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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REFERENCES Al-Awqati, Q. (1985). Rapid insertion and retrieval of pumps and channels into membranes by exocytosis and endocytosis. In “Regulation and Development of Membrane Transport Processes” (J. S. Graves, ed.), pp. 149-157. Wiley, New York. Aronson, P. S. (1983). Mechanisms of active H+ secretion in the proximal tubule. Am. J . Physiol. 245, F647-F659. Aronson, P. S., Nee, J., and Suhm, M. A. (1982). Modifier role of internal H+ in activating the Na+-H+ exchanger in revel microvillus membrane vesicles. Nature (London) 299, 161-163. Aronson, P. S., Suhm, M. A., and Nee, J. (1983). Interaction of external Na+-H+ exchanger in renal microvillus membrane vesicles. J . Biol. Chem. 258, 6767-6771. Becker, G. L., Fiskum, G., and Lehninger, A. L. (1980). Regulation of free Ca2+by liver mitochondria and endoplasmic reticulum. J. Biol. Chem. 255, 9009-9012. Berridge, M. J. (1984). Oncogenes, inositol lipids and cellular proliferation. Biorechnology 2, 541-546. Bishop, R., Martinez, R., Nakamura, K. D., and Weber, M. J. (1983). A tumor promoter stimulates phosphorylation on tyrosine. Biochem. Biophys. Res. Commun. 115, 536543. Blaustein, M. P., and Nelson, M. T. (1982). Sodium-calcium exchange: Its role in the regulation of cell calcium. In “Membrane Transport of Calcium” (E. Carafoli, ed.), pp. 217-236. Academic Press, London. Boonstra, J., Mummery, C. L., Tertoolen, L. G. J., Van Der Saag, P. T., and De Laat, S. W. (1981). Cation transport and growth regulation in neuroblastoma cells. Modulation of K+ transport and electrical membrane properties during the cell cycle. J. Cell. Physiol. 107, 75-83. Boron, W. F. (1977). Intracellular pH transients in giant barnacle muscle fibers. Am. J. Physiol. 233, C61-C73. Boron, W. F. (1983). Transport of H+ and of ionic weak acids and bases. J . Membr. Biol. 72, 1-16. Boron, W.F., and Boulpaep, E. L. (1983). Intracellular pH regulation in the renal proximal tubule of the salamander. Basolateral HCO; transport. J . Gen. Physiol. 81, 53-94. Boron, W. F., and De Weer, P. (1976). Intracellular pH transients in squid giant axons caused by COz, NHj and metabolic inhibitors. J . Gen. Physiol. 67, 91-112. Brazy, P., and Gunn, R. B. (1976). Furosemide inhibition of chloride transport in human red blood cells. J . Gen. Physiol. 68, 583-599. Breitwieser, G. E., and Russell, J. M. (1983). Sidedness of pH dependence of the sodium pump in dialyzed squid giant axons. Biophys. J. 41, 71a. Brown, A. M., and Lew, V. L. (1983). The effect of intracellular calcium on the sodium pump of human red cells. J. Physiol. (London) 343, 455-493. Burckhardt, B.-Ch., Sato, K., and Fromter, E. (1984). Electrophysiologic analysis of bicarbonate permeation across the peritubular cell membrane of rat kidney proximal tubule. I. Basic observations. PJlugers Arch. 401, 34-42. Bums, C. P., and Rozengurt, E. (1983). Serum, platelet-derived growth factor, vasopressin and phorbol esters increase intracellular pH in Swiss 3T3 cells. Biochem. Biophys. Res. Commun. 116,931-938. Cabantchik, Z . I., Knauf, P. A., and Rothstein, A. (1978). The anion transport system of the red blood cell. The role of membrane protein evaluated by the use of “probes”. Biochim. Biophys. Acta 515, 239-302. Cala, P. M. (1983a). Cell volume regulation by Amphiuma red blood cells. J. Gen. Physiol. 82,761-784.

1. MITOGENS AND ION FLUXES

49

Cala, P. M. (1983b). Volume regulation by red blood cells. Mechanisms of ion transport between cells and mechanisms. Mol. Physiol. 4, 33-52. Carpenter, G. (1981). Vanadate, epidermal growth factor and the stimulation of DNA synthesis. Biochem. Biophys. Res. Commun. 102, 1 1 15-1121. Carpenter, G . (1984). Properties of the receptor for epidermal growth factor. Cell 37, 357358.

Carpenter, G., and Cohen, S. (1979). Epidermal growth factor. Annu. Rev. Biochem. 48, 193-2 16. Cassel, D., Rothenberg, P., Zhuang, Y. X., Deuel, T. F., and Glaser, L. (1983). Platelet derived growth factor stimulates Na+/H+exchange and induces cytoplasmic alkalinization in NR6 cells. Proc. Natl. Acad. Sci. U . S . A . 80, 6224-6228. Cassel, D., Zhuang, Y.X., and Glaser, L. (1984). Vanadate stimulates Na+/H+exchange in A431 cells. Biochem. Biophys. Res. Commun. 118, 675-681. Cassel, D., Whitely, B., Zhuang, Y.X., and Glaser, L. (1985). Mitogen independent activation of Na+H+ exchange in human epidermoid carcinoma A431 cells. Regulation by medium osmolarity. J . Cell. Physiol., 122, 178-186. Cochet, C., Gill, G. W., Meisenhelder, J., Cooper, J. A., and Hunter, T. (1984). C-Kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor stimulated tyrosine protein kinase activity. J . Biol. Chem. 259, 2553-2558. Cooper, J. A., Sefton, B. M., and Hunter, T. (1984). Diverse mitogenic agents induce the phosphorylation of two related 42,000 dalton proteins on tyrosine in quiescent chick cells. Mol. Cell. Biol. 4, 30-37. Davis, R. J., and Czech, M. P. (1984). Tumor-promoting phorbol diesters mediate phosphorylation of the epidermal growth factor receptor. J. Biol. Chem. 259, 8545-8549. DiPolo, R., and BeaugC, L. (1983). The calcium pump and sodium-calcium exchange in squid axons. Annu. Rev. Physiol. 45, 313-324. Ellory, J. C., Dunham, D. B., Logue, P. J., and Stewart, G. W. (1982). Anion-dependent cation transport in erythrocytes. Philos. Trans. R . Soc. London Ser. B 299, 483-495. Fehlmann, M., Samson, M., Koch, K. S . , Leffert, H. L., and Freychet, P. (1981). The effect of amiloride on hormonal regulations of amino acid transport in isolated and cultured adult rat hepatocytes. Biochim. Biophys. Acra 642, 88-95. Forgac, M., Cantley, L., Wiedermann, B., Alstiel, L., and Branton, D. (1983). Clathrincoated vesicles contain an ATP-dependent proton pump. Proc. Natl. Acad. Sci. U . S . A . 80, 1300-1303. Friedman, B. A., Frackelton, A. R., Ross, A . H., Connors, J. M., Fujiki, H., Sugimura, T., and Rosner, M. R. (1984). Tumor promoters block tyrosine specific phosphorylation of the epidermal growth factor receptor. Proc. Natl. Acad. Sci. U . S . A . 81, 3034-3038. Frizzell, R. A., Field, M., and Schultz, S. G . (1979). Sodium-coupled chloride transport by epithelial tissues. A m . J . Physiol. 236, FI-F8. Gerson, D. F., and Kiefer, H. (1982). High intracellular pH accompanies mitotic activity in murine lymphocytes. J. Cell. Physiol. 112, 1-4. Gilmore, T., and Martin, G. S. (1983). Phorbol ester and diacylglycerol induce protein phosphorylation at tyrosine residues. Naiure (London) 308, 487-490. Gluck, S., Cannon, C., and Al-Awqati, Q . (1982). Exocytosis regulates urinary acidification in turtle bladder by rapid insertion of H+ pumps into the luminal membrane. Proc. Natl. Acad. Sci. U . S . A . 79, 4327-4331. Glynn, I. M., and Kalish, S. J. D. (1975). The sodium pump. Annu. Rev. Physiol. 37, 13-55. Goldman, D. E. (1943). Potential, impedance and rectification in membranes. J . Gen. Physiol. 27, 37-60. Grinstein, S . , DuprC, A., and Rothstein, A . (1982). Volume regulation by human lymphocytes. J. Gen. Physiol. 79, 849-868.

50

LUIS REUSS ET AL.

Grinstein, S., Cohen, S., and Rothstein, A. (1983a). Cytoplasmic pH regulation in thymic lymphocytes by an amiloride-sensitive Na+/H+antiport. J . Gen. Physiol. 83, 341-369. Grinstein, S., Clarke, C. A., and Rothstein, A. (1983b). Activation of Na+/H+exchange in lymphocytes by osmotically induced volume changes and by cytoplasmic acidification. J . Gen. Physiol. 82, 619-638. Grunberger, G., Zick, Y.,Taylor, S. I., and Gorden, P. (1984). Tumor-promoting phorbol ester stimulates tyrosine phosphorylation in U-937 monocytes. Proc. Narl. Acad. Sci. U.S.A.81,2762-2766. Hasselbach, W. (1981). Calcium-activated ATPase of the sarcoplasmic reticulum membranes. In “Membrane Transport” ( S . L. Bonting and J. J. H. H. M. De Pont, eds.), pp. 183-208. Elsevier, New York. Heinz, E. (1978). “Mechanics and Energetics of Biological Transport.” Springer-Verlag, Berlin and New York. Heldin, C.H., and Westermark, B. (1984). Growth factors: Mechanism of action and relation to oncogenes. Cell 37, 9-20. Heyworth, C. M., Whetton, A. D., Kinsella, A. R., and Housley, M. D. (1984). The phorbol ester TPA inhibits glucagon-stimulated adenylcyclase activity. FEBS Lett. 170, 38-42. Hille, B. (1984). “Ionic Channels of Excitable Membranes.” Sinauer, Sunderland, Massachusetts. Hodgkin, A. L., and Horowicz, P. (1959). The influence of potassium and chloride ions on the membrane potential of single muscle fibres. J. Physiol. (London) 148, 127-160. Hodgkin, A. L., and Katz, B. (1949). The effect of sodium ions on the electrical activity of the giant axon of the squid. J . Physiol. (London) 108, 33-37. Huang, J. S., Huang, S. S., and Deuel, T. F. (1984). Transforming protein of simian sarcoma virus stimulates autocrine growth of SSV-transformed cells through PDGF cell-surface receptors. Cell 39, 79-81. Hiilser, D. F., and Frank, W.(1971). Stimulation of embryonic rat cell in culture by a protein fraction isolated from fetal calf serum. Z. Naturforsch. 26b, 1045-1048. Hunter, T.,Ling, N., and Cooper, J. A. (1984). Protein kinase C phosphorylation of the EGF receptor at threonine residue close to the cytoplasmic face of the plasma membrane. Nature (London) 311, 480-483. Ives, H. E., Yee. V. J., and Warnock, D. G. (1983). Mixed type inhibition of the renal Na+/H+antiporter by Lit and amiloride. J . Biol. Chem. 258, 9710-9716. Iwashita, S . , and Fox, C. F. (1984). Epidermal growth factor and potent phorbol tumor promoters induce epidermal growth factor phosphorylation in a similar but distinctively different manner in human epidermal carcinoma A431 cells. J . Biol. Chem. 259, 25592561. Johnson, J. J., Epel, D., and Paul, M. (1976). Intracellular pH and activation of sea urchin eggs after fertilization. Nature (London) 262, 661-664. Kashiwagura, T., Deutsch, C. J., Taylor, J., Erecinska, M., and Wilson, D. F. (1984). Dependence of gluconeogenesis, urea synthesis, and energy metabolism of hepatocytes on intracellular pH. J . Biol. Chem. 259, 237-243. Kelleher, D. J., Pessin, J. E., Ruoho, A. E., and Johnson, G. L. (1984). Phorbol ester induces desensitization of adenylate cyclase and phosphorylation of the P-adrenergic receptor in turkey erythrocytes. Proc. Natl. Acad. Sci. U.S.A. 81, 4316-4320. Kinsella, J. L., and Aronson, P. S. (1980). Properties of the Na+-H+ exchanger in renal microvillous membrane vesicles. Am. J . Physiol. 238, F461-F469. Knauf, P. A. (1979). Erythrocyte anion exchange and the band 3 protein: Transport kinetics and molecular structure. Curr. Top. Membr. Transp. 12, 249-253. Koch, K. S., and Leffert, H. (1979). Increased sodium ion influx is necessary to initiate rat hepatocyte proliferation. Cell 18, 153- 163.

1. MITOGENS

51

AND ION FLUXES

L’Allemain, G . , Franchi. A., Cragoe, E., Jr., and Pouysskgur, J . (1984a). Blockade of the Na+/H+antiport abolishes growth factor-induced DNA synthesis in fibroblasts. J . B i d . Chem. 259,4313-4319. L’Allemain, G . , Paris, S., and PouyssCgur, J . (1984b). Growth factor action and intracellular pH regulation in fibroblasts. Evidence for a major role of the N a + / H +antiport. J. Biol. Chem. 259, 5809-5815. Larson, M., and Spring. K. R. (1984). Volume regulation by Necturus gallbladder: Basolateral KCI exit. J . Membr. B i d . 81, 219-232. Lau, K. R., Hudson, R. L., and Schultz, S. G. (1984). Cell swelling increases a bariuminhibitable potassium conductance in the basolateral membrane of Necturus small intestine. Proc. Natl. Acad. Sci. U . S . A . 81, 3591-3594. LeFevre, P. (1975). The present state of the carrier hypothesis. Curr. Top. Memhr. Transp. 7, 190-215. Lew, V. L . , and Ferreira, H. G. (1978). Calcium transport and the properties of a calciumsensitive potassium channel in red cell membranes. Curr. Top. Membr. Transp. 10, 217-271. Lindemann, B. (1984). Fluctuation analysis of sodium channels in epithelia. Annu. Rev. Physiol. 46,497-515. Lubin, M., Cahn, F., and Coutermarsh, B. A. (1982). Amiloride, protein synthesis, and activation of quiescent cells. J. Cell. Physiol. 113, 247-251. Macara, I. G . , Marinette, G . V., and Balduzzi, P. C. (1984). Transforming protein of avian sarcoma virus UR2 is associated with phosphatidylinositol kinase activity: Possible role in tumorogenesis. Proc. Natl. Acad. Sci. U . S . A . 81, 2728-2732. McNeil, P. L., McKenna, M. P., and Taylor, D. L. (1985). A transient rise in cytosolic calcium follows stimulation of quiescent cells with growth factors and is inhibitable with phorbol myristate acetate. J. Csll Biol. 101, 372-379. McNeil, P. L., Murphy, R. F., Lanni, R., and Taylor, D. L . (1984). A method for incorporating macromolecules into adherent cells. J . Cell Biol. 98, 1556-1564. Mancuso, D., and Glaser, L. (1985). Characterization of the Na+/H+exchanger in human epidemoid carcinoma A431 vesicles. J. Cell. Physiol. 123, 297-304. Mastro, A. M. (1983). Phorbol ester tumor promoters and lymphocyte proliferation. Cell Biol. Int. Rep. 7 , 881-893. Mix, L. L., Dinerstein, R., and Villereal, L. (1984). Mitogens and melittin stimulate an increase in intracellular free calcium concentration in human fibroblasts. Biochem. Biophys. Res. Commun. 119, 69-75. Moolenaar, W. H., De Laat, S. W., and Van der Saag. P. T. (1979). Serum triggers a sequence of rapid ionic conductance changes in quiescent membrane cells. Nature (London) 279, 721-723. Moolenaar, W. H., Mummery, C. L. Van der Saag, P. T., and De Laat, S. W. (1981). Rapid ionic events and the initiation of cell growth in serum-stimulated neuroblastoma cells. Cell 23, 789-798. Moolenaar, W. H., De Laat, S. W., Mummery, C. L., and Van der Saag, P. T. (1982). Na+/H+ exchange in the action of growth factors. I n “Ions, Cell Proliferation and Cancer” (A. L. Boynton, W. L. McKeehan, and J. F. Whitfield, eds.), pp. 151-162. Academic Press, New York. Moolenaar, W., Tsien, R., Van der Saag, P., and De Laat, S. (1983). Na+/H+exchange and cytoplasmic pH in the action of growth factors in human fibroblasts. Nature (London) 304,645-648. Moolenaar, W. H., Tertoolin, L. G. S., and de Laat, S . W. (1984a). Growth factors immediately raise cytoplasmic free Ca2+in human fibroblast. J . Biol. Chem. 259, 8066-8069. Moolenaar, W. H., Tertoolin, L. G. S., and de Laat, S. W. (1984b). Phorbol ester and ~

52

LUIS REUSS ET AL.

diacylglyceride mimic growth factor in raising cytoplasmic pH. Nature (London) 312, 371-374. Moore, R. D. (1981). Stimulation of Na: H exchange by insulin. Biophys. J . 33, 203-210. Mullins, L. J., and Noda, K. (1963). The influence of sodium-free solutions on the membrane potential of frog muscle fibers. J. Gen. Physiol. 47, 117-132. Mummery, C. L., Boonstra, J., Van Der Saag, T., and De Laat, S. W. (1981). Modulation of functional and optimal (Na+-K+) ATPase activity during the cell cycle of neuroblastoma cells. J. Cell. Physiol. 107, 1-9. Munson, R., Westermark, B., and Glaser, L. (1979). Tetrodotoxin-sensitive sodium channels in normal human fibroblasts and normal human glia-like cells. Proc. Natl. Arad. Sci. U.S.A. 76, 6425-6429. Mlirer, H., Hopfer, U., and Kinne, R. (1976). Sodium/proton antiport in brushbordermembrane vesicles isolated from rat small intestine and kidney. Biochem. J . 154,597604. Nishizuka, Y. (1983a). The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature (London) 308, 693-698. Nishizuka, Y. (1983b). Calcium, phospholipid turnover and transmembrane signalling. Philos. Trans. R. SOC.London Ser. B 302, 101-112. Okada, C. Y., and Rechsteiner, M. (1982). Introduction of macromolecules into cultured mammalian cells by osmotic lyses of pinocytic vesicles. Cell 29, 33-41. Okhuma, S.,and Poole, B. (1978). Fluorescence probe measurement of the intralysosomal pH in liver cells and the perturbation of pH by various agents. Proc. Natl. Acad. Sci. U.S.A. 75, 3327-3331. Owen, N. E., and Villereal, M. L. (1983). Na influx and cell growth in cultured human fibroblasts. Exp. Cell Rex. 143, 37-46. Palfrey, H. C., and Rao, M. C. (1983). Na/K/CI co-transport and its regulation. J . Exp. Biol. 106,43-54. Pardee, A. B. (1974). A restriction point for control of normal animal cell proliferation. Proc. Natl. Acad. Sci. U.S.A. 71, 1286-1290. Pardee, A. B., Dubrow, R., Hamlin, J. C., and Kletzien, R. F. (1978). Animal Cell Cycle. Annu. Rev.Biochem. 47,715-750. Pans, S . , and Pouyssbgur, J. (1984). Growth factors activate the Na+/H+ antiporter in quiescent fibroblasts by increasing its affinity for intracellular H+. J . Biol. Chem. 259, 10989- 10994. Petersen, K.-U., and Reuss, L. (1983). Cyclic AMP-induced chloride permeability in the apical membrane of Necturus gallbladder epithelium. J . Gen. Physiol. 81, 705-729. Pettigrew, D. W., and Frieden, C. (1979). Binding of regulatory ligands to muscle phosphofructokinase. A model for nucleotide binding as a function of temperature and pH. J . Biol. Chem. 259, 1887-1895. Pouyssbgur, J., Jacques, Y., and Lazdunski, M. (1980). Identification of a tetrodotoxinsensitive Na+ channel in a variety of fibroblast lines. Nature (London) 286, 162-164. Pouyssbgur, J., Sardet, C., Franchi, A., L’Allemain, G., and Paris, S . (1984). A specific mutation abolishing Na+/H+antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH. Proc. Natl. Acad. Sci. U.S.A. 81, 4833-4837. %chard, J. W., Alger, J. R., Behan, K. L., Pitroff, 0. H., and Shulman, R. G. (1983). Cerebral metabolic studies in vivo by IlP NMR. Proc. Natl. Acad. Sci. U.S.A.80,2748275 I . Pruss, R. M., and Henchman, H. R. (1977). Variants of 3T3 cells lacking mitogenic response to epidermal growth factor. Proc. Natl. Acad. Sci. U.S.A. 74, 3918-3921. Rasmussen, H. (1981). “Calcium and CAMPas Synarchic Messengers.” Wiley, New York.

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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.

REFERENCES Aickin, C. C., and Thomas, R. C. (1977). An investigation of the ionic mechanism of intracellular pH regulation in mouse soleus muscle fibers. J . Physiol. (London) 273, 295-3 16. Aronson, P. S., Nee, J., and Suhm, M. A. (1982). Modifier role of internal H+ in activating the Na+-H+ exchanger in renal microvillus membrane vesicles. Nature (London) 299, 161-163. Baker, P. F., Blaustein, M. P., Hodgkin, A. L., and Steinhardt, R. (1969). The influence of calcium on sodium efflux in squid axons. J. Physiol. (London) 200, 432-458. Benos, D. J. (1982). Amiloride: A molecular probe of sodium transport in tissues and cells. Am. J . Physiol. 242, C131-C145. Benos, D. J., and Saperstein, V. S . (1983). Characteristics of an amiloride-sensitive sodium entry pathway in cultured rodent glial and neuroblastoma cells. J . Cell. Physiol. 116, 213-220. Bentley, P. J. (1968). Amiloride, a potent inhibitor of sodium transport across the toad bladder. J . Physiol. (London) 195, 317-330. Bernstein, A., Hunt, V., Crichley, V., and Mak, T. W. (1976). Induction by ouabain of hemoglobin synthesis in cultured Friend erythroleukemic cells. Cell 9, 375-381. Besterman, J. M., May, W. S . , LeVine, H., Cragoe, E. J., and Cuatrecasas, P. (1985). Amiloride inhibits phorbol esters-stimulated Na+/H+exchange and protein kinase C. J. Eiol. Chem. 260, 1155-1 159. Blaustein, M. P., and Hodgkin, A. L. (1969). The effect of cyanide on the efflux of calcium from squid axons. J . Physiol. (London) 200, 491-527. Boonstra, J., Moolenaar, W. H., Harrison, H., Moed, P., van der Saag, P. T., and de Laat, S . W. (1983). Ionic responses and growth stimulation induced by nerve growth factor and epidermal growth factor in rat pheochromocytoma (PC12) cells. J . CeN Eiol. 97,9298. Brierley, G. P., Settlemire, C. T., and Knight, V. A. (1968). Ion transport by heart mitochondria. Arch. Biochem. Eiophys. 126,276-288. Brock, T. A., Lewis, L. J., and Smith, J. B. (1982). Angiotensin increases Na+ entry and Na+/K+pump activity in cultures of smooth muscle from rat aorta. Proc. Narl. Acad. Sci. U.S.A. 79, 1438-1442. Cassel, D., Rothenberg, P., Zhuang, Y.,Deuel, T., and Glaser, L. (1983). Platelet-derived growth factor stimulates Na+/H+exchange and induces cytoplasmic alkalinization in NR6 cells. Proc. Natl. Acad. Sci. U.S.A. 80, 6224-6228. Christensen, H. N., Akedo, H., Oxender, D. L., and Winter, C. G. (1962). Permeability and amino acid transport. On the mechanism of amino acid transport into cells. In “Amino Acid Pools” (T. J. Holden, ed.), pp. 527-538. Elsevier, Amsterdam. Crane, R. K., Miller, D., and Bihler, I. (1961). Restrictions on the possible mechanisms of intestinal active transport of sugars. I n “Membrane Transport and Metabolism” (A. Kleinzeller and A. Kotyk, eds.), pp. 439-449. Academic Press, New York. Cuthbert, A., and Cuthbert, A. W. (1978). Fertilization acid production in Psammechinus eggs under pH clamp conditions and the effects of some pyrazine derivatives. Exp. CeN Res. 114, 409-415.

2. ION EXCHANGE IN ACTIVATED CELLS

83

Cuthbert, A. W., and Edwardson, J. M. (1979).Synthesis, properties and biological activity caroxamide-a new ligand of tritiated N-benzylamidino-3,5-diamino-6-chloropyrazine for epithelial sodium channels. J. Pharm. Pharmacol. 31, 382-386. Davis, B. A., Schwartz, A., Samaha, F. J., and Kranias, E . G . (1983).Regulation of cardiac sarcoplasmic reticulum calcium transport by calcium-calmodulin-dependent phosphorylation. J. Biol. Chem. 258, 13587-13591. Deutsch, C., Holian, A., Holian, S. K., Daniele, R. P., and Wilson, D. F. (1979).Transmembrane electrical pH gradients across human erythrocytes and human peripheral lymphocytes. J. Cell. Physiol. 98, 137-144. Deutsch, C., Taylor, J. S . , and Wilson, D. F. (1982).Regulation of intracellular pH of human peripheral blood lymphocytes as measured by I9F-NMR. Proc. N a f l .Acad. Sci. U . S . A . 79, 7944-7948. Deutsch, C., Taylor, J. S., and Price, M. (1984).pH homeostasis in human lymphocytes: Modulation by ions and mitogen. J. Cell Biol.98, 885-894. Dicker, P., and Rozengurt, E. (1981).Phorbol ester stimulation of Na+ influx and Na+-K+ pump activity in Swiss 3T3 cells. Biochern. Biophys. Res. Commun. 100,431-433. Ehrlich, E. N., and Crabbe, J. (1968).The mechanism of action of amipramizide. P'ugers Arch. 302, 79-96. Eider, J., and Keifer, J. (1967). Effect of a new acylguanidine-amiloride HCI (MK870) on isolated amphibian skin. Klin. Wochenschr. 45, 737-738. Gerson, D. F., and Kiefer, H. (1982).High intracellular pH accompanies mitotic activity in murine lymphocytes. J . Cell. Physiol. 1l2, 1-4. Gerson, D. F., Kiefer, H., and Eufe, W. (1982).lntracellular pH of mitogen-stimulated lymphocytes. Science 216, 1009-1010. Grinstein, S., and Furuya, W. (1984).Amiloride-sensitive Na+/H+exchange in human neutrophils: Mechanism of activation by chemotactic factors. Biochem. Biophys. Res. Commun. l22, 755-762. Grinstein, S.,Clarke, C. A., Dupre, A,, and Rothstein, A. (1982).Volume-induced increase of anion permeability in human lymphocytes. J . Gen. Physiol. 80, 801-823. Grinstein, S., Clarke, C. A., and Rothstein, A. (1983).Activation of Na+/H+exchange in lymphocytes by osmotically induced volume changes and by cytoplasmic acidification. J . Gen. Physiol. 82, 619-638. Grinstein, S . , Cohen, S . , and Rothstein, A. (1984).Cytoplasmic pH regulation in thymic lymphocytes by an amiloride-sensitive N a + / H +antiport. J. Gen. Physiol. 83, 341-369. Gross, J. D., Bradbury, J., Kay, R. R., and Peacey, M. J. (1983).Nature (London)303,244245. Harold, F. M., and Pappineau, D. (1972).Cation transport and electrogenesis by Strepfococcus faecalis: Proton and sodium extrusion. J . Membr. B i d . 8, 45-62. Herchuelz, A., Sener, A., and Malaise, W. J. (1980).Regulation of calcium fluxes in rat pancreatic islets: Calcium extrusion by sodium-calcium countertransport. J. Membr. Biol. 57, 1-12. Home, W. C., and Simons, E. R. (1978).Probes of transmembrane potentials in platelets: Changes in cyanine dye fluorescence in response to aggregation stimuli. Blood 51,741749. Home, W. C., and Simons, E. R. (1979).Effects of amiloride on the response of human platelets to bovine thrombin. Thromb. Res. 13, 599-604. Home, W. C., Norman, N. E., Schwartz, D. B., and Simons, E. R. (1981).Changes in cytoplasmic p H and in membrane potential in thrombin-stimulated human platelets. Eur. J. Biochem. UO, 295-302. Ives, H. E.,Yee, V. J., and Warnock, D. G . (1983).Mixed type inhibition of the renal Na+/ H+ antiporter by Li' and amiloride. J. Biol. Chem. 258, 9710-9716.

84

MITCHEL L. VILLEREAL

Jamieson, G. J., Jr., and Villereal, M. L. (1985). Mitogen stimulated release of inositol phosphates in human fibroblasts. Arch. Biochem. Biophys. (submitted). Jamieson, G. A., Jr., Frazier, W. A., and Schlesinger, P. H. (1984). Transient increase in intracellular pH during Dictyostelium differentiation. J . Cell Biol. 99, 1183-1 187. Johnson, J. D., Epel, D., and Paul, M. (1976). Intracellular pH and activation of sea urchin eggs after fertilisation. Nature (London) 262, 661-664. Jurkowitz, M. S., Altschuld, R. A., Brierley, G. P., and Cragoe, E. J., Jr., (1983). Inhibition of Na+-dependent Ca2+efflux from heart mitochondria by amiloride analogues. FEBS Lett. 162, 262-265. Kinsella, J. L., and Aronson, P. S. (1981a). Amiloride inhibition of the Na+-H+ exchanger in renal microvillus membrane vesicles. Am. J. Physiol. 238, F374-F379. Kinsella, J. L., and Aronson, P. (1981b). Interaction of NH: and Li+ with the renal microvillus membrane Na+-H+ exchanger. Am. J . Physiol. 238, C220-C226. Koch, K. S., and Leffert, H. L. (1979). Increased sodium ion influx is necessary to initiate rat hepatocyte proliferation. Cell 18, 153-163. Korchak, H. M., and Weissmann, G. (1981). Stimulus-response coupling in the human neutrophil: Transmembrane potential and the role of extracellular Na+. Biochim. Biophys. Actu 601, 180-194. Krieger, N. S., and Tashjian, A. H., Jr. (1980). Parathyroid hormone stimulates bone resorption via a Na+-Ca2+ exchange mechanism. Nature (London) 287, 843-845. L’Allemain, G., Franchi, A., Cragoe, E., Jr., and Pouyssegur, J. (1984). Blockade of the Na+/H+antiport abolishes growth factor-induced DNA synthesis in fibroblasts: Structure-activity relationships in the amiloride series. J . Biol. Chem. 259, 4313-4319. Leffert, H. L., Koch, K. S., Fehlmann, M., Heiser, W., Lad, P. J., and Skelly, H. (1982). Amiloride blocks cell-free protein synthesis at levels attained inside cultured rat hepatocytes. Biochem. Biophys. Res. Commun. 108,738-745, Levenson, R., Housman, D., and Cantley, L. (1980). Amiloride inhibits murine erythroleukemia cell differentiation: Evidence for a CaZ+requirement for commitment. Proc. Natl. Acad. Sci. U.S.A. 77,5948-5952. Lindemann, B., and Van Driessche, W. (1977). Sodium-specificmembrane channels of frog skin are pores: Current fluctuation reveal high turnover. Science 195, 292-294. Lubin, M.,Cahn, F., and Coutermarsh, B. A. (1982). Amiloride, protein synthesis, and activation of quiescent cells. J . Cell. Physiol. 113, 247-251, Luttgau, H. C., and Niedegerke, R. (1958). The antagonism between Ca2+and Na+ ions on the frog’s heart. J . Physiol. (London) 143,486-505. McNeil, P. L., McKenna, M. P., and Taylor, D. L. (1984). Intracellular calcium increases transiently following mitogenic stimulation of quiescent fibroblasts with serum. J . Cell Biol. 99, 4a. Mager, D., and Bernstein, A. (1978). Early transport changes during erythroid differentiation of Friend leukemic cells. J. Cell. Physiol. 94, 275-285. Martin, F. T., and Rothman, F. G. (1980). Regulation of development in Dictyosrelium discoidium IV. Effects of ions on the rate of differentiation and cellular response to cyclic AMP. J . Cell Biol. 87, 823-827. Mitchell, P., and Moyle, J. (1969). Translocation of some anions, cations and acids in rat liver mitochondria. Eur. J . Biochem. 9, 149-155. Mitchell, P., and Moyle, J. (1967). Respiration-driven proton translocation in rat liver mitochondria. Biochem. J . 105, 1147-1 162. Mix, L. L., Dinerstein, R. J., and Villereal, M. L. (1984). Mitogens and melittin stimulate an increase in intracellular free calcium concentration in human fibroblasts. Biochem. Biophys. Res. Commun. 119,69-75.

2. ION EXCHANGE IN ACTIVATED CELLS

a5

Molski, T. F. P., Naccache, P. H., Volpi, M.. Wolpert, L. M., and Sha’afi, R. 1. (1980). Specific modulation of the intracellular pH of rabbit neutrophils by chemotactic factors. Biochem Biohys. Res. Commun. 94, 508-514. Moolenaar, W., Boonstra, J., van der Saag, P.. and de Laat, S. W . (1981a). Sodium/proton exchange in mouse neuroblastoma cells. J. B i d . Chrm. 256, 12883-12887. Moolenaar, W. H., Mummery, C. L., van der Saag, P. T., and de Laat. S. W. (1981b). Rapid ionic events and the initiation of growth in serum-stimulated neuroblastoma cells. Cell 23, 789-798. Moolenaar, W. H., de Laat, S. W., Mummery, C. L., and van der Saag, P. T. (1982). Na+/ H’ exchange in the action of growth factors. In “Ions, Cell Proliferation, and Cancer” (A. L . Boynton, W. L . McKeehan, and J. F. Whitfield, eds.). Academic Press, New York. Moolenaar, W. H., Tsien, R. Y., van der Saag, P. T., and de Laat, S. W. (1983). Na+/H+ exchange and cytoplasmic pH in the action of growth factors in human fibroblasts. Nature (London) 304,645-648. Moolenaar, W. H., Tertoolen, L., and de Laat, S. W. (1984a). Growth factors immediately raise cytoplasmic free Ca2+in human fibroblasts. J. B i d . Chem. 259, 8066-8069. Moolenaar, W. H., Tertoolen, L . G. J . , and de Laat, S. W . (1984b). Phorbol ester and deacylglycerol mimic growth factors in raising cytoplasmic pH. Nature (London) 312, 371-374. Moms, J., Metcalfe, J. C., Smith, G. A., Hesketh, R., and Taylor, M. V. (1984). Some mitogens cause rapid increases in free calcium in fibroblasts. FEBS Lett. 169, 189-193. Muldoon, L. L., and Villereal, M. L. (1985). Calcium uptake by permeabilized cultured fibroblasts: effect of mitochondria1 inhibitors, A23187, arachidonic acid, and inositol trisphosphate. Fed. Proc. 44, 1594. Muldoon. L. L., Dinerstein, R. J., and Villereal, M. L . (1985). Intracellular pH in cultured human fibroblasts: Effects of mitogens, A23187 and phospholipase activation. A m . J . Physiol. 249, C140-Cl48. Murer, H., Hopfer, U., and Kinne, R. (1976). Sodium/proton antiport in brush-bordermembrane vesicles isolated from rat small intestine and kidney. Biochem. J. 154, 597604. Naccache, P. H., Showell, H. J., Becker, E . L., and Sha’afi. R. 1. (1977). Transport of sodium, potassium and calcium across rabbit polymorphonuclear leukocyte membranes: Effect of chemotactic factor. J . Cell Biol. 73, 428-444. Naka, M., Nishikawa, M., Adelstein, R. S., and Hidaka, H. (1984). Phorbol ester-induced activation of human platelets is associated with protein kinase C phosphorylation of myosin light chains. Nature (London) 306,490, 492. Nishizuka, Y. (1984). The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature (London) 308, 693-697. O’Donnell, M. E., and Villereal, M. L. (1982). Membrane potential and Na’ flux in neuroblastoma x glioma hybrid cells: Effects of amiloride and serum. J. Cell. Physiol. 113, 405-4 I 2. O’Donnell, M. E., Becker, J., Cragoe, E . J., and Villereal, M. L . (1984). Inhibition of Na+ flux in Chinese hamster ovary cells and Swiss 3T3 cells by a potent new derivative of amiloride. Fed. Proc., Fed. Am. SOC.Exp. Biol. 43, 520a. Owen, N. E. (1984). Platelet-derived growth factor stimulates Na’ influx in vascular smooth muscle cells. Am. J. Physiol. 247, C50I-C505. Owen, N. E., and Villereal, M. L . (1982a). Effect of the intracellular Ca2+antagonist TMB-8 on serum-stimulated Na+ influx in human fibroblasts. Biochem. Biophys. Res. Commun. 109, 762-768.

86

MITCHEL L. VILLEREAL

Owen, N. E., and Villereal, M. L. (1982b). Evidence for a role of calmodulin in serum stimulation of Na+ influx in human fibroblasts. Proc. Natl. Acad. Sci. U . S . A .79, 35373541. Owen, N. E., and Villereal, M. L. (1983a). Efflux of 45Ca2+from human fibroblasts in response to mitogenic agents. J . Cell. Physiol. 117, 23-29. Owen, N. E., and Villereal, M. L. (1983b). Lys-bradykinin stimulates Na+ influx and DNA synthesis in cultured human fibroblasts. Cell 32, 979-985. Owen, N. E., and Villereal, M. L. (1985). Role of Ca2+in serum-stimulated Na+ influx in normal and transformed cells. Am. J . Physiol. 248, C288-C295. Paris, S., and Pouyssegur, J. (1983). Biochemical characterization of the amiloride-sensitive Na+/H+antiport in Chinese hamster lung fibroblasts. J . Biol. Chem. 258, 3503-3508. Pouyssegur, J., Chambard, J. C., Franchi, A,, Paris, S., and Van Obberghen-Schilling, E. (1982). Growth factor activation of an amiloride-sensitive Na+/H+exchange system in quiescent fibroblasts: Coupling to ribosomal protein S6 phosphorylation. Proc. Natl. Acad. Sci. U.S.A.79, 3935-3939. Reuter, H., and Seitz, N. (1967). Untersuchungen zur kinetik des 4SCa-effluxvon meerschweinchenvorhofen. Naunyn-Schmiedeberg’s Arch. Exp. Paihol. Pharmakol. 257, 324. Reuter, H., and Seitz, N. (1968). The dependence of calcium efflux from cardiac muscle on temperature and external ion composition. J . Physiol. (London) 195, 45 1-470. Rindler, M. J., Taub, M., and Saier, M.H. (1979). Uptake of 22Na+by cultured dog kidney cells (MDCK). J . Biol. Chem. 254, 11431-1 1439. Rink, T. J., Tsien, R. Y., and Pozzan, T. (1982). Cytoplasmic pH and free Mg2+in lymphocytes. J. Cell Biol. 95, 189-196. Roos, A., and Boron, W. F. (1981). Intracellular pH. Physiol. Rev. 61, 296-434. Rosoff, P. M., and Cantley, L. C. (1983). Increasing the intracellular Na+ concentration induces differentiation in a pre-B lymphocyte cell line. Proc. Null. Acad. Sci. U . S . A . 80,7547-7550. Rosoff, P. M., and Cantley, L. C. (1984). Phorbol esters induce differentiation in a pre-Blymphocyte cell line by enhancing Na+/H+ exchange. J . B i d . Chem. 259, 7056-7060. Rothenberg, P., Glaser, L., Schlesinger, P., and Cassel, D. (1983). Epidermal growth factor stimulates amiloride-sensitive 22Na+uptake in A431 cells: Evidence for Na+/H+ exchange. J . Biol. Chem. 258,4883-4889. Rozengurt, E., Gelehrter, T. D., Legg, A., and Pettican, P. (1981). Melittin stimulates Na+ entry, Na+-K+ pump activity and DNA synthesis in quiescent cultures of mouse cells. Cell 23, 781-786. Sandler, W. C., LeBreton, G. C., and Feinberg, H. (1980). Movement of sodium into human platelets. Biochim. Biophys. Acta 600,448-455. Saperstein, V. S., and Benos, D. J. (1984). Activation of amiloride-sensitive sodium transport in C6 glioma cells. J . Neurochem. 43, 1098-1105. Schuldiner, S., and Rozengurt, E. (1982). Na+/H+antiport in Swiss 3T3 cells: Mitogenic stimulation leads to cytoplasmic alkalinization. Proc. Narl. Acad. Sci. U.S.A. 79,77787782. Sha’afi, R. I., Molski, T. F. P., and Naccache, P. H. (1981). Chemotactic factors activate differentiable permeation pathways for sodium and calcium in rabbit neutrophils: Effect of arniloride. Biochem. Biophys. Res. Commun. 99, 1271-1275. Sha’afi, R. I., Naccache, P. H., Molski, T. F. P., and Volpi, M. (1982). Chemotactic stimuliinduced changes in the pHi of rabbit neutrophils. In “lntracellular pH: Its measurement, Regulation and Utilization in Cellular Functions” (R. Nuccetelli, ed.), pp. 513525. Liss, New York.

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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.
n.a.

ma.

ocsw

Furos. DIDS Furos.

n.a.

n.a.

n.a. NEM NEM

Furos. Furos. Furos.

4 0 - 4

ma. n.a.

Yes n.a. n.a.

n.a. n.a. n.a.

ocsw

Furos. Furos. MeZ+ + A23187

n.a. 10-3-10-5

(?)

n.a.

No Yes

-I5

Furos. Bumet.

4 0 - 3

Yes

n.a.

NEM

Toad lish (96) (Opsnnus tau)

Trout (19.20) (Salmo goirdneri)

Rcticulocytes Rabbit (130) ShCeP (95.99)

pie (108) Eoucleare red cells DO#(133) Low-K sheep (37-39)

Human (81,104,105,166)

K-CI

-17

CI >> 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

<10-4

" ma., Data not available: Furos.. furosemide; Prop.. propranolol: Bumet.. bumetanide: OSCH. osmotic cell shrinkage: Isop.. isoproterenol: Amil.. amiloride: Chol., cholera endotoxin; NE. norepinephrine: Cate.. catecholamines.

3. CI-DEPENDENT CATION COTRANSPORT AND CELL DIFFERENTIATION

103

transport studies on the early differentiation stages of these cells become feasible by the use of electron microprobe analysis (84,85). These studies have shown that the overall transport rates decrease at the late erythroblast stage, that is, long before enucleation occurs and hemoglobin synthesis reaches its maximum. This finding means that high-turnover transport systems must serve important functions, primarily in the earliest cell precursor, and, thereafter, are inactivated since they are no longer needed. Transport studies other than those conducted by the use of electron probe microanalysis are not available for the early generations of red cell precursors. Nevertheless, if one considers the nucleated bird and fish red cells as quasi-equivalents of late erythroblasts-early normoblasts, that is, as cells which just came out of the last mitosis and were prevented from further maturation by some mechanism, interesting insights into the properties of K-CI cotransport during the last steps of red cell differentiation and maturation can be obtained. B. Volume-Stimulated K-CI Cotransport

Table I1 reveals that Na-independent K-CI cotransport is present in nucleated red cells, in reticulocytes, and in the enucleate terminal red cells. From the data reported in the literature, it appears that the flux activity per cell is higher in the nucleated red cells, where maximum fluxes on the order of several hundred mmol/liter cells/hour may be measured. The activity is intermediate in reticulocytes and only marginal in the enucleate, mature red cells with fluxes of a few mmol/liter cells/hour or less. Moreover, in some suborders such as the ruminants, K-CI cotransport is present only in animals with genetically low-K (but not genetically high-K) red cells, although the reticulocyte precursors of both red cell types do possess K-Cl transport activities (99). From the work on muscovy duck and low-sheep red cells, it seems that the chemical gradients for the two ions are the driving forces, but much work is needed to establish the external and internal cation concentrations at which the net flux is zero. There are two features of K-Cl transport which are consistent throughout the three final red cell maturation stages. The values remain high, that is, the affinity for KOand Rb, (used as a K analog) is low, and the ICsovalues for the loop diuretics furosemide or bumetanide indicate a low affinity of the K-Cl transporter for these drugs. In comparison with C1-mediated Na or Na-K transport, the coincidence of these two parameters is significant but mechanistically not understood. An interesting relationship, however, has been reported between KO(using Rb,) and the ICso values for furosemide in low-sheep red cells, suggesting that the transported ions increase the affinity of the K-CI transporter for the drug by almost two orders of magnitude (101).

104

PETER K. LAUF

Although the comparative approach permits an extrapolation with respect to absolute transport activities to hemopoietic cells of earlier stages than given in Table 11, this cannot be easily used to answer the question of the functional role of this system during differentiation. Perhaps the acquisition of a K-Cl cotransport capacity responding at all stages of maturation (Table 11)to osmotic cell swelling occurred evolutionarily and ontogenically later than the development of the facility for RVD by K-amino acid release. The latter is found in red cells of bivalves (7,134) and is still present to some extent in those of flounders (48). Since ontogeny repeats evolution, one could make a case for a gradual transition to the development and utilization of K-Cl transport during the early phases of differentiation not covered by Table 11, a process destined to conserve or to replace amino acid not available any more for RVD. Still, in animals which are not exposed to tidal osmolarity changes as are the bivalves, hemopoietic cells maintain a K-Cl transporter capable of RVD into their most mature stage, the erythrocyte. One possibility is that K-Cl transport serves as an osmotic overflow valve at times when there is high Na-dependent nutritional solute uptake as, for example, in liver cells in vitro, where activation of RVD through uncoupled K and CI pathways has been reported (13,93). This proposition needs to be tested in proerythroblasts either in culture or with the electron probe. Another hypothesis to be tested is the possible role of outward K-Cl cotransport during the reticulocyte-to-erythrocyte transition. Several reports have shown that during reticulocyte maturation K rather than Na experiences the greatest turnover (108,130). In piglets C1-dependent K fluxes are highest in the early reticulocytes harvested 7 days after birth and are equivalent in magnitude to the Na-K pump activity, both parameters declining in parallel to almost adult cell levels over a short period of about 24 to 48 hours. Simultaneously, the cell volume is reduced by 50% (108). This work raises the important question of the functional coupling between KC1 fluxes and the Na-K pump during cellular differentiation and maturation, which has not been addressed satisfactorily either in thermodynamic or kinetic terms. This point is in particular very crucial for understanding the role of K-Cl transport v i s - h i s the development of genetically highand low-K ruminant red cells (see Section II1,C). C. Chemically Modified K-Ci Cotransport

Additional input about the nature as well as the function during differentiation of K-Cl transport comes from work using chemical modifiers such as NEM or methylmethane thiosulfonate (106). This work may be of particular value in understanding the mechanism of the high-K-low-K transition during differentiation and maturation of ruminant red cells (97)

3. CI-DEPENDENT CATION COTRANSPORT AND CELL DIFFERENTIATION

105

as well as in unmasking a low-activity Na-independent K-CI pathway in other mammalian red cells (81,104,105,165). Reticulocytes of rabbits (130) and piglets (108) possess K-CI transport, which in sheep reticulocytes responds to NEM (99).However, in high-K but not in low-K sheep maturation of reticulocytes involves reduction of both volume- and thiol-stimulated K-CI fluxes (99). This involution appears to contain a quantitative element because K-CI transport in the mature low-K cell (98) is clearly reduced as compared to reticulocytes (99). The mechanism by which K-CI cotransport is inactivated in high-cells is unknown. However, a metabolic control and perhaps divalent metal ions have to be considered because NEM is not able to stimulate K-CI transport in metabolically depleted low-K red cells (100). Divalent metal ion depletion of mature low-K cells in the presence of A23187 led to a severalfold activation of K-Cl fluxes which was metabolically dependent and not observed in NEM-treated cells exhibiting already apparently maximally activated K-CI transport (103,107). Metal ions other than Ca inhibited K-Cl transport in low-K red cells in the presence of A23187 with the following series of decreasing order of potency: Mn >> Ca > Mg > Sr >>> Ba, with Ba being practically ineffective (103). The importance of the studies with NEM is that they may lift the curtain from the modus operandi of K-CI transport, not only for membranes of ruminant red cells, but also for those of other species, thus helping us to understand why the early undifferentiated cells need high K-C1 flux activities and how they are turned on and inactivated by regulatory events throughout differentiation and maturation. For the development of the low-K ruminant red cells, an interesting possibility would be the utilization of an outward K-CI cotransport functionally coupled with a Na-H exchange flux, perhaps through the Na-Na countertransporter which is present in these cells at activities much higher than in other mature mammalian red cells (17,35). Ignoring the complexities in the kinetics of the Na-K pump in these cells (97), the passive carrier-mediated events envisioned above are depicted in Fig. 3. Of course, other schemes may be equally appropriate. The kinetic similarity of K-CI flux stimulated by NEM in human red cells (81) with that in sheep (98) is remarkable, a fact adding further support to the hypothesis that the OR K-CI fluxes remain kinetically rather constant throughout cellular differentiation and maturation. D. Na-CI or Na-K-2CI Transport

The analysis of Na-K-2CI transport and its changes during hemopoiesis are made more complex not only by the participation of an additional ion pair, Na-CI, but also by the multitude of physiologic and phar-

106

PETER K. LAUF RET ICULOCYTES NEY

NEH

CI-

ERYTHROCYTES

FIG.3. K-CI cotransport and cellular differentiation in hemopoietic cells of ruminants and the development of the high-K-low-K dimorphism. In this “working hypothesis” diagram the reticulocyte precursors of low-K (LK,left) and high-K (HK, right) mature erythrocytes are depicted to contain two defined, but not functional, volume pools. Both reticulocytes reduce cell volume by 30%, perhaps through SH-group-dependent and NEM-stimulated, probably volume-sensitive K-CI cotransport (99). The transition from the high-K reticulocyte to the mature low-K red cell may involve NEM-activated K-CI cotransport that operates in parallel with Na-H antiport, leading to a net exchange of cell K for extracellular Na accompanied by a functional electroneutral HCI exit since CI/HC03 exchange is operative (not shown). In reticulocytes destined to be high-K red cells, the SHdependent K-CI transporter is functionally inactivated (SR), perhaps by covalent bonding of the SH group, while the Na-H exchanger of the low-K line is converted to a futile Na-Na antiport that has been shown to be inhibited by NEM in both mature high-K and low-K red cells (35).

macologic ways of stimulating Na-K-2Cl cotransport. Using the same approach as in Section III,B,Table I11 reveals that C1-dependent Na or Na-K transport is present in cells as early as those from the embryonic chick (152,163), which still produce embryonic hemoglobin, as well as in all nucleated red cells. Hence this transport system must be evolutionarily (and ontogenetically) as old as the RVD K-CI transporter described before. A decrease during differentiation to the mature cell is noted in rat (34) and human (33,49,168) erythrocytes, the latter possessing the lowest activities recorded. There is evidence for a natural variant-ferret red cells which are low K, like those of ruminants, exhibit an extraordinarily high Na-K cotransport activity (44,45,114). There is a considerable problem in assessing the stoichiometry of the cation-anion cotransporter.

