation

Anion preference

K-CI K-CI

n.a. -30

0 1s NO, n.a.

ocsw ocsw

K-CI

ma.

CI >> NO,

ocsw

K-CI

-25

CI > Br >> 1 > NO, > acelate

K-CI(?) K-CI(?) K-CI(?)

-I5

n.a.

2&40 -10-12

CI >> NO, CI > NO?

K-CI K-CI

ma. 17-40

CI > Br >> SCN = F > NO, Br > CI >> HCO, = F >> NO3 = I

Type

ICso ( M )

n.a.

Metabolic dependency

[Klo at zero net flux

n.a. n.a.

-75 n.a.

n.a. Bumet.

5 x

n.a. n.a. > NO,

=

SCN

OCSW NEM A23187 NEM

4 0 - 3

ma., Data not available; Bumet., bumcIanide: Furos., furowmide; OCSW. osmotic cell swelling: NEM, Ncthylmaleimide; DIDS. 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid

TABLE Ill CHLORIDE-DEPENDENT Na OR Na-K TRANSPORT: PROPERTIES I N ERYTHROCYTES AT VARIOUSSTAGES OF DIFFERENTIATION^ Cell typekpecies differentiation (references) Nucleated red cells Embryonic chick (152.163) Pigeon (120,124)

Ions (Ratio)

Inhibitors

KO.C(mM)

Stimulus of activation

Anion preference

[Na'l,

[K+l,,

ICII,

Na-K

ma.

3.8

n.a.

n.a.

Care.. cAMP

Prop.

Na-K-CI (?)

n.a.

n.a.

n.a.

n.a.

OCSH, Cate.

ma.

Na-K-CI

ma.

6

n.a.

CI

=

Br >> I

Furos. Prop.

Na-K-CI

-17

-5

-75

CI

=

Br >> NO, = MeS04

n.a. ma.

-30 4-8

-500 -75

CI>>NOx CI > Br >> NO1 > SO4

OCSH NE. cAMP NaF, - 0 2 OCSH NE. cAMP OCSH, NE Isop.. NE Chol.. cAMP NaF. - 0 2 Cate.

Type

Metabolic dependency

[cation], ( m M ) at zero net flux

n.a.

n.a.

ma.

n.a.

Yes

n.a.

Yes

n.a.

40-3 10-~-10-*

n.a.

10-~-10-* 2.3 x lo-'

n.a. Yes

4.5 K 145 Na n.a. 4K I20 Na

ICm (MI

( 1 : I :2 ) ('!I

Muscovy duck (88-92.137) Pekin duck 6 - 6 7 . 112.149.151) Pekin duck (67) Turkey (6.16.51. 12&129,140.141. 162) Trout (15. 19.20)

=

NO,

=

SCN

= SO4

(I : 1 : 2 )

K-K-CI (?) Na-K-CI

n.a.

n.a.

n.a.

CI

=

=

I

Br >> I > NO, > acet.

=

SCN

(Sulmo gairdnen')

Enucleate red cells Ferret (44.45.1 14)

Rat and other rodents ( 2 2 3 . 6 9 ) Human (l,2,16. 19-31.33.36.49. 50.159.166.16R)

Na-K-CI (3: 1 :I ) Na-K-CI

(?)

>> N0,.S04.SCH

n.a.

0.35

n.a.

CI

65

3.5

n.a.

CI > > N O ,

24

&8

n.a.

CI

= Br

(l:l:2) Na-K-CI ( I :I :2)

=

Br >> SO4 > NO,

n.a.

OCSH Low-K diet OCSH (?)

Furos. Bumet. Bumet. Furos. Bumet.

2.5 x

ma.

n.a.

n.a.

n.a.

n.a.

n.a. n.a.

n.a.

5 x 5 x 10-7

Yes

2.5 K 140 Na 5K 140 Na

Furos. DIDS Amil.

2 x

Bumet. CaLt + A23187 cAMP Furos. Bumet. Furos. Bumet. cAMP Ca" + A23187

> NO, z gluconate n.a. CI = Br > NO, z i

CAMP n.a. CAMP

Furos. Furos. Furos. Burnt.

Na-(K)-CI 1 :( 1) : 2(l)1

4

4

20

CI >> gluconate

n.a.

CAMP, CGMP Funs.

Na-K-CI (l:l:2) Na-K-CI Na-CI

3 4

n.a.

49

n.a.

n.a. 26.6

n.a. -

n.a. 19.5

ma.

Na-K-CI (l:l:2) Na-(K)-CI [l:(l):I(2)1

9

9

49

n.a.

n.a.

n.a.

CI = Br >> I, Acet., NO,. SCN. SO, CI >> N&

Cate.

OCSH

~~

ma., Data not available; Bumet., bumetanide; Furos., funsemide; Cate., catecholamines: OCSH,osmotic cell sbrinkagc.

ICs (M)

n.a. ma. n.a.

40-4

n.a.

14 x

Metabolic dependency

10-4

Furos. Bumet. Furos . Bumet.

3 x 10-6 2 x 10-7 10-9

n.a. n.a.

Bumet. Furos. Bumet. Funs.

10-6

Yes

lo-' Br >> NO3 = SO, CI > Br >> NO3 = 1 = SO,

Ouabain Furosemide OCSW Furosemide 2-Deoxy-~-glucose Bumetanide Propranol, NEM

K-CI (1:2)

CI > acetate

Serum

ICx, ( M )

Hypothennia Bumetanide

Bumetanide Furosemide


> HCO3. NO3. acetate CI >> acetate

stimulus activation

Serum

Furos. Bumet. Bumet.

5 x 10-6 4 0 - 4

n.a.

Furos. Bumet. Bumet. FWOS. Bemet.

5 x 10-5 10-6 10-7 5 x 10-6 5 x 10-9

n.a.

PMA

> NO3

LTKd mouse fibroblasts (52,53.76,77)

Na-K-CI ( I : 1 :2) Na-K-CI

-45

-6

n.a.

CI >> NO3 >> SCN

OCSH, ouabain n.a.

15

3

*la0

CI > Br >> SCN. I, acetate, gluconale

Pept. (FGF. EGF)

Na-K-CI

33

n.a.

-60

Cl >> gluconate

Fetal serum

Vascular smooth muscle (80,122)

sx

n.a.

10-7

n.a.

Furos. Na-K-CI

n.a.

n.a.

n.a.

CI >> so,

n.a

Furos.