3. CI-DEPENDENT CATION COTRANSPORT AND CELL DIFFERENTIATION

107

From Table 111 it appears that during differentiation and maturation to the erythrocyte a dissociation occurs among the various stimuli which activate Na-K-2CI cotransport. Anucleate red cells no longer respond to catecholamines, a feature of transport in cells of earlier stages. Moreover, only rat red cells clearly show Na-K cotransport by induced cell shrinkage, a phenomenon operating in low-K-diet animals (34). Osmotic stimulation of Na-K cotransport in human red cells, if present, is small and needs to be investigated. This obvious reduction of the capability of Na-K cotransport to respond to pharmacologic and physiologic (osmotic) stimuli is associated with a loss of adenylate cyclase activity and suggests that the complex machinery mediating and transforming hormonal stimuli into greater Na-K cotransport activity has been at least functionally inactivated, if not in part structurally deleted (115). This aspect raises the question as to the mechanisms of functional and structural alterations of transport proteins during differentiation, an exciting area of research which only recently has drawn sufficient attention. Two main lines are being followed: first, metabolic downregulation of membrane transport, as evidenced by the reported ATP dependence of the rate of Na-K pump inactivation in reticulocytes (164), and of phosphorylation-linked reduction of Na-K pump turnover in the Friend-erythroid leukemia cell line (169); second, proteolytic breakdown and modification of cytosolic and membrane components involving ATP and ubiquitin. On the other hand, such processes of involution of membrane functions may be coupled to insertive processes which themselves not only add new functions such as band 3 protein (C1-Cl self-exchange) but may also alter existing ones. Analogous to the K-CI transporter (Section III,B), there is a marked constancy of kinetic parameters of Na-K-2CI cotransport during the later stages of hemopoiesis, suggesting an evolutionary conservation of these parameters throughout all biological membranes (Table 111). In general, the values for Na are always higher than those for K, the latter in turn being significantly lower than those reported for Na-independent K-CI transport. Based on this reasonable generalization, one should consider the K-K exchanges in Pekin red cells (65,67) at least to be a kinetic hybrid with the K-CI transporter of the RVD system. Furthermore, signatory for the presence of Na-K-2Cl cotransport is the generally high affinity for bumetanide and furosemide. Prior to learning about the topic of cotransport in differentiating hemopoietic cells, it is useful to address once more the biochemical regulation of cotransport, an area in which so little progress has been made since the early reports that there are relationships with membrane phosphorylation and cAMP levels (141) which still have to be considered noncausal in nature. Lately it has become apparent that cAMP may also exert an

108

PETER K. LAUF

inhibitory function since its addition to human red cells resulted in inactivation of furosemide-sensitive Na-K cotransport (50). Does this mean that the same biochemical regulator system that functioned in terms of activation in earlier red cell precursors now becomes an inhibitory regulator? New experimental approaches are necessary to answer this question. One possibility would be to study the effect of reconstitution of the adenylate cyclase system on the resurgence of Na-K cotransport, for example, by fusing red cells, which have the nucleotide regulatory proteins but not a functioning adenylate cyclase, with a cell possessing the latter but not the former. Another attractive but still untapped approach would be chemical modification in analogy to the work on K-Cl cotransport. N-Ethylmaleimide has been shown to abolish the furosemide-sensitive Na but not K fluxes in human red cells (104), suggesting that attention should also be given to the presence of SH groups in the Na-K-2Cl cotransporter. Some preliminary work suggests that treatment with p-chloromercuribenzene sulfonic acid (PCMBS) activates reversibly C1-dependent Na and K fluxes in human red cells, but their relationship to Na-K cotransport or the K-Cl cotransporter is yet to be established (68). IV. PROPERTIES IN DIFFERENTIATED EPITHELIAL CELLS

At the time of writing this review there was no work in the literature on C1-dependent cation cotransport in a differentiating epithelial cell line. Nevertheless, the presence of mainly K-Cl and Na-K-2Cl cotransport activities in a variety of epithelial cells from different organs and species suggests evolutionarily early utilization for specialized functions. These functions are basically cation-coupled C1 secretion and reabsorption (see Section 11,H). Accordingly, Table IV subdivides Na-Cl or Na-K-2Cl cotransport as reported for secretory and absorptive epithelia. Independent of function and topographic location, it appears that the ionic affinities for K are similar to those for Na, being higher than in the same transporter studied in red cells. However, in contrast to cells of the hemopoietic line, there is a much greater sensitivity to inhibition by furosemide, the basis of this difference being unknown. It has been amply documented that the overall processes of C1 secretion or reabsorption are hormonally and metabolically dependent. For example, C1 secretion through the apical membrane of the tracheal epithelium is manipulated by CAMP and interventions in the prostaglandin synthesis of the cell (156). The most satisfactory characterization of C1-dependent cation transport has been carried out using the MDCK cell line and the Ehrlich ascites tumor cell, both cells being of various epithelial origins (14,73,138). The

TABLE IV CHLORIDE-DEPENDENT Na OR Na-K TRANSPORT: PROPERTIES IN

DIFFERENTIATED

EPITHELIAL CELLS"

Ko.5 ( m M ) Cell typdspecies differentiation (references) Secretory epithelia Wla!eral location) SbarL rectal gland (41,62,71.153) Frog wrma (136) canine uachea (l26,l65,l67) Abmrptive epithelia (apicallocation) Flounder intestine (47.64.1 16)

R e d tubules Rabbit (dog) proximal (5&62,148) (thick ascending limb) Amphivma distal (118,119)

Necrurur gallbladder (4394.157) Other Madin-Darby canine kidney (MDCK) cells (24,25,113,138,139,147) Mouse Ehrlich ascites (54-56.73-75) ~~

ions (ratio)

ma],

inhibitors

[KI.

[CII,

Anion preference

stimulus activation

Type

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

4

21

75

n.a. n.a.

n.a. o.a.

n.a. n.a.

CI >> 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-' <2 x 10-3
Yes

n.a.

110

PETER K. LAUF

basolateral Na-K cotransport for C1 secretory function is preserved in the MDCK cell (138,147). The coupling ratio for the MDCK cell is 1 Na: I K : 2 C1(147), and the affinity of the transporter for any of the three ions is dependent on the presence of the other two (139). Hence the numbers given in Table IV for these cells are extrapolations, that is, they account for the saturating presence of the other ion whose transport was not measured. The dependence of Na-K cotransport on C1 and inhibition by any anions other than Br is best worked out for MDCK cells (4,113). Furthermore, metabolic dependence of Na-K cotransport has been demonstrated (113). Similar to the bird red cell system (140,141), it is proposed that the metabolic effects involve phosphorylation of a high-molecular-weight protein (MW = 240,000) by a protein-kinase-mediated mechanism and that the phosphorylated form is transport active (147). Ehrlich ascites cells have been the prototype cells for establishing the presence of Cl-dependent cation transport, which is activated by cell shrinkage (74). However, reports on the participation of three main ions were conflicting, with one group reporting Na-Cl transport only (74) and the other Na-K-2Cl transport (54-56). It is possible that this difference is caused by volume effects, but one should also consider differences in the cell strain (see below). Table V summarizes the evidence for the presence of K-C1 cotransport in epithelial cell types. The first evidence for electroneutral K-Cl transport effecting RVD came from work on cortical collecting tubules of the rabbit (32,57). While ouabain did not affect K-Cl loss, there was metabolic dependence. Furthermore, increasing extracellular K blocked RVD, which is consistent with a passive K transporter that responds to manipulations of ion gradients. Although there is a report on electroneutral K-CI transport in Necturus gallbladder (1 3 3 , others have reported a stoichiometry of 3 K : 2 C1(95), suggesting an electrogenic component. In Necturus hyposmotic media or blockade of the Na-K pump by ouabain, both lead to cell swelling and subsequent RVD by activation of basolateral K-Cl transport (95). The Ehrlich cell, when swollen in hyposmotic solutions, has been shown to be capable of RVD either through separate pathways for K and C1 (73) or through electroneutral K-Cl transport (158). The reason for these differences in cellular behavior is unclear and may be related to the cell strain. A recent report that NEM-activated K-Cl transport in Ehrlich cells suggests that indeed the cell is capable of and may have a lowactivity electroneutral K-Cl transport (86). Much work is needed to establish the relationship between the two mechanisms of RVD and to relate these observations to K self-exchange (10,ll).

TABLE V APPARENTLY Na-INDEPENDENT K-CI TRANSPORT: PROPERTIES IN DIFFERENTIATED EPITHELIAL AND NONEPITHELIAL CELLS Cell type/species differentiation (references) Epithelial Rabbit cortical collecting tubule (3537) Necturus gallbladder (95,136) MDCK cells, HeLa cells (3) Mouse ascites tumor cells (10,11,86,158)

Nonepithelial Mouse 3T3 fibroblasts (132)

OCSW, Osmotic cell swelling.

Ions (ratio)

Anion preference

Stimulus of activation

Inhibitor Type

K-CI K-CI (3 : 2) K-CI K-CI (K/K)CI

n.a. CI > ?

ocswo ocsw

CI > 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

<

5 x lo-’ 5 x

112

V.

PETER K. LAUF

NONEPITHELIAL CELLS AS MODELS FOR COTRANSPORT DURING DIFFERENTIATION

Among nonepithelial cells possessing C1-dependent cation cotransport are fibroblasts of various origins, human placenta cells, and cells of the neuromuscular tissues (Table VI). Fibroblasts are of particular interest because cell biologists have shown that in uitro growth of these cells may be accelerated or slowed down by the addition or removal of serum factors whose presence has been related to transient active and passive permeability changes preceding DNA and protein synthesis. One of the most intriguing questions that has been around for quite some time is whether or not the observed permeability changes are causal for the initiation of macromolecular events leading to cell growth and proliferation. Inspection of Table VI reveals that fetal or newborn calf sera are considered the prime stimulators of bumetanide-sensitive Na-K-2Cl cotransport, which has been reported for at least five fibroblast cell lines. More recently, hormones and factors which may be natural constituents of serum, such as insulin, fibroblast growth factor (FGF), and epidermal growth factor (EGF), have been shown to stimulate growth of fibroblasts and their C1-dependent cation transport system (8). Several lines of evidence, however, speak for a noncausal, rather pleiotropically parallel relationship of transport changes with intracellular macromolecular events of cell growth. First, as mentioned above, there are cell lines completely lacking furosemide-sensitive Na-K-2Cl cotransport, such as the HL-60 line (53). Second, I-M(TK-) cells can be selected to grow in low-K media (LTK-5 cells) by altering their furosemide-sensitive Na-K cotransport activities (52,761. Moreover, normal L-M(TK-) cells grow in low-K media so long as bumetanide is present to prevent cell K from running down its chemical gradient through the cotransport system (76,77). Third, although serum and a variety of factors (insulin, EGF, etc.) stimulated both cotransport and DNA synthesis, the latter, however, was unaffected by the presence of bumetanide (8). Fourth, if it is the function of Na-K-2C1 cotransport to participate in elevating internal K, which has been proposed by some to be required for DNA and protein synthesis, the latter should be reduced when intracellular K is kept at the level of resting cells or below. However, this was not found in Swiss 3T3 cells (8). The studies of serum effects on transport of quiescent and growing fibroblast cell lines seem to be besieged by problems similar to those encountered in the work with catecholamine-sensitive bird red cells, which have been shown to achieve the so-called “lower steady state” when withdrawn from and washed free of catecholamine-containing plasma (67,91). In fibroblasts the activity of Na-K cotransport seems to

TABLE VI CHLORIDE-DEPENDENT Na OR Na-K TRANSPORT: PROPERTIES IN DIFFERENTIATED NONEPITHELIAL CELLS" Inhibitors

KOJ (mM) Cell typelspecies differentiation (references)

(ratio)

Fibroblasts Quiescent mouse 3T3 cells (130) Growing mouse 3T3 cells (8.9.132)

Anion preference

Ions

"ale

[KI,

(Na?)-K-CI

n.a.

2.2

n.a.

Na-K-CI

n.a.

n.a.

n.a.

IC1l.

CI >> 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


n.a.

n.a.

ma.

n.a.

CI >> 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.


Na-K-CI (2 : I : 3) Na-K-CI

n.a.

n.a.

n.a.

CI >

n.a

<1o-s

n.a.

ma.

n.a.

CI >> gluconate

Serum, EGF,

Bumet. FUrOS. CAMP. Isop. Bumet. Furos.

PhOrb.

Rabbit uterus smooth muscle (78.79)

n.a.

n.a.

n.a.

Other Human placenta (21) Neuromuscular tissue Giant squid axon (142-146)

Metabolic dependency

n.a.

(Na?)-K-CI

BALB/c 3T3 preadipose cells (117)

ICw ( M )

Serum

SV-Transformed mouse 3T3 (5)

Human HWSP fibroblasts (123)

Type

Na-K-CI

20

2.2

-10

CI >> N@

n.a

40-5

ATP Yes

10-4

n.a.

110-3

a n.a., Data not available; Bumet.. bumetanide; Furos.. furosemide; Benzmeta., bcounetanide; Pept.. peptide mitogens such as fetal (FGF) or epidermis m wthfaaon (EGF); ~horb.. phorbol esters; Isop., isoproterenol; OCSH, osmotic cell shrinkage.

114

PETER K. LAUF

be a function of the adaptation of the cells to the quantities of serum or its factor present during culture. This means that the magnitude of flux stimulation depends on the basal flux activity (8). However, the kinetic properties of C1-dependent Na-K cotransport do not offer surprises in comparison to the same system discussed for other cells above. The Ko.5 for KOis low compared to that for Na,, and C1; furthermore, SCN, I, N03, acetate, and gluconate do not replace C1 or Br. There is no study detailing a metabolic dependence of this cotransport system. It appears then that C1-dependent Na-K transport cannot be assigned a triggering role for its activation during cell growth and proliferation but can only be seen in terms of the overall physiology of cell volume maintenance. Yet the interrelationship of Na-K-2Cl cotransport with other transporters activated during growth (such as the Na-H antiporter) has not been explored at all. For example, Na-H antiport resulting in an increase of cellular Na in exchange for protons may be accompanied not only by Na activation of the Na-K pump but also by stimulation or better utilization of a stimulated outward Na-K cotransport. This point illustrates the importance of establishing for the various cell lines the zero net flux conditions of the coupled transporters in order to assess their role in the maintenance of cell volume. Table VI also lists the presence of C1-dependent cation fluxes in other tissues not of epithelial or fibroblast origin and demonstrates the ubiquity of anion-cation coupled transport. Elegant studies on the inward (145) and outward (146) coupled Na-K-Cl transport of the giant squid axon reveal a deviation from the stoichiometry of 1 Na : 1 K : 2 C1 claimed for these transporters in other cellular systems. Of particular interest is the observation by Russell (142,144) that ATP and to a lesser degree ADP were required for sustenance of the bumetanide-sensitive Na-K-Cl fluxes in the squid axon, thus offering a model for studying the mechanism by which ATP may support Na-K-2Cl cotransport in other cell systems where its requirement had been inferred from metabolic depletion studies. Of great interest and potential for possible links between cellular differentiation and disease are the studies demonstrating Na-K-2Cl cotransport in smooth vascular muscle (80,122), where serum and phorbol esters have been found to activate, and cAMP to inhibit, the mediated transport (122). As in bird red cells, cAMP and hence the adenylate cyclase-dependent membrane phosphorylation which is regulated by various protein kinases, seem to parallel the catecholamine-induced activation of Na-K cotransport; the same biochemical system may also be involved in bringing about inhibition. On the other hand, there may be a complex feedback between the adenylate cyclase system and the phosphoinositol-oligophosphate cascade within which the phorbol ester-activated kinase C

3. CI-DEPENDENT CATION COTRANSPORT AND CELL DIFFERENTIATION

115

plays a crucial role (18). The discovery of the latter system, however, is too new to permit intelligent exploitation in terms of its relationship to Na-K cotransport, even though phorbol esters have been shown to inhibit significantly furosemide-sensitive Na-K-CI cotransport in BALB/c 3T3 preadipose fibroblasts (1 17). A good example for demonstrating the extraordinary difficulties in sorting out causes and effects is the known fact that Li, which is also transported by the Na-K cotransporter (29), is a strong inhibitor of kinase C-mediated phosphoinositol phosphorylation (18). This is a situation similar to that in the adenylate cyclase system, where fluoride stimulates the biochemical cascade but is not accepted by the transporter. Another potentially interesting study is the short report of the existence of a furosemide-sensitive Na-CI cotransport system in placental vesicles (21). The syncytiotrophoblast seems to be an intriguing model system with regard to the constancy or adaptability of this tissue to the need of the growing embryo. Furthermore, this tissue is a good model from the comparative point of view because there are at least five different types of placenta among mammals, distinguished from one another on the basis of the endothelial or syndesmotic nature of the link with the chorion epithelium. VI.

CONCLUSION

Based on work reported in the literature, no clear causal relationship is apparent between activation of Cl-dependent cation cotransport and cellular growth and differentiation. As pointed out at various occasions, cell lines which differentiate into a terminal cell with a particular differentiated function like the HL-60 cell line are rare, although a multitude of cell cultures derived from many sources exists (see the recent guide by the National Institute of General Medical Sciences on tissue cultures). From what I have learned I conclude that, unlike the Na-H or K-H antiports, the K-CI, Na-CI, and Na-K-2CI cotransporters do not play a primary role in the signal transduction for the cell to rise from the quiescent to the proliferating state and to grow and differentiate. Rather, the cotransport systems with their enormous mass transport capability may serve to reestablish quickly cellular volume perturbed by growth initiation events involving other transport systems. Perhaps one can say that such a function in vertebrates represents a useful adaptation of the osmoregulatory role these transporters exerted in evolutionarily older and perhaps simpler organisms of the invertebrates. From the compilation of the published data, we certainly can conclude that the kinetic properties of each system

116

PETER K. LAUF

are maintained fairly constant or stable, while only absolute activities are reduced during cellular differentiation and maturation. The quantitative aspects and the molecular basis of this process are unknown. In Ehrlich ascites cells the number of bumetamide binding sites presumably associated with volume regulatory Na-Cl cotransport has been estimated to be 5 x 106/cell(75) and in ferret red cells 1.2 x 104/cell(1 14). This means that the turnovedsite cannot be in the range of that reported for the fast C1-Cl exchanges in enucleate red cells but may be close to that of the Na-K pump at V,, . What is the fate of such a large number of transport sites during differentiation and maturation of hemopoietic cells? Is the down regulation of cotransport due to elimination or simply inactivation of a transport system whose activity in ontogenetically earlier cells seems to be associated with particular activity states of the adenylate cyclase system and perhaps of the inositol-oligophosphate cascade? And finally, what is the physiologic and molecular relationship of these involutionary changes to those of other transporters such as the Na-K pump and the alkali cation-proton antiports? I think we will be able to address these questions related to differentiations when we understand how biochemically, not thermodynamically, the C1-dependent cotransporters work and how cellular metabolites and divalent ions may modulate the activity status of C1-dependent cation cotransport. NOTE ADDED IN PROOF

While this manuscript was prepared for publication, I wrote and published a topical review on “K+-CI- Cotransport: Sulfhydryls, Divalent Cations, and the Mechanism of Volume Activation in a Red Cell,” in Journal of Membrane Biology 88(1), pp. 1-13 (1985). This paper deals with the possible mechanism of the activation and inactivation of K-Cl cotransport and has incorporated a unifying hypothesis based on the recent data from my laboratory. ACKNOWLEDGMENTS This work was supported by NIH Grant AM 28236. I am grateful to Ms. Gay Blackwell for patiently typing the manuscript and Dr. N. C. Adragna and Dr. D. K. Smith for carefully editing it, and I thank Drs. P. B. Dunham, J. Gargus, M. Haas, T. J. McManus, N. Owen, J. Russell, C. L. Slayman, and P. DeWeer for permitting me to quote in advance from their work in press. REFERENCES 1. Adragna, N. C., and Tosteson, D. C. (1984). Effect of volume changes on ouabaininsensitive net outward cation movements in human red cells. J . Membr. Biol. 78, 43-52.

3. CI-DEPENDENT CATION COTRANSPORT AND CELL DIFFERENTIATION

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2. Adragna, N. C., Perkins, C. M., and Lauf, P. K. (1985). Furosemide-sensitive Na+-K+ . cotransport and cellular metabolism in human erythrocytes. Biochim. B i o p h y ~Acta 812, 293-296. 3. Aiton, J . F., Chipperfield, A. R., Lamb, J . F., Ogden, P., and Simmons, N. L. (1981). Occurrence of passive furosemide-sensitive transmembrane potassium transport in cultured cells. Biochem. Biophys. Acta 646,389-398. 4. Aiton, J. F., Brown, C. D. A., Ogden, P., and Simmons, N. L. (1982). K’ transport in “tight” epithelial monolayers of MDCK cells. J. Membr. Biol. 65, 99-109. 5. Aiton, J. F., and Simmons, N. L. (1983). Effect of ouabain upon diuretic-sensitive K + transport in cultured cells. Evidence for separate modes of operation of the transporter. Biochim. Biophys. Acta 734, 279-289. 6. Alper, S. L., Beam, K. G., and Greengard, P. (1980). Hormonal control of Na+-K+ cotransport in turkey erythrocytes. J . Biol. Chem. 255, 4864-4871. 7. Amende, L. M . , and Pierce, S. K. (1980). Cellular volume regulation in salinity stressed molluscs: The response of Noetia ponderosa (Arcidae) red blood cells to osmotic variation. J . Comp. Physiol. 138, 283-289. 8. Amsler, K., Donahue, J. J., Slayman, C. W., and Adelberg, E . A. (1985). Stimulation of bumetanide-sensitive K + transport in Swiss 3T3 fibroblasts by serum and mitogenic hormones. J . Cell. Physiol. (in press). 9. Atlan, H., Snyder, D., and Panet, R. (1984). Ouabain-resistant Na+,K+transport system in mouse NIH 3T3 cells. J . Membr. Biol.81, 181-188. 10. Aull, F. (1981). Potassium chloride cotransport in steady-state ascites tumor cells. Biochim. Biophys. Acra 643, 339-345. I I . Aull, F. (1982). Specific drug sensitive transport pathways for chloride and potassium ions in steady-state Ehrlich mouse ascites tumor cells. Biochim. Biophys. Acta 688, 140-746. 12. Bakker-Grunwald, T. (1981). Hormone-induced diuretic-sensitive potassium transport in turkey erythrocytes is anion dependent. Biochim. Biophys. Acta 641,427-431. 13. Bakker-Grunwald, T. (1983). Potassium permeability and volume control in isolated rat hepatocytes. Biochim. Biophys. Acta 731, 239-242. 14. Barker, G., and Simmons, N. L. (1981). Identification of two strains of cultured canine renal epithelial cells (MDCK cells) which display entirely different physiological properties. J . Exp. Physiol. 66, 61-72. 15. Baroin, A., Garcia-Romeu, F., Lamarre, T., and Motais, R. (1984). Hormone-induced cotransport with specific pharmacological properties in erythrocytes of rainbow trout, Salmo gairdneri. J. Physiol. (London)350, 137- 157. 16. Beauge, L . A., and Adragna, N. (1971). The kinetics of ouabain inhibition and the partition of rubidium influx in human red blood cells. J . Gen. Physiol. 57, 576-592. 17. Becker, B. F., and Duhm, J. (1979). Studies on lithium transport across the red cell membrane. J. Membr. Biol. 51, 287-310. 18. Bemdge, M. J. (1984). Inositol trisphosphate and diacylglycerol as second messengers. Biochem. J. 220, 345-360. 19. Bourne, P. K., and Cossins, A. R. (1982). On the instability of K + influx in erythrocytes of the rainbow trout, Salmo gairdneri, and the role of catecholamine hormones in maintaining in uiuo influx activity. J . Exp. Biol. 101, 93-104. 20. Bourne, P. K., and Cossins, A. R. (1984). Sodium and potassium transport in trout (Salmo gairdneri) erythrocytes. J . Physiol. (London) 347, 361-375. 21. Boyd, C. A. R., Chipperfield, A. R., and Lund, E. K. (1980). NaCl cotransport by human placental plasma membrane vesicles. J . Physiol. (London) 307, 86P. 22. Brand, S. C., and Whittam, R. (1984). The effect of furosemide on sodium movements in human red blood cells. J . Physiol. (London) 348, 301-306.

118

PETER K. LAUF

23. Brazy, P. C., and Gunn, R. B. (1976). Furosemide inhibition of chloride transport in human red blood cells. J . Gen. Physiol. 68, 583-599. 24. Brown, C. D. A., and Simmons, N. L. (1981). Catecholamine-stimulationof C1- secretion in MDCK cell epithelium. Biochim. Biophys. Acta 649, 427-435. 25. Brown, C. D. A., and Simmons, N. L. (1982). A comparison of the actions of certain loop diuretics upon passive potassium fluxes and CI- secretion in a cultured epithelium of renal origin. J . Physiol. (London) 322, 25p. 26. Burg, M. B., and Stoner, L. (1976). Renal tubular chloride transport and the mode of action of some diuretics. Annu. Rev. Physiol. 38, 37-45. 27. Cala, P. M. (1977). Volume regulation by flounder red blood cell in anisotonic media. J . Gen. Physiol. 69, 537-552. 28. Cala, P. M. (1983). Volume regulation by red blood cells: Mechanisms of ion transport. Mol. Physiol. 4, 33-52. 29. Canessa, M. L., Bize, I . , Adragna, N., and Tosteson, D. C. (1980). Cotransport of lithium and potassium in human red cells. J. Gen. Physiol. 80, 149-168. 30. Chipperfield, A. R. (1981). Chloride dependence of furosemide- and phloretin-sensitive passive sodium and potassium fluxes in human red cells. J. Physiol. (London) 312, 435-444. 31. Dagher, G., Brugnara, C., and Canessa, M. (1984). The effect of metabolic depletion on the modes of the furosemide-sensitive Na+K+CI- cotransport inhuman erythrocytes. Biophys. J . 45, 18a. 32. Dellasega, M., and Grantham, J. J. (1973). Regulation of renal tubule cell volume in hypotonic media. A m . J . Physiol. 224, 1288-1294. 33. Duhm, J., and Gobel, B. 0. (1984). Role of the furosemide-sensitive Na+/K+transport system in determining the steady-state Na+ and K+ content and volume of human erythrocytes in vitro and in v i v a J. Membr. Biol. 77, 243-254. 34. Duhm, J., and Gobel, B. 0. (1984). Na+-K+ transport and volume of rat erythrocytes under dietary K+ deficiency. A m . J . Physiol. 46 (Cell Physiol. l5), C20-C29. 35. Duhm, J., Becker, B. F., and Lauf, P. K. (1980). Ouabain-insensitive sodium/lithium exchange and the effect of anti-L in low potassium sheep erythrocytes. Life Sci. 26, 1217- 1222. 36. Dunham, P. B., Stewart, G. W., and Ellory, J. C. (1980). Chloride-activated passive potassium transport in human erythrocytes. Proc. Nail. Acad. Sci. U . S . A . 77, 17111715. 37. Dunham, P. B., and Ellory, J. C. (1981). Passive potassium transport in low potassium sheep red cells: dependence upon cell volume and chloride. J . Physiol. (London) 318, 511-530. 38. Ellory, J. C., and Dunham, P. B. (1980). Volume-dependent passive potassium transport in LK sheep red cells. In “Membrane Transport in Erythrocytes” (U. V. Lassen, H. H. Ussing, and J. 0. Wieth, eds.), pp. 409-427. Munksgaard, Copenhagen. 39. Ellory, J. C., Dunham, P. B., Logue, P. J., and Stewart, G. W. (1982). Anion dependent cation transport in erythrocytes. Philos. Trans. R. Soc. London Ser. B 299, 483495. 40. Enyedi, A., Farago, A,, Sarkadi, B., Szasz, I., and Gardos, G. (1983). Cyclic AMPdependent protein kinase stimulates the formation of polyphosphoinositides in the plasma membranes of different blood cells. FEBS Lett. 161, 158-162. 41. Epstein, F. H., Stoff, J. S., and Silva, P. (1983). Mechanism and control of hyperosmotic NaC1-rich secretion by the rectal gland of squalus acanthias. J . Exp. Biol. 106, 25-41. 42. Field, M. (1978). Some speculation on the coupling between sodium and chloride

3. CI-DEPENDENT CATION COTRANSPORT AND CELL DlFFERENTlAflON

119

transport processes in mammalian and teleost intestine. I n “Membrane Transport Processes” (J. F. Hoffman, ed.), Vol. I, pp. 277-292. Raven, New York. 43. Fisher, R. S., and Spring, K. R. (1984). lntracellular activities during volume regulation by Necturus gallbladder. J . Membr. Eiol. 78, 187-199. 44. Flatman, P. W. (1983). Sodium and potassium transport in ferret red cells. J . Physiol. (London) 341, 545-557. 45. Flatman, P. W., and Andrews, P. L. R. (1983). Cation and ATP content of ferret red cells. Comp. Eiochem. Physiol. 74A,939-943. 46. Forbush, B., 111, and Palfrey, H. C. (1983). [3H]Bumetanide binding to membranes isolated from dog kidney outer medula: Relationship to the Na,K,Cl-cotransport system. J. Eiol. Chem. 258, 11787-1 1792. 47. Frizzell, R. A., and Field, M. (1983). NaCl cotransport across the apical membranes of flounder intestinal cells. Fed. Proc., Fed. Am. Soc. Exp. Eiol. 43, 2478-2479. 48. Fugelli, K. (1967). Regulation of cell-volume in flounder. (PleuronectesJlesus)erythrocytes accompanying a decrease in plasma osmolarity. Comp. Biochem. Physiol. 22, 253-260. 49. Garay, R., Adragna, N., Cannessa, M., and Tosteson, D. C. (1981). Outward Na and K cotransport in human red cells. J . Membr. Eiol. 62, 169-174. 50. Garay, R. P. (1982). Inhibition of the Na K cotransport system by cyclic AMP and intracellular Ca+ in human red cells (BBA 71229). Biochim. Eiophys. Acra 688,786792. 51. Gardner, J. D., Mensh, R. S., Kiino, D. R., and Aurbach, G. D. (1975). Effects of P-adrenergic catecholamines on potassium transport in turkey erythrocytes. J. Eiol. Chem. 250, 1155-1163. 52. Gargus, J. J . , and Slayman, C. W. (1980). Mechanism and role of furosemide-sensitive K+ transport in L cells: A genetic approach. J. Membr. Eiol. 52, 245-256. 53. Gargus, J. J., Adelberg, E. A., and Slayman, C. W. (1984). Coordinated changes in potassium fluxes as early events in the differentiation of human promyelocyte line HL-60. “Regulation and Development of Membrane Transport Processes.” Wiley, New York (in press). 54. Geck, P., Pietryzk, C., Burckhardt, B.-C., Pfeiffer, B., and Heinz, E. (1980). Electrically silent cotransport of Na+,K+ and CI- in Ehrlich cells. Biochim. Biophys. Acra 600,432-447. 55. Geck, P., and Heinz, E. (1980). Coupling of ion flows in cell suspension systems. Ann. N . Y. Acad. Sci. 341, 57-66. 56. Geck, P., Heinz, E., and Pfeiffer, B. (1981). Influence of high ceiling diuretics on ion fluxes and cell volume of Ehrlich ascites tumour cells. Scand. Audiol. Suppl. 14, 2537. 57. Grantham, J . , Lowe, C., Dellasega, M., and Cole, B. (1977). Effect of hypotonic medium on K and Na content of proximal renal tubules. A m . J . Physiol. 232 (Renal Fluid Electrolyte Physiol. I), F42-F49. 58. Greger, R., and Schlatter, E. (1981). Presence of luminal K’. a prerequisite for active NaCl transport in the cortical thick ascending limb of Henle’s loop of rabbit kidney. Pfiigers Arch. 392, 92-94. 59. Greger, R., Oberleithner, H., Schlatter, E., Cassola, A. C., and Weidtke, C. (1983). Chloride activity in cells of isolated perfused cortical thick ascending limbs of rabbit kidney. Pfugers Arch. 399, 29-34. 60. Greger, R., and Schlatter, E. (1983). Properties of the lumen membrane of the cortical thick ascending limb of Henle’s loop of rabbit kidney. PJliigers Arch. 396, 315-324. 61. Greger, R., Schlatter, E., and Lang, F. (1983). Evidence for electroneutral sodium

120

62. 63.

64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80.

PETER K. LAUF

chloride cotransport in the cortical thick ascending limb of Henle's loop of rabbit kidney. Pflugers Arch. 396,308-314. Greger, R., and Schlatter, E. (1984). Mechanism of NaCl secretion in the rectal gland of spiny dogfish (Squalus acanthias). Pfugers Arch. 402,63-75. Greengard, P. (1978). Phosphorylated proteins as physiologic effectors. Science 199, 146- 152. Guandalini, S., Roa, M. C., Smith, P. K., and Field, M. (1982). cGMP modulation of ileal ion transport: I n uitro effects of Escherichia coli heat stable enterotoxin. Am. J . Physiol. (Cell Physiol.) 243, G36-41. Haas, M., Schmidt, W. F., and McManus, T. J. (1982). Catecholamine-stimulated ion transport in duck red cells. Gradient effects in electrically neutral (Na + K + 2CI) cotransport. J . Gen. Physiol. 80, 125-147. Haas, M., and McManus, T. J. (1983). Bumetanide inhibits (Na + K + 2CI) cotransport at a chloride site. Am. J . Physiol. 245, C235-C240. Haas, M., and McManus, T. J. (1985). Effect of norepinephrine on swelling-induced potassium transport in duck red cells. J . Gen. Physiol. (in press). Haas, M., and Schmidt, W. F. (1985). PCMBS stimulation of chloride-dependent sodium and potassium transport in human red blood cells. Biochim. Biophys. Acta. (in press). Hall, A. C., and Willis, J. S. (1984). Differential effects of temperature on three components of passive permeability to potassium in rodent red cells. J. Physiol. (London) 348,629-643. Handler, J. S. (1983). Use of cultured epithelia to study transport and its regulation. J . EXP.Bi01. 106, 55-69. Hannafin, J., Kinne-Saffran, E., Fnedmann, D., and Kinne, R. (1983). Presence of a sodium-potassium chloride cotransport system in the rectal gland of squalus acanthias. J . Membr. Biol. 75, 73-83. Hoffman, J. F., and Kregenow, F. M. (1966). The characterization of new energy dependent cation transport processes in red blood cells. Ann. N.Y. Acad. Sci. 137, 566-576. Hoffman, E. K . (1982). Anion exchange and anion-cation cotransport systems in mammalian cells. Philos. Trans. R . SOC.London Ser. B 299, 519-535. Hoffman, E. K., SjZholm, C., and Simonsen, L. 0. (1983). Na+,C1- cotransport in Ehrlich ascites tumor cells activated during volume regulation (regulatory volume increase). J. Membr. Biol. 76, 269-280. Hoffman, E., Schifldt, M., and Dunham, P. (1986). The number of chloride-cation cotransport sites on Ehrlich ascites cells measured with 'H bumetanide. Am. J . Physiol. (Cell. Physiol.) 250 (in press). Jayne, D. W., Adelberg, E. A., and Slayman, C. W. (1981). Reduction of K' efflux in cultured mouse fibroblasts, by mutation or by diuretics, permits growth in K+-deficient medium. Proc. Natl. Acad. Sci. U . S . A . 78, 1057-1061. Jayme, D. W., Slayman, C. W., and Adelberg, E. A. (1984). Furosemide-sensitive potassium emux in cultured mouse fibroblasts. J . Cell. Physiol. 120, 41-48. Johns, A. (1980). Ouabain-sensitive thallium fluxes in smooth muscle of rabbit uterus. J . Physiol. (London) 309, 391-403. Johns, A., and Cutshaw, S. V. (1983). Furosemide-sensitive thallium fluxes in smooth muscle of rabbit uterus. A m . J . Physiol. SOC.245 (Renal Fluid Electrolyte Physiol. 14), F778-F783. Jones, A. W. (1973). Altered ion transport in vascular smooth muscle from spontaneously hypertensive rats. Circ. Res. 33, 563-572.

3. CI-DEPENDENT CATION COTRANSPORT AND CELL DIFFERENTIATION

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81. Kaji, D. M.. and Kahn, T. (1984). Kinetics of CI-dependent K influx of the human erythrocyte in the presence and absence of external sodium. Physiologist 27, 40.5

(Abstr.). 82. Karlish, S. J . D., Ellory, J . C., and Lew, V. L. (1981). Evidence against Na+-pump mediation of Ca2+-activatedK’ transport and diuretic-sensitive (Na+/K+)-cotransport. Biochim. Biophys. Acta 646,353-355. 83. Kevers, C., Pequeux, A,, and Gilles, R. (1979). Effects of an hypoosmotic shock on Na+,K+ and C1- levels in isolated axons of Carcinrts maenus. J . Comp. Physiol. 129, 365-371. 84. Kirk, R. G . , Andrews, S. B., and Lee, P. (1983). The correlation of composition and morphology during the high to low potassium transition single erythropoietic cells. J . Membr. Biol. 76, 281-287. 85. Kirk, R. G . , Andrews, S. B., and Lee, P. (1984). Rubidium uptake in single cells. J . Membr. Biol. 82, 137-143. 86. Kramhgift, B., Lambert, I. H., and Hoffmann, E. K. (1984). C1--dependent K+-transport in Ehrlich cells activated by N-ethylmaleimide and Hg’+. I n l . Congr. Comp. Physiol. Biochem.. I s l Liege Abstract B161. 87. Kregenow, F. M. (1971). The response of duck erythrocytes to nonhemolytic hypotonic media: Evidence for a volume controlling mechanism. J. Gen. Physiol. 58, 372395. 88. Kregenow, F. M. (1971). The response of duck erythrocytes to hypertonic media. J. Gen. Physiol. 58, 396-412. 89. Kregenow, F. M. (1973). The response of duck erythrocytes to norepinephrine and an elevated extracellular potassium. J. Gen. Physiol. 61, 509-527. 90. Kregenow, R. M. (1981). Osmoregulatory salt transport mechanisms: Control of cell volume in anisotonic media. Annu. Rev. Physiol. 43, 493-505. 91. Kregenow, F. M., Robbie, D. E., and Orloff, J. (1976). Effect of norepinephrine and hypertonicity on K influx and cyclic AMP in duck erythrocytes. Am. J. Physiol. 231, 306-312. 92. Kregenow, F. M., and Caryk, T. (1979). Co-transport of cations and C1 during the volume regulatory responses of duck erythrocytes. Physiologist 22, 73. and Folke, M. (1984). Volume-regulatory K’ efflux during concen93. Kristensen, L. 0.. trative uptake of alanine in isolated rat hepatocytes. Biochem. J. 221, 265-268. 94. Larson, M., and Spring, K. R. (1983). Bumetanide inhibition of NaCl transport by Necturus gallbladder. J . Membr. B i d . 74, 123-129. 95. Larson, M., and Spring, K. R. (1984). Volume regulation by Necturus gallbladder: Basolateral KCI exit. J. Membr. Biol. 81, 219-232. %. Lauf, P. K. (1982). Evidence for a chloride-dependent potassium and water transport induced by hypo-osmotic stress in erythrocytes of the marine teleost Opsunus mu. J. Comp. Physiol. 146,9-16. 97. Lauf, P. K. (1982). Active and passive cation transport and its association with membrane antigens in sheep erythrocytes. In “Membrane and Transport I ” (A. Martonosi, ed.), pp. 553-558. Plenum, New York. 98. Lauf, P. K. (1983). Thiol-dependent passive K/CI transport in sheep red cells: 1. Dependence on chloride and external K’ [Rb+] ions. J. Membr. Biol. 73, 237-246. 99. Lauf, P. K. (1983). Thiol-dependent passive K/CI transport in sheep red cells: 11. Loss of CI- and N-ethylmaleimide sensitivity in maturing high K’ cells. J. Membr. B i d . 73, 247-256. 100. Lauf, P. K. (1983). Thiol-dependent passive K+-CI- transport in sheep red blood cells. V. Dependence on metabolism. Am. J . Physiol. 245 (Cell Physiol. 14), C445-C448.

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101. Lauf, P. K. (1984). Thiol-dependent passive K/CI transport in sheep red cells: IV. Furosemide inhibition as a function of external Rb+,Na+, and C1-. J . Membr. Biol. 73, 57-62. 102. Lauf, P. K. (1984). Thiol-dependent passive K+Cl- transport in sheep red blood cells:

103. 104. 105.

106.

107.

108. 109.

110. 111.

112.

113.

114. 115. 116.

117.

VI. Functional heterogeneity and immunologic identity with volume-stimulated K+ (Rb+) fluxes. J . Membr. Biol. 82, 167-175. Lauf, P. K. (1985). Passive K+Cl- fluxes in low K+ sheep erythrocytes: Modulation by A23187 and bivalent cations. Am. J . Physiol. (Cell Physiol.) 249, C271-C278. Lauf, P. K., Adragna, N. C., and Garay, R. P. (1984). Activation by N-ethylmaleimide of a latent K+-Cl- flux in human red blood cells. Am. J . Physiol. 15, C385-C390. Lauf, P. K., Perkins, C. M., and Adragna, N. C. (1985). Cell volume and metabolic dependence of the N-ethylmaleimide activated K+CI- flux in human red cells. Am. J . Physiol. (Cell Physiol.), 249, C124-C128. Lauf, P. K., and Theg, B. E. (1980). A chloride dependent K+ flux induced by N-ethylmaleimide in genetically low K+ sheep and goat erythrocytes. Biochem. Biophys. Res. Commun. 92, 1422-1428. Lauf, P. K., and Mangor-Jensen, A. (1984). Effects of A23187 and Ca2+on volumeand thiol-stimulated, ouabain-resistant K’CI- fluxes in low K+ sheep erythrocytes. Biochem. Biophy. Res. Commun. 125, 790-796. Lauf, P. K., Zeidler, R. B., and Kim, H. D. (1984). Pig reticulocytes: V. Development of Rb+ influx during in vitro maturation. 3. Cell. Physiol. 121, 284-290. Letkowitz, R. J., Stadel, J. M., and Caron, M. G. (1983). Adenylate cyclase-coupled beta-adrenergic receptors: Structure and mechanisms of activation and desensitization. Annu. Rev. Biochem. 52, 159-186. Logue, P., Anderson, C., Kanik, C., Farquharson, B., and Dunham, P. (1983). Passive potassium transport in LK sheep red cells. J . Gen. Physiol. 81, 861-885. McManus, T. J., and Schmidt, W. F. (1977). Ion and co-ion transport in avian red cells. I n “Membrane Transport Process” (J. F. Hoffman, ed.), Vol. 1, pp. 79-106. Raven, New York. McManus, T. J., Haas, M., Starke, L. C., and Lytle, C. Y.(1985). The duck red cell model of volume-sensitive chloride-dependent cation transport. Ann. N. Y.Acad. Sci. (in press). McRoberts, J. A., Erlinger, S., Rindler, M. J., and Saier, M. H. Jr. (1982). Furosemide-sensitive salt transport in the Madin-Darby canine kidney cell line. Evidence for cotransport of Na+, K+, and C1-. J . Biol. Chem. 257,2260-2266. Mercer, R. W., and Hoffman, J. F. (1985). Bumetanide-sensitive Na/K cotransport in ferret red blood cells. Biophys. 3. 47, 157a. Montandon, J.-B., and Porzig, H. (1983). In vitro maturation of the rat reticulocyte beta-adrenoceptor adenylate cyclase system. Biomed. Biochim. Acta 42, 197-201. Musch, M. W., Orellana, S. A., Kimberg, L. S., Field, M., Halm, D. R., Krasny, E. J., and Frizzel, R. A. (1982). Na+-K+-CI- co-transport in the intestine of a marine teleost. Nature (London) 300,351-353. O’Brien, T. G., and Krzeminski, K. (1983). Phorbolester inhibits furosemide-sensitive potassium transport in BALB/c 3T3 preadipose cells. Proc. Natl. Acad. Sci. U.S.A.

80,4334-4338. 118. Oberleithner, H., Guggino, W., and Giebisch, G. (1983). The effect of furosemide on

luminal sodium, chloride and potassium transport in the early distal tubule of amphiuma kidney. Pflugers Arch. 396,27-33. 119. Oberleithner, H., Guggino, W., and Giebisch, G. (1983). Potassium transport in the

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123

early distal tubule of amphiuma kidney effects of potassium adaptation. Pfiigers Arch. 3%, 185-191. 120. Orskov, S . L. (1954). The potassium absorption by pigeon blood cells. A c f a Physiol. Scand. 31, 221-229. 121. Orskov, S. L. (1956). Experiments on the potassium absorption of the erythrocytes of rana esculenta and rana temporaria after bleeding and in hypertonic plasma. Acta Physiol. Scand. 33, 295-298. 122. Owen, N. E. (1984). Regulation of Na/K/CI cotransport in vascular smooth muscle cells. Eiophys. Biochem. Res. Commun. 125, 500-508. 123. Owen, N. E., and Prastein, M. L. (1985). Na/K/CI cotransport in cultured human fibroblasts. J. Eiol. Chem. (in press). 124. Palfrey, H. C. (1983). Na/K/CI cotransport in avian red cells: Reversible inhibition by ATP depletion. J. Gen. Physiol. 82, IOa. 125. Palfrey, H. C. (1984). Activation of the Na/K/CI cotransporter in ferret erythrocytes. Physiologist 27, 40.4 (Abstr.). 126. Palfrey, H. C., Feit, P. W., and Greengard, P. (1980). CAMP-stimulated cation cotransport in avian erythrocytes: Inhibition by ‘‘loop’’ diuretics. J. A m . Physiol. 238,C139c148. 127. Palfrey, H. C., and Greengard, P. (1980). Specific inhibition by “loop” diuretics of an anion-dependent Na+ + K+ cotransport system in avian erythrocytes. Ann. N . Y . Acad. Sci. 341, 134-138. 128. Palfrey, H. C., and Greengard, P. (1981). Hormone-sensitive ion transport systems in erythrocytes as models for epithelial ion pathways. Ann. N . Y . Acad. Sci. 372,291-308. 129. Palfrey, H. C., and Rao, M. C. (1983). Na/K/CI co-transport and its regulation. J. Exp. Eiol. 106, 43-54. 130. Panet, R., and Atlan, H. (1980). Characterization of a potassium carrier in rabbit reticulocyte membrane. J . Membr. Eiol. 52, 273-280. 131. Panet, R., Fromer, I., and Atlan, H. (1982). Differentiation between serum stimulation of ouabain-resistant and sensitive Rb influx in quiescent NIH 3T3 cells. J. Membr. Eiol. 70, 165-169. 132. Panet, R., Fromer, I . , and Alayoff, A. (1983). Rb+ influxes differentiate between growth arrest of cells by different agents. J . Membr. Biol. 75, 219-224. 133. Parker, J. C. (1983). Hemolytic action of potassium salts on dog red blood cells. J . A m . Physiol. 13, C313-C317. 134. Pierce, S. K. (1982). Invertebrate cell volume control mechanisms: A coordinated use of intracellular amino acids and inorganic ions as osmotic solute. Eiol. Bull. 163, 405419. 135. Reuss, L. (1983). Basolateral KCI co-transport in a NaC1-absorbing epithelium. Nature (London) 305, 723-726. 136. Reuss, L . , Reinach, P., Weinman, S. A,, and Grady, T. P. (1983). Intracellular ion activities and CI- transport mechanisms in bullfrog corneal epithelium. A m . J . Physiol. 244, C336-347. 137. Riddick, D. H., Kregenow, F. M., and Orloff, J. (1971). The effect of norepinephrine and dibutyryl cyclic adenosine monophosphate on cation transport in duck erythrocytes. J. Gen. Physiol. 57, 752-766. 138. Rindler, M. J . , Chuman, L. M., Schaffer, L., and Saier, M. H. (1979). Retention of differentiated properties in an established dog kidney epithelial cell-line (MDCK). J. Cell Eiol. 81, 635-648. 139. Rindler, M. J . , McRoberts, J. A., and Saier, M . H. (1982). (Na+,K+)-cotransportin the

124

140.

141.

142. 143. 144.

PETER K. LAUF

Madin-Darby canine kidney cell line. Kinetic characterization of the interaction between Na+ and K+. J. Biol. Chem. 257, 2254-2259. Rudolph, S. A., and Greengard, P. (1974). Regulation of protein phosphorylation and membrane permeability by P-andrenergic agents and cyclic adenosine 3‘ :5’monophosphate in the avian erythrocyte. J. Biol. Chem. 249, 5684-5687. Rudolph, S. A,, Schafer, D. E., and Greengard, P. (1977). Effects of cholera enterotoxin on catecholamine-stimulated changes in cation fluxes, cell volume, and cyclic AMP levels in the turkey erythrocyte. J. Biol. Chem. 252, 7132-7139. Russell, J. M. (1976). ATP-dependent chloride influx into internally dialyzed squid giant axons. J. Membr. Biol. 23, 335-349. Russell, J. M. (1979). Chloride and sodium influx: A coupled intake mechanism in the squid giant axon. J. Gen. Physiol. 73, 801-818. Russell, J. M. (1980). Anion transport mechanisms in neurons. Ann. N. Y. Acad. Sci.

341,510-523. 145. Russell, J. M. (1983). Cation-coupled chloride influx in squid axon. J. Gen. Physiol. 81, 909-925. 146. Russell, J. M. (1985). Reverse fluxes of CI through the Na: K : CI coupled transporter in squid axon. Biophys. J. 47, 275a. 147. Saier, M. H., and Boyden, D. A. (1984). Mechanism, regulation and physiological

148.

149.

150.

151.

152.

153.

154. 155.

156.

significance of the loop diuretic-sensitive NaCVKCI symport system in animal cells. Mol. Cell. Biochem. 59, 11-32. Schlatter, E., Greger, R., and Weidtke, C. (1983). Effect of “high ceiling” diuretics on active salt transport in the cortical thick ascending limb of Henle’s loop of rabbit kidney. Pflugers Arch. 3%, 210-217. Schmidt, W. F., 111, and McManus, T. J. (1977). Ouabain-insensitive salt and water movements in duck red cells. I. Kinetics of cation transport under hypertonic conditions. J. Gen. Physiol. 70, 59-79. Schmidt, W. F., 111, and McManus, T. J. (1977). Ouabain-insensitive salt and water movements in duck red cells. 11. Norepinephrine stimulation of sodium plus potassium cotransport. J. Gen. Physiol. 70,81-97. Schmidt, W. F., 111, and McManus, T. J. (1977). Ouabain-insensitive salt and water movements in duck red cells. 111. The role of chloride in the volume response. J. Gen. Physiol. 70, 99-121. Shanbaky, N. M., Wacholtz, M. C., and Sha’afi, R. I. (1981). Further studies of hormone-sensitive sodium and potassium transport in red cells form developing chick embryos. J. Cell. Physiol. 107, 303-308. Silva, P., Spokes, K., Epstein, J. A., Stevens, A., and Epstein, F. H. (1984). Cyclic AMP stimulates ouabain-insensitive ion movement in shark rectal gland. J. Comp. Physiol. B 154, 139-174. Simchowitz, L., and DeWeer, P. (1985). Chloride movements in human neutrophils: diffusion, exchange, and active transport. J. Gen. Physiol. (in press). Smith, L. H., and Pierce, S. K. (1983). Noetia ponderosa erythrocytes use intracellular ions for initial volume regulation during hypoosmotic stress. Am. Zool. 23, 994 (Abstr.). Smith, P. L., Welsh, M. J., Stoff, J. S., and Frizzell, R. A. (1982). Chloride secretion by canine tracheal epithelium. I. Role of intracellular CAMPlevels. J. Membr. Biol. 70,

2 17-226. 157. Spring, K. R., and Ericson, A. C. (1982). Epithelial cell volume modulation and regulation. J. Membr. Biol. 69, 167-176. 158. Thornhill, W. B., and Laris, P. C. (1984). KCI loss and cell shrinkage in the Ehrlich

3. CI-DEPENDENT CATION COTRANSPORT AND CELL DIFFERENTIATION

159. 160.

161. 162.

163. 164.

165.

166.

167. 168. 169.

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ascites tumor cell induced by hypotonic media. 2-deoxyglucose and propranolol. Biochim. Biophys. Acia 773, 207-218. Tosteston, D. C. (1981). Cation countertransport and cotransport in human red cells. Fed. Proc., Fed. A m . Soc. Exp. Biol. 40, 1429-1433. Tosteson, D. C . , and Johnson, J . (1957). The coupling of potassium transport with metabolism in duck red cells. J . Cell. Comp. Physiol. 50, 169-184. Tosteson, D. C., and Hoffman, J. F. (1960). Regulation of cell volume by active cation transport in high and low potassium sheep red cells. J. Gen. Physiol. 44, 169-194. Ueberschar, S . , and Bakker-Grunwald, T. (1983). Bumetanide-sensitive potassium transport and volume regulation in turkey erythrocytes. Biochim. Biophys. A r m 731, 243-250. Washoltz, M . , Chan, L.-N.. Sha’afi, R. 1. (1978). Development of hormonal regulation of ion transport in embryonic chick red cells. J . Cell. Physiol. 94, 1-12. Weigensberg, A. M., and Blostein, R. (1983). Energy depletion retards the loss of membrane transport during reticulocyte maturation. Proc. Nail. Acad. Sci. U . S . A . 80, 4978-4982. Welsh, M. J. (1983). Intracellular chloride activities in canine tracheal epithelium. Direct evidence for sodium-coupled intracellular chloride accumulation in a chloridesecreting epithelium. J . Clin. Invest. 71, 1392-1401. Wiater, L. A,, and Dunham, P. B. (1983). Passive transport of K’ and Nat in human red blood cells: Sulfhydryl binding agents and furosemide. Am. J. Physiol. 245 (Cell Physiol. 14), C348-C356. Widdicombe, J. H., Nathanson, I. T., and Highland, E. (1983). Effects of “loop” diuretics on ion transport by dog tracheal epithelium. A m . J . Physiol. 245, C388-396. Wiley, J. S., and Cooper, R. A. (1974). A furosemide-sensitive cotransport of sodium plus potassium in the human red cell. J. Clin. fnvesr. 53, 745-755. Yeh, L. A,, Ling. L., English, L., and Cantley, L. (1983). Phosphorylation of the (Na,K)-ATPase by a plasma membrane-bound protein kinase in Friend erythroleukemia cells. J . Biol. Chem. 258, 6567-6574.

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Part II

Triggers for Increased Transport during Activation

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CURRENT TOPICS IN MEMBRANES AND TRANSPORI'. VOLUME 27

Chapter 4 External Triggers of Ion Transport Svstems: Fertilization. Growth. and Hbrmone Systems JOAN BELL, LORETTA NIELSEN, SARAH SARIBAN-SOHRAB Y , A N D DALE BENOS Department of Physiology and Biophysics Laboratory of Human Reproducrion and Reproductive Biology Harvard Medical School Boston, Massrcchuser~,s 021 15

I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Ionic Responses to Fertilization . . . . . . . . . . . . . . . . . A. The Resting Potential ...................... .................... B. The Sperm-Egg Interaction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The Fertilization Potential . . . . . . . . . . . ............. D. Sperm-Gated Channels .............................................. E. Molecular Mechanism of Sperm-Induced Permeability Changes F. Fertilization-Evoked Intracellular Alkdlinization. . . . . . . . . . . . . . 111. Serum and Growth Factor Activation of Ionic Transport Systems in Cultured Cells and Lymphocytes.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Na+-H+ Exchange. .... B. Serum-Stimulated E ............................. C. Na+ Channel.. . . . . . . ........... D. Lectin-Induced Mitogenesis in Lymphocytes .......................... IV. Hormonal Stimulation of Ion Transport and Hormone Secretion . . . . . . . . . . . . . A. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The Cultured Pituitary Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Concluding Remarks ....... ... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.

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INTRODUCTION

The major focus of this chapter will be to review and examine critically the evidence that various extracellular molecules such as growth factors, 129 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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polypeptide hormones, and other external signals act as triggering mechanisms for the changes in ionic transport activity observed in several cells and tissues. Further, we will examine the possible relationship between ion transport changes and mitogenesis, excitation-secretion coupling, and hormone-induced adaptive responses. Our discussion, by necessity, will be limited to alterations in cationic transport characteristics, specifically those mediated by ion channels and sodium-proton antiporters. Our working hypothesis, schematically depicted in Fig. 1, is that there exists a potentially general physiological signaling mechanism, and that this mechanism is used repeatedly throughout the life cycle of a cell, with variations depending upon cell type and response required. Specifically, a receptor-mediated interaction between some external molecule and the plasma membrane of a cell is limited to a biochemical process such as membrane phosphatidylinositol turnover or activation of a protein kinase, which in turn produces a change in the ion transport properties of the responsive cell. This change in transport then modulates changes in intracellular pH or ion activities, signaling a specific alteration in some cellular function, such as metabolism, hormone secretion, or growth. We will begin with a general description of the ionic changes which accompany fertilization of the oocyte. This process, of course, initiates cellular growth and hence is pertinent to our concerns. Next, we will summarize the effect of serum, growth factors, phorbol esters, and hormones on the stimulation of ionic fluxes in cultured cells. Within the past 5 years, many laboratories have explored the relationship between these mitogenic compounds and membrane phosphatidylinositol metabolism. It appears that certain by-products of phosphatidylinositol metabolism, notably inositol triphosphate (IP3) and diacylglycerol (DAG), may act as messengers for the signal transduction of various hormones and growth factors. In fact, many naturally occurring growth factors and the phorbol ester tumor promoters can act through this system. This latter observation has led several investigators to suggest that this phosphatidylinositol pathway may be a potential target of oncogenes in eliciting their malignant transformation of cells (Kamata and Feramisco, 1984; Macara et al., 1984; Sugimoto et al., 1984). For example, the protein products of the Rous sarcoma virus src gene, pp60v-src,are capable of phosphorylating tyrosine residues of proteins as well as phosphorylating phosphatidylinositol and diacylglycerol (Sugimoto et al., 1984). Also, Kamata and Feramisco (1984) have shown that the GTP binding activity and phosphorylation of certain protein products of the ras transforming genes are enhanced by epidermal growth factor. The proteins encoded by the ras gene family are small and are associated with the inner surface of the plasma membrane. Ion transport systems at the plasma membrane acti-

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Sleroid Hormones

-

I U ~ II I orlapur L

CAMP. cGMP

ay>iuiiis

(Ion channels. Na-H antiport)

%L -

ca

111

f \

I \ I

4

Changes in intracellular ion activi t i e s

- Changes in cellular melabolism - Changes in rates of DNA end proleln synthesls - Hormone secretion - Biochemicel moalrication of channels

(e g , phosghoryletfon methylalion)

FIG.1. Proposed model for the activation of cellular responses to growth factors and peptide hormones. The binding of a growth factor or hormone to a membrane receptor (scheme I , top of figure) activates an enzyme (e.g., adenyl cyclase, methyltransferase) which eventually leads to an increase in some intracellular “messenger,” which in turn can activate ion transport systems, resulting ultimately in an alteration in intracellular ion activity. Steroid hormones with their intracellular receptors also fit into this scheme. Growth factor or peptide hormone receptor interaction (scheme 2, lower part of figure) activates an enzyme that catalyzes the conversion of phosphatidylinositol4,5-phosphate(PIP2) to inosito1 1,4,S-trisphosphate (IP3) and diacylglycerol (DAG). Phosphatidylinositol 4-phosphate (PIP) is in equilibrium with a plasma membrane pool of phosphatidylinositol (PI). IP3 causes calcium release from inIracellular storage sites or perhaps has a direct action on plasma membrane calcium channels. DAG activates protein kinase C, which in turn may activate directly or indirectly ion channels or Na+-H+ antiporters. These activated transport systems may then effect changes in the intracellular environment (e.g., pH, Ca2+,Na‘, K+), thereby initiating a physiological or biochemical response. Dotted lines indicate hypothetical pathways.

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vated by growth factors are potentially intimately associated with these biochemical processes. We conclude our chapter with a discussion of peptide hormone induction of ionic transport systems and their relationship to hormone secretion. It seems that no matter what the nature of the stimulus, there appears to be a causal correlation between cell division, hormone secretion, inositol lipid turnover, and ion transport. II. IONIC RESPONSES TO FERTILIZATION

In discussing triggers of increased ion transport, it is perhaps most appropriate to begin with the interaction of sperm and egg. The eggs of most organisms are generally quiescent: they do not divide, they have a very low respiratory rate, and they have low protein synthetic activity. Fertilization, however, changes all of this. The temporal sequence of events related to fertilization has been most extensively studied in the sea urchin and is outlined in Table I (see Epel, 1975; Whitaker and Steinhardt, 1982, for reviews). Fertilization evokes early events such as plasma membrane depolarization, transient calcium release from intracellular stores, and the so-called cortical granular reaction. These initial events are fol-

TABLE I TIMINGOF EARLYEVENTS AFTER FERTILIZATION OF S E A Event Membrane potential Ca+-Na+ action potential Na+ activation potential Increases in K+ conductance (remains at higher levels) Intracellular calcium release Cortical reaction Activation of NAD kinase Increases in reduced nicotinamide nucleotides (remains at higher levels) Acid emux Increases in intracellular pH (remains at higher levels) Increased oxygen consumption (initial burst) Initiation of protein synthesis Activation of amino acid transport Initiation of DNA synthesis Mitosis First cleavage (I

From Whitaker and Steinhardt (1982).

URCHIN

EGGS^

Time Before 13 sec 13- 120 sec 500-3000 sec 40- 120 sec 40- 100 sec 40-120 sec 40-900 sec 1-5 min 1-5 min 1-3 rnin 5 min onward

I5 rnin onward 20-40 min 60-80 rnin 85-95 rnin

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lowed by plasma membrane hyperpolarization and dramatic increases in protein and DNA synthesis and finally cellular division. Busa and Nuccitelli (1984) contend that these late events are due to intracellular alkalinization, perhaps resulting from the induction of a Na+-H+ exchange system at the plasma membrane. Upon contacting the egg, a single sperm is capable of inducing changes in the ionic permeability of the egg membrane, resulting in fluxes which may lead directly to these subsequent developmental events. In fact, in virtually every organism examined, the earliest measurable response to fertilization of the egg is a change in its membrane potential (Steinhardt et al., 1971; Jaffe, 1976; Gould-Somero et al., 1979; Hagiwara and Jaffe, 1979; Jaffe et al., 1979, 1983a,b; Kozuka and Takahashi, 1982; Miyazaki and Igusa, 1982; Charbonneau et al., 1983; Dale el al., 1983; Lansman, 1983). This change is designated the fertilization potential. The function of the fertilization potential in most organisms is unknown, although at least in the case of the sea urchin and probably other echinoderms, it most likely acts as a block to polyspermy (Jaffe, 1976; Jaffe et al., 1983a; DeFelice and Dale, 1979; Gould-Somero et al., 1979; Grey et ul., 1982; Whitaker and Steinhardt, 1982). This section will focus on the mechanisms by which sperm induce these changes in membrane permeability. To examine this problem we need to know three things. First, what is the state of the egg cell membrane before fertilization? Second, what is the molecular nature of the sperm-egg interaction? Third, what is the ionic basis for the fertilization potential? Because most experimental work has been done most successfully on the sea urchin, we will deal primarily with this organism. A. The Resting Potential

Until recently, there was some controversy regarding the magnitude of the physiological resting potential of sea urchin eggs, as well as other organisms (Chambers and de Armendi, 1979; Gould-Somero et ul., 1979; Whitaker and Steinhardt, 1982; Nuccitelli and Dale, 1984). The problem centers around the observation that for a given population of eggs, the measured resting potentials seem to fall into two groups: those that are close to 0 mV and those that have a negative value (the magnitude of which depends on the organism). Thus, when sea urchin eggs are suspended in seawater and are impaled with microelectrodes, some eggs have a resting potential around -10 mV and others around -70 mV (Higashi and Kaneko, 1971; DeFelice and Dale, 1979; Taglietti, 1980). It was important to resolve this issue because the two populations show

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different electrical responses to fertilization. Eggs with large resting potentials respond with an initial action potential which relaxes to a depolarized potential that is maintained for several minutes. The threshold for these action potentials is approximately -40 mV. Hence, eggs with resting potentials close to 0 mV do not generate an initial action potential upon fertilization. Nonetheless, both populations of eggs are capable of generating a sustained depolarizing fertilization potential and undergoing normal activation (Dale et al., 1983). However, in other organisms the eggs with small prefertilization resting potentials either died soon after impalement or appeared “unhealthy” (Jaffe et al., 1979). The most plausible explanation, that is, that the low potentials measured were due to leakage around the electrode, was confounded by noting that many laboratories using eggs from varied species, ranging from ascidians to hamsters, found this same dispersion in the measured resting membrane potential (Miyazaki and Igusa, 1982; Dale et d . ,1983). If leakage around the electrode were the correct explanation for the low resting potentials, one might have expected a distribution of values less negative than the lower measured value, rather than the bimodal distribution observed. However, the high resistance of these membranes (100-1000 ki2.cm2)might make even a “small” leak bring the potential close to 0 mV. In fact, eggs with small resting potentials do have lower membrane resistances, which would be expected if there were a leakage problem. Further evidence that the larger resting potentials were the physiologically accurate ones came from Chambers and DeArmendi (1979), who measured tracer fluxes of Na+ and K+ and then used the Goldman-Hodgkin-Katz potential equation to calculate the expected membrane resting potential. They obtained a value of -78 mV, which is quite consistent with the larger measured values of resting potential. The current view is that the low resting potentials are due to electrical leakage around the microelectrode and that the action potential elicited upon fertilization is indeed part of the normal response to insemination. Thus, in the sea urchin, Lytechinus uariegatus, the true resting potential in seawater is between -75 and -80 mV. Ascidian oocytes also have resting potentials close to -80 mV (Dale et al., 1983). By measuring the relationship between this potential and external K+concentration, Chambers and DeArmendi (1979) showed that the resting potential is almost entirely a K+diffusion potential. In contrast, the marine worm, Urechis caupo, has a resting potential normally around -35 mV, which is determined by both Na+ and K+ conductances (Gould-Somero et al., 1979).

Mammalian eggs also possess resting potentials in this range. Mice and hamster oocytes both maintain potentials around -40 mV, while rabbit

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eggs exhibit a resting potential close to -70 mV (Miyazaki and Igusa, 1982; McCulloh et al., 1983; Jaffe et al., 1983b). In general, the ionic basis for these potentials varies little from organism to organism. In starfish, amphibians, and mammals, the resting potential is largely determined by Na+ and/or K+ ion conductances. B. The Sperm-Egg Interaction What is the molecular nature of the sperm-egg interaction? There has been extensive work on both the morphological and biochemical events of sperm attachment to the egg surface (Vacquier and Moy, 1978; Veron and Shapiro, 1977; Chambers, 1980). In the sea urchin, the sperm must penetrate a thick jelly-like coat to reach the surface of the egg, where it appears to attach to the vitelline membrane, a thin proteinaceous membrane overlying the true plasma membrane. Figure 2A is a scanning electron micrograph showing a sperm adhering to the surface of a sea urchin egg. There is a species-specific sulphated glycoprotein in this jelly which can trigger the so-called acrosome reaction (Tyler, 1949; Segall and Lennarz, 1979). In this reaction the acrosomal vesicle in the head of the sperm fuses with the plasma membrane, thereby exposing its contents to the external environment. This reaction is an absolute requirement for fertilization (Takahashi and Sugiyama, 1973; Collins and Epel, 1977). The sole component of the acrosomal vesicle contents is a 31,000 MW protein which can aggregate sperm and egg (Vacquier and Moy, 1978). This protein, appropriately dubbed “bindin,” has been purified and shown to contain only amino acids, that is, no carbohydrate or lipid (Vacquier and Moy, 1977). Figure 2B shows bindin concentrated in the region of sperm-egg contact. Recently, the evidence has become quite convincing that there is a species-specific receptor for bindin in the vitelline membrane of the egg (Vacquier and Moy, 1977; Rossignol and Lennarz, 1983; Rossignol et al., 1984). Partial purification of the receptor yielded a large proteoglycan that binds species specifically to sperm only after they have undergone the acrosome reaction. Antibodies to this receptor are capable of attaching to the egg surface, and both antibody and sperm attachment can be prevented by treating the surface of the eggs with trypsin or pronase (Vacquier et al., 1973). Rossignol and Lennarz (1984) have purified a glycoprotein (MW > lo6) from the vitelline membranes of two species of urchin, Strongylocentrotus purpuratus and Arbacia punctulata, that can bind acrosome-reacted sperm and bindin antibody. The high molecular weight is due mostly to a carbohydrate-rich fragment containing hyaluronic acid, galactosamine, and sulphate, and hence is highly negatively charged.

FIG.2. (A) Scanning electron micrograph of a single spermatozoon at the egg surface of the sea urchin, Arbacia punctulafa, X90,OOO. (Photograph provided through the courtesy of Dr. Everett Anderson.) (B) Bindin localized at the sperm-egg bond (sea urchin). Arrows indicate immunoperoxide localization of antibindin antibody in area surrounding the acrosomal process of the sperm. Bindin also appears on the surface of the egg microvilli. S, spermatozoon; EC, egg cytoplasm; MV, microvillus. ~50,650.From Moy and Vacquier (1979). (Photograph kindly provided by Dr. V. D. Vacquier.) 136

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The discovery of bindin and the proteinaceous nature of the bindin receptor suggests that sperm may act as an agonist. That is, sperm may interact with a receptor, thereby mediating a permeability change which leads to the observed fertilization potential (Epel, 1978). However, sperm can still attach and induce a fertilization potential in eggs which have been stripped of the vitelline membrane, albeit with lowered efficiency and less species specificity (Rossignol et al., 1984). In addition, eggs treated with bindin or antireceptor antibody alone do not depolarize or activate. These observations have led some groups to believe that the bindin-receptor interaction is most likely involved in cementing the sperm of the proper species to the egg surface, and that the electrical events are actually triggered by the subsequent fusion of egg and sperm plasma membrane (Whitaker and Steinhardt, 1982). Unfortunately, because of the poor time resolution necessarily involved in documenting the morphological events of fertilization, it is difficult to distinguish cause and effect. It is thought that the complete reaction, from the acrosomal reaction to membrane fusion, may take from 7 to 9 seconds (at least in the annelid Hydroides and the meichordate Saccoglossus; Dale et al., 1983). The fertilization potential often occurs within 5 seconds of addition of sperm (Nuccitelli and Grey, 1984). In both cases, the time delays have been attributed to the time necessary for the sperm to swim to and penetrate the egg coating. Therefore, it is not yet clear what is the true chronology of events with respect to the moment at which the sperm contacts the egg vitelline membrane and when the fertilization potential is initiated. C. The Fertlllzatlon Potentlal

The voltage response to fertilization of the sea urchin is shown in Fig. 3A. After an initial depolarizing Ca2+-dependentaction potential, there is a slight repolarization which leads to a maintained plateau. Then a slow depolarization due to an increase in Na+ permeability is followed by a slow repolarization to resting potential. This general scheme, of an action potential which relaxes to a plateau level (which may be maintained for several minutes) and then gradually repolarizes, has been observed in many species of sea urchin as well as the marine worm Urechis (Jaffe, 1976; Chambers and DeArmendi, 1979). The true fertilization potential is considered to be the potential remaining after the action potential is subtracted out. This distinction is made because the action potential can be elicited by both current injection and sperm, while the maintained depolarization is elicited only by sperm. Also, sea urchin eggs with resting potentials more depolarized than the threshold of the action potential still

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40 Time

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-75

8

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44

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' I

I

I

I

520

540

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FIG.3. (A) Model of the time course of the fertilization potential of the sea urchin egg. The model incorporates the major phases seen thus far upon fertilization of sea urchin eggs. (1) Step depolarization; (2) Ca2+-dependent action potential; (3)-(5) plateau stage, which may include transient changes in Na+ permeability; (6) repolarization back to resting potential. [Reproduced with permission from Nuccitelli and Grey (1984).] (B) Fertilization potential of Lyrechinus uariegatus egg in low-Ca2+seawater, showing the step depolarization. FM indicates the interval during which the fertilization membrane lifted. [Reproduced with permission from Chambers and DeArmendi (1979).]

exhibit the slow depolarization and subsequent activation upon insemination (Nuccitelli and Grey, 1984). An interesting feature of the fertilization potential which can be seen when the action potential is suppressed (by using low-Ca2+ seawater, for example) is the step depolarization illustrated in Fig. 3B. By superimposing video images of sperm approaching

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an egg over an electrical recording, Schatten and Hulser (1983) showed that this step depolarization corresponds in time to the initial sperm-egg contact. The ionic basis for most of these changes remains poorly understood. In both sea urchin and sea worm, the fast action potential appears to be primarily Ca2+dependent. The maintained depolarization, and any fluctuation within that depolarization (see Fig. 3A), is at least partially determined by Na+. In Urechis, Jaffe et al. (1979) report a value of the maintained potential of about 30 mV, yet the Na+ equilibrium potential calculated from the experimental conditions is close to 90 mV. This implies that other ions are playing a large role in determining the magnitude of the fertilization potential. These results raise the possibility that there exists in the egg cell membrane ion channels which open or close in response to sperm, that is, sperm-gated ion channels. Gould-Somero (1981) showed that the magnitude of the fertilization potential (during the plateau) depends on the Na+ concentration in the vicinity of the spermegg interaction. In addition, DeFelice and Dale (1979) showed that during polyspermic fertilization each spermatozoon induced a step-like conductance change, suggesting that each spermatozoon activated a fixed number of channels. D. Sperm-Gated Channels

At this point we know that eggs depolarize when inseminated. Generally, the ions responsible for this depolarization are Naf and Ca2+.The question now remains as to how sperm cause the change in permeability to these ions. The response of several investigators to observing these potential changes was to speculate upon the location and number of ion channels involved (Jaffe, 1976; Gould-Somero, 1981 ; Hagiwara and Jaffe, 1979). This somewhat tacit assumption about the presence of ion channels in the egg cell was given some validity by Dale and DeFelice (1984). Using the patch clamp technique, they have reported the appearance of large, nonselective cation channels in the membrane of ascidian (Ciona intestinalis) eggs upon fertilization. Figure 4 shows the fluctuating nature of events which were recorded during the plateau phase of fertilization potential. These channels were not activated by current injection and appeared only when spermatozoa were added. However, these channels only appeared in about 20% of the experiments. Whether this low success rate was a technical problem or a physiological one due perhaps to a low density of channels in the membrane is not clear at this time. The fact remains that egg cells do appear to contain ion channels that may respond

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FIG.4. Voltage response and single-channel currents activated by sperm in ascidian oocytes. (a) Fertiliz,ition potential in an oocyte with a resting potential of about -80 mV. The vertical bar represents 40 mV, the horizontal bar 10 seconds. The lower trace in (a) shows the voltage noise (vertical bar = 1 mV). (b) Single-channelcurrents during the plateau phase of the fertilization potential. The pipet voltage trace, -20 mV, represents the voltage clamp step (a depolarization) at which the fluctuations were measured. Here, the vertical bar represents 20 pA and the horizontal bar represents 40 msec. [From Dale and DeFelice (1984). We thank Dr. L. DeFelice for kindly providing an original photop;raph.]

to the presence of sperm. Dale et al. (1978) suggested that the acrosomal vesicle may release a factor which activates channels in egg cell membranes. There are no reports in the literature of any attempt to try to evoke a fertilization potential with the vesicle contents, other than those using bindin. That channels are being inserted by fusion of the sperm membrane with the egg is unlikely because fertilization potentials can be evoked by treating eggs with trypsin alone (Jaffe et al. 1979). E. Molecular Mechanism of Sperm-Induced Permeability Changes

Although evidence that sperm may directly gate ion channels located in the plasma membrane of the egg is starting to appear, the mechanism by which a spermatozoon opens a channel is simply not known. If the spermatozoon is releasing a chemical factor, Dale and DeFelice (1984) point out that this factor must act via the cytoplasm of the egg to activate the channels because they were adding sperm outside of the patch pipette (whole cell patch), We can speculate that upon fusion of the egg and sperm membrane some messenger is released into the egg cytoplasm [in the region of fusion, as demonstrated by Gould-Somero (1981)l. Mechanisms analogous to those postulated for hormone effects on permeability (see below) may be involved. It has long been known that the Ca2+ ionophore A23187 can induced normal activation of sea urchin eggs (e.g., the cortical reaction, internal alkalinization, etc.). Although eggs can activate in the absence of a fertilization potential, it is possible that the Ca2+

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influx is initiating some step in the process which follows the initial membrane potential change. By further exploiting the patch clamp technique to determine the ion specificity and gating properties of channels found in egg cell membranes, it is likely that more insight into the molecular mechanism of how sperm alter the permeability properties of the egg cell membrane will be achieved. F. Fertlllratlon-Evoked lntracellular Alkallnlzatlon

Acid efflux following insemination has been noted in eggs of Urechis, in several species of sea urchin and mollusc, and in the only vertebrate species studied thus far, the toad Xenopus laevis (Johnson et al., 1976; Cuthbert and Cuthbert, 1978; Shen and Steinhardt, 1980; Nuccitelli et al., 1981). There is good evidence (at least in sea urchin and Urechis)that this so-called “fertilization acid” is protons (Paul, 1975; Holland et al., 1984). The acid release is accompanied by an increase in internal pH of the fertilized egg. The mechanism of this acid release and concommitant cellular alkalinization remains to be elucidated and is in fact a field of active debate. A number of laboratories have suggested that fertilization activates an electroneutral Na+-H+ exchange because the Na+ uptake and H+ release are similar in amount, require external Na+, and are both inhibited by amiloride, a diuretic that blocks Na+-H+ exchangers (Johnson et al., 1976; Benos, 1982; Payan et al., 1983). Furthermore, Whitaker and Steinhardt (1982) showed that voltage clamping the eggs to negative potentials did not inhibit the intracellular alkalinization, implying that the movement of H+ was not electrogenic. However, Gould and Holland (1984) showed that in Urechis eggs the stoichiometry of Na+ uptake and H+ release is not one-to-one under all conditions. These authors feel that H+ release is dependent on the magnitude of the membrane potential rather than on Na+ uptake. Because of ambiguities in the ionic basis of both the resting potential and the fertilization potential in this system, it is difficult to evaluate such a theory on the basis of experiments which simply vary the external Na+ or H+ concentrations, particularly when the possible role of a Na+-K+ pump is not taken into account. The role of the internal alkalinization in the subsequent activation of the egg is not entirely clear, although it has been shown that protein synthetic activity is greater at higher pH values (Brandis and Raff, 1979). Transferring fertilized sea urchin eggs to Na+-free seawater blocks the rise in cytoplasmic pH and arrests further development. Raising the external pH to 9 stimulates these fertilized eggs to cleave (Nishioka and Epel, 1977),

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suggesting that pH does play a regulatory role in the response to insemination. 111.

SERUM AND GROWTH FACTOR ACTIVATION OF IONIC TRANSPORT SYSTEMS IN CULTURED CELLS AND LYMPHOCYTES

The emergence of cell culture techniques in recent years has provided investigators with useful cell and epithelial model systems with which to study growth factor- and polypeptide-induced alterations in ion transport function. Rapid biochemical changes associated with growth factor action in cultured cells include modulation of ionic fluxes, protein phosphorylation, and ultimately cell division, just as we have seen with the fertilization process. We will now review some of the experiments designed to explore the possible relationship between growth factor and hormonal activation of ion transport systems and mitogenesis in cultured cells and lymphocytes. A. Na+-H+ Exchange

Stimulation of sodium, hydrogen, and potassium transport is observed within two minutes after growth-arrested cells have been in contact with serum or growth factors. Protein synthesis, DNA replication, and cell division follow these initial ionic events. The increased transport occurs in part via an activation of an amiloride-sensitive Na+-H+ antiporter located in the cell membrane. This exchange could itself be triggered by (1) biochemical reactions such as phosphorylation or methylation which either turn on the transporters or lead to the incorporation of new transporters in the plasma membrane from a cytoplasmic pool and/or (2) changes in intracellular ion activities (Ca2+,H+). A basic observation is that cultured cells do not proliferate in the absence of serum or appropriate growth factors in the culture medium (Holley, 1975). Cell proliferation following serum stimulation appears to be mediated by an increase in ion transport which is easily measurable, but the sequence of events leading from stimulus to enhanced transport remains to be clarified. A general scheme might involve binding of growth factors to plasma membrane receptors followed by a cascade of biochemical modifications of membrane lipid and protein, leading to a change in the functional properties of membrane-localized ion transport systems. Ion pumping activity thorough the Na+, K+-ATPasewas the first transport system to be studied as a possible target for growth action. Ro-

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zengurt and Heppel (1975) measured a four-fold increase in quiescent 86Rb+influx in quiescent 3T3 mouse fibroblasts 2 minutes after stimulation with serum. This increase was reversible and inhibited by ouabain. This stimulation of cation pumping activity was proposed as a requirement for the initiation of DNA synthesis (Rozengurt and Heppel, 1975). The stimulation of the Na+-K+ pump in serum-treated 3T3 cells was later correlated with an increase in Na+ entry as assessed by tracer fluxes (Smith and Rozengurt, 1978). In 1979 Koch and Leffert showed that amiloride, an inhibitor of Na+ transport, prevented rat hepatocyte proliferation in primary liver cell cultures as initiated by a combination of insulin, glucagon, and epidermal growth factor (EGF). The half-maximal inhibitory concentration of amiloride ( K I )was 0.2 mM (Koch and Leffert, 1978). The effect of amiloride was further investigated in human fibroblasts (Villereal, 1981). In these cells (HSWP) the serum-stimulated component of Na+ influx was inhibited by amiloride, with a K I of 0.2 mM (at 140 mM external "a+]). A 2.5-fold stimulation of the Na+ influx (i.e., equivalent to that induced by serum) could also be achieved in the absence of serum by preincubating HSWP cells with the Ca2+ionophore A12187 in the presence of external Ca2+.This stimulation was inhibited by amiloride, with a K I of 3 p M . The discrepancy between this number and the K1 for amiloride inhibition of the serum-stimulated Na+ influx was attributed to a reduced affinity of amiloride for its binding site in the presence of serum, perhaps due to a loss of surface Ca2+.From these data, Villereal speculated that a serum-stimulated increase in Ca2+influx led to the stimulation of an amiloride-sensitive Na+ pathway. Other possibilities included a serum-induced alteration in the rate of plasma membrane Ca2+-ATPaseactivity or a serum-induced mobilization of intracellular Ca2+stores. Unfortunately, A23187 activation of Na+ influx was not tested in the absence of extracellular Ca2+. In the absence of serum, A23187 by itself did not stimulate Na+ influx, a result at variance with the one reported by Villereal (1981). Benos and Sapirstein (1983) also reported that A23187 failed to elicit an amiloride-sensitive component of Na+ influx in cultured rat C6 glioma cells, although Na+ influx itself was increased after ionophore treatment. Investigating the growth factor regulation of the Na+-H+ antiporter, Paris and Pouyssegur (1984) showed that addition of thrombin and insulin to quiescent Chinese hamster lung fibroblasts (CCL 33) triggered a rapid increase in the affinity of the Na+-H+ antiporter for internal H+. At an external pH of 7.4, half-maximal stimulation of amiloride-sensitive 22Na+ uptake was observed at an internal pH of 6.65 in the absence of growth factors and at an internal pH of 6.95 in their presence. The intracellular pH was adjusted using the NHJ prepulse techique (L'Allemain et al.,

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1984). Since intracellular pH remained stable after growth factor addition, the initial alkalinization caused by H+ extrusion through the antiporter must be compensated by acidifying metabolic reactions in the cytoplasm. The authors proposed that the Na+-H+ antiporter conformation was modulated by growth factors, possibly at the level of an internal modifer H+ binding site. Within the past several months, three groups have demonstrated independently that tumor-promoting phorbol esters activate an amiloride-sensitive Na+-H+ exchange system in much the same way that serum or growth factors do (Besterman and Cuatrecasas, 1984; Whitely et al., 1984; Sapirstein and Benos, 1984). The phorbol esters initiate a plethora of diverse cellular events, ultimately affecting cellular proliferation and differentiation (see Abraham and Rovera, 1980, for a review). The most potent phorbol ester, phorbol 12-myristate 13-acetate (PMA), activates protein kinase C (a Ca2+-and lipid-requiring kinase) by substituting for diacylglycerol (a by-product of phosphoinositol metabolism and the physiological activator of protein kinase C; see Nishizuka, 1984). Protein kinase C is also the phorbol ester cellular receptor (Niedel et al., 1983; Leach et al., 1983; Ashendel et al., 1983). Epidermal growth factor, for example, activates amiloride-sensitive Na+-H+ exchange, stimulates phosphatidylinositol turnover, activates protein kinase C, and induces mitogenesis (Sawyer and Cohen, 1981). Activated protein kinase C in turn desensitizes the EGF receptor, suggesting a negative-feedback, regulatory interrelationship (Cochet et al., 1984; Iwashita and Fox, 1984; Whitely et al., 1984). In other words, Na+-H+ activation by EGF can be inhibited by prior addition of PMA through its effects on protein kinase C (Whitely et al., 1984). Both Besterman and Cuatrecasas (1984) and Whitely et al. (1984) found a good correlation between the ability of phorbol esters to increase Na+-H+ exchange and their ability to activate protein kinase C. Sapirstein and Benos (1984), in addition, showed that dioleylglycerol-the putative endogenous activator of protein kinase C-also increases specifically the amlioride-sensitiveNa+ entry pathway in cultured rat glial cells. B. Serum-Stimulated Electrical Events

Although the results summarized above involve a stimulation of Na+-Hf exchange by growth factors, measurements of the electrical behavior of the stimulated cell membrane by Moolenaar et ul. (1979) suggested other pathways for ion movement. These authors measured for the first time rapid ionic events following serum stimulation in mouse neuro-

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blastoma cells (NlE-115) using intracellular microelectrodes. Within a few seconds after addition of serum, the membrane depolarized and its resistance decreased eight-fold while the Na+ permeability increased. Tetrodotoxin, the voltage-dependent Na+ channel blocker, did not affect those electrical events, identifying the serum-activated Na+ conductance as different from the voltage-dependent Na+ channel underlying the generation of the neuroblastoma action potential (Moolenaar et al., 1979). Similarly, amiloride had no effect on the electrical properties of these cells. Therefore, these alterations in membrane potential and resistance after serum addition cannot be accounted for by an electrically silent transport system, that is, an antiporter. In 1981 Moolenaar et al. showed that the early serum-induced increase in Na+ conductance (depolarizationphase) reported previously was unaltered in the presence of ouabain, ruling out a direct participation of the Na+-K+ pump in mediating this response. Furthermore, when seruminduced Na+ influx was inhibited by amiloride, the serum-stimulated increase in pump rate was also inhibited. The depolarization phase was insensitive to changes in external “a+] or [Ca2+]and could be mimicked in the absence of serum by gramicidin, a substance that forms nonselective cation channels in lipid bilayers. These results suggested a relatively nonspecific initial increase in Na+, K+,and perhaps Ca2+permeabilities. The membrane depolarization was followed by a hyperpolarization phase due to increased K+ permeability. This phase remained unchanged at external [Ca2+]between 0.1 and I0 mM, indicating that enhanced Ca2+ influx was not involved in eliciting this hyperpolarization. At low external [Ca2+],A23 187 induced a transient hyperpolarization. This effect could be explained by an increased K+ permeability triggered by the release of Ca2+from mitochondria1 stores. A serum-induced transient depolarization of the apical plasma membrane was also observed in epithelial African green monkey kidney cells (BSC-1) (Rothenberg et al., 1982). These authors also measured an increased Na+ influx that was electrically silent, but the effect of A23187 was not tested. In summary, serum and growth factors seem to interact primarily with receptors at the plasma membrane level, although only an EGF receptor has been identified (Cohen et al., 1982).The rapidity of the serum stimulation as well as the very low (nanomolar range) concentrations of growth factors needed to trigger transport make plausible the receptor-mediated hypothesis. The first observable changes are twofold and include (1) a stimulation of an amiloride-sensitive Na+-H+ antiporter and (2) a stimulation of Na+, K+, and Ca2+conductances. The Na+-H+ antiporter could be triggered by an increase in intracellular Ca2+,as postulated by Villereal

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(1981), or by an increased affinity of the antiporter for internal H + , perhaps mediated by Ca2+-dependentmembrane changes, as proposed by Pans and Pouyssegur (1984). Increased Na+ transport through the antiporter results in both cytoplasmic alkalinization and stimulation of the Na+-K+-ATPase activity. The relationship between Ca2+,serum, and regulation of membrane transport is made even more complex by the fact that the cells could require a specific sequence of triggering factors in order for the transport to be stimulated (Tupper et al., 1982). In 1976 Rozengurt proposed a still up-to-date model to explain how binding of a growth factor to a receptor can elicit diverse biological responses. This model incorporated the dynamic fluid properties of cell membranes and the concept of mobile receptors. The model includes binding of a growth factor to its receptor, thereby changing the receptor's conformation so that it can interact directly with membrane effectors. The transmission of signals from receptor to effector may thus depend on the interactions between membrane lipid and protein components, which can be modulated by surface phosphorylation or methylation. Both reactions have been demonstrated in cultured cells (Mastro and Rozengurt, 1976; Sariban-Sohraby et al., 1984). It is possible that membrane-associated Ca2+(regulated by the presence of serum in the growth medium or by certain Ca2+-dependentreactions) could provide the appropriate localized increase in some intracellular Ca2+pool required for growth factor-induced transport activation.

C. Na+ Channel

As growth factor stimulation of the Na+-H+ antiporter has been linked to cell growth and dedifferentiation, hormonal stimulation of Na+ channels in contrast has been associated not with cell multiplication but with increased transport function in epithelia. In addition to the classical receptor-mediated pathway of steroid hormone action, another potential mechanism of action of these hormones, that is, a plasma membrane effect, has emerged (Duval et al., 1983). In epithelia, apical Na+ permeability is mediated by specific transport channels (Lindemann and Van Driessche, 1977). The observed increase in the number of conducting channels promoted by the mineralocorticoid aldosterone seems to result from the activation of nonconductive channels already present in the apical plasma membrane (Palmer and Edelman, 1981). This conclusion was reached from the following experiment. Apical membrane, channel-mediated Na+ transport in toad urinary bladder can be inhibited by diazosulfonic acid (DSA), an impermeant proteinmodifying reagent. Palmer and Edelman (1981) exposed the bladder to

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DSA for 1 hour, thereby inhibiting transport to 30-40% of the control value. Subsequent aldosterone addition increased Na+ transport (with an initial lag period of 1 hour) in both DSA-treated and control hemibladders. However, the absolute magnitude of the increase in Na+ transport was larger in the control bladder, while the fractional increases were the same. Thus, the inhibition of aldosterone-stimulated Na+ transport by DSA was the same as the DSA-induced inhibition of the basal rate of transport. The authors suggested that these results could be interpreted to mean that both conducting and nonconducting apical membrane channels were equally susceptible to DSA attack; hence, the aldosterone-activated channels preexisted in the apical membrane and were not inserted from some DSA-inaccessbile submembrane pool. This conclusion was later confirmed by Garty and Edelman, (1983), who showed that trypsinization of the apical membrane of toad urinary bladder resulted in an irreversible decrease in both basal and aldosterone-stimulated sodium transport as measured by short-circuit current. The findings indicate that the increase in apical sodium permeability elicited by aldosterone involved the opening of previously nonconductive, trypsin-accessible sodium channels present in the apical membrane. This result contrasts with the stimulating effect of anti-diuretic hormone (ADH) on Na+ transport in the toad urinary bladder, where the increased density of open channels after hormonal treatment (Lindemann, 1980) results from insertion of conductive channels in the membrane (Garty and Edelman, 1983). The induction of RNA (Rossier et al., 1974) and protein (Benjamin and Singer, 1974) synthesis by aldosterone was proposed as a requirement for increased Na+ transport mainly because of the observation that inhibitors of RNA and protein synthesis blocked the aldosterone-induced increase in Na+ transport (Chu and Edelman, 1972). However, citrate synthase, a regulatory enzyme of the tricarboxylic acid cycle in mitochondria and the only aldosterone-induced protein as yet identified, was not stimulated after aldosterone treatment in hormone-responsive cultured epithelia from toad bladder and kidney (Handler et al., 1981). Furthermore, in the amphibian urinary bladder, aldosterone caused an increase in the content of membrane phospholipid polyunsaturated fatty acid content by altering a deacylation-reacylation cycle (Goodman et al., 1971). The changes in membrane lipid metabolism were inhibited by the RNA and protein synthesis inhibitors cordycepin and cycloheximide, respectively (Lien et al., 1976). These results support the hypothesis that the changes in membrane phospholipid fatty acids may be related to the changes in sodium transport and raise the possibility that the role of aldosterone-induced proteins is related to changes in membrane lipid metabolism and not to synthesis or turnover of membrane transport proteins.

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Recent experiments by Sariban-Sohraby et al. (1984) provide further support to the hypothesis that steroid hormone-induced increases in ion transport result from specific membrane-associated biochemical reactions. These authors observed that aldosterone stimulation of Na+ influx into an apical membrane vesicle fraction prepared from cultured toad kidney cells could be mimicked by in uitro methylation of vesicle membrane lipid and protein. Further, in vesicles prepared from cells pretreated with aldosterone, both lipid and protein methylation were increased over controls. It is possible, then, that certain as of yet unidentified protein products resulting from aldosterone treatment produce their physiological effects on Na+ transport by catalyzing some membrane-associated biochemical process such as methylation. This reaction ultimately leads to an activation of silent channels already present in the membrane. In summary, membrane-localized biochemical modifications of lipid and protein appear as the key event in both growth factor stimulation of the Na+-H+ antiporter, leading to cell proliferation, and hormone stimulation of the Na+ channel, leading to increased transport. D. Lectin-Induced Mitogenesis in Lymphocytes

Plant lectins, such as concanavalin A (Con A) and phytohemagglutinin (PHA), stimulate mitogenesis in peripheral blood lymphocytes. These lectins also depolarize the lymphocyte membrane, increase 4sCa2+uptake, and increase internal calcium concentrations in a sodium-independent fashion. (Deutsch and Price, 1982; Tsien et al., 1982). Another mitogenic substance, the calcium ionophore A23 187, stimulates 86Rb+efflux from lymphocytes by 50% in 10 minutes in calcium-containing media but by only 20% in calcium-free, 1-mM EGTA medium. Quinine inhibits this &Rb+efflux in a dose-dependent manner, and at 75 p M , blocks cellular K+ loss as determined by flame spectrophotometry (Grinstein et al., 1982). Patch electrode, voltage clamp studies of lymphocyte membrane currents (Matteson and Deutsch, 1984; DeCoursey et al., 1984) have revealed the generation of an outward K+current upon membrane depolarization. No inward currents were detected. The magnitude of outward K+ current increased two-fold after PHA addition. In addition to the data cited above, evidence that the outward current is mediated by K+ channels include the observations that the reversal potential of the current is close to the K+ equilibrium potential and that K+ channel blockers, such as Cs+, quinine, TEA, and 4-aminopyridine, abolish this conductance. DeCoursey et al. (1984) observed voltage-dependent, single K+ chan-

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nels in lymphocyte membrane patches. These single channels had a conductance of 16 pS when bathed in normal Ringer solution. Addition of PHA altered K + channel gating by shifting the probability of finding these channels in an open state to more negative membrane potentials. These authors also found that TEA and 4-aminopyridine prevented r3H]thymidine incorporation into the lymphocyte DNA. These results suggest that these K+ channels may somehow be involved in growth. IV. HORMONAL STIMULATION OF ION TRANSPORT AND HORMONE SECRETION A. Introduction

W. W. Douglas and R. P. Rubin (1961) first used the phrase “stimulussecretion coupling” to describe the ability of acetylcholine (Ach) to elicit secretion of catecholamines from adrenal chromaffin cells. Douglas and colleagues showed that Ach depolarized the adrenal cell and increased membrane permeability to Ca2+and Na+. All of acetylcholine’s actions were blocked by atropine or hexamethonium, while a number of agents, including high external K + , could mimic the stimulatory effects of Ach. Removal of external Ca2+,but not external Na+, abolished catecholamine release. This concept and general characterization of stimulus-secretion coupling has proven to be applicable to many cell types (Douglas, 1968). All secretory cells must possess appropriate membrane effectors or systems which transduce the initial stimulus-receptor interaction into, eventually, exocytosis. In the neurosecretory cell, neurotransmitter release is regulated by Ca2+,which enters these cells through both Na+ and Ca2+ channels. The ability of hormones and other molecules to induce characteristic changes in plasma membrane electrical activity (and thus be linked to secretion) has also been documented in many endocrine and exocrine cells (see Douglas, 1968). For example, the characteristic electrical response of the pancreatic B cell to glucose consists first of membrane depolarization due to a decrease in K + conductance. Superimposed upon this depolarization are a series of fast spikes generated by the activation of voltage-dependent Ca2+channels (Atwater, 1980). The resultant increase in intracellular [Ca2+]as well as the depolarization itself activates a potassium conductance which repolarizes the cell and, in so doing, closes the Ca2+channels. As the concentration of Ca2+is decreased by intracellular sequestering systems, the potassium conductance is again decreased, and the cycle is completed. Somewhere within this sequence, these ionic changes result in insulin secretion.

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In this section we will cover in depth only one representative cell type, the growth hormone- and prolactin-secreting GH cell. The reader is referred to Petersen (1980), Hagiwara and Ohmori (1982), Maruyama and Petersen (1982), Maruyama et al. (l983), and Reuter (1983) for information on other systems. 5. The Cultured Pituitary Cell

In 1968 Tashjian and co-workers first described the establishment of clonal strains of growth hormone (GH)-secretingcells from a rat anterior pituitary tumor. One strain, GH3, subsequently elicited great interest because it secreted prolactin (PRL) in response to nanomolar concentrations of thyrotropin-releasing hormone (TRH) (Tashjian et al., 1971). Hinkle and Tashjian (1973) later demonstrated the presence of TRH receptors on these cells. Kidokoro (1975) studied the electrical properties of GH3 cells using intra- and extracellular microelectrode recordings of membrane potential differences. At room temperature, he recorded a resting potential of -41 mV in cells attached to the culture plate. Superimposed on the resting potential were small (4mV) periodic depolarizations, which occasionally initiated action potentials. Spontaneous action potentials fired at a rate of 52 per minute. When 30 nM TRH-acetate was applied to cells, the firing frequency increased to 91 per minute. Exposure to a low concentration of TRH (1 nM) or to [MeHis2]-TRH,an inactive TRH analog, had no effect on firing rate. The GH3action potential was not blocked by tetrodotoxin (TTX) nor by substitution of sodium by Tris in the external medium. However, addition of either La2+or Mn2+, calcium channel blockers, abolished the spikes. These data suggested a strong correlation between Ca2+action potential firing and hormone secretion in these cells. Unfortunately, hormone secretory rates were not directly quantified in these experiments. Ozawa and Miyazaki (1979) showed that the spontaneous action potentials had both Na+ and Ca2+components. In Na+-free medium the action potentials were unaffected by TTX and were completely abolished by 4 mM Co2+,and the rise time decreased from 7 to 2.5 V/second. Conversely, action potentials obtained in Ca2+-freemedium containing Co2 were eliminated with 3 p M TTX or when Naf was replaced with choline. When Sr2+or Ba2+were substitued for Ca2+,action potentials were still elicited, although they were of longer duration. One possible explanation was that Ca2+,but not Sr2+or Ba2+,turned on a late-activating potassium conductance. This explanation was supported by observations that in +

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high-Ca2+medium, the cell membrane would slowly hyperpolarize after repetitively induced firing, and that there was a decrease in the outward rectification of the current-voltage curve when Ba2+replaced external Ca2+.In addition, Biales et al. (1977) had previously shown that tetraethylammonium chloride (TEA), a blocker of voltage-sensitive potassium channels (Armstrong, 1971), delayed the repolarization of previously depolarized cells. Both Ozawa and Miyazaki (1979) and Tashjian et al. (1978) observed that elevated [K+](in the presence of Ca2+or Sr2+) more than doubled PRL release from the cells. Substitution of Ba2+for Ca2+stimulated PRL secretion to a level not significantly different from that measured in high-K+ medium, while addition of Co2+almost completely supressed the high-K+- and Ba2+-inducedchanges in PRL secretion. The authors did not compare their hormone secretory rates to those obtained after TRH stimulation. Further evidence for the role of K+ in hormone secretion came from Ozawa and Kimura (1979), who showed that the transient membrane hyperpolarization observed within seconds following TRH addition (and lasting for 5 to 15 seconds) persisted in either C1- or Na+-free medium. These results implicate an increase in membrane conductance to K+ as being responsible for this transient hyperpolarization. TRH stimulated PRL and GH secretion, while eliciting the same kind of hyperpolarization. The TRH-induced stimulation of hormone secretion could be completely blocked by external Co2+with no effect on the characteristic membrane hyperpolarization. Ozawa and Kimura (1979) concluded that TRH increased calcium entry through voltage-dependent calcium channels and that the resultant increase in intracellular Ca2+ triggered hormone secretion. Kaczorowski et al. (1983) compared the electrophysiological properties of the GH3 strain with a subclone, XG-10, which lacks TRH receptors. XG-10 cells failed to show stimulated PRL secretion in response to either TRH or TEA. In addition, the duration of spontaneous action potentials measured in XG-10 cells was increased as compared to those of GH3 cells and was unaffected by TRH and TEA. A high-K+ external solution (60 mM), however, enhanced PRL release in both strains, while calcium channel blockers such as verapamil and Co2+abolished all action potentials. When GH3 cells were treated with TRH, TEA, or TRH plus TEA, they exhibited electrical properties similar to those of the XG-10 cells, that is, action potentials with longer duration. The authors concluded that in GH, cells the TRH receptor regulates, or is part of, a TEAsensitive potassium channel. Binding of TRH to its receptor blocks this K+ channel, resulting in a depolarization of the cell membrane, which in turn causes voltage-sensitive calcium channels to open. They also postu-

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lated that XG- 10 cells lack these TEA-sensitive, voltage-dependentpotassium channels, but do have Ca2+channels and calcium-dependent K + channels responsible for membrane repolarization. The authors measured these electrical properties 10 minutes after high-K+ or TRH treatment. A series of flux experiments using 45Ca2+and the fluorescent Cat+ indicator quin-2 substantiated the theory that TRH stimulated a Co2+-sensitive uptake of calcium into the intracellular compartment (Tan and Tashjian, 1981, 1984a,b; Albert and Tashjian, 1984). Incubation of quin-2loaded GH cells with TRH resulted in a biphasic change in internal Ca2+ concentrations, [Car+]. Basal [Caf+]levels of 250 nM spiked in seconds to 1.7 pM, then rapidly declined to 330 nM, finally increasing over several minutes to a plateau value of 765 nM. Addition of high external K+ instead of TRH caused [Car+]to spike immediately to I .7 pM, followed by a decline over several minutes to a plateau of 630 nM. Several groups (Hagiwara and Ohmori, 1982, 1983; Dubinsky and Oxford, 1984; Matteson and Armstrong, 1984) have published studies of GH3 cell electrophysiological properties using the patch electrode voltage clamp technique (Hamill et al., 1981). Hagiwara and Ohmori (1982), using the whole-cell recording configuration, found that the membrane current following depolarizing voltage pulses consisted of a fast, Na+-dependent current (Na+ current) and a slower, Na+-independent current which they identified as a Ca2+current. The amplitude of the Na+ current varied from cell to cell, being almost absent in some cases. The magnitude of the Ca2+ current was relatively constant from cell to cell and increased with increasing external Ca2+concentration. This current could also be carried by either Sr2+or Ba2+.The maximal current carried per channel was 0.7 pA at 100 mM Ba2+,as calculated by ensemble noise analysis. Hagiwara and Ohmori (1983) studied single-channel properties of this Ca2+ channel, using patch clamp. The mean channel open time, which was nearly voltage independent in the range -20 to 40 mV, was 1.3 millisecond, and the single-channel conductance was 7 picosiemens. Matteson and Armstrong (1984) and Dubinsky and Oxford (1984) performed almost identical studies using the whole-cell patch clamp technique. Depolarization to 20 mV elicited a rapid inward current, a slow inward current (in accordance with Hagiwara and Ohmori's results), and a sustained outward current (Matteson and Armstrong, 1984). The rapid inward current was absent in Na+-freemedium, was blocked by TTX, and displayed reversal potentials which agreed with those calculated from the Nernst equation for a Na+-selective channel. The amplitude of the slow inward current depended upon external [Ca2'1 and increased when Ba2+ replaced Ca2+.The sustained outward current was abolished by replace-

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ment of internal K+ with Cs+ and was blocked by internal Ba2+.Dubinsky and Oxford (1984) identified the same three currents in both GH3 and GH4 cells. From the inactivation kinetics of the Na+ current, they calculated that only 35% of the sodium channels would be able to activate and contribute to the action potential at a typical cell resting potential of -43 mV. Also, although GH4 cells secrete prolactin, they exhibit very little sodium current during action potential generation. Therefore, sodium channels are probably not necessary for hormone secretion in GH cells. The slow inward calcium current was examined by Dubinsky and Oxford (1984). This current characteristically declined during a depolarizing voltage pulse. This decline could be due either to a decrease in inward Ca2+conductance because of Ca2+channel inactivation or to an increase in outward current. To distinguish between these possibilities, they measured tail currents under conditions where the Cs+ (substituted for K+) and C1- concentrations were adjusted so that their equilibrium potentials were at the potential where the tail currents were measured. [Note: A tail current is the current measured following a step change of voltage clamp potential to a level different from the pulse value.] Under these conditions the magnitude of the measured tail current should decrease with the duration of the Ca2+ channel activating pulse only if the Ca2+channels inactivate. The results showed that the magnitude of the tail current was unaffected by the length of the depolarizing pulse. They concluded that the decrease of slow inward current with time was due to the activation of an outward, TEA-sensitive K+-Cs+ current. When EGTA was added to the internal solution, the slow inward current still inactivated. Matteson and Armstrong (1984) reached somewhat different conclusions from a similar experiment. These investigators found that the outward current was abolished when internal K + was replaced with Cs+. Therefore, they concluded that the decline in the slow inward current following a depolarizing pulse was due to Ca2+channel inactivation. Thus, because internal EGTA had no effect on slow inward current inactivation, accumulation of intracellular Ca2+ does not produce channel inactivation. The nature of the observed decline in slow inward current is thus a point open to debate. To examine the possible involvement of C1- in the apparent outward current, Dubinsky and Oxford (1984) substituted aspartate for C1- in both intra- and extracellular solutions. No changes in the membrane currents were observed. An analysis of the kinetics and pharmacological properties of the K+ current suggested that both voltage-dependent and calciumactivated K+ channels were present. The effects, if any, of TRH on Na+, K+ or Ca2+currents have not yet been reported. As we have indicated, the earliest measured response of the cultured GH cell to TRH is a rapid increase in [Cat']. The peak increase in [Cat+]

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occurs within 5 seconds of TRH exposure. Depolarizing the GH cell with elevated extracellular [K+],that is, 60 mM, also results in a rapid increase in [Caf+].This same time course of increased [Caz+] is seen in hepatocytes stimulated with epinephrine or vasopressin (Thomas et al., 1983). Phorbol esters stimulated both prolactin synthesis and release (Osborne and Tashjian, 1981). This stimulation of hormone release was a Ca2+dependent process; phorbol ester-induced prolactin release could be completely blocked by 2 mM Co2+.TRH also stimulates the hydrolysis of polyphosphatidylinositides to form inositol polyphosphates and diacylglycerol (A. H. Tashjian, personal communication). Enhanced PI hydrolysis is maximal with 10 to 15 seconds of TRH addition. One product of this reaction, IP3, has been shown to induce a rapid release of Ca2+ from nonmitochondrial stores (presumably the endoplasmic reticulum) in permeabilized nonpituitary cells (Streb et al., 1983; see Fig. 1). This Ca2+ release is transient, as the IP3 is quickly converted to an inactive form, and the released Ca2+is again sequestered into storage pools. In GH cells, TRH produces a biphasic change in intracellular IP3 content: an initial spike (5-10 seconds), coinciding with the onset of enhanced PI hydrolysis, followed by a prolonged (50 minute) plateau of elevated (with respect to basal levels) IP3. The initial spike in IP3 may mediate the observed changes in [Caf+]following TRH addition. However, the mechanism(s) by which IP3 releases intracellular Ca2+is (are) unknown. Also, the relationship between diacylglycerol and TRH and the activation and inactivation of both Ca2+and K+ channels in the plasma membrane of the GH cell remains to elucidated. In summary, there is substantial evidence from work on GH3 and related strains of rat anterior pituitary tumor cells to support a model of hormone stimulation-secretion coupling as outlined in Fig. 1 . Continued research on these cells promises to elucidate a mechanism of hormone secretion with general applicability to many endocrine glands. V.

CONCLUDING REMARKS

The growth and development of many cells are regulated through the control of periods of proliferation and differentiation. An important initial event in the transition of cells from a quiescent to a proliferative state seems to be the alkalinization of the intracellular medium caused by the induction of an amiloride-sensitive sodium-proton exchange system. This particular ion transport system can be stimulated by the addition of sperm to quiescent eggs (Johnson et al., 19761, by volume perturbations (Cala, 1983; Grinstein et al., 1984; Whitely et al., 1984), by serum or growth

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factor addition to quiescent cells in culture (e.g., Villereal, 1981; Moolenaar et al., 1981), or by tumor promoters (Besterman and Cuatrecasas, 1984; Whitely et al., 1984; Sapirstein and Benos, 1984). In addition, mitogenic agents can also induce changes in ion transport systems such as ion channels (Matteson and Deutsch, 1984; DeCoursey et al., 1984). It is also evident that even peptide-releasing hormones like TRH may induce hormone secretion via alterations in membrane transport systems (Kaczorowski et al., 1983). The involvement of phosphatidylinositol in this process suggests at least partially coincident mechanisms for mitogenesis and hormone secretion. In this regard, it would be informative to investigate the effects of phorbol ester tumor promoters in the fertilization process. Both Cala in Amphiuma red cells and Grinstein in lymphocytes have found that phorbol esters (like cell volume decreases) stimulate Na+-H+ exchange (S. Grinstein, personal communication). Turner et al. (1984) recently reported that the polyphosphoinositol content of sea urchin eggs increases just after fertilization, and this increase precedes all events except membrane depolarization. Based on the available data, it is likely that phosphoinositol turnover may be initiated by certain mitogens, growth factors, and hormones, with the net result being the elevation of cytoplasmic Ca2+,diacylglycerol, and protein kinase C activity. These changes can in turn activate or induce various ion transport systems which ultimately influence cellular behavior, for example, proliferation, differentiation, volume regulation, or secretion. ACKNOWLEDGMENTS We thank Drs. E. Anderson, V. Vacquier, and L. DeFelice for providing original micrographs and tracings. We especially thank Ms. Kayte Taudel for her patience and excellent secretarial assistance. The preparation of this review was supported by NIH Grants AM25886, HD12353, and T32HD07130. REFERENCES Albert, P. R., and Tashjian, A. H. (1984). Thyrotropin releasing hormone-induced spike and plateau in cytosolic free Ca2+concentrations in pituitary cells: Relation to prolactin release. J . Biol. Chem. 259, 5827-5832. Abraham, J., and Rovera, G . (1980). The effect of tumor-promoting phorbol diesters on terminal differentiation of cells in culture. Mol. Cell. Biochem. 31, 165-175. Armstrong, C. M. (1971). Interaction of tetraethylammonium ion derivates with the potassium channels of giant axons. J . Gen. Physiol. 58, 413-437. Ashendel, C. L., Staller, J. M., and Boutwell, R. K. (1983).Identification of a calcium- and phospholipid-dependent phorbol ester binding activity in the soluble fraction of mouse tissues. Biochem. Biophys. Res. Commun. 111, 340-345. Atwater, I. (1980). Control mechanisms for glucose-induced changes in the membrane potential of mouse pancreatic B-cell. Clin. Biol. Portugal 5, 299-315.

4. FERTILIZATION, GROWTH, AND HORMONE SYSTEMS

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Benjamin, W. B., and Singer, I. (1974). Aldosterone-induced protein in toad urinary bladders. Science 186, 269-272. Benos. D. J . (1982). Amiloride: A Molecular probe of Na+ transport in tissues and cells. Am. J . Physiol. 245, C131-CI45. Benos, D. J., and Sapirstein, V. S. (1983). Characteristics of an amiloride-sensitive sodium entry pathway in cultured rodent glial and neuroblastoma cells. J. Cell. Physiol. 116, 213-220. Besterman, J . M., and Cuatrecasas, P. (1984). Phorbol esters rapidly stimulate amiloridesensitive Na+/H+exchange in a human leukemic cell line. J . Cell Eiol. 99, 340-343. Biales, B., Dichter, M. A., and Tischler, A. (1977). Sodium and calcium action potential in pituitary cells. Nature (London) 267, 172-174. Brandis, J. W., and Raff, R. A. (1979). Elevation of protein synthesis is a complex response to fertilization. Narure (London) 278, 467-469. Busa, W. B., and Nuccitelli, R. (1984). Metabolic regulation via intracellular pH. Am. J. Physiol. 246, R409-R438. Cala, P. M. (1983). Volume regulation by red blood cells. Mechanisms of ion transport between cells and mechanisms. Mol. Physiol. 4, 33-52. Chambers, E. L. (1980). Fertilization and cleavage of the egg of the sea urchin. Lytechinus variegatus in Ca2+free sea water. Eur. J. Eiol. 22, 476-490. Chambers, E. L., and De Armendi, J. (1979). Membrane potential, action potential and activation potential of the eggs of the sea urchin Lytechinus variegatus. Exp. Cell Res. 122, 203-218. Charbonneau, M., Moreau, M., Picheral, B., Vilain, J. P., and Guenier, P. (1983). Fertilization of amphibian eggs: A comparison of electrical responses between anurans and urodeles. Dev. Biol. 98, 304-318. Chu, L. L. H., and Edelman, I. S. (1972). Cordycepin and a-amanitin: Inhibitors of transcription as probes of aldosterone action. J . Membr. Eiol. 10, 291-310. Cochet, C., Gill, G. N., Meisenhelder, J., Cooper, J. A., and Hunter, T. (1984). C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity. J . Eiol. Chem. 259, 2553-2558. Cohen, S . , Ushiro, H., Stoscheck, C., and Chinkers, M. (1982). A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J . Eiol. Chem. 257, 1523. Collins, F., and Epel, D. (1977). The role of calcium ions in the acrosome reaction of sperm: Regulation of exocytosis. Exp. Cell Res. 106, 21 1-222. Cuthbert, A., and Cuthbert, A. W. (1978). Fertilization acid production in Psammechinus eggs under pH clamp conditions, and the effect of some pyrazine derivatives. Exp. Cell Res. 114, 409-415. Dale, B., and DeFelice, L. (1984), Sperm-activated channels in ascidian oocytes. Dev. Eiol. 101, 235-239. Dale. B., DeFelice, L. J., and Taglietti, V. (1978). Membrane noise and conductance increase during single spermatozoon-egg interactions. Nature (London) 275, 217219. Dale, B., Desantis. A., and Ortolani. G. (1983). Electrical response to fertilization in Ascidian oocytes. Dev. Eiol. 99, 188-193. DeCoursey, T. E., Chandy, K. G., Gupta, S., and Cahalan. M. (1984). Voltage-gated K + channels in human T lymphocytes: A role in mitogedesis? Nature (London) 307,465468. DeFelice, L. J . , and Dale, B. (1979). Voltage response to fertilization and polyspermy in sea urchin eggs and oocytes. Dev.Eiol. 72, 327-341.

158

JOAN BELL ET AL.

Deutsch, C., and Price, M. A. (1982). Cell calcium in human peripheral blood lymphocytes and the effect of mitogen. Biochim. Biophys. Actu 687, 211-218. Douglas, W. W. (1968). Stimulus-secretion coupling: The concept and clues from chromaffin and other cells. Br. J . Pharrnacol. 34,451-474. Douglas, W . W., and Rubin, R. P. (1961). The role of calcium in the secretory response of the adrenal medulla to acetylcholine. J . Physiol. (London) 159, 40-57. Dubinsky, J. M., and Oxford, G. S. (1984). Ionic currents in two strains of rat anterior pituitary tumor cells. J . Gen. Physiul. 83, 309-339. Duval, D., Durant, S., and Homo-Delarche, F. (1983). Non-genomic effects of steroids. Interactions of steroid molecules with membrane structures and functions. Biochim. Biophys. Acra 737, 409-441. Epel, D. (1975). The program of and mechanisms of fertilization in the echinoderm egg. A m . ZOO/.15, 507-522. Epel, D. (1978). Mechanisms of activation of sperm and egg during fertilization of sea urchin gametes. Curr. Top. Dev. Biol. 12, 185-246. Garty, H., and Edelman, I. S. (1983). Amiloride-sensitive trypsinisation of apical sodium channels. Analysis of hormonal regulation of sodium transport in toad bladder. J. Gen. Physiol. 81, 785-803. Goodman, B. P., Allen, J. E., and Rasmussen, H. (1971). Studies on the mechanism of action of aldosterone hormone-induced changes in lipid metabolism. Biochemistry 10, 3825-3831, Gould-Somero, M . (1981). Localized gating of egg Na+ channels by sperm. Nulitre (London) 291, 254-356. Could, M., and Holland, L. Z. (1984). Fertilization acid release in Urechis eggs. 11. The stoichiometry of Na+ uptake and H+ release. Dev. B i d . 104, 329-335. Could-Somero, M.,Jaffe, L. A., and Holland, L. Z. (1979). Electrically mediated fast polyspermy block in eggs of the marine worm Urechis cuupo. J . Cell Biol.83,426440. Grey, R. D., Bastiani, M. J., Webb, D. J., and Schertel, E. R. (1982). An electrical block is required to prevent polyspermy in eggs fertilized by natural mating of Xenupus laeuis. Dev. Biol. 89, 475-484, Grinstein, S . , Dupre, A., and Rothstein, A. (1982). Volume regulation by human lymphocytes. J . Gen. Physiul. 79, 848-868. Grinstein, S., Cohen, S., and Rothstein, A. (1984). Cytoplasmic pH regulation in thymic lymphocytes by an amiloride-sensitive Na+/H+antiport. J . Gen. Physiol. 83, 341-369. Hagiwara, S., and Jaffe, L. A. (1979). Electrical properties of egg cell membranes. Annu. Rev. Biophys. Bioeng. 8, 385-416. Hagiwara, S . , and Ohmori, H. (1982). Studies of calcium channels in rat clonal pituitary cells with patch electrode voltage clamp. J . Physiol. (London) 331, 231-252. Hagiwara, S . , and Ohmori, H. (1983). Studies of single calcium channel currents in rat clonal pituitary cells. J . Physiol. (London) 336, 649-661. Hamill, 0. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. PJlugers Arch. 391, 85-100. Handler, J. S., Preston, A. S., Perkins, F. M., Matsum, M., Johnson, J. P., and Watlington, C. 0. (1981). The effect of adrenal steroid hormones on epithelia formed in culture by A6 cells. Ann. N.Y. Acad. Sci. 372, 442-454. Higashi, A., and Kaneko, H. (1971). Membrane potential of sea urchin eggs and effect of external ions. Annot. Zuol. Jpn. 44, 65-75. Hinkle, P. M . , and Tashjian, A. H. (1973). Receptors for thyrotropin-releasing hormone in prolactin-producing rat pituitary cells in culture. J. B i d . Chem. 248, 6180-6186.

4. FERTILIZATION, GROWTH, AND HORMONE SYSTEMS

159

Holland, L. Z., Gould-Somero, M., and Paul, M. (1984). Fertilization acid release in Urechis eggs. I. The nature of the acid and the dependence of acid release and egg activation on external pH. Dev. Biol. 103, 343-354. Holley, R. W. (1975). Control ofgrowth of mammalian cells in cell culture. Nature (London) 258, 487-490. Iwashita, S., and Fox, C. F. (1984). Epidermal growth factors and potent phorbol tumor promoters induce epidermal growth factor receptor phosphorylation in a similar but distinctively different manner in human epidermoid carcinoma A 43 I cells. J. Biol. Chem. 259, 2559-2567. Jaffe, L. A. (1976). Fast block to polyspermy in sea urchin eggs is electrically mediated. Nature (London) 261, 68-71, Jaffe, L. A., Gould-Somero, M.,and Holland, L. (1979). Ionic mechanism of the fertilization potential of the marine worm, Urechis ctrupo (Echuria). J . Gen. Physiol. 73, 469492. JaEe, L. A., Cross, N. L., and Picheral, B. (1983a). Studies of the voltage-dependent polyspermy block using cross-species fertilization of amphibians. Dev. Biol. 93, 3 17323. Jaffe, L. A., Sharp, A. P., and Wolf, D. P. (1983b). Absence of an electrical polyspermy block in the mouse. Dev. Biol. 96, 317-323. Johnson, J. D., Epel, D., and Paul, M. (1976). lntracellular pH and activation of sea urchin eggs after fertilization. Nature (London) 262, 661 -664. Kaczorowski, G . J., Vandlen, R. L., Katz, G. M., and Reuben, J. P. (1983). Regulation of excitation-secretion coupling by thyrotropin-releasing hormone (TRH): Evidence for TRH receptor-ion channel coupling in cultured pituitary cells. J . Membr. Biol. 71, 109118. Kamata, T., and Feramisco, J. R. (1984). Epidermal growth factor stimulates guanine nucleotide binding activity and phosphorylation of ras oncogene proteins. Nature (London) 310, 147-150. Kidokoro, Y . (1975). Spontaneous calcium action potentials in a clonal pituitary cell line and their relationship to prolactin secretion. Nature (London) 258, 741-743. Koch, K. S., and Leffert, H. L. (1979). Increased sodium ion influx is necessary to initiate rat hepatocyte. Cell 18, 153-163. Kozuka, M., and Takahashi, K. (1982). Changes in holding and ion-channel currents during activation of an ascidian egg under voltage clamp. J . Physiol. (London) 323, 267-286. L'Allemain, G., Paris, S., and Pouyssegur, J. (1984). Growth factor action and intracellular pH regulation in fibroblasts. J . Biol. Chem. 259, 58099-5815. Lansman, J. B. (1983). Voltage-clamp study of the conductance activated at fertilization in the starfish egg. J. Physiol. (London)345, 353-372. Leach, K. L., James, M. L., and Blumberg. P. M. (1983). Characterization of a specific phorbol ester aporeceptor in mouse brain cytosol. Prac. Narl. Acad. Sci. U.S.A. 80, 4208-42 12. Lien, E. L., Goodman, D. B. P., and Rasmussen, H. (1976). Effects of inhibitors of protein and RNA synthesis on aldosterone-stimulated changes in phospholipid fatty acid metabolism in the toad urinary bladder. Biochim. Biophys. Acta 421, 210-217. Lindemann, B. (1980). The beginning of fluctuation analysis of epithelial ion transport. J . Membr. Biol. 51, 5-1 I. Lindemann, B., and Van Driessche, W. (1977). Sodium-specific membrane channels of frog skin are pores: Current fluctuations reveal high turnover. Science 195, 292-294. McCulloh, D. H., Rexroad, C. E., and Levitan, H . (1983). Insemination of rabbit eggs is associated with slow depolarization and repetitive diphasic membrane potentials. Deu. Biol. 95, 312-377.

160

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Macara, I. G., Marinetti, G. V., and Balduzzi, P. C. (1984). Transforming protein of avian sarcoma virus UR2 is associated with phosphatidylinositol kinase activity: Possible role in tumorigenesis. Proc. Natl. Acad. Sci. U.S.A. 81, 2728-2732. Maruyama, Y., and Petersen, 0. H. (1982). Cholecystokinin activation of single-channel currents is mediated by internal messenger in pancreatic acinar cells. Nature (London) 300,61-63. Maruyama, Y.,Petersen, 0. H., Flanagan, P., and Pearson, G. T. (1983). Quantification of Ca2+activated K+ channels under hormonal control in pancreas acinar cells. Nature (London) 305,228-232. Mastro, A., and Rozengurt, E. (1976). Endogenous protein kinase in outer plasma membrane of cultivated 3T3 cells. J . Riol. Chem. 251, 7899-7906. Matteson, D. R., and Armstrong, C. M. (1984). Na+ and Ca2+channels in a transformed line of anterior pituitary cells. J. Gen. Physiol. 83, 371-394. Matteson, D. R., and Deutsch, C. (1984). K+ channels in T lymphocytes: A patch clamp study using monoclonal antibody adhesion. Nature (London) 3U7, 468-471. Miyazaki, S., and lgusa, Y. (1982). Ca2+-mediatedactivation of a K+ current at fertilization of golden hamster eggs. Proc. Natl. Acad. Sci. U.S.A. 79,931-935. Moolenaar, W. H., de Laat, S. W.,and Van Der Saag, P. T. (1979). Serum triggers a sequence of rapid ionic conductance changes in quiescent neuroblastive cells. Nature (London) 279,721-723. Moolenaar, W.H., Mummery, C. L., Van Der Saag, P. T., and de Laat, S. W. (1981). Rapid ionic events and the initiation of growth in serum-stimulated neuroblastoma cells. Cell 23,789-798. Moy, G. W., and Vacquier, V. D. (1979). Immunoperoxidase localization of bindin during sperm-egg interaction. Curr. Top. Dev. Biol. W, 31-44. Niedel, J. E., Kuhn, L. J., and Vandenbark, G. R. (1983). Phorbol diester receptor copurifies with protein kinase C. Proc. Natl. Acad. Sci. U.S.A. 80, 36-40. Nishioka, D., and Epel, D. (1977). Intracellular pH and activation of sea urchin eggs at fertilization. J . Cell Biol. 75, 40a (Abstr.). Nishizuka, Y. (1984). The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature (London) 308, 693-698. Nuccitelli, R., and Grey, R. D. (1984). Controversy over the fast, partial block to polyspermy in sea urchins. Dev. Biol. 103, 1-17. Nuccitelli, R., Webb, D. J., Lagier, S. T., and Matson, G. B. (1981). "P-NMR reveals increased intracellular pH after fertilization in Xenopus eggs. Proc. Natl. Acad. Sci. U.S.A. 78,4421-4425. Osborne, R., and Tashjian, A. H., Jr. (1981). Tumor-promoting phorbol esters affect production of prolactin and growth hormone by rat pituitary cells. Endocrinology 108, 1164-1 170. Ozawa, S., and Kimura, N. (1979). Membrane potential changes caused by thyrotropinreleasing hormone in the clonal GH3cell and their relationship to secretion of pituitary hormone. Proc. Natl. Acad. Sci. U.S.A. 76, 6017-6020. Ozawa, S.,and Miyazaki, S. (1979). Electrical excitability in the rat clonal pituitary cell and its relation to hormone secretion. Jpn. J . Physiol. 29, 41 1-426. Palmer, L. G., and Edelman, I. S. (1981). Control of apical sodium permeability in the toad urinary bladder by aldosterone. Ann. N . Y . Acad. Sci. 372, 1-14. Paris, S., and Pouyssegur, J. (1984). Growth factors activate the Na+/H+antiporter in quiescent fibroblasts by increasing its affinity for intracellular H+. J . Biol. Chem. 259, 10989- 10994. Paul, M. (19750. Release of acid and changes in light-scattering properties following fertilization of Urechis capuo egg. Dev. Biol. 43, 299-312.

4. FERTILIZATION, GROWTH, AND HORMONE SYSTEMS

161

Payan, P., Girand, J. P., and Ciapa, B. (1983).Mechanisms regulating intracellular pH in sea urchin eggs. Dev. Biol. 100, 29-38. Petersen, 0. H. (1980).“The Electrophysiology of Gland Cells.” Academic Press, London. Reuter, H.(1983).Calcium channel modulation by neurotransmitters, enzymes, and drugs. Nature (London) 301, 569-574. Rossier. B. C., Wilce, P. A., and Edelman, I. S. (1974).Kinetics of RNA labeling in toad bladder epithelium: Effects of aldosterone and related steroids. Proc. Natl. Acad. Sci. U.S.A. 71, 3101-3105. Rossignol, D. P., and Lennarz, W. J. (1983).The molecular basis of sperm-egg interaction in the sea urchin. Ciba Found. Symp. 98,268-285. Rossignol, D. P., Earles, B. J., Decker, G. L., and Lennarz, W. J . (1984).Characterization of the sperm receptor on the surface of the eggs of Strongylocentrotus. Dev. B i d . 104, 308-32 1. Rothenberg, P., Reuss, L., and Glaser, L. (1982).Serum and epidermal growth factor transiently depolarize quiescent BSC-1 epithelial cells. Proc. Natl. Acud. Sci. U . S . A . 79, 7783-7787. Rozengurt, E. (1976). Coordination of early membrane changes in growth stimulation. J . Cell. Physiol. 89, 627-631. Rozengurt, E.,and Heppel, L. A. (1975). Serum rapidly stimulates ouabain-sensitive B6Rb influx in quiescent 3T3 cells. Proc. Natl. Acad. Sci. U.S.A. 72, 4492-4495. Sapirstein, V. S ., and Benos, D. J. (1984).Activation of amiloride-sensitive sodium transport in C6 glioma cells. J. Neurochem. 43, 1098-1 105. Sariban-Sohraby, S., Burg, M. B., Chiang, P. K., Wiesmann, W., and Johnson, J. (1984). Methylation increases sodium transport into A6 apical membrane vesicles: Possible mode of aldosterone action. Science 225, 745-746. Sawyer, S. T., and Cohen, S. (1981). Enhancement of calcium uptake and phosphatidylinositol turnover by epidermal growth factor in A 431 cells. Biochemistry 20, 62806286. Schatten, G., and Hulser, D. (1983).Timing the early events during sea urchin fertilization. Dev. Biol. 100, 244-248. Segall, G. K.,and Lennarz, W. J. (1979).Chemical characterization of the component of the jelly coat from sea urchin eggs responsible for induction of the acrosome reaction. Dev. Biol. 71, 33-49. Shen, S.S.,and Steinhardt, R. A. (1980).lntracellular pH controls the development of new potassium conductance after fertilization of the sea urchin egg. Exp. Cell Res. l25,5561. Smith, B. J., and Rozengurt, E. (1978).Serum stimulates the Na+,K+ pump in quiescent fibroblasts by increasing Na+ entry. Proc. N a f l . Acad. Sci. U.S.A. 75, 5560-5564. Steinhardt, R. A., Lundin, L., and Mazia, D. (1971).Bioelectric responses of the echinoderm egg to fertilization. Proc. Natl. Acad. Sci. U . S . A . 68, 2426-2430. Streb, H., Irvine, R. F., Benidge, M. J., and Schulz, I. (1983).Release of Ca2+from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-l,4,5-triphosphate. Nature (London) 306, 67-69. Sugimoto, Y.,Whitman, M., Cantley, L. C., and Erikson, R. L. (1984).Evidence that the Rous sarcoma virus transforming gene product phorphorylates phosphatidylinositol and diacylglycerol. Proc. Natl. Acad. Sci. U.S.A. 81, 21 17-2121. Taglietti, V. (1980).Early electrical responses to fertilization in sea urchin eggs. Exp. Cell Res. 1u),448-45 1. Takahashi, I., and Sugiyama, M. (1973).Relation between the acrosome reaction and fertilization in the sea urchin. I. Fertilization in Ca-free seawater with egg-water-treated spermatoza. Dev. Growth Differ. 15, 261-267.

162

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Tan, K. N . , and Tashjian, A. H. (1981). Receptor mediated release of plasma membrane Cat+ uptake by thyrotropin-releasing hormone in pituitary cells in culture. J . B i d . Chem. 256, 8894-9002. Tan, K. N., and Tashjian, A. H. (1984a). Voltage-dependent calcium channels in pituitary cells in culture. I. Characterization by 45Cafluxes. J. Biol. Chem. 259, 418-426. Tan, K. N., and Tashjian, A. H. (1984b). Voltage-dependent calcium channels in pituitary cells in culture. 11. Participation in thyrotropin-releasing hormone action on prolactin release. J . Biol. Chem. 259, 427-434. Tashjian, A. H., Yasumua, Y ., Levine, L., Sato, G. H., and Parker, M. L. (1968). Establishment of clonal strains of rat pituitary tumor cells that secrete growth hormone. Endocrinology 32, 342. Tashjian, A. H., Barowsky, N. J., and Jensen, D. K. (1971). Thyrotropin releasing hormone: Direct evidence for stimulation of prolactin production by pituitary cells in culture. Biochem. Biophys. Res. Commun. 43, 516-523. Tashjian, A. H., Bancroft, F. C., and Levine, L. (1978). Production of both prolactin and growth hormone by clonal strains of rat pituitary tumor cells in culture. Biochem. Biophys. Res. Commun. 81,793-805. Thomas, A. P., Marks, J. S., Coll, K. E., and Williamson, J. R. (1983). Quantitation and early kinetics of inositol lipid changes induced by vasopressin in isolated and cultured hepatocytes. J. Biol. Chem. 258, 5716-5725. Tsien, R. Y.,Pozzan, T., and Rink, T. J . (1982). T-cell mitogens cause early changes in cytoplasmic free CaZ+and membrane potential in lymphocytes. Nature (London) 295, 68-71. Tupper, J. T.,Ryals, W. T., and Bodine, P. V. (1982). Membrane transport properties differ following return of serum-deprived versus Ca2+human fibroblasts to a proliferate state. J . Cell. Physiol. 110, 29-34. Turner, P. R.,Sheetz, M. P., and Jaffe, L. A. (1984). Fertilization increases the polyphosphoinositide content of sea urchin eggs. Nature (London) 310, 414-415. Tyler, A. (1949). Properties of fertilizin and related substance of eggs and sperm of marine animals. A m . Nut. 83, 195-210. Vacquier, V. D., and Moy, G. W. (1977). Isolation of bindin: The protein responsible for adhesion of sperm to sea urchin eggs. Proc. Nail. Acad. Sci. U.S.A.74, 2456-2460. Vacquier, V. D., and Moy, G. W. (1978). Macromolecules mediating sperm-cell recognition and adhesion during sea urchin fertilization. In “Cell Reproduction” (E. R. Dirkson, D. M. Prescott, and C. F. Fox, eds.). Academic Press, New York. Vacquier, V. D., Tegner, M. J., and Epel, D. (1973). Protease released from sea urchin eggs at fertilization alters the vitelline layer and aids in preventing polyspermy. Exp. Cell Res. 80, 111-119. Veron, M., and Shapiro, B. M. (1977). Binding of concanavalin A to the surface of sea urchin eggs and its alteration upon fertilization. J. Biol. Chem. 252, 1286-1292. Villereal, M. L. (1981). Sodium fluxes in human fibroblasts: Effect of serum, Ca2+ and amiloride. J . Cell. Physiol. 107, 359-369. Whitaker, M. J., and Steinhardt, R. A. (1982). Ionic regulation of egg activation. Q. Rev. Biophys. 15, 593-666. Whiteley, B., Cassel, D., Zhuang, Y.-X., and Glaser, L. (1984). Tumor promoter phorbol 12-myristate 13-acetate inhibits mitogen-stimulated Na+/H+exchange in human epidermal carcinoma A 431 cells. J. Cell Biol. 99, 1162-1 166.

CURRENT TOPICS IN MEMBRANES A N D TRANSPORT. VOLUME 27

Chapter 5 Early Stimulation of Na+-H+ Antiport, Na+-K+ Pump Activity, and Ca2+Fluxes in Fibroblast Mitogenesis ENRIQUE ROZENGURT A N D STANLEY A . MENDOZA’ Imperial Cancer Research Fund London WC2A 3PX, England

......................................... I. Introduction. . . . . . . . . . . . . 11. Ionic Responses Elicited by Growth Factors in Quiescent Cells A. Monovalent Ion Transport in S B. Ion Fluxes and Initiation of DNA Synthesis . . . 111. Protein Kinases and Ion Fluxes . . . . . . . . . . . . . . A. Cyclic AMP, Cell Growth, and B. Protein Kinase C, Cell Growth ........ ............. IV . Calcium Fluxes.. ... V. Conclusions and Perspectives . . . . . References . . . . . . . . ...............................................

1.

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The cells of many tissues and organs exist in a nonproliferating state in Go/G, in which they remain viable and metabolically active. They retain the capacity to respond to extracellular signals such as hormones, peptide factors, and antigens by increasing their rate of proliferation. Features of the phenomenon of stimulation of cell proliferation can be studied in cell culture (Pardee et al., 1978; Rozengurt, 1979a). Thus, normal fibroblasts in general and mouse 3T3 cells in particular (Todaro and Green, 1963) t Present address: Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla, California 92093.

163 Copyright 0 I9Rh hy Academic I’m\\, Inc. All right5 of rrproductiun in any furm re\erved.

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become quiescent in the Go/G1phase of the cell cycle when they deplete the serum present in the medium of an essential growth factor(s). The arrest of growth is reversible; addition of fresh serum or purified growth factors to such quiescent cultures stimulates reinitiation of DNA synthesis and cell division (Holley, 1975; Pardee et al., 1978; Rozengurt, 1979a, 1980). In recent years it has become apparent that the proliferation of normal cells can be regulated by a variety of extracellular factors (Rozengurt, 1980). Thus, quiescent 3T3 cells can be stimulated to reinitiate DNA synthesis by a variety of exogenous agents, including the platelet-derived growth factor (PDGF), a potent mitogen present in serum but not in plasma (Ross and Vogel, 1978), the peptides epidermal growth factor (EGF) and insulin (Rozengurt, 1980), the neurohypophyseal hormone vasopressin and its related peptides (Rozengurt et al., 1979, 1981a), the tumor-promoting agents of the phorbol ester family (Dicker and Rozengurt, 1978, 1979a) and certain modulators of membrane permeability such as melittin and amphotericin B (see Section II,B,2), compounds that disrupt the microtubule organization (Friedkin et al., 1979, 1980; Friedkin and Rozengurt, 1981), vitamin A derivatives (Rozengurt, 1980; Dicker and Rozengurt, 1979b), and agents that elevate the intracellular level of CAMP (see Section 111,A). Studies carried out with combinations of defined growth-promoting molecules have revealed an important aspect of their action: the existence of defined patterns of synergistic interactions (Rozengurt, 1980, 1984, 1985). Specific combinations of mitogenic molecules can be as effective as whole serum in eliciting a complex set of biochemical events (Rozengurt, 1979b) and in stimulating DNA synthesis (Rozengurt, 1980). Tumor cell lines display a marked reduction in their dependence on exogenous growth factors for proliferation and produce growth factors which could contribute to their autonomous growth (see Rozengurt, 1983; Heldin and Westermark, 1984). For example, a virus-transformed cell line releases into the medium a polypeptide (fibroblast-derived growth factor, FDGF) which is a potent mitogen for 3T3 cells (Rozengurt et al., 1982) and exhibits many properties in common with PDGF (Dicker et al., 1981; Stroobant et al., 1985). A link between growth factors and the expression of malignant transformation has been strikingly reinforced by the findings of a remarkable homology between the amino acid sequence of PDGF and the transforming protein of the simian sarcoma virus encoded by the cys28 oncogene (Doolittle et al., 1983; Waterfield et al., 1983; Heldin and Westermark, 1984) and between the EGF receptor and the erb-B oncogene (Downward et al., 1984). All these findings suggest that growth factors play a critical role in modulating normal and abnormal cell prolif-

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eration. In accord with this, our effort has been directed toward the understanding of the mechanism by which these diverse external signals modulate the proliferation of fibroblastic cells. In particular, our attention has been focused on the initial cellular responses associated with the interaction of mitogenic factors and hormones with the cell in the expectation that the early events will provide useful clues to defining primary regulatory mechanisms (Rozengurt, 1979b, 1980, 1983). The first step in the action of many growth factors is to bind specific receptors located at the cell surface (Rozengurt and Collins, 1983). A central problem in understanding the mechanism of action of growth factors is to elucidate how, after binding to specific surface receptors, such factors trigger the generation of internal signals capable of eliciting a mitogenic response. The purpose of this chapter is to review our evidence suggesting that increases in ion fluxes are among the earliest events associated with mitogenesis and that they may contribute to signaling the initiation of DNA synthesis in quiescent fibroblastic cells. II. IONIC RESPONSES ELICITED BY GROWTH FACTORS IN QUIESCENT CELLS A. Monovalent Ion Transport in Swiss 3T3 Cells

1. Na+-K+ PUMPACTIVITY The initial observation linking ion transport and reinitiation of cell proliferation was the finding by Rozengurt and Heppel(l975) that addition of serum stimulated the rate of s6Rb+influx into quiescent Swiss 3T3 cells. This isotope serves as a K+ tracer. This stimulation occurred within 2 minutes after the addition of serum. Most of the increase in s6Rb+uptake after the addition of serum was inhibited by ouabain, indicating that the enhanced uptake of this cation was mediated by the plasma membrane Na+-K+ pump. By contrast, addition of serum to 3T3 cells had no effect on the rate of s6Rb+efflux from radioactively preloaded cells (Rozengurt and Heppel, 1975; Smith and Rozengurt, 1978a).Accordingly, the stimulation of the Na+-K+ pump resulted in a significant increase in the steadystate concentration of cellular K+ (Rozengurt and Heppel, 1975; Mendoza et al., 1980a; Lopez-Rivas et al., 1982). Subsequently, a variety of agents which are mitogenic in Swiss 3T3 cells were shown to stimulate Na+-K+ pump activity. These include PDGF (Mendoza et al., 1980a; Lopez-Rivas et al., 1984), FDGF (Bourne and Rozengurt, 1976), vasopressin (Mendoza et al., 1980b), phorbol esters (Moroney et al., 1978; Dicker and Ro-

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zengurt, 1981a), melittin (Rozengurt et al., 1981b), amphotericin B (Rozengurt and Mendoza, 1980), and bombesin (Mendoza et al., 1986). Furthermore, the stimulation of the Na+-K+ pump could be demonstrated in quiescent cells from many other sources (see below). These findings clearly show the generality of the stimulation of Na+K+ pump activity in quiescent cells stimulated to proliferate. It was therefore important to elucidate the mechanism of the activation of the pump by growth-promoting factors in quiescent cells. A set of experiments on Na+, Li+, K+, and 8aRb+fluxes suggested a unifying model to explain the regulation of the pump by exogenous mitogenic agents: (1) that the activity of the Na+-K+ pump is restricted by the supply of Na+ across the plasma membrane; (2) that serum and growth-promoting factors increase Na+ entry into the cell; and (3) that the cells respond to increased Na+ entry by augmented activity of the Na+-K+ pump (Smith and Rozengurt, 1978a,b; Mendoza et al., 1980a,b; Rozengurt and Mendoza, 1980; Rozengurt, 1981a,b). An important notion that emerges from this model is that a rapid increase in the rate of Na+ influx constitutes a primary effect of serum and growth factors in quiescent cells. 2. EFFECTOF INCREASED Na+ ENTRYON Na+-K+ PUMP

THE

ACTIVITYOF THE

Smith and Rozengurt (1978b) provided evidence that the activity of the Na+-K+ pump was highly sensitive to small changes in intracellular Na+. Monensin, which catalyzes the electroneutral exchange of Na+ for H+ across membranes, caused a dose-dependent increase in the activity of the Na+-K+ pump and in the intracellular Na+ content (Smith and Rozengurt, 1978b). The polyene antibiotic amphotericin B (Rozengurt and Mendoza, 1980) and the Na+-K+ ionophore gramicidin also increased intracellular Na+ and Na+-K+ pump activity, while the K+ ionophore valinomycin and the Ca2+ ionophore A23187 had no effect on either (Smith and Rozengurt, 1978b; Gelehrter and Rozengurt, 1980). Although monensin, gramicidin, and amphotericin B interact with the cell membranes in different ways, a common action of each is their ability to increase the flux of Na+ into the cell. The striking increases in Na+-K+ pump activity produced by raising cellular Na+ provide direct support to the proposition that the Na+-K+ pump activity is limited by the availability of Na+ and raise the possibility that changes in the rate of Na+ entry play a primary role in controlling the rate of Na+-K+ pump activity. A corollary of these findings is that other substrates of the pump, namely, K+, Mg2+,and ATP, are present in intact quiescent cells at concentrations which do not restrict the activity of the pump.

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3. EFFECTOF MITOGENS ON Naf ENTRY INTO SWISS3T3 CELLS Smith and Rozengurt (1978a) reported that serum stimulated the uptake of Lit by Swiss 3T3 cells. One component of Lit uptake (40%) which was ouabain sensitive and required Na+ was presumably mediated by the Na+-K+ pump. A second component (about 50%) of Lit uptake was ouabain insensitive but was inhibited by amiloride or external Na+. This component apparently occurred by a Na+-specific transport system. Serum stimulated Li+ uptake through both the Na+-K+ pump and the Na+specific transport system. These studies suggest that serum stimulates the entry of Na+ into the cell. Smith and Rozengurt (1978b) provided direct evidence for this possibility. Addition of fresh serum to quiescent cultures of Swiss 3T3 cells stimulated 22Na+uptake and net Na+ entry in the presence of ouabain, indicating that the effect was not secondary to stimulation of the Na+-K+ pump. That an increase in the rate of Na+ influx is one of the earliest events elicited by serum and other mitogens was confirmed by a considerable body of subsequent work. Mendoza et al. (1980a) provided evidence that regulation of Na+-K+ pump activity by Na+ entry was not unique to Swiss 3T3 cells. They studied seven additional fibroblastic types of murine, hamster, and human origin. In each cell type, addition of the ionophore monensin increased intracellular Na+ and stimulated Na+-K+ pump activity. Furthermore, addition of fresh serum to quiescent cultures of these various cell types produced a similar pattern of effects. In the absence of ouabain, serum stimulated 86Rb+uptake and increased intracellular K+.In the presence of ouabain, serum had little or no effect on 86Rbt uptake but increased intracellular Na+. Thus, in these different fibroblastic cells, as in Swiss 3T3 cells, the primary effect of serum was to increase Na+ entry into the cells. The resulting increase in intracellular Na+ stimulated the Na+-K+ pump, increasing intracellular K+ and reestablishing the electrochemical gradient for Na+. Similar results have been reported from other laboratories using a variety of cell types. Serum, EGF, vasopressin, and lys-bradykinin all stimulated 22Na+influx in human foreskin (HSWP cells) and lung (WI-38) fibroblasts (Villereal, 1981; Owen and Villereal, 1983a). Similarly, EGF stimulated Na+ influx and Na+-K+ pump activity in neonatal foreskin cells (Moolenaar et al., 1982). In hepatocytes, the mitogenic combination of insulin, glucagon, and EGF rapidly stimulated amiloride-sensitive 22Na+influx (Koch and Leffert, 1979). In quiescent dog kidney epithelial cells (MDCK), serum increased Na+ uptake and Na+-K+ pump activity (Reznik et al., 1983) as well as stimulated DNA synthesis. It has subsequently been shown that a variety of mitogenic agents,

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including partially purified PDGF (Mendoza et al., 1980a) as well as homogeneous preparations of PDGF (Lopez-Rivas e? al., 1984), vasopressin (Mendoza et al., 1980b), bombesin (Mendoza et al., 1986), phorbol esters (Dicker and Rozengurt, 1981a),amphotericin B (Rozengurt and Mendoza, 1980), and melittin (Rozengurt et al., 1981b), all stimulated the Na+-K+ pump in Swiss 3T3 cells by increasing Na+ entry into the cells. A similar effect was observed with mitogenic combinations such as PDGF, vasopressin, and insulin (Schuldiner and Rozengurt, 1982); EGF, vasopressin, and insulin (Burns and Rozengurt, 1984); and a-thrombin and insulin (Pouyssegur et al., 1982). All these findings demonstrated that increased Na+ influx is a primary effect of growth factors in quiescent cells. Thus, it was of considerable importance to identify the mitogen-sensitive pathway($ involved in Na+ transport. CELLS 4. Na+-H+ ANTIPORTSYSTEMIN CULTURED The translocation of Na+ across the plasma membrane of a variety of cell types is mediated, at least in part, by an amiloride-sensitive electroneutral Na+-H+ antiport system which is driven by the Na+ electrochemical gradient across the plasma membrane (Roos and Boron, 1981). Recent reports from various laboratories suggest the presence of an amiloride-sensitive Na+-H+ antiport in cultured cells. Schuldiner and Rozengurt (1982) have produced evidence indicating the presence of a functional Na+-H+ antiport system in substratum attached Swiss 3T3 cells: (1) the maintenance of intracellular pH (pHJ required the presence of Na+ in the extracellular medium; (2) addition of Naf to Na+-depleted cells increased pHi ; (3) the effect of Na+ was concentration dependent; (4) Na+ could be replaced by Li+ but not by choline or K+ ; (5) the Na+-induced increase in pHi was blocked by amiloride, which also inhibited the influx of Na+ into the cells at a comparable half-maximal concentration; and (6) increased extracellular pH led to a significant enhancement in Na+ entry which was sufficient to stimulate the activity of the Na+-K+ pump. All these observations are consistent with the presence of a functional Na+H+ antiport in Swiss 3T3 cells. The existence of a Na+-H+ antiport system has also been demonstrated in renal epithelial cells (Rindler et al., 1979; Rindler and Saier, 1981), neuroblastoma cells (Moolenaar et al., 1981a), human fibroblasts (Moolenaar e? al., 1982, 1983, 1984a), Chinese hamster fibroblasts (Paris and Pouyssegur, 1984), human leukemic cell line (Besterman and Cuatrecasas, 1984), and murine pre-B lymphocyte cell line (Rosoff et al., 1984). In general, the Na+-H+ antiport system of cultured cells is sensitive to amiloride, is electrically silent, and operates

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in either direction depending on the transmembrane gradients for Na+ and H+. 5 . EFFECTOF MITOGENS ON

THE

Na+-H+ ANTIPORT

Since Na+-H+ exchange can operate in either direction, the stimulation of Na+ entry via the Na+-H+ antiport by mitogenic agents could result from at least two alternative mechanisms. Growth factors could directly activate the membrane-bound antiport system, leading to an increase in Na+ for H+exchange and subsequent increase in pHi . Alternatively, the enhancement of Na+-H+ antiport activity by mitogens could represent a compensatory mechanism for the secretion of excess protons generated by cellular metabolism which is enhanced by growth factors. Indeed, an increase in the internal concentration of H+ can activate in a cooperative fashion the Na+-H+ antiport of plasma membrane vesicles of renal proximal tubule cells (Aronson et al., 1982) and of intact human fibroblasts (Moolenaar et al., 1982), hamster cells (Paris and Pouyssegur, 1984), and BALB/c cells (Frelin et al., 1983). To distinguish between these possibilities it was necessary to measure the effect of mitogens on pHi . Schuldiner and Rozengurt (1982) demonstrated that incubation of quiescent Swiss 3T3 cells in the presence of PDGF, vasopressin, and insulin, a potent mitogenic combination, increases the pHi by 0.16 0.04 pH units, as derived from the uptake of weak acids. Subsequent work showed that mitogenic stimulation of different cell types by different factors leads to cytoplasmic alkalinization. Thus, the combination of insulin, EGF, and vasopressin (Burns and Rozengurt, 1983), phorbol esters (Burns and Rozengurt, 1983), vasopressin (Burns and Rozengurt, 1983), human PDGF (Burns and Rozengurt, 1983), homogeneous porcine PDGF (Lopez-Rivas et al., 1984), serum (Burns and Rozengurt, 1983), and bombesin (Mendoza et d . , 1986) also stimulated the Na+-H+ antiport, leading to an increase in pHi in Swiss 3T3 cells. Similarly, L’Allemain et al. (1984a) demonstrated that mitogenic stimulation of CHO fibroblast cells by athrombin and insulin increases pHi . The preceding findings, based on measurements of weak acid distributions across the plasma membrane, are in good agreement with studies using internalized fluorescent indicators, which show cytoplasmic alkalinization in human fibroblasts treated with fresh serum or with EGF and insulin (Moolenaar et al., 1983) and in 3T3 cells treated with human PDGF (Cassel et al., 1983). The ability of growth factors to induce cytoplasmic alkalinization strongly suggests that the activation of Na+-H+ exchange is a primary effect of the mitogens rather than a secondary mechanism for

*

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the extrusion of excess protons resulting from a growth factor-induced acceleration of cellular metabolism.

6. THENa+ CYCLE Taken together, the above data suggest that the fluxes of Na+, H+, and K+ across the membrane of Swiss 3T3 cells and other cell types can be envisaged as a Na+ cycle. The influx of Na+ is linked to H+ efflux occurring through an amiloride-sensitive, electroneutral Na+-H+ antiport. The efflux of Na+ is linked to K+ influx through the ouabain-sensitive Na+-K+ pump. Furthermore, the activity of the Na+-K+ pump is markedly dependent upon intracellular Na+ concentration. Thus, stimulation of the Na+H+ antiport by mitogens increases Na+ influx and H+ efflux. This raises intracellular Na+ and pH. The increase in intracellular Na+ stimulates the Na+-K+ pump, increasing intracellular K+ and restoring the electrochemical gradient for Na+. As will be discussed in the following section, these linked changes in ion fluxes and concentration appear to play a significant role in the regulation of cell proliferation. B. ion Fluxes and initiation of DNA Synthesis

Changes in ion fluxes and redistributions have been proposed to play a central role in the initiation of cell proliferation (Rozengurt, 1981a,b). The recent findings on the coupled movements of Na+, K+, and H+ in mitogen-stimulated 3T3 cells described in the preceding section provide support for the proposition that changes in monovalent ion fluxes may act as internal signals in the stimulation of quiescent cells. In fact, these ionic events could have multiple and profound effects on cell metabolism and organization (Rozengurt, 198la; Nuccitelli and Deamer, 1982). For example, changes in pHi have been demonstrated in other biological systems and, in some of these, the increase in intracellular pH has been implicated as a crucial part of the control mechanism (Nuccitelli and Deamer, 1982). However, to prove that a given early event in growth stimulation is causally related to the subsequent events leading to DNA replication is a very difficult task. In addition to the generality of rapidly increased Na+, K+, and H+ fluxes after mitogenic stimulation (see Table I for a summary), the possibility that increased ionic fluxes may play a significant role in mitogenesis is supported by the following lines of evidence: (1) the concentration of Na+ and K+ in the culture medium markedly influences the development of the mitogenic response; and (2) stimulation of Na+ influx by hormonal peptides, tumor promoters, or membrane permeability modulators initiates DNA synthesis, acting synergistically with other growth-

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TABLE 1 GENERALITY OF INCREASED MONOVALENT CATION F1 U X E S I N QUIESCENT CELLS THEYARE STIMULATED TO PROLIFERATE Species Mouse

Hamster

Cell type Swiss 3T3 Swiss 3T6 BALBIc 3T3 Secondary cultures of embryo fibroblasts Neuroblastoma NR6 Nil-8 BHK CHO (CCL 39)

Rat

Hepatocytes

Chicken

Pheochromocytoma Glioma Embryo fibroblasts

Human

Monkey Dog Sea urchin Brine shrimp Pig

Embryo skeletal myoblasts Lymphocytes Fibroblasts Epidermoid carcinoma (A 431) BSC-I epithelial cells MDCK Egg Embryos (cysts) Lymphocytes

Mitogen(s)

AFTER

References"

See text Serum Serum Serum

See text I. 2 I. 3 I, 2

Serum PDG F Serum Serum Serum, thrombin, and insulin Insulin + glucagon + EGF Nerve growth factor, EGF Serum Serum, multiplicationstimulating activity Serum

4. 5 6 I

I 7, 8, 9 10

II 5 12, 13 14

P h y to hemagglu tin in Serum, lys-bradykinin. EGF, vasopressin Serum. EGF

21, 22, 23

Serum, EGF Serum Fertilization Aerobic rehydration Concanavalin A

24 25 26, 27 28 29

15

I . 16-20

Key to references: (1) Mendoza et a / . (1980); (2) Smith and Rozengurt (1978b); (3) Tupper er a / . (1977); (4) Moolenadr et d.(1981b); ( 5 ) Benos and Sapirstein (1983); (6) Cassel et al. (1983); (7) Pouyssegur et a/. (1982); (8) Paris and Pouyssegur (1984); (9) L'Allemain et al. (1984a), (10) Koch and Leffert (1979);( I 1 ) Boonstra et a / . (1983); (12) Johnson and Weber (1980); (13) Smith (1977); (14) Vandenburgh (1984); (15) Segal ct a / . (1979); (16) Villereal (1981); (17) Owen and Villereal (1983a); (18) Moolenaar et ul. (1982); (19) Moolenaar et al. (1983); (20) Moolenaar et a / . ( 1 9 8 4 ~ )(21) ; Whiteley et a / . (1984); (22) Rothenberg et a / . (1983a); (23) Rothenberg et a / . (1983b); (24) Rothenberg at a / . (1982); (25) Reznik et a / . (1983); (26) Whitaker and Steinhardt (1982); (27) Dube ct a / . (1984); (28) Busa and Crowe (1983); (29) Felber and Brand (1983).

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promoting factors. These points will be the subject of the following sections. 1. INHIBITION OF ION FLUXESPREVENTS THE INITIATION OF DNA SYNTHESIS The stimulation of DNA synthesis by serum in quiescent cultures of 3T3 cells depends on the concentration of Na+ and K+ in the nutrient medium. A decrease in the K+ concentration in the medium inhibited the stimulation of DNA synthesis by serum (Rozengurt and Heppel, 1975). Subsequently, Lopez-Rivas et al. (1982) found that the stimulation of DNA synthesis by insulin, EGF, and vasopressin in a serum-free medium was strongly dependent on the intracellular K+ content or K+ concentration. The relationship between DNA synthesis and intracellular K+ content was sigmoid. An increase in the intracellular K+ above a certain threshold level (0.56 pmoles/mg protein; 90 mM) was required to sustain the proliferative response of quiescent fibroblasts to peptide growth factors. The effects of K+on the GI-S transition are, at least in part, exerted via its control of protein synthesis (Lopez-Rivas et al., 1982). The findings suggested that a relatively small change in the intracellular K+ level influenced the ability of 3T3 cells to initiate DNA synthesis in response to peptide factors in serum-free medium. Reducing external Na+ below 100 mM inhibited serum-stimulated DNA synthesis until at 20 mM it was completely blocked (Smith and Rozengurt, 1978b). Recently, Burns and Rozengurt (1984), using Swiss 3T3 cells stimulated by EGF, vasopressin, and insulin, demonstrated that there was a striking dependence of the initiation of DNA synthesis on the Na+ concentration of the medium. The relationship was approximately sigmoidal, with a plateau of 70 mM and a half-maximal point at 35 mM Na+. Thus, removal of Na+ from the medium blocked the stimulation of DNA synthesis induced by peptide growth factors in a serum-free medium. Several early studies (Koch and Leffert, 1979; Rozengurt and Mendoza, 1980; Moolenaar et al., 1982) demonstrated that amiloride prevents the mitogenic response in different cell types. However, the possibility that this treatment interferes with the proliferative response by mechanisms other than its effect on Na+ influx is difficult to rule out completely (Rozengurt, 1981a; Lubin, 1982; Balk and Polimeni, 1982). In a recent study with amiloride analogs (with half-maximal inhibitory concentration Ki varying from loT5to 5 x M), L’Allemain et al. (1984b) found that the functioning of the Na+-H+ antiport system is required for stimulation of DNA synthesis in Chinese hamster fibroblasts maintained in HC0;-

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free medium. Pouyssegur et al. (1984) confirmed this conclusion using a mutant cell line defective in Na+-H+ exchange activity. The mutant was able to proliferate only when the medium was adjusted to an alkaline pH. To test further the possibility that ionic events are involved in cell proliferation, we focused our attention on the effects of hormones, ionophores, and toxins which promote Na+ influx in cultured cells and investigated whether such substances can be mitogenic to quiescent cells. 2. INCREASED IONFLUXES PROMOTE INITIATION OF DNA SYNTHESIS

If ionic events at the plasma membrane mediate the effect of growthpromoting factors, substances which alter ion transport should be mitogenic for quiescent cells. This constitutes an important prediction of the hypothesis that increased monovalent ion fluxes play a signaling role in mitogenesis. However, a major problem in determining whether an ion flux modulator has mitogenic activity is that the assay for initiation of DNA synthesis requires a long (about 20-40 hour) exposure of the cells to the agent. During this time, side effects of the agent might inhibit the proliferative response. An ideal substance for testing this hypothesis would confine its effects to the plasma membrane without modifying the permeability of other cellular membranes. In view of the existence of synergistic relationships between mitogens, it is likely that promoters of ion fluxes would induce DNA synthesis only when added with other growth-promoting factors which alter cell metabolism in a complementary fashion through other mechanisms. We have found that peptides which induce cation fluxes in their target cells, such as vasopressin or bombesin or the membrane permeability modulators melittin and amphotericin B , can act as mitogens in a serum-free medium. Some of the effects of these agents on ion fluxes have been discussed earlier. Their mitogenic effects constitute important evidence that changes in ion fluxes play a role in signaling a mitogenic response. a . Vasopressin, Phorbol Esters, and Bombesin. The addition of vasopressin to quiescent cultures of Swiss 3T3 cells rapidly stimulates ion movements across the plasma membrane (Rozengurt and Mendoza, 1980; Mendoza et al., 1980b) and acts synergistically with insulin (Rozengurt et al., 1979; 1981a; Rozengurt, 1979b), EGF (Dicker and Rozengurt, 1980), FDGF (Rozengurt and Mendoza, 1980), PDGF (Dicker et al., 1981), and CAMP-elevating agents (Rozengurt, 1982b) to stimulate DNA synthesis in Swiss 3T3 cells. These cells exhibited a striking specificity in their response to neurohypophyseal hormones. Vasopressin was lo2- 103-fold more potent than oxytocin in stimulating ion fluxes and DNA synthesis in

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quiescent cultures of 3T3 cells (Rozengurt and Mendoza, 1980; Rozengurt, 1979b; Dicker and Rozengurt, 1980). Dicker and Rozengurt proposed that the mitogenic actions of vasopressin and the potent tumor-promoting agents of the phorbol ester family (Dicker and Rozengurt, 1978, 1979b, 1981a; Collins and Rozengurt, 1982a) are mediated via a common mechanism (Dicker and Rozengurt, 1980). Vasopressin and phorbol esters can substitute for each other in synergistically stimulating DNA synthesis when added in the presence of other growth factors but show neither synergistic nor additive effects with each other (Dicker and Rozengurt, 1980). Since these agents bind to different receptors (Collins and Rozengurt, 1982a,b, 1983),the convergence of their action must occur at a postreceptor step. Phorbol esters, like vasopressin, caused an increased rate of Na+ entry into ouabain-blocked 3T3 cells (Dicker and Rozengurt, 1981a). The stimulation of Na+ influx was produced only by phorbol ester derivatives, which are potent inducers of DNA synthesis (Collins and Rozengurt, 1982a; Dicker and Rozengurt, 1981b), whereas biologically inactive analogs did not stimulate Na+ flux (Dicker and Rozengurt, 1981a). The significance of phorbol ester stimulation of ion fluxes is further discussed in Section II1,B. Recently we found that the regulatory tetradecapeptide bombesin was a potent mitogen for Swiss 3T3 cells (Rozengurt and Sinnett-Smith, 1983; Zachary and Rozengurt, 1985). At low (nanomolar) concentrations, bombesin stimulated initiation of DNA synthesis in Swiss 3T3 cells maintained in a serum-free medium. The mitogenic effect was dose and time dependent, specific, and markedly enhanced by insulin and other growthpromoting agents. Neither vasopressin nor phorbol esters enhanced the maximal level of DNA synthesis induced by bombesin. These findings suggested that vasopressin, phorbol esters, and bombesin share common pathways in stimulating DNA synthesis in Swiss 3T3 cells (Rozengurt and Sinnett-Smith, 1983). Recent experiments demonstrated that bombesin causes a potent stimulation of Na+ entry, Na+-H+ exchange, and Na+-K+ pump activity in Swiss 3T3 cells (Mendoza et al., 1986). All these findings provided further support to the hypothesis that ion fluxes are involved in signaling the initiation of DNA synthesis induced by regulatory peptides, hormones, and tumor promoters, However, these agents stimulate a complex array of cellular responses (Rozengurt, 1980; Dicker and Rozengurt, 1980; Collins and Rozengurt, 1982a), some of which may be elicited by other, as yet undiscovered, signals. b. Permeability Modulators. Melittin, the principal component of bee venom, is a water-soluble, strongly amphipathic 26-amino acid polypeptide (Haberman, 1972). At micromolar concentrations, this toxin binds to

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phospholipids (Knoppel et al., 1979) and increases permeability to ions in liposomes and artificial bilayer membranes, presumably by forming ionconducting channels (Tosteson and Tosteson, 1981). This amphipathic polypeptide provided a tool for selectively perturbing the ion permeability of the plasma membrane (rather than intracellular membranes) and thereby for testing further whether ion fluxes may signal the initiation of mitogenesis. Rozengurt er al. (1981b) found that addition of melittin to quiescent 3T3 cells stimulated Na+ entry and caused a several-fold increase in the initial rate of ouabain-sensitive 86Rb+ uptake. The toxin, at concentrations which promoted ion fluxes, stimulated DNA synthesis in a serum-free medium when added in the presence of insulin, EGF, or FDGF (Rozengurt et d . ,1981b). The concentration dependence of the melittin stimulation of DNA synthesis paralleled that of increases of 86Rb+uptake (Gelehrter and Rozengurt, 1980). Interestingly, melittin did not act synergistically with either vasopressin or phorbol esters in stimulating DNA synthesis (Rozengurt er al., 1981b), further suggesting that the toxin, the hormone, and the tumor promoter might act through a common mechanism. Nevertheless, the effects of melittin on pHi in the absence and in the presence of insulin and other synergistic factors requires investigation. Polyene antibiotics such as amphotericin B interact with both artificial and biological sterol-containing membranes to form channels or pores which permit the movement of monovalent cations (Kobayashi and Medoff, 1977; De Kruiff et al., 1974; Van Hoogvest and de Kruijff, 1978). A subtoxic concentration of amphotericin B increased Na+ influx and enhanced the activity of the Na+-K+ pump in 3T3 cells (Rozengurt and Mendoza, 1980). Interestingly, the addition of amphotericin B stimulated DNA synthesis in quiescent cultures of 3T3 cells, acting synergistically with insulin (Rozengurt, 1981b). The dose-response relationship for the stimulation of DNA synthesis was very similar to that found for the stimulation of 86Rb+uptake in quiescent 3T3 cells (Rozengurt and Mendoza, 1980; Rozengurt, 1981b). The level of DNA synthesis induced by amphotericin B was considerably less pronounced than that induced by serum or a combination of growth factors. Since the transmembrane channel formed by amphotericin B allowed both the inward movement of Na+ and the efflux of K+ from the cell (Rozengurt and Mendoza, 1980), it did not reproduce the selective increase in Na+ ion permeability caused by serum, hormones, and growth factors in 3T3 cells. In summary, considerable evidence supports the hypothesis that cation fluxes act as signals in initiating mitogenesis in cultured cells. This hypothesis poses a considerable number of important questions. In particular, the mechanism by which this ionic signal is translated into the initia-

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ENRIQUE ROZENGURT AND STANLEY A. MENDOZA

tion of DNA synthesis constitutes an important area of future research. Similarly, it will be of interest to define the role of other ionic pathways, for example, Cl--HCO; exchange, in the control of pHi in mitogenstimulated cells. In the following section we will discuss some of the possible molecular mechanisms responsible for the increased monovalent ion fluxes. 111.

PROTEIN KINASES AND ION FLUXES

As discussed in preceding sections, there is a wide range of mitogenic factors that stimulate Na+-H+ antiport activity in quiescent cells. Since these factors bind to separate and specific receptors (Rozengurt and Collins, 1983), it is of great interest to elucidate how such an array of receptors can modulate the Na+-H+ antiport system in the presence of their respective ligands. It is not known whether the tyrosine-directed protein kinase activity associated with the receptors for EGF (Cohen et al., 1982), PDGF (Ek and Heldin, 1982), IGF (Kasuga et al., 1982), and insulin (Petruzzelli et al., 1984) plays any role in the elicitation of the early ionic response. In contrast, CAMP, presumably acting via CAMP-dependent protein kinase, has been shown to affect the activity of the Na+-K+ pump. More germane to the preceding sections, the Ca2+-sensitive, phospholipid-dependent protein kinase (protein kinase C) may play a significant role in the control of the Na+-H+ antiport system. In what follows we will discuss the role of these protein kinases in the control of ion fluxes. A. Cycllc AMP, Cell Growth, and Ion Fluxes The role of cyclic nucleotides, cyclic AMP (CAMP),and cyclic GMP in the control of the proliferative response of quiescent fibroblastic cells has been the subject of a large and controversial literature (Chlapowski et al., 1975; Pastan et al., 1975; Friedman et al., 1976;Rozengurt, 1981~).In 3T3 cells and other fibroblastic cells, increased levels of cAMP were thought to reduce the rate of growth and inhibit the stimulation of DNA synthesis promoted by adding serum to quiescent cells. An objection to many of these studies has been that these effects were elicited by high concentrations of analogs of cAMP and could be regarded as nonspecific (see Rozengurt, 1981c, for review). Because a definitive conclusion on the effects of cAMP on the initiation of DNA synthesis of 3T3 cells was not possible, we evaluated the effects of CAMP-elevating agents on the initiation of DNA synthesis of 3T3 cells. In contrast to previous reports, we found that

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increased cellular concentrations of cAMP act synergistically with other growth-promoting agents to stimulate DNA synthesis in quiescent cultures of 3T3 cells (Rozengurt et al., 1981c, 1983a,b; Rozengurt 1982a,b). This evidence has recently been reviewed in detail (Rozengurt, 1984, 1985). Paris and Rozengurt (1982) found that an increased cellular level of cAMP stimulated the Na+-K+ pump-mediated uptake of *6Rb+into Swiss 3T3 cells. The increase in Na+-K+ pump activity occurred whether cAMP was generated endogenously by stimulation of the adenylate cyclase activity by cholera toxin (Rozengurt et al., 1981c), 5’-N-ethylcarboxamideadenosine (NECA) (Rozengurt, 1982a), or PGE, (Rozengurt et al., 1983b) or added exogenously as 8-Br-CAMP (Rozengurt, 1982b).The stimulatory effect of these compounds on 86Rb+uptake was potentiated by inhibitors of cyclic nucleotide phosphodiesterase activity (Paris and Rozengurt, 1982). Several lines of evidence suggested that the mechanism by which cAMP regulated Na+-K+ pump activity was fundamentally different from that of other mitogenic agents. In contrast to the rapid stimulation of the Na+-K+ pump caused by addition of Na+ flux modulators (serum, PDGF, vasopressin, phorbol esters, etc.), the stimulation of ouabain-sensitive 86Rb+uptake by CAMP-elevating agents was maximal only after hours of incubation. Further, increased intracellular cAMP failed to augment Na+ influx into 3T3 cells, whereas under identical conditions serum markedly increased Na+ entry (Rozengurt and Courtenay-Luck, 1982; Paris and Rozengurt, 1982). Further evidence suggesting that cAMP and Na+ fluxes regulate the Na+-K+ pump by independent mechanisms was furnished by the observation that phorbol esters and vasopressin did not affect the cellular content of cAMP under conditions which caused rapid stimulation of the Na+-K+ pump (Collins and Rozengurt, 1983; Rozengurt et al., 1983a, and unpublished results). These findings indicated that the timedependent stimulation of Na+-K+ pump activity caused by increased cAMP levels differed mechanistically with the rapid control of pump activity by serum, which is primarily mediated by increased Na+ entry into the cells. We suggested that CAMP, presumably acting through CAMPdependent protein kinase, activated a pathway leading to mitogenesis that was separate from that utilized by growth factors and mitogenic factors that enhanced amiloride-sensitive Na+ fluxes (Rozengurt, 1984, 1985). 6. Proteln Klnase C, Cell Growth, and Ion Fluxes

It is increasingly recognized that Ca2+-sensitive,phospholipid-dependent protein kinase (protein kinase C), which is stimulated by unsaturated

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ENRIQUE ROZENGURT AND STANLEY A. MENDOZA

diacylglycerol and serves as a major receptor for the tumor promoters of the phorbol ester family (see Nishizuka, 1984, for review), may play an important role in signaling a variety of cellular responses, including cell growth. It has been hypothesized that the high-affinity specific receptor for phorbol esters which was detected in a wide variety of cells and tissues is a complex formed between protein kinase C, Ca2+,and membrane phospholipids, and that unsaturated diacylglycerols generated by phospholipid breakdown may represent the endogenous analogs of phorbol esters (Kikkawa et al., 1983; Nishizuka, 1984). Substantial evidence for this hypothesis has come from recent studies in intact cells demonstrating that the synthetic diacylglycerol 1-oleyl-2-acetylglycerol(OAG) directly competes with 3H-PBt2for binding to specific receptors in intact 3T3 cells, rapidly stimulates protein kinase C in these cells, and is a potent mitogen for Swiss 3T3 cells, acting as a phorbol ester agonist (Rozengurt et al., 1984). In addition, prolonged treatment of Swiss 3T3 cells with phorbol esters decreases the number of high-affinity phorbol ester receptors in intact cells (Collins and Rozengurt, 1982a, 1984) and reduces the activity of protein kinase C measured in cell-free preparations (Rodriguez-Pena and Rozengurt, 1984) or in intact cells (Rozengurt et al., 1983~).This treatment blocks the mitogenic response to a subsequent addition of either phorbol esters (Collins and Rozengurt, 1982a,b, 1984) or OAG (Rozengurt et al., 1984). In view of these results, it was of considerable importance to define whether activation of protein kinase C elicits monovalent ionic fluxes in quiescent Swiss 3T3 cells. Recently, Vara et al. (1985) showed that OAG rapidly stimulates ouabain-sensitive 86Rb+uptake. The effect is dose dependent and can be detected as early as 1 minute after the addition of OAG. Further, OAG or PBtz increases 22Na+entry into 3T3 cells, an effect prevented by amiloride. These findings strongly suggest that OAG and PBt2 enhance amiloride-sensitive Na+-H+ antiport activity, leading to stimulation of the Na+-K+ pump which is highly sensitive to small increases in cell Na+, as discussed previously. These recent data are consistent with previous reports from our laboratory showing that the biologically active phorbol esters PBt2 and TPA stimulated Na+ entry and the Na+-K+ pump in 3T3 cells (Dicker and Rozengurt, 1981a); the half-maximal concentration of PBtz to elicit this effect was virtually identical to the Kd of 3H-PBt2for its high-affinity receptor in these cells (Collins and Rozengurt, 1982a). If the activators of protein kinase C, for example, PBt2 and OAG, stimulate Na+ influx through an increased Naf-H+ antiport system, these agents should increase pHi . Vara et al. (1985) found that OAG caused a significant increase in the steady-state concentration of labeled DMO, and the pHi was increased by 0.15 pH units. Burns and Rozengurt (1983)

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reported that PBt, also causes cytoplasmic alkalinization in Swiss 3T3 cells. These findings are in good agreement with recent reports using a murine pre-B lymphocyte cell line (Rosoff et al., 1984),a human leukemic cell line (Besterman and Cuatrecases, 1984),a rat glial cell line (Sapirstein and Benos, 1984), and human fibroblasts (Moolenaar et al., 1984a) in which phorbol esters enhance amiloride-sensitive Na+-H+ exchange. Prolonged pretreatment with PBt2 markedly reduces the number of high-affinity phorbol ester binding sites (Collins and Rozengurt, 1982a, 1984),reduces the specific activity of protein kinase C in cell-free preparations (Rodriguez-Pena and Rozengurt, 1984), and desensitizes the cell to further biological effects of phorbol esters (Collins and Rozengurt, 1982a,b, 1984; Rozengurt et al., 1983c, 1984). It is noteworthy that the magnitude of stimulation of the Na+-H+ antiport system by an acid load achieved by a transient exposure to NH4CI and the enhancement of Na+K+ pump activity induced by the Na+ ionophore monensin were virtually identical in control and PBt2-treated cells (Vara el al., 1985). These important controls indicate that the Na+-H+ antiport system and the Na+-K+ pump are present and functional in PBt2-desensitized cells. A salient feature of the results reported by Vara et al. (1985) is that prolonged treatment of the cells with PBt2 abolished the stimulation of &Rb+ or 22Na+uptake of a subsequent addition of either PBt, or OAG. This striking loss of ionic responses to OAG and PBt2 seen in 3T3 cells with a greatly reduced number of high-affinity phorbol ester receptors and activity of protein kinase C (see above) implicates this phosphotransferase system in the stimulation of monovalent cation fluxes. The above findings strongly suggest that activation of protein kinase C leads either directly or indirectly to increased activity of the Na+-H+ antiport system which, in turn, promotes Na+ influx, increases pH,, and stimulates the Na+-K+ pump activity. In this manner, protein kinase C may represent an important molecular link in the sequence of events triggered by the binding of growth-promoting factors to their respective receptors. This possibility takes on added interest in view of the fact that addition of a variety of mitogens, including serum, PDGF, FDGF, and the peptides vasopressin and bombesin, to quiescent 3T3 cells rapidly activates protein kinase C (Rozengurt et al., 1983c, 1984; Rodriguez-Pena and Rozengurt, 1985; Zachary et al., 1986) as judged by the rapid enhancement in the phosphorylation of a M , = 80,000 cellular protein (80 kDa). As expected, PBt2 and OAG cause a rapid (15 second) increase in 80-kDa phosphorylation. Thus, it is likely that activation of protein kinase C may mediate, at least in part, the early ionic response elicited by these growthpromoting factors. The use of PBt2-desensitized 3T3 cells may provide an experimental approach to evaluating the contribution of protein kinase C

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in mediating the ionic responses elicited by growth factors and mitogenic hormones (Vara and Rozengurt, 1985). IV. CALCIUM FLUXES

Although Ca2+has been frequently implicated in the control of cell proliferation, little attention has been given to the early changes in Ca2+ fluxes in quiescent fibroblastic cell lines stimulated to proliferate. Recently, Lopez-Rivas and Rozengurt (1983) reported that addition of serum caused a rapid (less than 15 second) increase in the rate of 45Ca2+efflux from radioactively loaded quiescent Swiss 3T3 cells. Serum also increased 4sCa2+release from quiescent Rat-1 cells, mouse 3T6 cells, baby hamster kidney cells (BHK), and secondary cultures of mouse embryo fibroblasts. The stimulation of 45Ca2+efflux by serum occurred in the absence of extracellular Ca2+,indicating that 4sCa2+-40Ca2+ exchange was not the major cause of the increased Ca2+efflux. Further exposure to serum led to a fall in the intracellular Ca2+content of the cells. These findings indicated that serum mobilizes Ca2+from an intracellular store. Changes in Ca2+distribution as a result of mitogenic stimulation are not restricted to serum-stimulated cells, since the nonapeptide vasopressin also stimulated 4sCa2+efflux from 4sCa2+preloaded 3T3 cells (LopezRivas and Rozengurt, 1984). The effect of vasopressin on 45Ca2+efflux was also seen in secondary cultures of mouse embryo fibroblasts which are responsive to this hormone (Rozengurt et al., 1981a). Vasopressin It appeared likely that caused a 52% decrease in total intracellular 45Ca2+. vasopressin caused release of 45Ca2+from an intracellular store into the cytosol from which it was pumped out of the cells by the plasma membrane Ca2+-ATPase.In addition to vasopressin, PDGF, FDGF, and bombesin all stimulate 4SCa2+efflux, while tumor-promoting phorbol esters do not (Rozengurt and Mendoza, 1985; Mendoza et al., 1986). The stimulation of 45Ca2+efflux by vasopressin was mediated by a pressor-type (Vl) receptor, as indicated by the relative potency of vasopressin analogs, by the use of a specific antagonist of V1 receptors (Lopez-Rivas and Rozengurt, 1984), and by the failure of the hormone to stimulate adenylate cyclase activity in membranes from Swiss 3T3 cells (Collins and Rozengurt, 1983).In other cell types, the interaction between vasopressin and pressor-type receptors causes breakdown of inositol phospholipids, producing diacylglycerols (see Section II1,B) and inositol polyphosphates (Thomas et al., 1984). Inositol triphosphate (IP3) has been implicated as a second messenger in the action of vasopressin and other ligands that induce Ca2+release (Berridge and Irvine, 1984). Interestingly, PDGF caused increases in the level of inositol triphos-

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phate in Swiss 3T3 cells (Berridge et al., 1984). In intact cells, PDGF caused 4sCa2+release while IP3did not. In cells made ‘‘leaky’’ by saponin treatment in a high-K+ medium, Ca2+release was stimulated by IP3 and not by PDGF. Inositol 1,4,5-triphosphate was the most potent stimulator of 4sCa2+release from leaky cells, but other inositol derivatives with phosphates in the 4 and 5 position also stimulated Ca2+release. Inositol 1,Cbiphosphate had no effect (Irvine et al., 1984). It was suggested that PDGF causes phosphoinositol breakdown, releasing IP3 intracellularly, which is then believed to cause Ca2+mobilization. Intracellular calcium concentrations were measured in Swiss 3T3 cells using the fluorescent indicator quin-2 (Morris et al., 1984; Rozengurt and Mendoza, 1985; Mendoza et al., 1986). They found that vasopressin and bombesin caused a rapid increase in intracellular Ca2+concentration. This effect occurred within 10 seconds and was maximal at 60-90 seconds. Neither insulin nor TPA had an effect on cytosolic Ca2+. Recently, changes in calcium fluxes and in intracellular calcium concentration have been demonstrated following mitogenic stimulation of other types of cells. Owen and Villereal (1983b) reported that serum and the combination of EGF, Lys-bradykinin, vasopressin, and insulin cause a marked increase of 45Ca2+release from quiescent human fibroblasts. Subsequently, Mix et al. (1984) demonstrated that the same mixture of mitogens causes a rapid, transient rise in intracellular Ca2+activity. Similar results were reported by Moolenaar et al. (1984b).They found that serum, PDGF, or EGF increased intracellular free Ca2+ concentration, while insulin and TPA did not. It was suggested that the increase in Ca2’ concentration might be caused by the release of inositol triphosphate. This hypothesis was supported by the recent report that serum, bradykinin, and vasopressin all rapidly stimulate inositol phosphate release (Vicentini and Villereal, 1984). Thus, stimulation of mitogenesis is associated with changes in calcium fluxes and/or cytosolic Ca2+concentration. These changes appear to be mediated by the release of inositol polyphosphates from phospholipids, as was found with many ligands that induce Ca2+ mobilization in their respective target cells (Berridge and Irvine, 1984). There is a paucity of information concerning the role of Ca2+fluxes and redistributions in signaling the mitogenic response of quiescent cells. In particular, it is known that many mitogens, including vasopressin, must be present in the incubation medium for several hours before they commit the cell to DNA synthesis and division (Dicker and Rozengurt, 1981b). Therefore, it is difficult to envisage a major role for the transient change in Ca2+flux that has been demonstrated so far. An intriguing possibility is that a persistent depletion of Ca2+from an intracellular pool may play a role in the elicitation or modulation of the long-term mitogenic response

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rather than a transient change in cytosolic Ca2+which reflects the exit of the cation from that pool. V. CONCLUSIONS AND PERSPECTIVES Understanding of the mechanisms of action of proliferative stimuli requires the identification of the intracellular signals which initiate or modulate the mitogenic response. Ion fluxes and redistributions have been suggested to play a role in mediating the action of mitogenic agents. In recent years, it has become apparent that changes in cation fluxes are among the earliest events observed in quiescent cells stimulation to divide. The pattern of changes, originally described in mouse Swiss 3T3 cells, can be described as Na+ cycle composed of Na+ influx through an amiloride-sensitive Na+-H+ antiport system which leads to intracellular alkalinization and Na+ efflux via the ouabain-sensitive Na+-K* pump, which results in active K+ uptake, and to the restoration of the electrochemical gradient of Na+ across the plasma membrane (see Fig. 1). BeArniloride

,-I

Ouabain

I

a I

8

cAMPlntracellular

Kinase

FIG. 1. Ion fluxes stimulated by growth factors in quiescent cells. The mitogenic enhancement of Na+, K+, and H+ fluxes in quiescent cells can be envisaged as a Na+ cycle composed of Na+ influx via an amiloride-sensitive Na+-H+ antiport which modulates intra' cellular pH and Na+ efflux via the ouabain-sensitive Na+-K+ pump, which leads to K accumulation. Growth factors also cause a rapid mobilization of Ca2+from an intracellular The molecular mechanism by pool. This process appears to be mediated by Ins 1,4,5-P3. which the binding of the mitogenic ligand elicits the stimulation of monovalent ion fluxes may involve the activation of protein kinase C. For further details see text.

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cause these changes have been demonstrated in a wide range of quiescent cell types stimulated to proliferate by a variety of growth-promoting factors (summarized in Table I), it is reasonable to suggest that increased ionic fluxes represent one of the key events that take place when quiescent cells are stimulated to reinitiate DNA synthesis and cell division. There are many aspects of the role of monovalent cation fluxes in cell proliferation that remain unsolved and warrant further experimental work. The nature of the molecular steps that link receptor occupancy with the elicitation of the ionic response is only beginning to be understood. Recent data suggest that one of the pathways that stimulates the Na+-H+ antiport system involves the activation of protein kinase C. This takes place as a result of receptor-mediated phospholipid breakdown and generation of diacylglycerol or by addition of phorbol esters or synthetic diacylglycerols (Fig. I). The possibility that the Na+-H+ antiport system is directly phosphorylated by protein kinase C, leading to a change in kinetic properties, is an attractive hypothesis. A direct test of this possibility must await the isolation and purification of the Na+-H+ antiport system itself. It is noteworthy that other mechanisms of control of the Na+-H+ antiport, that is, not involving protein kinase C, are not excluded. It is of interest that increased cAMP levels, which activate protein kinase A, enhance the activity of the Na+-K+ pump but fail to activate Na+-H+ exchange. Thus, protein kinases C and A modulate the Na+ cycle at different points (Fig. 1). It is not known whether cAMP influences intracellular pH by another pathway, for example, CI--HCO; exchange. In addition to changes in monovalent cation fluxes, certain mitogens cause rapid and transient changes in Ca2+flux and cytosolic concentration which may be mediated by inositol 1,4,5-P3, a product of inositol phospholipid breakdown. Any consideration about the role of this change in the elicitation of other cellular responses must take into account the characteristic transient time course of the Ca2+flux. Finally, the elucidation of the molecular mechanisms by which increased ionic fluxes are translated into a mitogenic response is an important area that requires further experimental work. It is conceivable that the Na+ cycle induced by mitogens represents a primitive regulatory system linked to cell multiplication which has been conserved during evolution and plays a permissive role in the development of the proliferative response. Alternatively, ion fluxes may signal intracellular events leading to the initiation of cell proliferation, for example, by controlling energy metabolism, cytoskeletal arrangements, the rate of macromolecular synthesis, and/or the expression of critical genes (e.g., oncogenes) required for initiation of cell proliferation. It is important to bear in mind that one of the salient features in the action of most mitogenic agents, added to

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quiescent cells in serum-free media, is that they exhibit striking synergistic effects when presented in specific combinations (see Rozengurt, 1985, for review). It is increasingly recognized that the stimulation of quiescent cells into DNA synthesis is a complex process that requires the synergistic interaction of complementary signals. The results discussed in this chapter support the possibility that ionic fluxes may represent one of the signals that synergistically lead to cell proliferation. ACKNOWLEDGMENTS This manuscript was written during the tenure of an American Cancer Society Eleanor Roosevelt International Cancer Fellowship awarded by the International Union Against Cancer to S. A. M. REFERENCES Aronson, P. S., Nee, J., and Suhm, M. A. (1982). Modifier role of internal H+ in activating the Na+/H+exchanger in renal microvillus membrane vesicles. Nature (London) 299, 161-163. Balk, S. D., and Polimeni, P. I. (1982). Effect of reduction of culture medium sodium using different sodium chloride substitutes on the proliferation of normal and rous sarcoma virus-infected chicken fibroblasts. J . Cell. Physiol. 112, 25 1-256. Benos, D. J., and Sapirstein, V. S. (1983). Characteristics of an amiloride sensitive sodium entry pathway in cultured rodent glial and neuroblastoma cells. J . Cell. Physiol. 116, 213-220. Benidge, M. J., and Irvine, R. F. (1984). Inositol triphosphate, a novel second messenger in cellular signal transduction. Nature (London) 312, 315-321. Berridge, M. J., Heslop, J. P., Irvine, R. F., and Brown, K. D. (1984). Inositol triphosphate formation and calcium mobilization in Swiss 3T3 cells in response to platelet-derived growth factor. Biochem. J . 222, 195-201. Besterman, J. M., and Cuatrecasas, P. (1984). Phorbol esters rapidly stimulate amiloridesensitive Na+/H+exchange in a human leukemia cell line. J . Cell B i d . 99, 340-343. Boonstra, J., Moolenaar, W. H., Harrison, P. H., Moed, P., van der Saag, P. T., and de Laat, S. W. (1983). Ionic responses and growth stimulation induced by nerve growth factor and epidermal growth factor in rat pheochromocytoma (PC12) cells. J . Cell B i d . 97992-98. Bourne, H. R., and Rozengurt, E. (1976). An 18,000 molecular weight polypeptide induces early events and stimulates DNA synthesis in cultured cells. Proc. Natl. Acad. Sci. U.S.A. 73,4555-4559. Bums, C. P., and Rozengurt, E. (1983). Serum, platelet-derived growth factor, vasopressin and phorbol esters increase intracellular pH in Swiss 3T3 cells. Biochem. Biophys. Res. Commun. 116,931-938. Bums, C. P., and Rozengurt, E. (1984). Extracellular Na' and initiation of DNA synthesis: Roles of intracellular pH and K+. J. Cell B i d . 98, 1082-1089. Busa, W. B . , and Crowe, J. H. (1983). Intracellular pH regulates transitions between dormancy and development of brine shrimp (Artermia salina) embryos. Science 221,366368. Cassel, D., Rothenberg, P., Zhuang, Y.-X., Deuel, T. F., and Glaser, L. (1983). Platelet-

5. STIMULATION OF ION TRANSPORT IN FIBROBLAST MITOGENESIS

185

derived growth factor stimulates Na+/H+exchange and induces cellular alkalinization

in NR6 cells. Proc. Natl. Acad. Sci. U . S . A . 80, 6224-6228. Chlapowski, F. J., Kelly, L. A., and Butcher, R. W. (1975). Cyclic nucleotides in cultured cells. Adv. Cyclic Nucleotide Res. 6 , 245-338. Cohen, S., Ushiro, H., Stoscheck, C., and Chinkers, M. (1982). A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J . Biol. Chem. 257, 1523-1531. Collins, M. K . L., and Rozengurt, E . (1982a). Binding of phorbol esters to high-affinity sites on murine fibroblastic cells elicits a mitogenic response. J. Cell. Physiol. 112, 42-50. Collins, M., and Rozengurt, E . (1982b). Stimulation of DNA synthesis in murine fibroblasts by the tumor promoter teleocidin: Relationship to phorbol esters and vasopressin. Biochem. Biophys. Res. Commun. 104, 1159-1 166. Collins, M. K. L., and Rozengurt, E. (1983). Vasopressin induces selective desensitization of its mitogenic response in Swiss 3T3 cells. Proc. Natl. Acad. Sci. U . S . A . 80, 19241928. Collins, M. K. L., and Rozengurt, E. (1984). Homologous and heterologous mitogenic desensitization of Swiss 3T3 cells to phorbol esters and vasopressin: Role of receptor and postreceptor steps. J . Cell. Physiol. 118, 133-142. Dawson, A. P., and Irvine, R. F. (1984). Inositol(1,4,5) triphosphate-promoted Ca2+release from microsomal fractions of rat liver. Biochem. Biophys. Res. Commun. 120,858-864. de Kruijff, B., Genitsen, W. J., Oerlemans, A,, Demel, R. A,, and van Deenen, L. L. M. (1974). Polyene antibiotic-sterol interactions in membranes of Acholeplasma ladiawii cells and lecithin liposomes. Specificity of membrane permeability changes induced by polyene antibiotics. Biochim. Biophys. Acta 339, 30-43. Dicker, P., and Rozengurt, E. (1978). Stimulation of DNA synthesis by tumour promoter and pure mitogenic factors. Nature (London) 276, 723-726. Dicker, P., and Rozengurt, E. (1979a). Synergistic stimulation of early events and DNA synthesis by phorbol esters, polypeptide growth factors and retinoids in cultured fibroblasts. J. Supramol. Struct. 11, 79-93. Dicker, P., and Rozengurt, E. (1979b). Retinoids enhance mitogenesis by tumour promoter and polypeptide growth factors. Biochem. Biophys. Res. Commun. 91, 1203-12 10. Dicker, P., and Rozengurt, E. (1980). Phorbol esters and vasopressin stimulation DNA synthesis by a common mechanism. Nalure (London) 287, 607-612. Dicker, P., and Rozengurt, E. (1981a). Phorbol ester stimulation of Na influx and Na-K pump activity in Swiss 3T3 cells. Biochern. Biophys. Res. Commun. 100, 433-441. Dicker, P., and Rozengurt, E . (1981b). Stimulation of DNA synthesis by transient exposure of cell cultures to TPA or polypeptide mitogens: Induction of competence or incomplete removal? J. Cell. Physiol. 109,99-109. Dicker, P., Pohjanpelto, P., Pettican, P., and Rozengurt, E. (1981). Similarities between fibroblast-derived growth factor and platelet-derived growth factor. Exp. Cell Res. 135, 22 1-227. Doolittle, R. F., Hunkapiller, M. W., Hood, L. E., Devane, S. G., Robbins, K. C., Aaronson, S. A., and Antoniades, H. N. (1983). Simian sarcoma virus oncogene, u-sis, is derived from the gene (or genes) encoding a platelet-derived growth factor. Science 221, 275-277. Downward, J., Yarden, Y., Mayes, E., Scrace, G., Totty, N., Stockwell, P., Ullrich, A , , Schlessinger, J., and Waterfield, M.D. (1984). Close similarity of epidermal growth factor receptor and V-erb-B oncogene protein sequences. Nature (London) 307, 52 I 527. Dube, F., Schmidt, T., Johnson, G. H., and Epel, D. (1984). The rise of intracellular pH is

186

ENRIQUE ROZENGURT AND STANLEY A. MENDOZA

only transiently required for initiation of early development and increased protein synthesis in sea urchin embryos. J . Cell Biol. 99, 266a. Ek, B., and Heldin, C.-H. (1982). Characterization of a tyrosine-specific kinase activity in human fibroblast membrane stimulated by platelet-derived growth factor. J . Biol. Chem. 257, 10486-10492. Felber, S . M., and Brand, M. 0. (1983). Concanavalin A causes an increase in sodium permeability and intracellular sodium content of pig lymphocytes. Biochem. J . 210, 893-897. Frelin, C., Vigne, P., and Lazdunski, M. (1983). The amiloride-sensitive Na+/H+antiport in 3T3 fibroblasts. J . Biol. Chem. 258, 6272-6276. Friedkin, M., and Rozengurt, E. (1981). The role of cytoplasmic microtubules in the regulation of the activity of peptide growth factors. Adv. Enzyme Regul. 19, 39-59. Friedkin, M., Legg, A., and Rozengurt, E. (1979). Antitubulin agents enhance the stimulation of DNA synthesis by polypeptide growth factors in 3T3 mouse fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 76, 3909-3912. Friedkin, M., Legg, A., and Rozengurt, E. (1980). Enhancement of DNA synthesis by colchicine in 3T3 mouse fibroblasts stimulated with growth factors. Exp. Cell Res. 129, 23-30. Friedman, D. L., Johnson, R. A., and Zerlig, C. E. (1976). The role of cyclic nucleotides in the cell cycle. Adv. Cyclic Nucleotide Res. 7, 69-1 14. Gelehrter, T. R., and Rozengurt, E. (1980). Stimulation of monovalent ion fluxes and DNA synthesis in 3T3 cells by melittin and vasopressin is not mediated by phospholipid deacylation. Biochem. Biophys. Res. Commun. 92,716-724. Haberman, E. (1972). Bee and wasps venoms. Science 177, 314-322. Heldin, C. H., and Westermark, B. (1984). Growth factors: Mechanism of action and relation to oncogenes. Cell 37, 9-20. Holley, R. W. (1975). Control of growth of mammalian cells in cell culture. Nature (London) 258,487-490. Irvine, R. F., Brown, K. D., and Benidge, M. J. (1984). Specificity of inositol triphosphateinduced calcium release from permeabilized Swiss mouse 3T3 cells. Biochem. J . 221, 269-272. Johnson, M. A., and Weber, M. J. (1980). Serum stimulation of potassium fluxes, ouabain binding and sodium fluxes in quiescent chicken embryo fibroblasts. J . Cell. Physiol. 103, 363-370. Kasuga, M., Zick, Y., Blith, D. L., Karlsson, F. A., Haring, H. U., and Kuhn, C. R. (1982). Insulin stimulation of phosphorylation of the B subunit of the insulin receptor. J . Biol. Chem. 257,9891-9894. Kikkawa, U., Takai, Y.,Tanaka, Y.,Miraka, R., and Nishizuka, Y. (1983). Protein kinase C as a possible receptor protein of tumour promoting phorbol esters. J . Biol. Chem. 258, 1 1442-1 1445. Kobayashi, G . S., and Medoff, G. (1977). Antifungal agents: Recent developments. Annu. Rev. Microbiol. 31, 291-308. Koch, K. S., and Leffert, H. L. (1979). Increased sodium ion influx is necessary to initiate rat hepatocyte proliferation. Cell 18, 153-163. Knoppel, E., Eisenberg, D., and Wickner, W. (1979). Interactions of melittin, a peptide model with detergents. Biochemistry 18, 4177-4181. L’Allemain, G., Paris, S . , and Pouyssegur, J. (1984a). Growth factor action and intracellular pH regulation in fibroblasts. Evidence for a major role of the Na+/H+antiport. J. Biol. Chem. 259, 5809-5815.

5. STIMULATION OF ION TRANSPORT IN FIBROBLAST MITOGENESIS

187

L’Allemain, G., Franchi, A., Cragoe, E., Jr., and Pouyssegur, J. (1984b). Blockade of the Na+/H+antiport abolishes growth factor induced DNA synthesis in fibroblasts. J. Biol. Chem. 259,4313-4319. Lopez-Rivas, A., and Rozengurt, E . (1983). Serum rapidly mobilizes calcium from an intracellular pool in quiescent fibroblastic cells. Biochem. Biophys. Res. Commun. 114,240247. Lopez-Rivas, A., and Rozengurt, E. (1984). Vasopressin rapidly stimulates Ca2+efflux from an intracellular pool in quiescent Swiss 3T3 cells. Am. J . Physiol. 247, cl56-cI62. Lopez-Rivas, A,, Adelberg, E . A,, and Rozengurt, E. (1982). lntracellular K t and the mitogenic response of 3T3 cells to peptide factors in serum-free medium. Proc. Natl. Acad. Sci. U . S . A . 79, 6275-6279. Lopez-Rivas, A., Stroobant, P., Waterfield, M. D., and Rozengurt, E. (1984). Ionic responses rapidly elicited by porcine platelet-derived growth factor in Swiss 3T3 cells. EMBO J . 3, 939-944. Lubin, M. (1982). The potassium-sensitivity shift and other matters. I n ‘‘Ions, Cell Proliferation and Cancer” (A. L. Boynton, W. L . McKeehan, and J. P. Whitfield, eds.), pp. 131-150. Academic Press, New York. Mendoza, S. A., Wigglesworth, M. M., Pohjanpelto, P., and Rozengurt, E . (1980a). Na entry and Na-K pump activity in murine, hamster and human cells. Effect of monensin, serum, platelet extract and viral transformation. J . Cell. Physiol. 103, 17-27. Mendoza, S. A., Wigglesworth, N. M., and Rozengurt, E. (1980b). Vasopressin rapidly stimulates Na entry and Na-K pump activity in quiescent cultures of mouse 3T3 cells. J . Cell. Physiol. 105, 153-162. Mendoza, S. A., Schneider, J. A., Lopez-Rivas, A., Sinnett-Smith, J. W., and Rozengurt, E. (1986). Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. I1 Changes in Na+ and Ca2+fluxes, Na+-K+ pump activity and intracellular pH. J . Cell. Biol. (in press). Mix, L. L., Dinerstein, R. J., and Villereal, M. L . (1984). Mitogens and melittin stimulate an increase in intracellular free calcium concentration in human fibroblasts. Biochem. Biophys. Res. Commun. 119, 69-75. Moolenaar, W. H., Boonstra, J., van der Saag, P. T., and de Laat, S. W. (1981a). Sodium/ proton exchange in mouse neuroblastoma cells. J . Biol. Chem. 256, 12883-12887. Moolenaar, W. H., Munnery, C. L., van der Saag, P. T., and de Laat, S. W. (1981b). Rapid ionic events and the initiation of growth in serum-stimulated neuroblastoma cells. Cell 23, 789-798. Moolenaar, W. H., Yarder, Y., de Laat, S. W., and Schlessinger, J. (1982). Epidermal growth factor induces electrically silent Na’ influx in human fibroblasts. J . Biol. Chem. 257, 8502-8506. Moolenaar, W. H., Tsein, R. Y., van der Saag, P. T . , and de Laat, S. W. (1983). N a + / H + exchange and cytoplasmic pH in the action of growth factors in human fibroblasts. Nature (London) 304,645-648. Moolenaar, W. H., Tertoolen, L. G . J., and de Laat, S. W. (1984a). Phorbol ester and diacylglycerol mimic growth factors in raising cytoplasmic pH. Nature (London) 312, 371-374. Moolenaar, W. H., Tertoolen, L. G. J., and de Laat, S. W. (3984b). Growth factors immediately raise cytoplasmic free CaZ+in human fibroblasts. J. Biol. Chem. 259, 8066-8069. Moroney, J., Smith, A., Tomei, L. D., and Wenner, C. E. (1978). Stimulation of 86Rb+and ’*PI movements in 3T3 cells by prostaglandins and phorbol esters. J . Cell. Physiol. 95, 287-294.

188

ENRIQUE ROZENGURT AND STANLEY A. MENDOZA

Moms, J. D. H., Metcalfe, J. C., Smith, G. A., Hesketh, T. R., and Taylor, M. V. (1984). Some mitogens cause rapid increases in free calcium in fibroblasts. FEBS Lett. 169,189193. Nishizuka, Y. (1984). The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature (London) 308, 693-698. Nuccitelli, R., and Deamer, D. W. (eds.) (1982). I n “Intracellular pH: Its Measurement, Regulation and Utilization in Cellular Function.” Liss, New York. Owen, N. E., and Villereal, M. E. (1983a). Lys-bradykinin stimulates Na+ influx and DNA synthesis in cultured human fibroblasts. Cell 32, 979-985. Owen, N. E., and Villereal, M. L. (1983b). Efflux of 45CaZ+from human fibroblasts in response to serum or growth factors. J. Cell. Physiol. 117, 23-29. Pardee, A. B., Dubrow, R., Hamalin, J. L., and Kletzein, R. F. (1978). Animal cell cycle. Annu. Rev. Biochem. 47, 715-750. Paris, S., and Pouyssegur, J. (1984). Growth factors activate the Na+/H+antiporter in quiescent fibroblasts by increasing its affinity for intracellular H+. J. Biol. Chem. 259, 10989- 10994. Paris, S . , and Rozengurt, E. (1982). Cyclic AMP stimulation of Na-K pump activity in quiescent Swiss 3T3 cells. J. Cell. Physiol. 112, 273-280. Pastan, I. H., Johnson, G. S., and Anderson, W. B. (1975). Role of cyclic nucleotides in growth control. Annu. Rev. Biochem. 44,491-522. Petruzzelli, L., Hervera, R., and Rosen, 0. M. (1984). Insulin receptor is an insulin-dependent protein kinase: Copurification of insulin-binding activity and protein kinase activity to homogeneity from human placenta. Proc. Natl. Acad. Sci. U.S.A.81,3327-3331. Pouyssegur, J., Chambard, J. C., Franchi, A., Pans, S . , and van Oblerghen-Schilling, E. (1982). Growth factor activation of an amiloride-sensitive Na+/H+exchange system in quiescent fibroblasts: Coupling to ribosomal protein S6 phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 79, 3935-3939. Pouyssegur, J., Sardet, C., Franchi, A,, L’Allemain, G., and Pans, S. (1984). A specific mutation abolishing Na+/H+antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH. Proc. Natl. Acad. Sci. U.S.A. 81, 4833-4837. Reznik, V. M., Villela, J., and Mendoza, S. S. (1983). Serum stimulates Na entry and the Na-K pump in quiescent cultures of epithelial cells (MDCK). J . Cell. Physiol. 117,211214. Rindler, M. H., and Saier, M. H., Jr. (1981). Evidences for Na+/H+antiport in cultured dog kidney cells (MDCK). J. Biol. Chem. 256, 10820-10825. Rindler, M. J., Taub, M., and Saier, M. H., Jr. (1979). Uptake of 22Na+by cultured dog kidney cells (MDCK). J . Biol. Chem. 254, 11431-11439. Rodriquez-Pena, A., and Rozengurt, E. (1984). Disappearance of Ca2+sensitive, phospholipid-dependent protein kinase in phorbol ester treated 3T3 cells. Biochem. Biophys. Res. Commun. 120, 1053-1059. Rodriguez-Pena, A., and Rozengurt, E. (1985). Serum, like phorbol esters, rapidly activates protein kinase C in intact quiescent fibroblasts. EMBO J . 4, 71-76. Roos, A., and Boron, W. F. (1981). Intracellular pH. Physiol. Rev. 61, 296-434. Rosoff, P. M., Stein, L. F., and Cantley, L. C. (1984). Phorbol esters induce differentiation in a pre-B lymphocyte cell line by enhancing Na+/H+exchange. J. Biol. Chem. 259, 7056-7060. Ross, R., and Vogel, A. (1978). The platelet-derived growth factor. Cell 14, 203-210. Rothenberg, P., Reuss, L., and Glaser, L. (1982). Serum and epidermal growth factor transiently depolarize quiescent BSC-I epithelial cells. Proc. Natl. Acad. Sci. U . S . A . 79, 7783-7787.

5. STIMULATION OF ION TRANSPORT IN FIBROBLAST MITOGENESIS

189

Rothenberg, P., Glaser, L., Schlesinger, P., and Cassel, D. (1983a). Epidermal growth factor stimulates amiloride-sensitive zzNa+uptake in A 431 cells. J. Biol. Chem. 258, 4883-4889. Rothenberg, P., Glaser, L., Schlesinger, P., and Cassel, D. (1983b). Activation of N a + / H + exchange by epidermal growth factor elevates intracellular pH in A 431 cells. J . B i d . Chem. 258, 12644-12653. Rozengurt, E. (1979a). Early events in growth stimulation. In “Surface Properties of Normal and Neoplastic Cells” (R. Hynes, ed.), pp. 323-353. Wiley, New York. Rozengurt, E. (1979b). Biochemical basis of the early events stimulated by serum and mitogenic factors in cultures of quiescent cells. Cold Spring Harbor Conf. Cell Pro/$. 6, 773-788. Rozengurt, E. (1980). Stimulation of DNA synthesis in quiescent cultured cells: Exogenous agents, internal signals and early events. Curr. Top. Cell. Regul. 17, 59-88. Rozengurt, E. (1981a). Stimulation of Na influx pump activity and DNA synthesis in quiescent cultured cells. Adv. Enzyme Regul. 19, 61-85. Rozengurt, E. (1981b). Na influx as a mitogenic signal for cultured fibroblasts. In “Cellular Responses to Molecular Modulators” (C. W. Mozes, J. Schultz, W. A. Scott, and R. Werner, eds.), pp. 149-168. Academic Press, New York. Rozengurt, E. (1981~).Cyclic AMP: A growth-promoting signal for mouse 3T3 cells. Adv. Cyclic Nucleotide Res. 14, 429-442. Rozengurt, E. (1982a). Adenosine receptor activation in quiescent Swiss 3T3 cells: Enhancement ofcAMP levels. DNA synthesis and cell division. Exp. CellRes. 139, 71-78. Rozengurt, E . (1982b). Synergistic stimulation of DNA synthesis by cyclic AMP derivatives and growth factors in mouse 3T3 cells. J. Cell. Physiol. 112, 343-350. Rozengurt, E. (1983). Growth factors, cell proliferation and cancer: An overview. Mol. B i d . Med. 1, 169-181. Rozengurt, E. (1984). Role of ion fluxes and cyclic nucleotides in signalling mitogenesis in 3T3 cells. Horm. Cell Regul. 8, 17-36. Rozengurt, E. (1985). The mitogenic response of cultured 3T3 cells: Integration of early signals and synergistic effect in a unified framework. In “Molecular Mechanisms of Transmembrane Signalling” (P. Cohen and M. Housley, eds.), Vol. 4, pp. 429-452. Elsevier, Amsterdam. Rozengurt, E., and Collins, M. (1983). Molecular aspects of growth factor action: Receptors and intracellular signals. J . Parhol. 141, 309-331. Rozengurt, E., and Courtenay-Luck, N. (1982). Signalling mitogenesis in 3T3 cells: Role of monovalent ion fluxes and cyclic nucleotides. Biosci. Rep. 2, 589-595. Rozengurt, E., and Heppel, L. A. (1975). Serum rapidly stimulates ouabain-sensitive n6Rb uptake in quiescent 3T3 cells. Proc. Narl. Acad. Sci. U . S . A . 72, 4492-4495. Rozengurt, E., and Mendoza, S. A. (1980). Monovalent ion fluxes and the control of cell proliferation in cultured fibroblasts. Ann. N. Y. Acad. Sci. 337, 175-190. Rozengurt, E., and Mendoza, S. A. (1985). Synergistic signals in mitogenesis: role of ion fluxes, cyclic nucleotides and protein kinase C in Swiss 3T3 cells. J. Cell. Sci., Suppl. 3, 229-242. Rozengurt, E., and Sinnett-Smith, J. (1983). Bombesin stimulation of DNA synthesis and cell division in cultures of Swiss 3T3 cells. Proc. Natl. Acad. Sci. U . S . A . 80, 29362940. Rozengurt, E., Legg, A., and Pettican, P. (1979). Vasopressin stimulation of mouse 3T3 cell growth. Proc. Natl. Acad. Sci. U . S . A . 76, 1284-1287. Rozengurt, E . , Brown, K. D., and Pettican, P. (1981a). Vasopressin inhibition of epidermal growth factor binding to cultured mouse cells. J. B i d . Chem. 256, 716-722.

190

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Rozengurt, E., Gelehrter, T. D., Legg, A., and Pettican, P. (1981b). Melittin stimulates Na entry, Na-K pump activity and DNA synthesis in quiescent cultures of mouse cells. Cell 23, 781-788. Rozengurt, E., Legg, A., Strang, G., and Courtenay-Luck, N. (1981~).Cyclic AMP: A mitogenic signal for Swiss 3T3 cells. Proc. Nail. Acad. Sci. U.S.A. 78, 4392-4396. Rozengurt, E., Collins, M., Brown, K. D., and Pettican, P. (1982). Inhibition of epidermal growth factor binding to mouse cultured cells by fibroblast-derived growth factor. J. Biol. Chem. 257, 3680-3686. Rozengurt, E., Stroobant, P., Waterfield, M. D., Deuel, T. D., and Keehan, M. (1983a). Platelet derived growth factor elicits cyclic AMP accumulation in Swiss 3T3 cells: Role of prostaglandin synthesis. Cell 34, 265-272. Rozengurt, E., Collins, M. K. L., and Keehan, M. (1983b). Mitogenic effect of prostaglandin E in Swiss 3T3 cells: Role of cyclic AMP. J . Cell Physiol. 116, 379-384. Rozengurt, E., Rodriguez-Pena, M., and Smith, K. A. (1983~).Phorbol esters, phospholipase C and growth factors rapidly stimulate the phosphorylation of a Mr 80,000 protein 80, 7244-7248. in intact quiescent 3T3 cells. Proc. Naif. Acad. Sci. U.S.A. Rozengurt, E., Rodriguez-Pena, A., Coombs, M., and Sinnett-Smith, J. (1984). Diacylglycerol stimulates DNA synthesis and cell division in mouse 3T3 cells. Role of Ca2+sensitive phospholipid-dependent protein kinase. Proc. Nail. Acad. Sci. U.S.A. 81, 5148-5152. Sapirstein,.V. S., and Benos, D. J. (1984). Activation of amiloride-sensitive sodium transport in C6 glioma cells. J. Neurochem. 43, 1098-1105. Schuldiner, S., and Rozengurt, E. (1982). Na+/H+antiport in Swiss 3T3 cells: Mitogenic stimulation leads to cytoplasmic alkalinization. Proc. Nail. Acad. Sci. U.S.A.79,77781182. Segal, G . B., Simon, S., and Lichtman, M. A. (1979). Regulation of sodium and potassium transport in phytohemagglutinin-stimulatedhuman blood lymphocytes. J . Clin. Invest. 64, 834-841. Smith, G. L. (1977). Increased ouabain-sensitive %ubidium uptake after mitogenic stimulation of quiescent chicken embryo fibroblasts with purified multiplication stimulating activity. J . Cell Biol. 73, 761-763. Smith, J. B., and Rozengurt, E. (1978a). Lithium transport by fibroblastic mouse cells: Characterization and stimulation by serum and growth factors in quiescent cultures. J . Cell. Physiol. 97, 441-450. Smith, J. B., and Rozengurt, E. (1978b). Serum stimulates the Na+/K+pump in quiescent fibroblasts by increasing Na+ entry. Proc. Natl. Acad. Sci. U . S . A . 75, 5560-5564. Stroobant, P., Gullick, W. J., Waterfield, M. D., and Rozengurt, E. (1985). Highly purified fibroblast-derived growth factor, an SV40-transformed fibroblast-secreted mitogen, is closely related to platelet-derived growth factor. EMBO J. 4, 1945-1949. Thomas, A. P., Alexander, J., and Williamson, J. R. (1984). Relationship between inositol polyphosphate production and the increase cytosolic free CaZ+induced by vasopressin in isolated hepatocytes. J. Biol. Chem. 259, 5574-5584. Todaro, G.J., and Green, H. (1963). Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J. Cell Biol. 17, 299-313. Tosteson, M. T., and Tosteson, D. C. (1981). The sting mellitin forms channels in lipid bilayers. Biophys. J . 36, 109-116. Tupper, J. T., Zorgniotti, F., and Mills, B. (1977). Potassium transport and content during G , and S phase following serum stimulation of 3T3 cells. J. Cell. Physiol. 91,429-440. Vanderburgh, H. H. (1984). Relationship of muscle growth in viiro to sodium pump activity and transmembrane potential. J . Cell. Physiol. 119, 283-295.

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191

Van Hoogvest, P., and de Kruijff, B. (1978). Effect of amphotericin B on cholesterolcontaining liposomes of egg phosphatidylcholine and didocosenoyl phosphatidylcholine. Biochim. Biophys. Acta 511, 397-407. Vara, F.,and Rozengurt, E. (1985). Stimulation of Na+/H+antiport activity by epidermal growth factor and insulin occurs without activation of protein kinase C. Biochem. Biophys. Res. Commun. WO, 646-653. Vara, F., Schneider, J. A., and Rozengurt, E. (1985). Ionic responses rapidly elicited by activation of protein kinase C in quiescent Swiss 3T3 cells. Proc. Natl. Acad. Sci. U.S.A.02, 2384-2388. Vicentini, L. M.,and Villereal, M. L. (1984). Serum, bradykinin and vasopressin stimulate release of inositol phosphates from human fibroblasts. Biochem. Biophys. Res. Commun. ll3,663-670. Villereal, M. L. (1981). Sodium fluxes in human fibroblasts: Effect of serum, Ca2+ and amiloride. J . Cell. Physiol. 107, 359-369. Waterfield, M. D., Scrace, G. T., Whittle, N., Stroobant, P., Johnsson, A , , Wasteson, A , , Westermark, B., Heldin, C.-H., Huang, J. S . , and Deuel, T. F. (1983). Platelet-derived growth factor is structurally related to the putative transforming protein p28sis of simian sarcoma virus. Nature (London) 304,35-39. Whitaker, M. J., and Steinhardt, R. A. (1982). Ionic regulation of egg activation. Annu. Rev. Biophys. 15, 593-666. and Glaser, L. (1984). Tumor promoter phorbol Whiteley, B., Cassel, D., Zhuang, Y.-X., 12-myustate 13-acetate inhibits mitogen stimulated Na+/H+exchange in human epidermoid carcinoma A 431 cells. J . Cell Biol. 99, 1162-1 166. Zachary, J., and Rozengurt, E.(1985). High-afhity receptors for peptides of the bombesin family in Swiss 3T3 cells. Proc. Natl. Acad. Sci. U.S.A. 82, 7616-7620. Zachary, J., Sinnett-Smith, J. W. and Rozengurt, E. (1986). Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. I Activation of protein kinase C and inhibition of epidermal growth factor binding. J . Cell Biol. (in press).

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CURRENT TOPICS IN MEMBRANES A N D TRANSPORT. VOLUMF. 27

Chapter 6

Volume-Sensitive Ion Fluxes in Amphiuma Red Blood Cells: General Principles Governing Na-H and K-H Exchange Trans ort and CI-HC03 Exchange Coup ing

P

PETER M. CALA Department of Human Physiology School of Medicine University of California, Davis Davis, California 95616

I. Introduction: The Role of Alkali Metal-H Exchange in Cell Regulatory

111.

IV. V. VI.

Conductive Alkali A. General Princ .......................... B. The Forces Driving Conductive and Electroneutral Transport Can Be Equal.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Volume-Sensitive Ion Fluxes in Amphiidma Red Blood Cells . . . . . . . . . A. Identification of Transport as Electroneutral Alkali Metal-H Exchange: Consideratio ........... and Membrane Voltage B. Characteristics of Net T CI-HCO, Exchangers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ca2+-Dependent Alkali Meta The Nature of Net Na Flux by Amphiuma Red Blood Cells in Hyperosmotic Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CI-HC03 Exchange and Its Functional Relationship to Alkali Metal-H Exchange, Alkali Metal-C1 Cotransport, and Parallel Alkali Metal and H or CI Conductance Pathways. ...................................... A. Stoichiometry between Alkali Metal and CI Ions and Buffer Power of Cells and Medium.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

195 197 198

198

205

207 207

193 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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PETER M. CALA

B. Alkali Metal-H Exchange versus Alkali Metal-C1 Cotransport. ........ VII. Activation and Control of Alkali Metal-H Exchange in Amphiuma Red Blood Cells.. ......................................................... A. Alkali Metal-H Exchange in Amphiuma Red Blood Cells: Volume-Dependent Ca2+Sensitivity. ................................ B. Protein Kinase C: The Basis for Synergy between Ca2+and Cell Volume? ......................................................... VIII. Summary. ............................................................ References ...........................................................

209 210 210 213 215 216

I. INTRODUCTION: THE ROLE OF ALKALI METAL-H EXCHANGE IN CELL REGULATORY PROCESSES

Following osmotic perturbation, animal cells, in general, are able to regulate volume to normal prestress levels (Fugelli, 1967; Kregenow, 1971a,b;Dellasega and Granthan, 1973; Parker, 1973a,b;Hendil and Hoffm a n , 1974; Weissenberg and Katz, 1975; Cala, 1977, 1980; Siebens and Kregenow, 1978; Vislie, 1980; Ellory and Dunham; Fisher et al., 1981; Grinstein et af., 1982a; Lauf, 1982). Since animal cell membranes are unable to support (or exert) hydrostatic forces, these regulatory volume changes are referable to changes in the number of intracellular osmotically active particles. In invertebrate cells having large free amino acid pools, changes in organic molecules are often responsible for alterations in intracellular particle number and therefore cell volume. Since in vertebrate cells the primary osmotically active particles are alkali metal and C1 ions, it is these ions that necessarily serve as osmotic effectors during vertebrate cell volume regulation. The recent interest in vertebrate cell volume regulation stems not only from the fact that it is a fundamental biological process, but that the volume-sensitive ion fluxes are large, dynamic, and versatile. With regard to the versatility of the ion transport pathways, it is generally observed that during volume regulation ions traverse the membrane by electroneutral pathways, predominantly alkali metal-H exchange or alkali metal-C1 cotransport. Functionally, similar pathways have been observed to participate in a variety of important regulatory and homeostatic processes which include (1) regulation of cell volume, (2) regulation of cell pH (Thomas, 1977), (3) fluid and electrolyte transport by epithelia (White, 1980; Weinman and Reuss, 1982), (4) regulation of the ionic milieu in the central nervous system, and (5) cell growth and development (L’Allemain et al., 1981; Rosoff and Cantley, 1983; Rothenberg et al., 1983; Vincentini et al., 1984).

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195

With regard to point (3, reports by a number of laboratories have shown that agents which produce an increase in cell Na and pH also cause increases in cell growth, division, and differentiation (Rosoff and Cantley, 1983; L’Allemain et al., 1984; Rosoff et al., 1984; Vicentini et al., 1984). It is generally believed that the Na transport resulting in increased cell Na and pH is via an Na-I-f exchange mechanism. The systems share characteristics which include amiloride sensitivity and Na-dependent changes in pH (Rindler et al., 1981; Rothenberg et al., 1983; L’Allemain et al., 1984; Rosoff et al., 1984). Further, there are numerous reports that the alkali metal-H exchange pathways associated with cell growth and development respond to changes in [Cali or exposure to phorbol esters (L’Allemain et al., 1984; Rothenberg et al., 1983; Rosoff Pt d.,1984; Vicentini et al., 1984; Berridge, 1984). In this chapter we will attempt to outline general principles of electroneutral alkali metal-H (Am-H) exchange and alkali metal-C1 (Am-CI) cotransport pathways. Particular attention will be given to (1) force-flow analyses, (2) the nature of H involvement, (3) the role of anions and the CI-HC03 exchange pathway, and (4) stimulus-response coupling. The data and examples presented will be drawn primarily from studies of volume-sensitive, electroneutral fluxes in erythrocytes. Due to the generality of the principles, analogy with transport processes and, indeed, stimulus-response coupling as described for developing cells should be apparent. II. THERMODYNAMIC PRINCIPLES OF ION TRANSPORT: ELECTRONEUTRAL VERSUS CONDUCTIVE ALKALI METAL ION FLUXES A. General Prlnclples of Force-Flow Coupllng

In order to predict the magnitude and direction of solute flux, we must identify the force to which the flix is coupled. The force is the rate of change of energy with distance. In the case of electrolyte transport by current-carrying pathways, the relevant energy term is that for electrochemical potential; El. = p + zFE, where is the chemical and E the electrical potential, while z and F have their usual meanings. When discussing membrane transport, the distance x over which /sL changes is the membrane thickness, and the force driving conductive ion flux is therefore -d& = = -R T d C

dx

C dx

+ zF-dE dx

196

PETER M. CALA

where R and T have their usual meanings, c i s the local concentration of the transported species, and d c / d x represents the local concentration gradient. Since the flux of an ion, Ji, is equal to (mobility) x (concentration) x (driving force), we have

where wi is the mobility of the ion in (cm/second)/(dyne/mol), Ci the concentration in mo1e/cm3, and dr;i/dx the force in dyne.cm/(mol/cm). Substituting Eq. (2) into Eq. (l),

dC

Ji

=

-wiRT

ZFCidE

(-fx + RT -) dx

(3)

where WiRT is the diffusion coefficient Di , in cm*/second. Therefore

Ji

=

-Di

(4 dCi + RzFC~dE X

T dx

(4)

Expression (4) is the Nernst-Planck equation, which describes the flux of ion i in terms of chemical and electrical forces. The above expression shows that the flux of i is a function of the chemical or diffusional drift of i driven by dC/dx and the electrical or imposed drift due to the electric field (dEldx). In the case of electrolyte transport when the membrane voltage is zero or when considering the transport of a nonelectrolyte, where the voltage is not a relevant driving force, the flux of i is solely due to the chemical drift and Eq. (4) reduces to

which is Fick’s Law. In addition to the processes described above, there are obligatorily (as opposed to functionally) coupled transport processes. This chapter will focus upon obligatorily 1 : 1 coupled (electroneutral) K-H and Na-H exchange transport. Since ion flux by such pathways is not capable of producing a current, the flux will not contribute directly to the membrane voltage, nor do the ions moving through such pathways respond to voltage as a driving force. As such, the force driving an electroneutral Na-H exchange is

Thus, the flux of Na by a Na-H exchange pathway ( J N ~ H can ) be expressed as

6. VOLUME-SENSITIVE ION FLUXES IN Amphiuma RED BLOOD CELLS

197

or

Equations (4) and (7) are analogous in that they are expressions describing net Na flux in terms of its conjugate driving forces. In both expressions the Na flux is a function of the chemical or diffusional drift of Na as reflected by the inclusion of the derivative of [Na] with distance. The electroneutral and conductive flux of Na differ with respect to the forces responsible for imposed drift. In the case of electroneutral Na-H exchange, the imposed drift results from a chemical force which is a reflection of d[H]ldx. In contrast, the imposed drift of Na transport by conductive routes is due to electrical energy and is represented by dEldx in Eq. (4). Both expressions (4) and (7) will reduce to that for Fick diffusion when the terms for imposed drift go to zero. A more important characteristic from an analytical perspective is the fact that when d[Na]ldx, = 0, the conductive flux of Na can be driven only by voltage, while an electroneutral Na-H exchanger can be driven by hydrogen ions. As the terms for imposed drift increase in magnitude relative to d[Na]ldx, the direction of net Na flux will be opposite that dictated by the Na gradient. In their present form expressions (4) and (7) are of little use for precise determina~ JN~H In. order for them to be useful analytically, it is tion of J N or necessary to integrate the expressions. The assumptions made in order to integrate Eqs. (4) and (7) will be highly model dependent. The expressions as they stand are, however, useful in that they illustrate principles governing transport. Thus, in the absence of analytical solutions to the formal expressions, it is possible to evaluate the mode of transport based upon the correspondence to the transmembrane difference in the potential energy terms. Since the potential energy is changing over the membrane H the forces driving thickness (distance), Aii or ApNa - A ~ constitute conductive Na flux and Na-H exchange flux, respectively. Thus, if the magnitude and direction of Na flux corresponded to A b N a and not A p N a A ~ Hthe , flux would be conductive. Correspondence to ApNa- A ~ and H not to A f i N a indicates that the flux is electroneutral Na-H exchange. B. The Forces Driving Conductive and Electroneutral Transport Can Be Equal

Before proceeding with examples, it is important to point out that there are circumstances where the force driving the conductive (ApAm+ zFE,)

198

PETER M. CALA

and electroneutral (Aph - A ~ Htransport ) of an ion are indistinguishable. Using the example of Na conductance, the relevant driving force is A p N a + ZFEm. In contrast, if Na flux is via Na-H exchange, the relevant force driving Na flux is A ~ -N A p~H . If, however, H is at electrochemical equilibrium, then by definition A j i ~= 0 = A ~ + H zFE,,,. Therefore, ZFE;, = - A p H and A p N a + ZFEm = A ~ -NA p ~H . In other words, there will be equality between the driving forces for Na conductance and electroneutral Na-H exchange when H is at electrochemical equilibrium. Similarly, the force driving Na conductance ( A p N a + #Em) will be equal to that driving electroneutral Na-Cl cotransport ( A p N a + ApcJ when C1 is at electrochemical equilibrium. At electrochemical equilibrium for C1, A ~ C=I 0 = A p a + ZFEm and (because zc1= - 1) Apc1 = ZFEm. Thus, if Cl is distributed at equilibrium, A p N a + Apc~(the driving force for Na-C1 cotransport) is equal to A p N a + ZFE, (the driving force for Na conductive flux). More generally, the forces driving electroneutral Am flux and Am conductance are indistinguishable if the ion to which the Am ion is coupled is at electrochemical equilibrium. It is possible to distinguish between conductive and electroneutral transport employing the force-flow analysis outlined above even in the case when the co- or counter-ion is normally equilibrium distributed if (1) the membrane potential is altered using a nonequilibrium distributed ion and (2) Am flux is measured before the co- or counter-ion is able to relax to its new equilibrium position. Indeed, this approach was employed in order to establish that volume and catecholamine-activated alkali metal and C1 fluxes in duck red cells are via electroneutral Am-Cl cotransport pathways (Haas et al., 1982) and that volume and Ca2+-sensitive ion fluxes in Amphiuma red blood cells are the result of electroneutral Am-H exchange (Cala, 1983b). 111.

VOLUME-SENSITIVE ION FLUXES IN Arnphlurna RED BLOOD CELLS

A. ldentlflcatlon of Transport as Electroneutral Alkall Metal-H Exchange: Conslderatlons Based upon Measurements of Net Ion Flux and Membrane Voltage

Subsequent to suspension in hypotonic medium, Amphiuma red blood cells swell osmotically, while suspension in hypertonic medium results in osmotic shrinkage. Following the osmotic phase there is a “regulatory” phase which results in the return of cell volume to normal levels (Siebens and Kregenow, 1978). In response to both swelling and shrinkage the

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199

"regulatory" water fluxes are secondary to net Am and C1 ion flux. In response to cell swelling, regulatory volume decrease (RVD) is mediated by net loss of K and C1 (Fig. 1A). In contrast, subsequent to osmotic shrinkage (hypertonic media), regulatory volume increase (RVI) is the result of net uptake of Na, C1, and osmotically obliged H20 (Fig. IB). The volume-sensitive Na and K fluxes are orders of magnitude greater than the steady-state fluxes of these ions in isotonic media and are unaffected

, 60

30

90

240 ' 0

\

'

I4O

1

I

60 40

30 60 Minutes

90

u

2o 0

30

60

90

Mlnutor

FIG.1. The ion and water content of Amphiuma red blood cells as a function of time after suspension in (A) hypotonic and (B) hypertonic medium. The content is expressed as liters or mmol/kg dry cell solid (dcs). Immediately following cell swelling (A) the cells begin to lose K, CI, and osmotically obliged water. The K loss is often in excess of CI. Following The Na uptake is often sigmoidal with time cell shrinkage (B) the cells gain Na, CI, and H20. and is inhibitable by the diuretic drug amiloride over the range of 5 x lo-' to M .Neither the response to swelling nor that to shrinkage are altered by exposure of cells to ouabain. The volume-induced K (A) and Na (B) fluxes are orders of magnitude greater than the dissipative fluxes of these ions in isotonic medium, whereas inhibition of the Na,K-ATPase results in net Na uptake and K loss on the order of 3 to 5 mmole/kg dcs in an hour. The osmolarity of isotonic medium is 240 m o m , while the hypotonic medium (RVD)is 150 mosm and the hypertonic medium is 360 mosm. Unless otherwise specified, these are the osmolarities used for isotonic, hypotonic, and hypertonic media, respectively. (From Cala, 1985.)

200

PETER

M. CALA

by ouabain (Cala, 1980). As such, some event associated with changes in cell volume results in the induction of robust net Na or K flux pathways. In order to understand the nature of the volume-sensitive ion fluxes, electrical studies were performed upon cells in iso-, hypo-, and hypertonic media in the presence and absence of the K ionophore valinomycin (Cala, 1980). Briefly, when cells were osmotically swollen, net K flux was as large as 100 mmole/kg dry cell solid (dcs) in 15 minutes, yet the membrane potential, measured directly with microelectrodes, was unchanged. Similarly, Na fluxes of the same magnitude by shrunken cells were without effect upon Em.Yet exposure of cells to valinomycin, which increases K conductance, caused the membrane to hyperpolarize from its normal value of -23 mV to between -40 and -50 mV. This 30-mV hyperpolarization was the result of a valinomycin-induced K flux of 2-5 mmole/kg dcs in 15 minutes. Further, the valinomycininduced K flux and membrane hyperpolarization were the same regardless of cell volume. Since the pathway that carries the most current will contribute most to the membrane potential and Emresponds to the modest valinomycin-induced K flux but not the much larger volume-induced fluxes, we must conclude that the latter do not contribute to the membrane current. Also, since the valinomycin-induced AEm is the same at all volumes (regardless of the magnitude or direction of volume-induced cation flux), we conclude that the ratio of the valinomycin-induced current to total membrane current is constant, as is the ratio of the valinomycininduced membrane conductance to total membrane conductance. Since the valinomycin-induced current and conductance are constant regardless of cell volume (as seen from the volume-independence of the valinomycin-induced AE,,,), so too are the total membrane current and conductance. In light of the above, the volume-induced K, Na, and C1 fluxes must be nonconductive and the membrane conductance must be constant and independent of volume (Cala, 1980, 1983b). While the above observations argue that the volume-sensitive fluxes are electroneutral, the basis for electroneutrality was unclear. That is, are cation and anion fluxes electroneutral because of obligatory 1 : 1 coupled cation-anion cotransport or cation-cation and anion-anion exchange pathways? If net ion flux were due to alkali metal-C1 cotransport, the stoichiometry between Am and C1 ion fluxes should be 1 : 1. Yet net Am and C1 fluxes are only equal under very special circumstances (see Section VI). Further, if the process is by Am-Cl cotransport, then inhibition of net C1 flux should stop net Am flux, yet net Am flux persists when C1 flux is blocked by the anion transport inhibitor DIDS. In addition, when net C1 flux is inhibited by DIDS, net Na uptake by osmotically shrunken cells is exactly equal to H extrusion. In summary, (1) electrical studies in

6. VOLUME-SENSITIVE ION FLUXES IN Arnphiurna RED BLOOD CELLS

201

the absence and presence of valinomycin established that the volumesensitive fluxes were electroneutral but did nor distinguish between AmH exchange and Am-CI cotransport; (2) the studies of net Am flux and its correlation with net H and CI fluxes in the presence and absence of DIDS established that the pathways were electroneutral by virtue of obligatory exchange of Am for H ions.

B. Characteristics of Net Transport by Parallel Alkali Metal-H and CI-HCOa Exchangers The models depicted in Figs. 2A and B were proposed to explain the experimental results described above. Am and H ions are obligatorily coupled, accounting for the electroneutral flux of Am ions. As a result of the H fluxes consequent to such a pathway, pH and therefore [HC03] is

A CELL

OUTSIDE

CELL

OUTS I DE

ncoj n

C

0

3

T

H

C

0

3

-

ANION

ANION

C1Nei K

aC\ Fluxes

OjNET K

CYCLIC

Net NO

OjNET

FLUXES OF H+

a CL F l u x e s

IjNET

Cl

IjNET

NO

a nco3-

CYCLIC

FLUXES OF

c1

n+ a iico,-

FIG.2. Depiction of the details of the volume regulatory ion flux pathways as inferred from ion flux and electrical measurements performed on osmotically perturbed Amphiuma red blood cells. The model would have alkali metal ions obligatorily coupled to H and CI coupled to HCO,. While H and HCO, serve as counterions for alkali metal and CI ions in an electrical sense, H and HCO, are without osmotic consequence because they cycle through the membrane alternately as H and HCO? or C 0 2 and H20. The electroneutral anion and cation transport pathways are functionally coupled via H rather than the membrane potential, as is the case for conductive anion and cation flux. Pathway (A) represents the obligatorily coupled K-H exchange responsible for volume regulation following osmotic swelling (regulatory volume decrease, RVD). Pathway (B) represents the Na-H exchange responsible for volume regulation subsequent to osmotic shrinkage (regulatory volume increase, RVI). The correspondence between net Na and CI fluxes during RVI is generally closer than that between K and CI during RVD, where net K loss can exceed that of CI by a factor of 5 (for discussion, see Section VI).

202

PETER M. CALA

altered. To the extent that [HC03] is altered, the force driving the C1HCO3 exchanger [(AFHCO~ - Apd/dx] is displaced from zero, and net anion flux through the anion exchanger occurs. Finally, H+ and HCO;, while serving as counter ions for Am and C1 in an electrical sense, are without osmotic consequence, because they cycle through the membrane alternatively as H and HC0;or HzO and COZ(there is no net change in [HI or [HC03] on either side of the membrane). If the proposed model is correct, one would expect that to the extent that Am flux exceeds that of C1, changes in pH of poorly buffered media should occur. This prediction, which is consistent with, yet not unique, to the model, was observed experimentally. It was also observed that increasing HC03from < 1 to 42 mM (at fixed pH,) resulted in stimulation of net Am flux (Cala, 1980). Initially, this observation was interpreted in terms of HCO3 flux via the HC03-Cl exchange and its importance as a means of buffering changes in pH generated via the Am-H exchangers. Recent studies showing that Am flux is stimulated by HC03 even when the C1-HCO3 exchanger is inhibited by DIDS have prompted the alternative interpretation that the Am-H exchange pathways are HCOTsensitive (Cala, unpublished observations). Finally, reports by Siebens and M was able to Kregenow (1978) had shown that amiloride at lop4to inhibit Na flux in osmotically shrunken Amphiuma red blood cells, and our studies confirmed this initial observation and identified the Na flux pathway as a Na-H exchange (Cala, 1980).

IV. Ca2+-DEPENDENTALKALI METAL ION FLUX IN Arnphiurna RED BLOOD CELLS

Having determined that the volume-sensitive ion fluxes were by electroneutral alkali metal-H and Cl-HCO3 exchangers, attention was directed to understanding their control. Based upon previous observations by Lassen et al. (1974, 1976, 1978, 1980) which showed that (1) K loss by Amphiuma red cells was Ca2+sensitive and (2) net K loss was larger than expected based upon estimates of membrane conductance, it was hypothesized that Caf+may stimulate electroneutral K-H exchange. Interpretation of studies evaluating the role of Cq2+ was more complicated than the previously described studies on volume-perturbed cells (Section II1,A) because, like many other cell types, the Amphiuma red blood cell has a Caf+-activated K conductance (Gardos, 1956; Gardos et al., 1976). As such, Caf+-activatedK loss is accompanied by membrane hyperpolarization, which is dependent upon [K],. Because the Em of cells exposed to the Ca2+ ionophore A23187 varies by 40 mV/decade A[K], yet in the

6. VOLUME-SENSITIVE ION FLUXES IN Amphiuma RED BLOOD CELLS

203

absence of A23187 E m is virtually K independent, it is clear that A23187 exposure (increased [Ca2+Ii) activates a K conductance pathway. Thus, in order to evaluate the ability of increased [Ca2+]ito activate K-H exchange, it was necessary to distinguish between K flux via conductive versus electroneutral K-H routes. While not without utility, measurements of Em in A23187-treated cells are less useful (more ambiguous) as a means of distinguishing between conductive and electroneutral transport than was the case for swollen cells (Cala, 1980, 1983b). In order to avoid such ambiguity, a force-flow analysis (Cala, 1983a,b; see also Section 11) was employed as a means of testing the hypothesis that increasing [Ca2+Ii of Arnphiurna red blood cells induces potassium flux via both conductive and electroneutral routes, the latter being responsible for measurable net K flux. Briefly, it was reasoned that if the Ca2+-activatedK flux occurred via K-H exchange, net K flux (Jp) should respond to A ~ -K ApH.If, however, the A23187-induced JFet was conductive, the flux should be coupled to A l ; l ~as its driving force. The flux data in Figs. 3A and B were obtained from osmotically swollen A23187-treated cells. In Fig. 3A the flux plotted against A ~ isKseen to decrease with decreasing A ~ Kyet , the flux changes direction before A / i K changes sign. In contrast, plotting the same values of Jf;l"'against h p -~ A ~ shows H that Jp (1) decreases with A p K - A ~ H (2) ; is zero when ApK - A ~ isH zero; and (3) reverses direction as A ~ -KA ~ changes H sign. The decrease in Jpt (Figs. 3A and B) with both A ~ (where K A f i ~= A ~ +K ZFEm) and A ~ -K A ~ isH a reflection of the fact that the magnitudes of both expressions depend upon A ~ (RT K In[K]i/[K],), which is the dominant term when [K], is small (large positive values for A j i K and A ~ -K A ~ H )As . A ~ . decreases K [and ( A ~ K- A ~ H equals ) zero] Jp = 0. In contrast, decreasing A ~ so K that A ~ +KzFE, approaches zero results in a reversal of the direction of net K flux before A ~ + K iFEm goes to zero. Under conditions where net K uptake occurs (negative values for Jp in Figs. 3A and B), both A p K and zFEmfavor net K loss, while both the magnitude and direction of A p H are such to promote net K uptake. As such, the net K uptake is driven solely by A ~ and H must therefore be the result of K-H exchange. Since, as previously shown (Lassen et al., 1976, 1980; Cala, 1983b), increased [Ca2+]i causes a K-dependent membrane hyperpolarization, we conclude that there are two components of Caf+-activated net K flux: an electroneutral component due to K-H exchange which is responsible for the measurable net K flux and a conductive component which, while too small to produce a measurable net K flux, is capable of hyperpolarizing the membrane. In Amphiurna red cells H, C1, OH, and HC03 are all distributed at electrochemical equilibrium. By definition, the chemical potential differences for all of these ions are equal in magnitude to zFE,. Consequently,

A HYPOTONIC MEDIA 7pM A23187 500pM CO [K], =3-70mM

40-

30 -20

--

lo -I

-2000

2000

-30 -40

'

4&0(jou1es APK mole-')

t

JN: (rnrnoles/kg dcsxl5 r n i n )

J (mrnoles/ka d c s x l 5 rnin)

HYPOTONIC MEDIA 7aM A23187 5OOpM CO [K], =3-70mM

B

,,i 20

-30

t

-401

J (mrnoles/kg dcsxlb rnin)

FIG.3. Force-flow relations for net K flux associated with cells osmotically swollen in the presence of A23187. In both (A) and (B) the K flux is the same, yet in (A) flux is plotted against A/.iK, the force driving K conductance; (B) the same flux data are plotted against A p K - A p H ,the force driving K flux via K-H exchange. The magnitudes of A/iK and A ~ -KA p H are varied by replacing "a], with [K], at fixed osmolarity. (From Cala, 1985.)

6. VOLUME-SENSITIVE ION FLUXES IN Arnphiurna RED BLOOD CELLS

205

as discussed in Section II,B the forces driving K-H exchange ( A ~ KA ~ H ) K-CI , cotransport ( A ~ K+ A p a ) , and conductive K flux (ApK + ZFE,) are equal. In the above example a K-CI cotransport as a possible route for net K loss is eliminated because K loss can occur in the absence of net C1 flux. Distinction between net K flux by conductance and K-H exchange pathways based upon force-flow analysis is possible because, as a result of Caf+-dependentmembrane hyperpolarization, - A ~ H# zFE, and therefore A ~ -K A ~ #H A ~ +K zFE, (see Section 11,B). Furthermore, since the time for H redistribution (via the H conductance) is long with respect to experimental time, H is away from electrochemical equilibrium ( - A ~ H# zFE,) and virtually unchanged throughout the experiment (see Cala, 1983a,b). As such, the force-flow analysis as applied above is performed in accordance with conditions outlined in Section II,B. In keeping with these principles all subsequent studies employing force-flow analysis were performed upon cells in which Em is altered by exposure to valinomycin. V. THE NATURE OF NET Na FLUX BY Amphiuma RED BLOOD CELLS IN HYPEROSMOTIC MEDIA

The force-flow analysis illustrated above can also be applied to Na uptake by osmotically shrunken cells. Previously, based upon electrical measurements and ion flux, it was concluded that net Na flux in shrunken cells was via electroneutral Na-H exchange (Cala, 1980). The validity of this conclusion can be tested using force-flow analysis. Figure 4 depicts H A j i for ~ ~osmotically the relationship between JE;t and ApNa - A ~ and shrunken cells. Note that relative to Fig. 3 the axis in Fig. 4 has been rotated by 180". The sign convention is the same in both Figs. 3 and 4 so that a negative sign denotes force and flow directed into the cell. The magnitude of ApNa - A ~ and H A/i;i~~ were varied by replacing external Na ("a], 5 to 140 mM) with tetramethylammoniumand valinomycin (2 p M ) was added to the cell suspensions to hyperpolarize the membrane (for reasons outlined in Sections II,B and IV,A) so that - A ~ H# zFE,. Because [K]i was constant at = 155 mM and [K], was constant at 3 mM, Em was -50 mV at all values of "a], . The curve describing the relationship H that JE;' is near zero when ApNa between JgEt and A ~ - NA ~~ shows A ~ isH zero. In contrast, the relationship between J!;' and A/iNa shows that JE;' is zero when A/iNa = -2100 J mol-'. Since a flow must go to zero when its conjugate force is zero and reverse direction when the force changes sign, the above correspondence argues that ApNa - A ~ isH the force to which Na flux couples and therefore, that net Na flux by

PETER M. CALA

206 in JN,

(mmolelkg dcsx20 min)

'No

-140--

ValI

-120-100-

- 80- 60 --40-

1

-3000

I

-5000

I

, (J mol-1)

-7000

FIG.4. Force-flow relation for Na flux by osmotically shrunken Amphiuma red blood ~ attempts to fit the net Na flux to the relevant driving cells. The curve labeled A , i i ~represents force for Na conductance. The curve labeled ApNa - A p H shows relation of net Na flux to are varied by replacing the relevant driving force for Na-H exchange. AbNa and A p N a "a], with [K],at fixed osmolarity.

shrunken cells is via Na-H exchange. It is also possible to vary the value of A ~ atN which ~ there is zero net Na flux by varying A ~ H (data , not shown). Since ApH is normally directed out of the cell, net Na uptake continues to occur even when A l ; i ~=~0 ([Nali = [Na],). If, however, A ~ H is set to zero, net Na uptake ceases when A ~ =N0. In ~ contrast to its dependence upon A ~ HJ3Et , is insensitive to the magnitude and sign of Em.Given the conditions described above, the correlation between J#Et and A ~ -NApH ~ is unique to Na-H exchange. While more complicated from an experimental point of view, the interpretation of such studies is not clouded by the uncertainties associated with attempts to identify Na-H exchange based upon measurements of pH or amiloride sensitivity. Amiloride or pH sensitivity of Na flux, while consistent with the operation of a Na-H exchange mechanism, does not rule out other possibilities such as parallel Na and H conductive fluxes (see Section VI).

6. VOLUME-SENSITIVE ION FLUXES IN Arnphiume RED BLOOD CELLS

207

VI. CI-HCOs EXCHANGE AND ITS FUNCTIONAL RELATIONSHIP TO ALKALI METAL-H EXCHANGE, ALKALI METAL-CI COTRANSPORT, AND PARALLEL ALKALI METAL AND H OR CI CONDUCTANCE PATHWAYS A. Stofchlometry between Alkali Metal and CI Ions and Buffer Power of Cells and Medium 1. ALKALIMETAL-H A N D Cl-HCO3 NET TRANSPORT: THEROLEOF BUFFERS

In contrast to conductive transport where cations and C1 are coupled through membrane voltage, the coupling between Am and C1 ions observed studying Am-H exchange processes is via H or, more accurately, changes in [HCO3] and pH. At fixed PCO2, changes in cell or medium pH will result in altered [HC03]. Because net flux via the anion exchanger is O ~A p c ~ ,any changes in dependent upon a nonzero value for A ~ H C [HC03]will result in the net exchange of C1 for HC03 until a new equilibrium is reached. Conversely, [HC03] will remain unchanged and there will be no force to drive the Cl-HC03 exchanger as long as cell and medium buffers are able to buffer the H moving through the Am-H exchanger. As such, if the net Am and CI fluxes are via parallel Am-H and Cl-HCO3 exchangers, the stoichiometric coupling of net Am and CI fluxes will be a function of the nonbicarbonate buffer power of the system. As the buffer power is increased, changes in pH and [HC03]as a result of H flux through the Am-H exchange will be minimized and net Am and CI fluxes will uncouple. Conversely, as the buffer power is decreased, Am and C1 ions will couple at 1 : 1.

2. ALKALIMETAL-Cl COTRANSPORT While in the case of parallel Am-H and CI-HC03 exchangers Am : CI stoichiometry will be determined by factors affecting C1-HCO3 exchange, the anion exchange will also affect the ratio of net Am to C1 fluxes if AmCl cotransport is in parallel with CI-HC03 exchange. In the example of , parallel Am-H and Cl-HCO3 exchangers, H flux alters A ~ H c o ~displacing A p ~ c o ~A p c I (the driving force for CI-HC03 exchange) from zero and causing the anion exchange to cycle. In the case of parallel Am-CI and C1-HCO3 exchangers, net C1 flux via the cotransport pathway alters A ~ Cand I therefore the value of A ~ H C O ~A p c l . The net result with respect to net Cl-HC03 exchange flux will be the same as that described

208

PETER M. CALA

for Am-H exchange, in that the side of the membrane from which net Am transport occurs will be acidified due to HC03 removal (see Fig. 5 ) . The magnitude of the net Cl-HC03 exchange flux necessary to restore A ~ C-I ApHco3 to zero will be a function of the system buffer capacity. As buffer capacity is increased the magnitude of net Cl-HC03 exchange flux necessary to change pH and [HC03] ( A ~ H c o ~will ) increase because the H equivalents generated by HC03 removal will be buffered. At fixed PCO;! HC03 will be regenerated (on the side of the membrane from which it is being removed by the Cl-HCO3 exchanger) and a large net Cl-HCO3 exchange flux will be necessary to reestablish equilibrium for the CI-HC03 exchanger (Apcl - A ~ H C = O 0). ~ In contrast, as buffer capacity is decreased, C1 back flux through the CI-HCO3 exchange will alter pH and therefore [HC03] with minimal net Cl-HCO3 exchange flux. Consequently¶ net flux via the Cl-HCO3 exchange will be small and the ratio of net Am to C1 fluxes via Am-Cl cotransport will approach its limiting value of unity. In summary, the parallel operation of a CI-HCO3 exchanger with a Am-H exchanger or a Am-Cl cotransport pathway will result in a buffer power-dependent , apparent Am-to-C1 stoichiometry . However, the magnitude of net Cl-HCO3 exchange flux relative to net Am-H exchange or Am-C1 cotransport will be a different function of buffer capacity. In the case of parallel Am-H and Cl-HCO3 exchanges, the net C1-HCO3 exchange flux (relative to Am-H) will be increased as buffer capacity is decreased. In contrast, the net Cl-HCO3 exchange transport relative to that of net Am-Cl cotransport will increase as buffer capacity increases. The implications of this behavior as it relates to changes in pH associated with net Am flux and identification of the pathway responsible for net Am flux will be discussed in Section VI,B,l. ¶

3. MEMBRANES WITH HIGHALKALIMETALAND H CONDUCTANCE Similar considerations regarding the correspondence between net Am and C1 fluxes as well as changes in pH will govern the stoichiometry of net Am-to-C1 fluxes if the membrane has high conductance to Am+ and H+ and a robust Cl-HCO3 exchanger. As such, identification of transport mode based upon pH changes or Am : C1 stoichiometry is far from conclusive. Electrophysiological and/or force-flow analyses are necessary in order to eliminate conductive transport as a possibility (Cala, 1980, 1983a,b; Grinstein et al., 1982b; Hass et al., 1982). In the case of all modes of net Am flux described above, the net flux of Am is dependent upon Ap.,t,,,,. Identification of the nature of net Am transport as conduc-

6. VOLUME-SENSITIVE ION FLUXES IN Amphiuma RED BLOOD CELLS

CI Am ( 4 pH)

<x -,::I

209

( ? pH)

HCO3

FIG.5 . Diagram depicting coupling between K-CI cotransport pathway and CI-HCO, exchanger in a membrane having both pathways. As a result of net KCl cotransport, ApcI will change and the Cl-HC03 exchange will be driven. As a consequence the cell will regain CI (lost by the K-CI cotransport pathway) in exchange for HCO, and the suspension medium will become alkaline.

tive, Am-H exchange, or Am-CI cotransport relies upon establishing zFE,,,, A ~ Hor, A g a , respectively, as the coupled force.

B. Alkali Metal-H Exchange versus Alkali M e t a l 4 Cotransport 1. INHIBITION OF CI-HCO3 EXCHANGE TRANSPORT AND CHANGES IN pH

Assuming that a conductive mode of transport has been eliminated, it is possible to distinguish between Am-H exchange and Am-CI cotransport based upon pH changes associated with net Am flux if CI-HC03 exchange is inhibited. As first pointed out by Kregenow (1981), if Am flux were via Am-H exchange, inhibition of CI-HC03 exchange would decrease net C1 flux relative to net Am flux and increase the apparent net H flux (based upon measurements of pH) relative to net Am flux. In contrast, if transport were by Am-CI cotransport, inhibition of CI-HC03 exchange would increase net C1 flux relative to net Am flux and decrease net H flux relative to net Am flux. That is, in the case of parallel Am-H and Cl-HC03 exchangers, the C1-HCOJ exchanger will tend to bufferpH changes resulting from net Am-H exchange flux. In contrast, if Am-CI cotransport and CI-HC03 exchange are operating in parallel, the ClHCOJ exchange will give rise to changes in pH resulting from net CI backflux through the CI-HC03 exchanger. In light of the above, it is possible to distinguish between parallel AmH and C1-HC03 exchange fluxes and parallel Am-CI cotransport and CIHC03exchange based upon pH changes secondary to net Am flux in the presence and absence of CI-HC03 exchange inhibition.

210

PETER M. CALA

A MEANSOF IDENTIFYING ELECTRONEUTRAL ALKALIMETALION TRANSPORT ARE IF ALKALI METALIONFLUXES DIFFICULTTO INTERPRET AREVOLUMEAND/OR pH SENSITIVE

2. ANIONREPLACEMENT STUDIES AS

Studies attempting to determine transport mode by ion replacement, especially in volume- and pH-sensitive systems having a Cl-HC03 exchanger, must be carefully controlled and interpreted with caution. If the anion used as a C1 replacement is unable to move on the Cl-HC03 exchanger, the cell will lose C1 in exchange for HC03 uptake. As a result, both cell volume and internal pH will change as volume decreases and pHi becomes more alkaline. One such example of an anion unable to move on the Cl-HCO3 exchange is gluconate, In addition to pH and volume changes resulting from gluconate replacement, gluconate complexes Ca2+ (unpublished observation). Thus, studies using gluconate as a C1 replacement must be controlled for changes in volume, pH, and free Ca2+.Two other ions commonly used to replace C1 are SCN and NO3. These anions will move on the Cl-HCO3 exchanger but will also permeate the membrane via conductive routes. Our experience with Amphiuma red blood cells has shown that substitution of these ions for Cl result in inhibition of the rate of Am-H exchange. Because SCN and NO3 are inhibitory even when the C1-HC03 exchanger is blocked by DIDS, it would appear that the inhibitory effects are not so much due to C1 removal but to a more direct interaction of NO3 and SCN with Am-H exchange. In this regard Parker, studying Na-H exchange in dog red blood cells, has recently made similar observations concerning the inhibitory effects of SCN (Parker, 1984). Based upon cleverly designed studies he concludes that SCN inhibits activation of Na-H exchange but not its operation once activated. In light of the above, inhibition of net Am flux by replacement of C1 with NO3 or SCN does not constitute proof that Am flux is via AmC1 cotransport. VII. ACTIVATION AND CONTROL OF ALKALI METAL-H EXCHANGE IN Amphiuma RED BLOOD CELLS A. Alkall Metal-H Exchange In Amphiuma Red Blood Cells: Volume-Dependent Ca2+ Sensltlvlty

The Am-H exchange pathways in Amphiuma red blood cells can be activated by C q + . If C$+ is increased with A23187, Am-H exchange is stimulated. The Caf+-dependent stimulation is much greater for K-H than Na-H exchange. Caf+-dependent K-H exchange by swollen cells

6. VOLUME-SENSITIVE ION FLUXES IN Arnphiurna RED BLOOD CELLS

21 1

can result in a loss of almost 80% of the cell K in 15 minutes. In contrast, exposure of shrunken cells to A23 187 in medium containing 500 pM Ca2+ can result in a 20% stimulation of Na-H exchange, but only under conditions when net Na-H exchange flux is modest. As the Na-H exchange rate is increased by progressively decreasing cell volume, the ability of Cat+ to stimulate Na-H exchange is decreased (Fig. 6). Thus, while Cat+ is a stimulus for both Na-H and K-H exchange, the latter pathway exhibits a more profound Ca2+-sensitivity. Cat+ is able to activate K-H exchange in swollen cells and cells at normal volume. Figures 7A and B depict K loss from cells in is0 (A) and hypotonic medium (B) when exposed to a fixed concentration of A23187 (7 pM)and Ca:+ is varied from 50 to 1000 pM.Clearly, the Caf+stimulation is greater for the swollen cells than for those cells in isotonic medium. Thus, it appears as if the physiological stimulus (cell swelling) primes the cells to respond to Cat+ (Cala et al., 1986). As has been reported for other Cat+-mediated responses, most notably those studied in platelets (Sanchez et d , 1983), there is a synergy between Cat+ and the “physiological” stimulus. Assuming that A23 187-mediated Ca2+transport is volume independent and not rate limiting, then at fixed [A231871and a given [Ca2+],, intracellular [Ca2+]is the same regardless of cell volume. As such, we can compare the Cat+-induced K-H exchange rates in swollen cells and with those in isotonic medium. Figure 8 is a plot of 1 / @ ’ versus 1/[Ca2+],. These data show that the apparent K , for Cat+ is decreased by a factor of approximately 7 in swollen cells. Thus, while Cat+ is clearly a mediator of K-H exchange flux in Amphiumu red blood cells, there are other bio-

loo

r

R V I + A 2 3 / 8 7 / / 5RI

RVI+A23/87//3RI

Minutes

FIG.6. The effect of A23187 and progressively hypertonic medium upon cellular Na content of osmotically shrunken cells. Curves labeled 1.3 R represent cells in medium which is 1.3 times more concentrated than isotonic (240 mosm). The curves labeled 1.5 R represent results obtained from cells placed in medium with osmolarity 1.5 times that for control cells.

21 2

PETER M. CALA

A 220 in

[Co], p M

[A23187], p M

500

0

50

7

250

7

500

7

1000

7

200

0

U

2

180

\

Y

2 160

E

140 I20

15

0

200

30

60 Minutes

90

t

7

-"O

15

30

60 Minutes

90

FIG.7. Change in K content of cells suspended in (A) isotonic and (B) hypotonic medium and exposed to fixed [A231871 and variable [Ca],. For both swollen cells and those in isotonic medium, exposure to 500 p M Ca in the absence of A23 187 (control) is indistinguishable from exposure to 50 p M Ca and 7 p M A23187. (From Cala, 1983b.)

6. VOLUME-SENSITIVE ION FLUXES IN Amphiuma RED BLOOD CELLS

21 3

I R ( r = 99) Km’=l.9mM

[K], = 3 mM

5

RVD ( r = . 9 6 7 ) Km’= 0 2 7 m M

--

~ J ~ a X ~ 1 3 0 m m o l e dcs / k g

&

-5 - 4 -3 - 2 -I

0 I 2 3 4 5 6 7 8

FIG.8. Double reciprocal plot of net K flux against external [Ca]. The data were obtained in paired experiments on cells from the same animal. One group of cells was osmotically swollen in hypotonic medium (RVD), while the other group of cells was exposed to isotonic medium (IR). In both groups 7 pM A23187 was present and medium Ca was vaned from 250 to loo0 pM. While [Ca], # [Cali, it is assumed that Cai is dependent only upon [Ca], and [A23187]. As such the absolute values of K6 are without meaning. yet their relative values are significant. (From Cala et a / . , 1986.)

chemical events associated with changes in cell volume which increase the Cqz’ sensitivity of the K-H exchange apparatus. B. Protein Klnase C: The Basis for Synergy between Ca2+and Cell Volume?

A variety of Ca2+-stimulatedmembrane events associated with secretion, cell growth and development, and ion transport appear to be dependent upon phospholipid turnover and protein kinase C (Nishizuka, 1981; for review, see Berridge, 1984). As suggested by Sanchez ef al. (1983), the synergy between the receptor and Ca2+-activatedresponses may be referable to the kinase C. Given our observations with Ca2+,we investigated the possible role for kinase C by exposing cells to analogs of diacylglycerol (diacylglycerol is rate limiting in kinase C-dependent protein phosphorylation). Figure 9 depicts the effect(s) of the diacylglycerol analog 13-acephorbol myristol acetate (PMA), 12-0-tetradecanoyl-4-p-phorbol

PETER M. CALA

21 4

140

10-7

5

I20

Na

100

10-8

80 60 40

-

*O 0

10-7

10-8

30 60 MINUTES

90

or lo-' M 12-0FIG.9. K and Na content of cells in isotonic medium exposed to tetradecanoyl 4p-phorbol 13-acetate (PMA) in the presence and absence of 5 p M A23187. All media contain 500 pM Ca2+and all suspensions are at a hematocrit of 10%.

tate, in the presence and absence of A23187, on the ion content of Amphiuma red blood cells. These data show that PMA is able to stimulate large net Na and K fluxes and that the addition of Ca results in further stimulation. Based upon the thermodynamic criteria previously outlined in this chapter and elsewhere (Cala, 1983a,b), we have determined that these fluxes are via Na-H and K-H exchange. In addition to the data shown in Fig. 9, similar studies have been performed upon shrunken and swollen cells and have yielded qualitatively similar results. In all cases, exposure of cells to 4a-phorbol 12,13-didecanoate, an inactive diacylglycerol analog, is without stimulatory effect upon Na-H and K-H exchange, while the p or active form is stimulatory. That exposure of cells to PMA is able to activate both Na-H and K-H exchange suggests a role for kinase C in

6. VOLUME-SENSITIVE ION FLUXES IN Amphiurna RED BLOOD CELLS

215

the activation of Am-H exchange. If kinase C-dependent protein phosphorylation plays a role in the activation of Am-H exchange associated with osmotic swelling and shrinkage, then other events must also be involved. That is, where osmotic swelling or shrinkage activate either KH or Na-H exchange, exposure to PMA activates both pathways simultaneously. Given the above, if diacylglycerol and kinase C play a role in the activation of Am-H exchange associated with volume perturbation, then other events must be involved in determining Am selectivity. VIII.

SUMMARY

This chapter describes volume-sensitive ion fluxes in Amphiumu red blood cells. When Amphiuma red blood cells are swollen they lose K, CI, and osmotically obliged H20, and when shrunken the cells gain Na, CI, and HzO. Based upon direct measurements of the membrane potential and net ion fluxes, it was determined that the volume-sensitive alkali metal ion fluxes are the result of an obligatorily I : 1 coupled (electroneutral) exchange with H+. Further, these alkali metal-H exchange fluxes are functionally coupled to net CI-HCO3 exchange fluxes such that net alkali metal and C1 fluxes proceed in the same direction. The principles governing the relative turnover rates of the alkali metal-H and CI-HC03 exchangers are discussed, as are the implications with regard to the “apparent” alkali metal-H stoichiometry. In addition, data addressing the basis for the control of the volumesensitive alkali metal-H fluxes are presented. There is evidence that Ca2+ is involved in activation of the K-H exchange but not the Na-H exchange pathway. Yet, since the Ca2+effects upon K-H exchange are a function of volume, it must be concluded that there are other events (volume sensitive) involved that prime the system to respond to Ca2+. In this regard there are preliminary data based upon phorbol ester activation of Na-H and K-H exchange which suggest a role for the ubiquitous Cadependent enzyme kinase C. The data obtained from cells stimulated with phorbol ester show that the volume sensitivity of the alkali metal-H exchange pathway is decreased, yet so too is the selectivity with respect to activation and Ca sensitivity. The theme woven through all of the above studies is that of identification of electroneutral alkali metal-H exchange flux pathways. The chapter outlines a force-flow approach to identifying alkali metal-coupled H exchange flux pathways which is based upon thermodynamic principles. Within this framework, criteria are established and evaluated to delineate electroneutral alkali metal-H exchange.

216

PETER M. CALA

ACKNOWLEDGMENT The author acknowledges the technical assistance of Karen S. Hoffmann and the word processing powers of Phoebe Ling. He also thanks Dr. Joseph Adorante for his stimulating input. This work was supported by NIH Grant HL-21179. REFERENCES Bemdge, M. J. (1984). Inositol triphosphate and diacylglycerol as second messengers. Biochem. J . 220, 345-360. Cala, P. M. (1977). Volume regulation by flounder red blood cells in anisotonic media. J. Gen. Physiol. 69, 537-552. Cala, P. M. (1980). Volume regulation by Amphiuma red blood cells: The membrane potential and its implications regarding the nature of the ion-flux pathways. J. Gen. Physiol. 76, 683-708. Cala, P. M. (1983a). Volume regulation by red blood cells: Mechanisms of ion transport. Mol. Physiol. 4, 33-52. Cala, P. M. (1983b). Cell volume regulation by Amphiuma red blood cells: The role of Ca as a modulator of alkali rnetal/H+ exchange. J . Gen. Physiol. 82, 761-784. Cala, P. M. (1985). Volume regulation by Amphiuma red blood cells: Strategies for identifying alkali metal/H+ transport. Fed. Proc., Fed. Am. SOC.Exp. Biol. 44, 2500-2507. Cala, P. M.,Mandel, L. J., and Murphy, E. (1986). Measurement of cytosolic free Ca2+ during volume regulation in Amphiuma red blood cells. Am. J. Physiol. Cell 19, C423429. Dellasega, M., and Grantham, J. J. (1973). Regulation of renal tubule cell volume in hypotonic media. Am. J. Physiol. 224, 1288-1293. Ellory, J. C.,and Dunham, P. B. (1980). Volume-dependent passive potassium transport in LK sheep red cells. I n “Membrane Transport in Erythrocytes, Alfred Benzoni Symposium 14” (U. V. Lassen, H. H. Ussing, and Wieth, J. O., eds.), pp. 409-437. Munksgaard, Copenhagen. Fisher, R. S., Persson, B. E., and Spring, K. R. (1981). Epithelial cell volume regulation: Bicarbonate dependence. Science 214, 1357-1359. Fugelli, K. (1967). Regulation of cell volume in flounder (PleuronecresJesus) erythrocytes accompanying a decrease in plasma osmolarity. Comp. Biochem. Physiol. 22,253-260. Gardos, G . (1956). The permeability of human erythrocytes to potassium. Acta Physiol. Acad. Sci. Hung. 10, 185-189. Gardos, G., Lassen, U. V., and Pape, L. (1976). Effect of antihistamines and chlorpromazine on the calcium-induced hyperpolarization of the Amphiuma red cell membrane. Biochim. Biophys. Acta 448, 599-606. Grinstein, S., DuPre, A., and Rothstein, A. (1982a). Volume regulation by human lymphocytes. Role of Calcium. J. Gen. Physiol. 79, 849-868. Grinstein, S., DuPre, A., and Rothstein, A. (1982b). Volume regulation by human lymphocytes: Role of calcium. J . Gen. Physiol. 79, 849-868. Hass, M., Schmidt, W. F., 111, and McManus, T. J. (1982). Catecholamine-stimulated ion transport in duck red cells. Gradient effects in electrically neutral [Na + K + 2C1] cotransport. J. Gen. Physiol. 80, 125-147. Hendil, K. B., and Hoffmann, E. K. (1974). Volume regulation by Ehrlich ascites tumor cells. J . Physiol. (London) 84, 115-126.

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Kregenow, F. M. (1971a). The response of duck erythrocytes to nonhemolytic hypotonic media. Evidence for a volume-controlling mechanism. J . Gen. Physiol. 58, 372-395. Kregenow, F. M. (1971b). The response of duck erythrocytes to hypertonic media. Further evidence for a volume-controlling mechanism. J . Gen. Physiol. 58, 396-412. Kregenow, F. M. (1981). Osmoregulatory salt transporting mechanisms: control of cell volume in anisotonic media. Physiol. Rev. 43, 493-505. L’Allemain, G., Franchi, A., Cragoe, E., Jr., and Pouyssegur, J. (1984). Blockade of the Na/H antiport abolishes growth factor-induced DNA synthesis in fibroblasts. J . Biol. Chem. 259,4313-4319. Lassen, U. V., Pape, L., Vestergaard-Bogind, B., and Bengtson, 0. (1974). Calcium related hyperpolarization of the Amphiuma red blood cell membrane following micropuncture. J. Membr. Biol. 18, 125-144. Lassen, U. V., Pape, L., and Vestergaard-Bogind, B. (1976). Effect of calcium on the membrane potential of Amphiuma red cells. J . Membr. B i d . 26, 51-70. Lassen, U. V., Pape, L., and Vestergaard-Bogind, B. (1978). Chloride conductance of the Amphiuma red cell membrane. J . Membr. Biol. 39, 27-48. Lassen, U. V., Pape, L., and Vestergaard-Bogind, B. (1980). Calcium related transient changes in membrane potential of red cells. In “Membrane Transport in Erythrocytes: Relations between Function and Molecular Structure. Alfred Benzon Symposium 14” (U. V. Lassen, H. H. Ussing, and J. 0. Weith, eds.), pp. 255-273. Munksgaard, Copenhagen. Lauf, P. K. (1982). Evidence for chloride-dependent potassium and water transport induced by hyposmotic stress in erythrocytes of the marine teleost, Opsanus tau. J . Comp. Physiol. 146, 9-16. Nishizuka, Y. (1981). The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature (London) 308, 693-698. Parker, J . C. (1973a). Dog red blood cells. Adjustment of density in uivo. J . Gen. Physiol. 61, 146-157. Parker, J. C. (1973b). Dog red blood cells. Adjustment of salt and water content in viuo. J. Gen. Physiol. 62, 147-156. Parker, J. C. (1984). Gluleraldehyde fixation of sodium transport in dog red blood cells. J. Gen. Physiol. 84, 789-803. Rindler, M. J., and Saier, M. H., Jr. (1981). Evidence for Na/H antiport in cultured dog kidney cells (MDCK). J . Biol. Chem. 256, 10820-10825. Rosoff, P. M., and Cantley, L. C. (1983). Increasing the intracellular Na+ concentration induces differentiation in a pre-B lymphocyte cell line. Proc.. Narl. Acad. Sci. U . S . A . 80, 7547-7550. Rosoff, P. M., Stein, L. F., and Cantley, L. C. (1984). Phorbol esters induce differentiation in a pre-B-lymphocyte cell line by enhancing Na/H exchange. J. Biol. Chem. 259,70567060. Rothenberg, P., Glaser, L., Schlesinger, P., and Cassel, D. (1983). Epidermal growth factor stimulates amiloride sensitive 2ZNauptake in A 431 cells. J. Biol. Chem. 258, 48834889. Sanchez, A., Hallam, T. J., and Rink, T. J . (1983). Trifluoperazine and chlorpromazine block secretion from human platelets evoked at basal cytoplasmic free calcium by activators of C-kinase. FEES Lett. 164, 43-46. Siebens, A. W., and Kregenow, F. M. (1978). Volume regulatory responses of salamanaer red cells incubated in anisotomic media: Effect of amiloride. Physiologist 21, 110 (Abstr.).

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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. Vincentini, L. M., Miller, R. J., and Villereal, M. L. (1984). Evidence for a role of phospholipose activity in the serum stimulation of Na+ influx in human fibroblasts. 1. Biol. Chem. 259,6912-6919. Vislie, T. (1980). Hyper-osmotic cell volume regulation in uiuo and in uitro in flounder (PlatichthysJesus) heart ventricles. J . Comp. Physiol. 140, 185-191. Weinman, S. A., and Reuss, L. (1982). Na+-H+ exchange at the apical membrane of Necturus gallbladder. Extracellular and intracellular pH studies. J . Gen. Physiol. 80, 299321. Weissenberg, J., and Katz, U. (1975). Effect of osmolality and salinity adaptation on cellular composition and on potassium uptake, of erythrocytes from the euryhaline toad Bufo uirdis. Comp. Biochem. Physiol. 52A, 165-169. White, J. F. (1980). Bicarbonate-dependent chloride absorption in small intestine: Ion fluxes and intercellular chloride activities. J . Membr. Biol. 53, 95-107.

Part 111

Consequences of the Alterations in Ion Transport Observed during Activation

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 27

Chapter 7 Intracellular Ionic Chanaes and Cell Activation: Regulation 6f DNA, RNA, and Protein Synthesis KATHI GEERING Institut de Pharmacologie Universitt! de Lausanne CH-1005Lausanne, Switzerland

I. Introduction . . . . . . . . . . 11. DNA Synthesis.. . . . . . ....................................... A. Relation between I B. Influence of Ions on Bioch 111. RNA Synthesis.. . . . . . . . . . . . . .................... A. Relation between Ionic Ch nthesis Induction.. . . B. Influence of Ions o and Processing ................................... IV. Protein Synthesis ..... ............................... A. Relation between I B. Influence of Ions on Biochemical Processes Related to Protein Synthesis . V. Posttranslational Event Expression of Proteins .......................... A. Ions and Intracellu ............. B. Ions and Expression o VI. Conclusions.. ................... ..................... References ................................ ............

1.

22 I 224 224 23 1 232 232 234 231 231 243 241 241 248 249 250

INTRODUCTION

In general terms, cell activation can be defined as a process which induces a particular altered state of cellular activity. Depending on the nature of the activation stimulus and the cell type, cell activation can ultimately lead to such diverse outcomes as cell growth, cell differentiation, or adaptation to changed environmental conditions. Despite the di221 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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versity of the end result, the process of cell activation often involves changes in the same biochemical processes, and this fact could suggest that a common mechanism might underlie the induction of the various cellular activity states. An understanding of the sequence of biochemical events leading ultimately to cell growth and differentiation would be a major breakthrough in basic research. In addition, it could have important consequences for the applied medical sciences, in particular for the treatment of cancer and viral infections. So far a multitude of observations exist on specific changes of biochemical pathways, but it has not yet been possible to integrate the isolated phenomena into a unifying model describing the cell activation process. One of the earliest events upon cell activation is a rapid modulation of the intracellular ionic composition. After the emergence of newer analytical methods, the generality of this phenomenon was established and several transport systems potentially related to the observed ionic changes were identified. These facts have led to the hypothesis that specific ions might act as primary triggers of biochemical cellular responses involved in the activation process and leading finally to cell growth or differentiation. The present state of knowledge on intracellular ionic changes accompanying cell activation by serum and related growth factors, as well as by some chemical agents, favors two candidates which could assume such a role, that is, pH changes and Ca2+. In a variety of systems such as sea urchin eggs (for review see Epel, 1980), fibroblasts of various origins (Mendoza et al., 1980; Moolenaar et al., 1982; Smith and Rozengurt, 1978), lymphocytes (Gerson, 1982), mouse neuroblastoma cells (Mummery et al., 1983), and rat pheochrornocytoma cells (Boonstra et al., 1983), a rapid increase in Na+ influx coupled to a Na+-H+ exchange system is observed upon cell stimulation with mitogens such as epidermal growth factor (EGF), nerve growth factor (NGF), etc. Because the rise in intracellular Na+ is rapidly dissipated by stimulation of Na,K-ATPase activity, the slight increase in intracellular pH was proposed as the primary event triggering the biochemical response of quiescent cells to mitogenic factors (for recent review see Busa and Nuccitelli, 1984). In this respect it is also interesting that tumor cells, which in many ways can be regarded as normal cells in a particular state of activation, exhibit a more alkaline intracellular pH than normal quiescent cells (for review see Gillies, 1981). Ca2+has also been proposed as an essential factor in growth or differentiation. Increased intracellular Ca2+concentration, due to stimulated Ca2+ influx (Tupper et al., 1978) or to mobilization of potential intracellular Ca2+ stores such as mitochondria or endoplasmic reticulum (for review see Somlyo, 1984), has been held responsible for early events occurring upon mitogen stimulation of fibroblasts (Mix et al., 1984; Owen and Ville-

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real, 1983). Also in favor of this hypothesis is the observation that neoplastic cells have elevated Ca2+levels and continue to grow with suboptimal concentrations of extracellular Ca2+(for review see Hickie et al., 1983). Since extracellular and intracellular Mg2f influences the effect of Ca2+on the initiation of cell replication, it has been proposed that the two ions act in concert and that Mg2+thus has an important role in cellular growth control (Rubin and Sanui, 1979; Vidair and Rubin, 1982). Many of the pleiotropic effects of Ca2t are mediated through its interaction with the intracellular Ca2+-bindingprotein calmodulin (for recent review see Levine and Dalgarno, 1983; Means et al., 1982; Stevens, 1983). An increase of this Ca2+mediator in cancer cells is also positively related to tumor growth rates (for review see Hickie et al., 1983). The important role of Ca2' in cell differentiation is particularly well documented in murine erythroleukemia cells. Certain chemical agents induce a rise in Ca2+ influx into these cells via a Na+-Ca2+ exchange system (Smith et al., 1982). The increased activity of this transporter is thought to be controlled by an increase in the intracellular Na+ concentration brought about by an inhibition of the Na,K-ATPase through phosphorylation (Ling and Cantley , 1984). Initiation of commitment to terminal differentiation is ultimately related to a secondary mobilization of Ca2+from depolarized mitochondria (Levenson et nl., 1982). Besides alkalinization and Ca2+,monovalent cations such as Na+ and K+ have also been implicated in the control of cellular processes leading to cell proliferation. Na,K-ATPase is responsible for the rigorous control of the low Na+ and high K+ levels of eukaryotic cells (for review see Jgrgensen, 1980, 1982). The transport activity of this enzyme is very sensitive to changes in intracellular Na+ concentrations (for review see Katz, 1982), and it is thus not surprising that increased Na' entry into mitogen-treated cells leads to an elevated intracellular K+ level, which is consequently held responsible for initiation of cell activation (LopezRivas et nl., 1982). Results obtained with ouabain, which through inhibition of the Na,K-ATPase decreases the Kt-Naf ratio in the cell, indeed suggest that such changes affect growth rates in normal cells but not in virus-transformed cells (Lubin, 1980). Growth control, however, has also been attributed to an increase in intracellular Na+ per se, since intracellular Na+ content is consistently elevated in tumor cells compared to their normal counterparts and thus is associated with a high mitosis rate (for review see Gillies, 1981). Both increased and decreased intracellular K+ levels have been implicated in the control of cell differentiation. In fact, since ouabain can induce erythroid differentiation in Friend erythroleukemia cells, it was suggested that a decrease in intracellular Kt might play a role in the stimulation of hemoglobin synthesis (Bernstein et al., 1976). On the other

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hand, high extracellular Ca2+, which induces terminal differentiation of epithelial cells to keratinocytes, leads to an increase in intracellular K+ concentration which could act as the cellular mediator for later stages of epidermal differentiation (Hennings et al., 1983a,b). From this brief summary on the observed intracellular ionic changes occurring upon cell activation, it already appears evident that it is not possible to put forward a hypothesis which would designate one particular ion as a universal cellular trigger for initiation of cell growth and development. In various chapters of this volume, several cellular activities of ions during cell activation will be discussed, including the influence of ions on cell metabolism or the modulation by specific ions of their own or the transport system of other ions. In the following some other biochemical processes will be considered which are of particular importance in cell activation and which could be potential targets for ionic control. A salient feature of activated cells, ultimately undergoing mitosis and growth, is the replication of their DNA. Control of this event, for example, by ions, is conceivable at different levels. In fact, ions might stimulate the activity of enzymes involved in the supply and/or assembly of nucleotides, thus exerting a direct effect on DNA synthesis. On the other hand, ions might have indirect effects on DNA synthesis by controlling biochemical events at the transcriptional, translational, or posttranslational level which lead to the expression of specific gene products that are responsible for the induction of DNA synthesis. While ionic effects on DNA synthesis concern, of course, only activated cells undergoing mitosis, the action of ions on RNA and protein synthesis might be of a more general importance and might also play a role in activated cells undergoing differentiation or hormone-induced adaptation. The following review will thus summarize the evidence for ionic control of DNA, RNA, and protein synthesis. A first point of discussion will be the relationship between the expression of these cellular processes and the intracellular changes of particular ions upon cell activation. These general considerations on the possible role of ions in cell activation will be followed by a more detailed discussion of specific biochemical reactions involved in DNA, RNA, and protein synthesis which could be potential targets for ionic control. II. DNA SYNTHESIS A. Relatlon between ionic Changes and DNA Synthesls Induction

Though ionic changes observed during cell activation and implicated in mitogenesis must by definition have an effect on DNA synthesis, several

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criteria should be fulfilled in order to define a particular ion as an intracellular trigger of biochemical pathways leading directly or secondarily to DNA synthesis. Hence (1) it should be demonstrated that induction of DNA synthesis by modulation of extracellular ion concentrations is accompanied by a change in the intracellular ion contents (at first sight, this statement seems trivial, but disregard of this point or lack of appropriate analytic methods have led to many conflicting results in the past); (2) a close relation should exist between the observed ionic change and the initiation or amplitude of DNA synthesis with respect to time and concentration, respectively; (3) depletion of the cell of the particular ion under study by omission from the medium or by inhibition of the appropriate transport system should lead to a concentration-dependent block of DNA synthesis; (4) concomitant changes of other ions should have no effects on DNA synthesis; (5) membrane insertion of ionophors which can bypass a particular transport system and can artificially increase the intracellular concentration of a specific ion should mimic the mitogen-induced change in DNA synthesis induction. Finally, comparison with the situation in experimental models, such as tumor or virus-infected cells, which reflect a particular state of cell activation has been found to be revealing for the determination of the role of intracellular ions as triggers of cell growth. On the basis of these criteria (which, of course, also hold true for the other biochemical processes discussed later, namely, RNA and protein synthesis), the following paragraphs will review the evidence that the various ions serve as intracellular mediators of biochemical pathways leading ultimately to mitogenesis.

1. CALCIUM AND MAGNESIUM Rubin and associates have reported that both Ca2+and Mg2+are necessary to initiate DNA synthesis and have therefore attributed a central regulatory role to Mg2+in the proliferation of mammalian cells (for review see Rubin and Sanui, 1979). Lowering of Mg2+and Ca2+concentrations in the medium indeed reduces the rate of progress of cultured cells through the GIperiod, and readdition of Mg2+alone to these cells is sufficient to induce onset of DNA synthesis after 4 hours in chick embryo fibroblasts and after 10 hours in mouse 3T3 cells. An effect of the accompanying changes in monovalent cation levels could be excluded. A role for Mg2+ in the regulation of intracellular Ca2+ content and distribution was suggested on the basis of the following two observations: (1) small changes in Mg2+content in BALB/c 3T3 cells are correlated with large changes in their CaZ+content (Rubin et al., 1981) and (2) Mg2+ deprivation of spontaneously transformed 3T3 cells restores their ele-

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vated Ca2+content to the level of nontransformed cells. That Mg2+ is indeed involved in mechanisms of cell cycle control has recently been questioned since the reductions in growth requirement for Mg2+and Ca2+, respectively, in transformed 3T3 cells do not seem to occur simultaneously. These results suggest that the two events are not linked (Ribeiro and Armelin, 1984). This observation, together with the fact that in contrast to Ca2+, Mg2+ is found at constantly high cellular levels and is needed throughout the cell cycle, support the hypothesis that Mg2+is a permissive or function-maintaining factor rather than a function-initiating factor (Whitfield et al., 1979). On the other hand, Ca2+fulfills some necessary requirements to be a regulator in the cell proliferation mechanism. Ca2+reduction to 0.1 mM or less inhibits DNA synthesis and proliferation in actively cycling hepatocytes, and a subsequent increase of extracellular Ca2+concentration to normal values restores these processes, probably by acting in late GI phase just prior to DNA synthesis (Armato et al., 1983; Whitfield et al., 1979). High Ca2+ in the medium has also been shown to stimulate cell division of quiescent fibroblasts synergistic with serum (Dulbecco and Elkington, 1975). Certain mitogens such as vasopressin and EGF indeed provoke a 2-3-fold transient increase in intracellular Cat+ within 15 to 90 seconds in quiescent 3T3 fibroblasts (Morris et al., 1984) and hepatocytes (Charest et al., 1983). In lymphocytes (Hesketh et al., 1983) and in fertilized sea urchin eggs (Steinhardt e! al., 1977), these rapid changes in intracellular Ca2+ concentration are followed by an onset of DNA synthesis within 1 hour (Steinhardt and Epel, 1974; Whitfield et al., 1979). The effect of Ca2+ionophores (e.g., A23187) has been studied in lymphocytes and in quiescent fibroblasts. Addition of such drugs indeed lead to initiation of DNA synthesis in the former but not in the latter cell type (for ref., see Rozengurt, 1979). As pointed out by the author, care should be taken in the interpretation of these results since, in addition to translocating divalent cations across membranes, such ionophores might have other pharmacological effects on cell metabolism. A final argument for the role of Ca2+ in the mitogenic response is the fact that the intracellular concentration of calmodulin, a receptor and signal transducer of Ca2+, also transiently increases during the prereplicative phase of various cells (Chafouleas et al., 1982; Whitfield et al., 1982). Moreover, calmodulin antagonists prevent the initiation of DNA synthesis in Chinese hamster ovary K-1 cells to an extent which correlates with the affinity of the compounds for their receptor (Hidaka et al., 1981) and can inhibit tumor growth in certain cancer cells (for review see Hickie et al., 1983).

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2. pH On the basis of the observation that intracellular alkalinization precedes mitosis and DNA synthesis in slime molds, ciliates, and lymphocytes (for review see Busa and Nuccitelli, 1984; Gillies, 1982; Gerson, 1982) or in quiescent fibroblasts (Schuldiner and Rozengurt, 1982), cytoplasmic pH has been proposed as a putative mitogenic signal. Indeed, as shown in Fig. 1, a close correlation exists between intracellular pH changes upon mitogenic stimulation of spleen lymphocytes and the rate of [ 3H]thymidine incorporation (Gerson, 1982). Manipulation of intracellular pH in

I

+LPS

o Control

7.1

7.2

7.5 7.6 7.7 lntracellular pH

7.3 7.4

FIG. 1. The relation between [3H]thymidine incorporation rate and intracellular pH over the time period from 24 to 78 hours following the mitogenic stimulation of BALB/c spleen lymphocytes. Con A, Concanavalin A; LPS, bacterial lipopolysaccharide. (From Gerson, 1982.)

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lymphocytes (Gerson, 1982) or in unfertilized sea urchin eggs (Mazia and Ruby, 1974) results in similar changes of DNA synthesis as upon mitogenic activation or fertilization, respectively, thus supporting the hypothesis that the two processes are physiologically linked. Upon stimulation with growth factors of quiescent fibroblasts, a Na+-H+ antiporter in the plasma membrane is rapidly activated (PouyssCgur et al., 1982; Rozengurt, 1981), and it is very likely that this transporter activation directly reflects the mitogen-induced alkalinization of the intracellular medium by 0.2-0.3 pH units. Both effects are indeed inhibited by amiloride and are Na+ dependent (L'Allemain et al., 1984a). Recently, it was shown that a close correlation exists between the half-maximal concentration of various amiloride analogs (Ki ranging from 4 x lo-* to 2 X M) to block the Na+-H+ exchange (measured by Na+ uptake) and the percentage of cells entering the S phase upon growth factor action (L'Allemain et al., 1984b) (Fig. 2). In addition, growth factors fail to promote DNA synthesis in Na+-H+ transport-deficient mutants (L'Allemain et al., 1984b). Taken

(000

!j

/

i

Y

" /4,

i

Y 8 l

FIG. 2. Correlation between the inhibition by arniloride and its analogs of Na+-H+ exchange and growth factor-induced DNA replication in fibroblasts. The concentration of amiloride analogs inhibiting 50% of Na+-H+ exchange at 50 mM N$ are plotted against the correspondingICs for growth factor-inducedDNA replication (concentrationinhibiting 50% of labeled nuclei). (From L'Allernain et al., 1984a).

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together, there is convincing evidence that reinitiation of DNA synthesis is indeed tightly linked to changes in intracellular pH. 3. SODIUM AND POTASSIUM

Changes in intracellular concentrations of monovalent cations have also been proposed as triggers for initiation of DNA synthesis. Indeed, activation of the Na+-H+ exchange system upon growth factor stimulation not only decreases intracellular pH but might as well increase intracellular Na+ concentration. Serum and EGF stimulate Na+ uptake into various cells (Koch and Leffert, 1979; Rozengurt, 1981). The early increase in Na+ uptake (8-60 minutes after growth factor addition) is followed by an increase in DNA synthesis, with a 12-hourdelay in hepatocytes (Koch and Leffert, 1979). Na+ influx is abolished in human fibroblasts by amiloride analogs at concentrations which are compatible with inhibition of DNA synthesis (O’Donnell et al., 1983). However, such inhibitor studies may not irrevocably prove a role for Na+ influx in cell growth since, as mentioned by the authors, amiloride is known to exert nonspecific effects on protein synthesis (Lubin et al., 1982). It has frequently been pointed out that stimulation of DNA synthesis might be mediated by an increment in net Na+ fluxes rather than intracelMar Na+ concentration since small amounts of extracellular Na+ can stimulate DNA synthesis (Toback, 1980) and increased Na+ uptake is not accompanied by a significant rise in total intracellular Na+, for example, in fibroblasts, since a rapid stimulation of the Na,K-ATPase maintains intracellular Na+ unchanged (for review see Rozengurt, 1981). On the other hand, in confluent African green monkey kidney cells, growth stimulation after a medium change is indeed accompanied by an increase in cellular Na+ content between 90 minutes and 2 hours, followed by cell multiplication after 4 days (Walsh-Reitz et al., 1984). Cell growth inhibitors produced by confluent kidney cells were able to attenuate the increase in Na+ content as well as the growth of activated cells. High levels of intracellular Na+ have also been observed in transformed cells, and it has been postulated that they might be involved in the cancer state of cells (for review see Pool et al., 1981). Amiloride indeed decreases significantly intranuclear Na+ content in H6 hepatoma cells concomitant with a decrease in tumor cell proliferation (Sparks et al., 1983). These latter results would speak for a role of intracellular Na+ per se in the triggering of processes involved in DNA synthesis. However, this inference is not supported by experiments which artificially manipulate intracellular Na+ concentration. Ouabain, which by inhibition of the Na+ pump increases intracellular Na+ concentration, as well as monensin, a

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Na+ ionophore, in fact block initiation of DNA synthesis in hepatocytes (Koch and Leffert, 1979). As pointed out by the authors, these results can only be explained if one assumes that monensin blocks the Na,K-ATPase in a nonspecific way and that increased bidirectional Na+ fluxes are necessary to initiate DNA synthesis. A rapid stimulation of the Na,KATPase upon growth stimulation has indeed been observed by measuring ouabain-sensitive 86Rb(a K+ tracer) influx into various cells (Moolenaar et al., 1982; Reznik et al., 1983; Rozengurt and Heppel, 1975; Smith, 1977). DNA synthesis is maximally stimulated after about 12 hours, with the same concentrations of serum needed to maximally induce 86Rbinflux (Smith, 1977). Increased Na+ entry into mitogen-treated cells might thus lead to a stimulation of the Na,K-ATPase and consequently to an elevation in intracellular K+(up to 20% over controls) rather than Na+ concentration (de Laat et af., 1982). It has indeed been shown that initiation of DNA synthesis upon growth factor stimulation in 3T3 cells is closely related to intracellular K+ concentration (Lopez-Rivas et af., 1982). A sigmoid dependence of DNA synthesis on intracellular K+ levels is observed (Fig. 3), but during the early G I phase rather than at the GI-S boundary. Therefore, these results attribute a role for K+ only in processes preceding initiation of DNA synthesis. Indications for a specific role in mitogenesis for intracellular K+ fluctuations independent of Na,K-ATPase activity come from recent studies

IWTRACELLULAR K * (pmol/mg protoin 1 FIG. 3. DNA synthesis in 3T3 cells stimulated by growth factors as a function of the internal concentration of K + . The arrow and the hatched area represent the mean ? SEM of the intracellular K+in cultures incubated with 5 mM K+ medium for 40 hours in the absence of growth factors. (From Lopez-Rivas et al., 1982.)

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which report the existence of a voltage-gated K' channel in human T lymphocytes (De Coursey ez al., 1984; Matteson and Deutsch, 1984). Phytohemaglutinin (PHA), at concentrations that produce mitogenesis, indeed alters K+channel gating within I minute, causing channels to open more rapidly and at more negative potentials. Various specific K + channel blockers inhibit [ 3H]thymidine incorporation by T lymphocytes following PHA stimulation at doses found to block K + channels (De Coursey et al., 1984). B. Influence of Ions on Biochemical Processes Related to DNA Synthesis

The brief summary on the relations between ionic changes observed upon cell growth stimulation and the initiation of DNA synthesis clearly shows that each of the various ions discussed fulfills at least some of the requirements stated at the beginning of this chapter. In most experimental systems, however, there exists a considerable temporal gap between the intracellular ionic changes and the onset of DNA synthesis, which makes it very unlikely that ions might have a primary effect on biochemical processes directly linked to DNA synthesis. According to available data only Ca2+,acting through its intracellular receptor calmodulin, or pH changes could eventually assume such a role in certain experimental models. 1. THYM~DYLATE SYNTHASE INDUCTION

The observation that alterations in thymidine utilization are often the most striking effects after Ca2+depletion of normal but not of tumor cells led to the conclusion that CaZ+might act during the late GI phase on nucleotide synthesis (Parsons et al., 1983; Whitfield et al., 1979). Indeed, brief exposure of quiescent lymphoblasts to high concentrations of thymidine can mimic Ca2+,causing initiation of DNA synthesis. Since, on the other hand, a putative source of thymidilic acid, for example, deoxyuridine, cannot stimulate DNA synthesis unless CaZ+is in the medium, it was deduced that CaZ+might stimulate the dihydrofolate reductase-thymidylate synthase complex, which is inactive in quiescent cells (Whitfield et al., 1976; Youdale and MacManus, 1975). Supporting this hypothesis are the observations that Ca2+stimulates [ 14C]formateincorporation exclusively into thymidine residues of DNA and that the stimulatory action of Ca2+is completely blocked by inhibitors of thymidylate synthase (e.g., methotrexate) (Whitfield et al., 1976). The molecular mechanism underlying this enzyme stimulation has not been explored and it is thus not

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known whether protein phosphorylation mediated through Ca2+-dependent protein kinases plays a role in this biochemical event. 2. DNA POLYMERASE ACTIVITY

Gerson (1982) has pointed out that a key enzyme in DNA synthesis, DNA polymerase, has very alkaline pH optima in a number of species and could be expected to increase its activity during an increase of intracellular pH. Such an effect of pH could at least play a role in certain cell species, including Physarum and lymphocytes, where DNA synthesis coincides temporally with intracellular alkalinization upon cell activation. As mentioned by Busa and Nuccitelli (1984), such a mechanism is, however, much less likely to be important in species such as yeast and Tetrahymena, where onset of DNA synthesis is delayed with respect to intracellular pH maximum. Besides these two examples, involving Ca2+or pH, there is little evidence that ions might act on biochemical processes directly linked to DNA synthesis. As suggested by the frequently observed time lag between ionic changes upon cell activation and the initiation of DNA replication, it is more likely that processes linked to protein synthesis either at the transcriptional, translational, or posttranslational level precede the onset of DNA synthesis. In the following paragraphs, the evidence for the involvement of ions in such processes will therefore be discussed. 111.

RNA SYNTHESIS

A. Relation between Ionic Changes and RNA Synthesis lnductlon

Few data are available on the relation between ionic changes and induction of RNA expression, although increased RNA synthesis or expression of specific genes is very frequently observed after cell activation with growth factors or hormones. Several years ago Rossow et al. (1979) proposed that the control of growth at the initiator point leading to the DNA synthesis phase depends on a serum-dependent accumulation of a specific labile protein in 3T3 cells. The hypothesis was soon put forward that such proteins might play a role in uncontrolled growth observed in tumor cells and that the induced proteins during cell activation might resemble species encoded for by viral genes such as src (Oppermann et al., 1979) or onc (Duesberg and Vogt, 1979). Epidermal growth factor-induced cytoplasmic proteins have indeed been shown to activate DNA replication in isolated quiescent nuclei from frog spleen cells (Das, 1980). A direct correlation between the

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levels of a nuclear protein of 36,000 Da (cyclin) and the proliferative state of normal skin biopsis and skin fibroblasts could also be established (Celis and Bravo, 1984). Expression of specific genes upon mitogen stimulation has been particularly well studied in PDGF-stimulated cells. Thus, in quiescent BALB/c 3T3 cells, as in lymphocytes, addition of PDGF leads to a rapid increase in c myc mRNA, a species which codes for a nuclear protein (for review see Heldin and Westermark, 1984). Abnormal expression of the c myc gene has also been implicated in human malignancy (Klein, 1983) and, taken together, the observations suggest that this oncogene might be involved in the response to growth factors as an intracellular mediator. A number of other mRNAs whose abundance is specifically increased after addition of PDGF to quiescent BALB/c 3T3 cells have been identified using molecular cloning of cDNA representing gene sequences regulated by growth factors (Cochran and Stiles, 1982; Linzer and Nathans, 1983). As suggested recently, there is some evidence that different growth factors specifically stimulate separate domains in the genome (Shipley et al., 1984). The expression of none of these mRNAs or proteins potentially implicated in the triggering of the proliferation response has been related to intracellular ionic changes, and in the following it can thus only be shown on the ground of other examples that ions indeed could play a role in such events. 1. CALCIUM

Activation of RNA synthesis by Ca2+ has been reported in several tissues or cultured cells, including GH3 cells (White et al., 1981) and cultures of chicken pectoralis muscle (Wu et al., 1981) and in conjunction with the action of hormones such as prolactin, ACTH, and TSH (for ref. see Cameron and Rillema, 1983). Ca2+ranging from 0.01 to 4 mM stimulates the uptake of [ 3H]uridine into total RNA of rat myocardium with a peak at physiological concentration of 2 to 2.5 mM (Kaplan and Richman, 1973). The data indicate that Ca2+is necessary for optimal RNA biosynthesis in this tissue and that the Ca2+stimulation of RNA synthesis might be a factor in the observed increase in protein synthesis by the cation. Extracellular Ca2+concentrations between 5 and 10 pM are necessary for prolactin (PRL) to stimulate an increase in RNA synthesis in mouse mammary glands (Cameron and Rillema, 1983). Since both Ca2+and Mg2+ have been shown to enhance PRL binding to its cell surface receptors and since only Ca2+but not Mg2+or Mn2+can restore increased RNA synthesis after EGTA treatment, it has been concluded that the effect of extra-

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cellular Ca2+ on RNA synthesis cannot simply be explained by altered PRL binding. However, there is also no evidence that Ca2+ influx is involved in the hormone-mediated process, since Ca2+ ionophors were not able to mimic the hormone effect on RNA synthesis. As mentioned by the authors, it is thus not yet clear whether Ca2+ is an intracellular mediator for PRL’s action. The role of Ca2+ as a trigger for the expression of specific proteins which could be either molecular regulators of the transition between proliferation and differentiation or which are markers of the differentiated state has been particularly well studied. One example is differentiation of skeletal muscle, which only proceeds at a critical level of extracellular Ca2+(Shainberg et al., 1969). Lowering the Ca2+concentration prevents fusion into myotubes and proliferation of myoblasts continues. The use of Ca2+ionophores has now revealed that an mRNA species coding for an 80,000-Da protein is selectively increased in a number of different cells, including cultured chicken pectoralis muscle cells (Wu et al., 1981).These results suggest that Ca2+-mediated regulatory mechanisms of RNA synthesis might play a role during development. 2. SODIUM Koch and Leffert (1979) have studied the role of increased Na+ fluxes in overall RNA synthesis rates after growth stimulation of rat hepatocytes. They report a biphasic increase of [ 3H]uridine incorporation into total RNA upon growth factor addition which precedes DNA synthesis. The later peak at 8 to 12 hours is sensitive to amiloride, but again the results do not completely rule out a direct effect of this drug on RNA synthesis rather than through an inhibition of Na+ fluxes. RNA synthesis is, however, clearly needed during the prereplicative interval since actinornycin D blocks growth initiation. B. Influence of Ions on Biochemical Processes Related to RNA Synthesls and Processing

A well-characterized system with respect to increased RNA synthesis during cell growth is the regenerating liver. Although the influence of ions has not been studied, it seems worthwhile in the context of this review to mention some characteristics of this experimental model with respect to RNA synthesis. They might indeed turn out to be of general validity for the understanding of regulated growth. After partial hepatectorny the rate of synthesis of poly(A)+ mRNA increases twofold after 6 hours, thus more rapidly and to a greater extent than total RNA (for review see

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Fausto, 1984). The pattern of alterations of mRNA during regeneration is quite different from that occurring during liver development or as a consequence of hormonal changes in the adult liver. It appears, however, that the changes are only of a quantitative nature, for example, the abundance of existing mRNAs is changed without alterations of the spectrum of transcripts produced by the cell. It is logical to assume that DNA sequences which become active at the early stages of regeneration must include genes which are essential for the triggering of DNA synthesis and that changes in the abundance of certain transcripts must be of critical importance for the triggering and progression of the regenerative response. Several mechanisms have been suggested which might be implicated in the regulation of RNA expression. They include control at the transcriptional and/or posttranscriptional level. At the nuclear level, phosphorylation of chromosomal proteins (for review see Jungmann and Kranias, 1977), histone acetylation (McKnight et al., 1980; Truscello et al., 1983), and DNA methylation (for review see Shay, 1983) seem to play an important role in gene transcription. In addition, the activity of the enzyme ADP-ribosyl transferase, which might be implicated in DNA breaks and rearrangement of genetic material and thus in altered gene expression, has been related to differentiation in cardiac muscle, Friend erythroleukemia cells, and Xenopus laeuis embryos (for review see Williams and Johnstone, 1983). Processes most likely involved in posttranscriptional regulation of RNA expression concern changes in RNA stability (Andrews et al., 1982; Moore et al., 1980), or eventually processing of messenger ribonucleoprotein precursors. Only a few of these processes have been shown to be ion-dependent, and none has yet been correlated to ionic changes occurring during cell growth or differentiation. 1. CHROMOSOMAL PROTEIN PHOSPHORYLATION

Non-histone proteins are thought to influence the structure of DNA, affect the interaction of RNA polymerase with the DNA template, modulate the activity of RNA polymerase, and serve to transmit physiological signals for gene activation or repression in response to stimuli by hormones and cyclic nucleotide. Phosphorylation of non-histone proteins have been implicated in the expression of specific genes (Park et al., 1977), while dephosphorylation inhibits transcription (Offenbacher and Kline, 1979). The Ca-calmodulin complex is known to stimulate directly or indirectly a variety of protein kinases (for review see Means et al., 1982), and

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the generality of the phenomenon suggests that Ca-calmodulin can modulate most if not all of the protein phosphorylation in the cell. In addition, Ca-calmodulin regulates phosphatase activity, including the dephosphorylation of histones (Wolff et al., 1981). Thus it appears that Ca-calmodulin may influence the degree of protein phosphorylation through regulation of both phosphatases and kinases. Studies on protein phosphorylation during the cell cycle have revealed that both histone and non-histone chromosomal proteins exhibit cell-cycle-specific patterns (De Morales et al., 1974; Gurley et al., 1978). Consistent with the idea that Ca2+,through its cellular mediator calmodulin, might indeed be involved in chromosomal protein phosphorylation and thus in altered RNA expression during cell activation are the following observations: (1) Ca2+ stimulates phosphorylation of various age-specific non-histone chromosomal proteins in the rat brain (Kanungo and Thakur, 1979), (2) Cacalmodulin levels increase in the GI phase (Chafouleas et ul., 1982), ( 3 ) Ca-calmodulin is found in increased amounts in nuclei of certain hormone-treated cells (Harper et al., 1980), and (4) Ca-calmodulin-dependent protein kinases have been identified in rat liver nuclei (Sikorska et al., 1980). That the RNA-activating action of Ca2+ in intact tissue is probably a more complex process than simple interaction of the cation with a nuclear protein is, however, suggested by studies with isolated nuclei. Under these experimental conditions Ca2+concentrations as low as 1 p M inhibit rather than stimulate RNA synthesis (Pardo and Fernandez, 1982). 2. POSTTRANSLATIONAL MODIFICATIONS OF mRNA

mRNA is synthesized as a high-molecular-weight precursor containing nontranslatable RNA sequences. Procesing of this precursor to the active mRNA species occurs in the nucleus and in the cytoplasm, including polyadenylation, methylation, and splicing (for review see Darnell, 1982). Unfertilized eggs of the sea urchin contain a large amount of “masked” messenger ribonucleoproteins (Kaumeyer et al., 1978; Jenkins et al., 1978) which are not translatable in uitro unless modified. pH changes have been made responsible for the recruitment of this oogenetic mRNA and thus at least in part for the increase in active RNA expression as observed after fertilization or alkalinization by NH&l of unfertilized eggs (Brandis and Raff, 1979). A possible mechanism of action is discussed in Section IV,A,2 in connection with pH effects on protein synthesis. Alternatively, it has been reported that maternal message of amphibia oocytes has a sequence organization which does not permit its translation unless structurally modified (for review see Richter e f al., 1984). Nothing is known, however, about how this interspersed mRNA is activated during fertilization.

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IV. PROTEIN SYNTHESIS A. Reiatlon between lonlc Changes and Protein Synthesis induction

Net protein accumulation as well as a change in the overall pattern of cell proteins are features of cell activation by growth factors, which in most studied cases precede DNA synthesis and cell division (Rossow ef a / . , 1979; Thomas ef al., 1981). In addition, differentiation of cells is also accompanied by particular changes in the cellular protein set. The role of ions in the overall expression of protein synthesis is a well-documented research field. On the basis of the observation that eukaryotic cells maintain high K+concentration and low Na+ and Ca2+levels compared to the extracellular medium, it has been assumed for a long time that these ions might play an important role in such fundamental metabolic processes as protein synthesis and that even small intracellular changes in the concentration of these ions could significantly alter the activity of this process, leading finally to an altered cell state. 1. CALCIUM

Ca2+has been reported to affect the synthesis of proteins in different tissues. Wilde el al. (1981) observed that depletion in Ca2+with EDTA from cultured mammary gland tissue markedly reduces cytosolic protein synthesis, including casein synthesis. Ca2+also provokes a fourfold stimulation of precursor incorporation into proteins of rat adrenal slices (Farese, 1971). An interesting observation in this context is that abnormally high intracellular concentrations of Ca2+,,which often accompany cataract formation, inhibit protein synthesis in cultured lenses (Hightower, 1983). Concerning the mechanism by which Ca2+might stimulate the formation of proteins, it has been postulated that Ca2+may (1) stimulate the transcription of RNA as discussed above, (2) increase the transport of amino acids into the intracellular pool available for protein synthesis (Kypson and Hait, 1971), or finally (3) stimulate the synthesis of proteins directly at the translational level. In support of this latter hypothesis are recent data reported by Brostrom et al. (1983). In C6 glial tumor cells, protein synthesis as measured by the incorporation of several radiolabeled amino acids is markedly reduced by Ca2+depletion with 1 mM EGTA. Repletion with Ca2+at concentrations of 300 pM in excess of the chelator induces a I0-fold increase in the incorporation of radioactivity into the trichloroacetic acid-insoluble fraction (Fig. 4). Other physiologically occurring ions than Ca2+have no effect on protein synthesis in these

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il

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8 12 1 6 2 0 MINUTES FIG.4. ['TILysine uptake (A, A) and incorporation (0, 0)by Ca2+-depleted(A,0) and Ca2+-restored(A, 0)C6 cells. (From Brostrom et al., 1983.) 0

4

tumor cells. The requirement for Ca2+of protein synthesis was also demonstrated in embryonic tissues as well as in normal adult tissue. In support of the hypothesis that Ca2+acts directly at the translational level of protein synthesis are the following observations: (1) restoration by CaZ+of protein synthesis activity within minutes after EGTA treatment is observed, (2) suppression of protein synthesis by chelators occurred while amino acid uptake (Fig. 4), purine nucleotide content, protein catabolism, and aminoacetylation of methionyl-tRNA were largely unchanged, and (3) actinomycin D, a transcriptional inhibitor, did not block the stimulatory effect of Ca2+.In addition, Ca2+up to 5 pM was also found to markedly stimulate incorporation of precursors into proteins in cell-free systems. The physiological significance of these results must, however, be confirmed by the demonstration of intracellular Ca2+changes in a physiological range. Although it is tempting to assign a role to Ca2+as a coupling factor between cellular activation and the rate of protein synthesis, this hypothesis is poorly documented. Hormones have, however, been shown to stimulate in a Ca2+-dependentmanner protein synthesis in numerous tissues, including adrenals (Dazord et al., 1981) and mammary glands (Wilde et al., 1981). In addition, it has been reported that protein synthesis of

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unfertilized eggs treated with NH4CI can only be elevated to levels comparable to fertilized eggs when intracellular free Ca2+ is concomitantly increased either by treatment with ionophores or by external Ca2+addition (Winkler, 1982). These results, however, are contested since other authors (Dubt and Guerrier, 1983) were not able to demonstrate an enhancing effect of increased Ca2+influx on protein synthesis stimulated by an intracellular pH increase. Mouse keratinocyte cultures are a model system in which extracellular Ca2+regulates growth and differentiation (Hennings et al., 1980). In medium containing 0.05-0.1 mM Ca2+(“low Ca2+”), mouse keratinocytes proliferate rapidly with a high growth rate, do not stratify, and are separated by widened intercellular spaces. When the extracellular Ca2+concentration is switched to 1.2 mM (“high Ca*+”),cell-to-cell contact occurs, cells stratify, and DNA synthesis is inhibited. These changes mimic in many ways the transition of keratinocytes in uiuo from the proliferative basement membrane-anchored basal cell layer to stratified nonproliferating dflerentiating cells (Stanley and Yuspa, 1983). While the electrophoretic profiles of the major proteins of epidermal cells grown in low or high Ca2+medium are similar, the level of several specific proteins changes in parallel with the Ca2+-induceddifferentiation. Thus, the synthesis of pemphigoid antigen, a basement membrane protein of about 220,000 Da whose function is still unknown, is selectively repressed in high Ca2+medium, while the glycoprotein pemphigus antigen, which is part of the glycocalyx of stratified cells, is only synthesized in high Ca2+(Stanley and Yuspa, 1983). In addition, appearance of keratohyalin granules is one of the morphological changes associated with Ca2+induced keratinocyte differentiation. While keratin biosynthesis is not altered by extracellular Ca2+concentration (Hennings et al., 1980), the synthesis of filaggrin, a 27,000-Da histidine-rich protein which functions as a keratin matrix protein, is indeed induced in high Ca2+(Dale el al., 1983). In addition, the temporal appearance of this protein coincides with the appearance of phase-dense cytoplasmic granules. Several possibilities have been proposed, but not yet substantiated, for the role of Ca2+in the stimulation of filaggrin synthesis, including a stabilization of the highly phosphorylated precursor of filaggrin by direct ionic interaction, by exerting an effect on the enzymes responsible for posttranslational modifications of filaggrin precursor, or finally by stimulation of filaggrin precursor synthesis. This latter mechanism has also been suggested to explain the increase in epidermal trans-glutaminase activity observed upon switch to high Ca2+(Hennings et al., 1980). This enzyme is critical for maturation of keratinocytes since it catalyzes (E-y-glutamy1)lysinecrosslinkages, which are responsible for cornified envelope formation (Buxman and Wuepper,

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1976). Finally, high extracellular Ca2+stimulates the activity of ornithine decarboxylase (ODC), the first enzyme involved in the biosynthesis of the polyamines putrescine and spermine in keratinocytes (Landgon et al., 1983). In this case, stimulation of ODC activity by high Ca2+is not dependent on RNA synthesis, but on the other hand requires protein synthesis. Recently, it has been suggested that an increase in intracellular K+ rather than Ca2+ might be responsible for the observed effects of highCa2+medium on protein synthesis (Hennings et al., 1983a). Indeed, ouabain as well as the Ca2+ionophore A23187 inhibit terminal differentiation in mouse epidermal cells concomitant with a decrease in intracellular K+, but not Na+. In contrast to the expression of particular proteins which are rather implicated in the later stages of differentiation, early ultrastructural changes such as desmosome formation are independent of Na+ and K+ and might instead be modulated by increased Ca2+levels at the cell surface (Hennings et d.,1983b). 2. pH The role of intracellular pH in protein synthesis and growth has been recognized for a long time (Mackenzie et al., 1961). The best studied model is again the sea urchin egg (Epel et al., 1974; Grainger et al., 1979), where alkalinization of the cell interior in the range of 0.4 pH units accounts by about 40% for the 5-30-fold increase in the rate of protein synthesis occurring after fertilization (Winkler, 1982). In cell-free systems the rate of protein synthesis also sharply rises at pH 6.9 (close to the pH of unfertilized eggs), with an optimum at pH 7.4 (close to the pH of fertilized eggs). The elongation rate of nascent polypeptides could (Winkler, 1982) or could not (Brandis and Raff, 1979) be implicated in the pH effect on protein synthesis. However, agreement exists that an increase in pH results in a more efficient translation of certain “masked” ribonucleoprotein particles. A model was proposed which suggests that an RNA masking factor exists in sea urchin eggs which, upon an increase in pH, decreases its binding affinity for mRNA, thus permitting mRNA translation (Winkler, 1982). AND SODIUM 3. POTASSIUM

Twenty years ago it was observed that reduction in intracellular K+ induced by omission of extracellular K+ or by treatment with ouabain decreases cell growth in bacteria (for review see Lubin, 1964)as well as in mammalian cells (Ledbetter and Lubin, 1977; Lubin, 1967; Kuchler, 1967), concomitant with an inhibition of protein synthesis. Reducing cell K+in human fibroblasts to 60-80% of control values in fact decreases the

7. REGULATION OF DNA, RNA, AND PROTEIN SYNTHESIS

24 1

rate of protein synthesis in proportion to further reduction of K+ without affecting RNA synthesis, and restoration of intracellular K+ levels reestablishes protein synthesis as well as cell growth (Ledbetter and Lubin, 1977). Small changes in extracellular K+ also affect protein synthesis in hippocampal slices from guinea pig. In this tissue a two-fold increase in the intracellular K+-Na+ ratio is accompanied by a three-fold increase in the rate of protein synthesis (Lipton and Heimbach, 1978). The effect of K+ does not involve RNA synthesis or changes in the precursor pool but requires aerobic glycolysis (Lipton and Robacker, 1983). No effect of extracellular K+ on protein synthesis, however, was observed in noncerebra1 tissues of this species (Lipton and Heimbach, 1978). Ouabain and omission of K+ from the extracellular medium not only decrease intracellular K+ concentrations but increase in parallel intracellular Na+ concentrations through the well-documented inhibition by these two treatments of Na,K-ATPase activity. In none of the mentioned studies is it well established which of the two ionic changes might be responsible for the observed effects on protein synthesis. Thus, it has indeed been demonstrated that incubation of cells in high Na+ medium inhibits the initiation of protein synthesis (Saborio et al., 1974). On the other hand, however, initiation of translation of cellular proteins is also inhibited by incubating cells in low-Na+ medium (Garry et al., 1979). The reason for these apparently contradictory results could be that initiation of translation of mRNAs is inhibited because high-Na+ medium results in an increase in intracellular Na+ and low-Na+ medium results in a decrease in intracellular K+ (Garry et al., 1979). Such an interpretation assigns an inhibitory role to high Na+ and a stimulating role to high K+ on protein synthesis, and this assumption is in agreement with the physiological condition characterized by a high intracellular K+-Na+ ratio. However, there has been some doubt about the quantitative aspects of the relation between decreased K+-Na+ ratios and decreased protein synthesis on the one hand and inhibition of cell growth on the other hand. Indeed, Lubin (1980) reported that the decrease in the K+-Na+ ratio required to inhibit [3H]leucineincorporation into polypeptides is too large to account for the actual decrease in cell growth of 3T3 fibroblasts. As pointed out by the author, this could mean that protein synthesis is not the mechanism by which K+-Na+ inhibits cell growth or, alternatively, that small decreases in protein synthesis might be sufficient to inhibit proliferation. This latter hypothesis is indeed supported by the recent observation that when intracellular K+ concentrations are modulated in early G I phase of 3T3 fibroblasts, 50% inhibition of DNA synthesis only required 10-20% reduction of protein synthesis (Lopez-Rivas ef al., 1982). On the other hand, however, curves which relate DNA synthesis to protein synthesis (when the

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latter is inhibited by decreased K+) in late rather than in early GI phase reveal a linear correlation between the two parameters, with 50% reduction of DNA synthesis at 50% inhibition of protein synthesis. Since doses of K+and of cycloheximide giving identical reductions of protein synthesis reduce DNA synthesis to a similar extent, it was concluded that the effects of K+ on DNA synthesis are at least in part exerted through its control of protein synthesis. The inhibitory effect of high extracellular Na+ concentration on protein synthesis has been found to be accompanied by a complete breakdown of polyribosomes in HeLa cells (Saborio et al., 1974). The same phenomenon is observed in rat kidney after intraperitonal injection of hypertonic NaCl and is accompanied by an increased activity of renal ribonuclease (Murty et al., 1982). It has thus been suggested that high Na+ concentrations inhibit protein synthesis selectively at the level of chain initiation, a hypothesis which is further supported by the facts that elongation and termination of nascent polypeptide chains proceed normally in high Na+ medium (Saborio et al., 1974). A breakdown of polyribosomes is also observed after addition of KC1, NH&l, or sucrose to the culture medium, suggesting that the inhibition of protein synthesis is due to an effect of hypertonicity rather than to a particular ion. In support of a role for K+-Na+ ratios in protein synthesis are several studies on virus-infected cells. Lytic virus infections lead to inhibition of host cell proteins, and this phenomenon seems to involve the initiation step of protein synthesis since host mRNA is not degraded and can be translated in cell-free systems (for review see Carrasco, 1977). Inhibition of host proteins in virus-infected cells is frequently accompanied by an increase in intracellular Na+ concentration (from 20 mM to over 60 mM) and a decrease in intracellular K+ concentrations (from I50 mM to less than 60 mM) (for ref. see Garry et al., 1982). The change in intracellular K+-Na+ ratio and termination of cellular protein synthesis correlates temporally with an inhibition of the Na,K-ATPase in Sindbis virus-infected avian fibroblasts (Garry et al., 1982), indicating that the observed ion changes are not due to an increase in nonspecific membrane permeability. The alterations in Na+ and K+ concentration seem to be sufficient to account for the decreased amount of host-specified proteins since there exists a good correlation between the ability of a cellular protein to be synthesized during lytic infection and cells with altered intracellular concentrations of Na+ and K+. On the other hand, synthesis of virus-specific proteins seem to be much more resistant than cell-specific protein synthesis to an initiation block by altered Na+ and K+levels (Garry et al., 1979; Saborio et al., 1974). Changes in intracellular K+-Na+ ratios can probably not be regarded as

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a general mechanism by which the cell protein synthesis machinery discriminates between cell-specific and viral proteins. It has been shown that infection of cells with Semiliki Forest (Gray et af., 1983) vaccinia (Norrie et al., 1982) or herpes simplex viruses (Hackstadt and Mallavia, 1982) does in fact lead to decreased host-protein synthesis, but this event is temporally not related to changes in the intracellular ion concentrations. It was thus suggested that increased Na+ concentrations are a consequence rather than a cause of altered protein synthesis in virally infected cells (Gray et al., 1983). Another experimental system, the heat-shock-treated cell, has also served to study the effect of intracellular ion changes on protein synthesis. Hyperthermic treatment results in a number of cellular effects (for ref. see Boonstra et al., 1984), but one of the most rapid and drastic effects is an inhibition of protein synthesis. Changes in membrane fluidity or membrane composition (Dennis and Yatvin, 1981; Cress et al., 1982) have been correlated to the sensitivity of cells to hyperthermia, and thus, in analogy with the role of the plasma membrane in growth regulation, it was proposed that intracellular ions might have an important mediator role in the shutoff of protein synthesis in heat-treated cells. A recent report has studied this question in heat-treated rat hepatoma cells (Boonstra et al., 1984). Treatment for 30 minutes at 42°C indeed inhibits protein synthesis in these cells but has no effect on intracellular K + and Na+ concentration. In fact, increased temperature affects K+ influx via stimulation of the Na,K-ATPase but also passive K+ efflux, leaving the intracellular ionic balance unchanged. These data thus rule out a direct inhibitory action of Na+ or K + on protein synthesis in heat-shock-treated hepatoma cells. It is clear from this overview that Na+ and K+ might be involved in processes affecting protein synthesis but that they are surely not the only factors which could explain altered protein synthesis in all activated cell states. Despite the conflicting results, it is uncontested that K+ is an important cofactor in protein synthesis as assessed in cell-free systems. Some of the biochemical steps which are influenced by K+ and Na+ are discussed in the next paragraph. B. Influence of Ions on Biochemical Processes Related to Protein Synthesis

Cell-free translation systems are indispensable tools for the study of the regulatory mechanisms of protein synthesis by ions. By using this experimental approach, it has been shown that K+ probably influences elongation of nascent polypeptides (Cahn and Lubin, 1978). Furthermore, it was

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demonstrated that different mRNAs have different K+ requirements for their translation (Sonenshein and Brawerman, 1976), for example, the range of KCI optima in cell-free systems is from 60 mM for globin and unmethylated reovirus mRNA to 120 mM for encephalomyocarditis virus RNA (Brender et al., 1981). The fact that under a particular set of Mg2+ and K+ concentrations the same order of initiation efficiences for different mRNAs is observed in vitro and in vivo suggests that the in vitro system indeed mimics the in uivo situation and that the sites of regulation of protein synthesis are the same in both cases. 1. mRNA COMPETITION

Excess mRNAs can compete with one another in binding to a limiting message-discriminatory component during an early stage in the initiation process (Godefroy-Colburn and Thach, 1981), and this form of translational control has been proposed as a putative regulator of on-off switches of key proteins in altered physiological situations. Interestingly, the order of competitive efficiencies of mRNAs can be modulated by changes in the K+ concentration, probably by changing the affinity of the mRNA for the discriminatory factor (Brendler et al., 1981). 2. RECOGNITION OF mRNA CAP STRUCTURE

The 5-termini of most eukaryotic mRNAs have a unique structure of 7methylguanosine (m7G), which is a recognition signal necessary for efficient translation of various mRNAs (Kemper and Stolarsky, 1977; Kozak, 1978). The sensitivity of capped mRNA translation to competition by 7methylguanosine 5’-phosphate (pm7G)was found to be a function of K+ concentration (Kemper and Stolarsky, 1977). In fact, increasing K+ concentration increases inhibition of translation of globin mRNA by pm7Gor, in other words, the affinity for a putative receptor protein of the m7G group increases at lower K+ concentration. At K+concentrations optimal for translation of m7G-containing mRNA, unmethylated mRNA, on the other hand, is translated at an efficiency of only 15-20% (Weber et al., 1977). The dependence of translation on the cap structure is, in addition to K+, strongly affected by temperature (Weber et al., 1977) and by mRNA concentrations and ionic strength (Chu and Rhoads, 1978) but also by pH changes in the range of pH 6.8-7.8 (Rhoads et al., 1983). This pH dependence has been explained by the existence of a zwitterionic form of the active cap and an increase in the enolate form with increasing pH, which would preferentially interact with cap-binding proteins. The mechanism underlying the discrimination between viral and cellu-

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lar protein synthesis has recently been investigated in high-salt-treated MDCK cells infected with influenza B and vesicular stomatitis viruses (VSV) (Tsurumi et al., 1983). Particular attention was given to the role of the 5' terminal structure of capped mRNA and its dependence on NaCl concentration. The hypothesis was tested whether the synthesis of influenza virus, which has a host-specific 5' terminal structure, would be suppressed by high salt treatment to the level of cellular protein synthesis, while VSV protein synthesis, which has a virus-specific 5' structure, would be unaffected. The synthesis of both virus proteins, however, was depressed to a similar extent by hypertonic media (about 170 mM NaCI), although less than that of host proteins, indicating that at least in this cell line high salt probably has no effect on the function of the cap structure but rather may inhibit the translation of cellular mRNA at another initiation site (Tsurumi et af., 1983).

3.

s 6 PHOSPHORYLATiON

In recent years considerable evidence has accumulated that covalent modifications are major mechanisms which control the activity of regulatory proteins involved in protein synthesis and hence the efficiency of the biochemical reactions associated with them. Phosphorylation of initiation factors (Ranu, 1982; Schatzman et al., 1983; Siekierka et a / . , 1984) and ribosomal proteins (for review see Gordon et af., 1982) is well documented and occurs in many eukaryotic cells. For our purposes, phosphorylation of the s6 protein of the small ribosomal subunit is of particular interest. Phosphorylation of this protein is indeed highly correlated with growth-promoting stimuli in a wide variety of systems, including serum and growth factor-induced cell proliferation, viral transformation, fertilization, and oocyte maturtion (for ref. see Gordon ef af., 1982). In addition, in most systems, increased protein synthesis accompanies s6 phosphorylation. Unfortunately, conclusive evidence for the involvement of s 6 in the initiation of protein synthesis is not available, but recent studies suggest that phosphorylation of s6 protein induces a conformational change of the ribosome involving several proteins on the small and large ribosomal subunits (Kisilevsky et al., 1984). The proteins identified on the small subunit have been shown to be topographically close to s6 (Tolan and Traut, 1981) and have been implicated in initiation. Thus s6 phosphorylation might, through a conformational change of the small ribosomal subunit, permit correct interaction of ribosomes with initiation factors and mRNA (Traugh, 1981). In 3T3 cells, phosphorylation of S60ccurs in a sequential manner, probably involving CAMP-dependent (MartinPerez et al., 1984) and CAMP-independent steps (Leader, 1980).

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In many systems s6 phosphorylation has been observed together with an increase in intracellular pH during cell activation (Ballinger and Hunt, 1981; PouyssCgur et al., 1982), and the hypothesis has thus been formulated that increased intracellular pH might lead directly to increased s 6 phosphorylation. In support of this hypothesis are the observations that (1) serum-stimulated s 6 phosphorylation in Chinese hamster fibroblasts is abolished by amiloride at a concentration that inhibits serum-stimulated Na+ influx through the Na+-H+ exchange system and (2) intracellular acidification with weak acids abolishes growth factor-induced s6 phosphorylation (PouyssCgur et al., 1982). In Arbaciu punctualata eggs, s6 phosphorylation also coincides with the onset of increased protein synthesis after fertilization and with the loss of a specific s 6 phosphatase found in unfertilized eggs (Ballinger et al., 1984). However, alkalinization of the cell interior, which leads to half-maximal increase in the rate of protein synthesis, neither causes s6 phosphorylation nor inactivates the specific s6 phosphatase. s 6 phosphorylation seems to require both a Ca2' transient and pH changes and is not triggered by either signal alone. In Xenopus leuuis oocytes it has recently also been shown that s 6 phosphorylation can be dissociated from changes in intracellular pH. Cholera toxin treatment indeed increases s6 phosphorylation with no change in intracellular pH, and progesterone treatment of small oocytes increases intracellular pH with no increase in s 6 phosphorylation (Stith and Maller, 1984). As pointed out by the authors, caution should be applied to results obtained by the use of weak bases to alter intracellular pH. A wide variety of cationic drugs have indeed been shown to induce maturation in some oocytes, likely by a nonspecific interaction with the plasma membrane (Schorderet-Slatkine et al., 1977), which could well trigger an increase in intracellular pH but also other intracellular changes through nonspecific alterations of membrane permeability. This overview clearly shows that in order to definitely assign a role of intracellular pH in s6 phosphorylation, more work is needed. One approach might be to microinject substances which modify intracellular pH into cells and measure s 6 phosphorylation. In addition, attempts should be made to purify protein kinases from resting and activated cells and to study their activation patterns by pH in the intact cell and in cell-free systems.

FACTOR (eIF-2) PHOSPHORYLATION 4. INITIATION Initiation factor eIF-2 has been identified as one of the regulatory components of protein synthesis (for review see Gupta, 1982). It forms a ternary complex with methionyl-tRNA and is responsible for the binding

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of this translation initiater to the ribosome. Phosphorylation of the a subunit of eIF-2 mediated by self-phosphorylation of various protein kinases leads to inhibition of protein synthesis. Recently the effect of Ca2+ on the self-phosphorylation of the cAMP-independent kinases and the heterophosphorylation of the eIF-2 has been studied in cell-free systems (Ranu, 1982). Interestingly, Ca2+inhibits both the self-phosphorylation of the kinase and the heterophosphorylation of eIF-2. As pointed out by the author, protein synthesis might thus be stimulated by Ca2+by acting at two levels, first by inhibition of the activation of the protein kinase and second by inhibition of the phosphorylation of eIF-2. V. POSTTRANSLATIONAL EVENTS INFLUENCING INTRACELLULAR TRAFFIC AND CELL SURFACE EXPRESSION OF PROTEINS

Several posttranslational modifications of proteins dependent on ionic conditions have already been described in this chapter. In particular, it was shown that phosphorylation reactions may modify the activity of certain proteins implicated in DNA and protein synthesis and could thus be trigger events in cell activation. In the following the influence of ions on some other types of posttranslational protein modifications which might have effects on protein targeting and on protein expression at the cell surface will be discussed. Such processes are potentially important in an altered cellular state, in particular during differentiation and cell growth arrest but also upon hormonal stimulation. A. Ions and lntracellular Protein Traffic

There is increasing evidence that differential expression of glycoproteins plays an important regulatory role in cellular processes involved in cell growth inhibition (Holley et al., 19831, cell differentiation (Robinson et al., 1984), morphogenesis (Edelman, 1984), and embryonic development (Lennarz, 1983; Shur, 1984). Although no studies exist which have looked at the relation between ion fluxes upon alterations in cell states and the cellular expression of glycoproteins, several lines of evidence indeed suggest that this aspect might deserve some consideration. Complex-type N-glycosylation of plasma membrane or secretory proteins implies that the polypeptide has to follow a specific cellular route starting with the synthesis and concomitant coreglycosylation in the rough endoplasmic reticulum, followed by vesicular transport through the Golgi complex, where trimming of mannoses and terminal glycosylation takes place.

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Finally the glycoprotein is targeted to the plasma membrane, where it may remain as an integral membrane protein or might then be released from the cell as a secretory glycoprotein (for review see Hubbard and Ivatt, 1981). It is now clear that the ability of eukaryotic cells to conduct this protein traffic is highly sensitive to even slight perturbations of cellular Na+ and K+ levels. Evidence for this finding is mainly derived from the experimental use of the drug monensin, which easily inserts into cellular membranes and acts as a Na+-K+ ionophore, leading to a transient change in the intracellular ionic balance (Pressman and Fahim, 1982). The most striking effects of moderate doses of monensin (0.11pM) in a variety of normal, malignant, and virus-infected cells is indeed a drastic slowing of the intracellular transport of newly synthesized secretory proteins, proteoglycans, and plasma membrane proteins (for review see Tartakoff, 1983). Since monensin treatment does not affect intracellular pH, ATP levels, or protein synthesis, and since its effects can be partially mimicked by ouabain or a deacrease in extracellular K+, the altered intracellular ion levels themselves seem to be responsible for the observed effects (Tartakoff, 1982). Protein transport seems to be arrested in the proximal part of the Golgi compartment (Johnson and Schlesinger, 1980), thus preventing glycoproteins from acquiring terminal sugars such as galactose, glucose, and sialic acid, which are normally attached in the more distal portion of the Golgi stacks. It is not known how changes in intracellular Na+-K+ levels bring about these effects, but under the influence of monensin the Golgi cisternae become dilated (Tartakoff and Vassalli, 1978), and it is likely that these morphological perturbations hinder the correct transport of proteins. Considering these data it is tempting to speculate that processes involved in vesicular protein transport might be potential targets for changes in intracellular K+-Na+ ratios observed upon cell activation. An increase in cellular Na+, through increased Na+ influx or K+ or through secondary stimulation of the Na,K-ATPase could lead through its effects on the Golgi complex to suppression or alternatively preferential expression of specific glycoproteins playing a role in the altered state of cells. 8. Ions and Expresslons of Latent Plasma Membrane Proteins

In addition to its effects on glycoprotein traffic, monensin treatment also increases the release of secretory products from storage granules, including catecholamine secretion from chromaffin cells (Suchard et al., 1982). This event was related to a secondary liberation by increased Na+ fluxes of Caz+from intracellular stores, which in turn facilitates exocytosis. That increased intracellular Ca2+may indeed play a role in exocytosis

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is supported by the observation that release of vasopressin from rat neurohypophysis can be provoked by Ca2+addition to Ca2+-depletedtissue. In addition, the response to Ca2+is greatly enhanced after inhibition of the Na+ pump by ouabain and depends on external Na+ (Sorimachi, 1983). In this case, the results suggest that an increase in intracellular Na+ stimulates a Na+-dependent Ca2+influx, which mediates vasopressin secretion. In analogy to the fusion of secretory vesicles with the plasma membrane, it is possible that cell surface expression of plasma membrane proteins might as well be influenced by a Na+-Ca2+-dependent fusion process of a latent protein pool contained in intracellular vesicles. Such a hypothesis could at least explain results obtained on the increase in Na,KATPase activity in aldosterone-treated target epithelia. Aldosterone stimulates transepithelial Na+ reabsorption in the toad urinary bladder and in the mammalian kidney (for review see Marver and Kokko, 1983). The cellular mechanisms which mediate aldosterone's action include a rapid increase in the Na+ permeability of the apical membrane and in turn a stimulation of the pump rates of Na,K-ATPase, localized at the basolateral membrane. In addition, the relative rate of biosynthesis of Na,KATPase increases 2-4-fold over controls after 6 hours of hormone treatment in the toad urinary bladder (Geering et af., 1982). On the other hand, however, an increased expression of functionally active pump sites in the plasma membrane of renal collecting tubules could already by observed 90 minutes after hormone treatment of adrenalectomized rabbits (Petty et al., 1981). A possible explanation of these apparently contradictory results might be found in the fact that amiloride, which blocks the apical Na+ entry in these tissues, abolishes the increased expresssion of Na+ pumps in the plasma membrane (Handler et af., 1981; Petty et al., 1981) but not the aldosterone-induced increase in the biosynthesis rate of this enzyme (Geering et al., 1982). These data indeed suggest that increased Na+ fluxes across the apical membrane upon aldosterone stimulation might directly (or indirectly through a secondary intracellular increase in Ca2+)be responsible for a rapid mobilization of Na+ pumps from an intracellular pool. On the other hand, the delayed increase in the biosynthesis rate is not affected by intracellualr Na+ and is probably mediated by a direct effect of aldosterone at the transcriptional level. VI.

CONCLUSIONS

Particular consideration has been given in this review to the role of ions (Ca2+,H+, K + , Na+) in the triggering of biochemical processes related to cell activation, namely, of DNA replication and of transcriptional, trans-

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lational, and posttranslational events. The data are consistent with a model which looks upon cell activation as a pleiotropic event mediated at the biochemical level through the effect of various ions acting in parallel and/or in succession. Indeed, a good correlation exists in various experimental systems between intracellular ionic changes occurring upon cell activation and the induction of DNA synthesis leading to mitosis as well as of RNA and protein synthesis related to cell growth and differentiation. In addition, several biochemical reactions tightly linked to these metabolic processes have been shown to depend on and/or be controlled by particular ionic conditions. However, in most cases evidence has not yet been provided for a causal link between these biochemical reactions and the cell activation process; in other words, the question whether the biochemical reactions controlled by ions are indeed key events in the cell activation process often remains unsettled. To solve this issue more work is needed which relates the biochemical data obtained in vitro to the biochemical situation as it is observed under cell activation conditions in vivo. REFERENCES Andrews, G. K., Janzen, R. G., and Tamaoki, T. (1982). Stability of a-fetoprotein messenger RNA in mouse yolk sac. Deu. Biol. 89, 11 1-1 16. Armato, U.,Andreis, P. G., and Whitfield, J. F. (1983). The calcium-dependence of the stimulation of neonatal rat hepatocyte DNA synthesis and division by epidermal growth factor, glucagon and insulin. Chem. Biol. Interact. 45, 203-222. Ballinger, D. G., and Hunt, T. (1981). Fertilization of sea urchin eggs is accompanied by 40 S ribosomal subunit phosphorylation. Deu. Biol. 87, 277-285. Ballinger, D. G . , Bray, S. J., and Hunt, T. (1984). Studies of the kinetics and ionic requirements for the phosphorylation of ribosomal protein S6 after fertilization of Arbacia punctulata eggs. Dev.Biol. 101, 192-200. Bernstein, A., Hunt, D. M.,Crichley, V.,and Mak, T. W. (1976). Induction by ouabain of hemoglobin synthesis in cultured Friend erythroleukemic cells. Cell 9, 375-381. Boonstra, J . , Moolenaar, W.H., Harrison, P. H., Moed, P., Van Der Saag, P. T., and De Laat, S. W. (1983). Ionic responses and growth stimulation induced by nerve growth factor and epidermal growth factor in rat pheochromocytoma (PC12) cells. J . Cell Biol. 97992-98. Boonstra, J., Schamhart, D. H. J., De Laat, S. W., and Van Wijk, R. (1984). Analysis of K+ and Na+ transport and intracellular contents during and after heat shock and their role in protein synthesis in rat hepatoma cells. Cancer Res. 44, 955-960. Brandis, J. W., and Raf€, R. A. (1979). Elevation of protein synthesis is a complex response to fertilisation. Nature (London) 278, 467-469. Brendler, T., Godefroy-Colburn, T., Yu, S., and Thach, R. E. (1981). The role of mRNA competition in regulating translation. 111. Comparison of in vitro and in uiuo results. J . Biol. Chem. 256, 11755-11761. Brostrom, C. O., Bocckino, S. B., and Brostrom, M. A. (1983). Identification of a Ca2+ requirement for protein synthesis in eukaryotic cells. J. Biol. Chem. 258, 14390-14399.

7. REGULATION OF DNA, RNA, AND PROTEIN SYNTHESIS

251

Busa, W. B., and Nuccitelli, R. (1984). Metabolic regulation via intracellular pH. A m . J . Physiol. 246, R409-R438. Buxman, M.M., and Wuepper. K. D. (1976). Isolation, purification and characterization of bovine epidermal transglutaminase. Biochim. Biophys. Acta 452, 356-369. Cahn, F., and Lubin, M. (1978). Inhibition of elongation steps of protein synthesis at reduced potassium concentrations in reticulocytes and reticulocyte lysate. J . Biol. Chem. 253, 7798-7803. Cameron, C. M., and Rillema, J. A. (1983). Extracellular calcium ion concentration required for prolactin to express its actions on casein, ribonucleic acid, and lipid biosynthesis in mouse mammary gland explants. Endocrinology 113, 1596-1600. Carrasco, L. (1977). The inhibition of cell functions after viral infection. A proposed general mechanism. FEES Lett. 76, 11-15. Celis, J. E., and Bravo, R. (1984). Synthesis of the nuclear protein cyclin in growing, senescent and morphologically transformed human skin fibroblasts. FEES Lett. 165, 21-25. Chafouleas, J. G., Bolton, W. E., Hidaka, H., Boyd, A. E., 111, and Means, A. R. (1982). Calmodulin and the cell cycle: Involvement in regulation of cell-cycle progression. Cell 28, 41-50. Charest, R., Blackmore, P. F., Berthon, B., and Exton, J. H. (1983). Changes in free cytosolic Ca2+ in hepatocytes following a,-adrenergic stimulation. Studies on quin-2loaded hepatocytes. J . Biol. Chem. 14, 8769-8773. Chu, L. Y.,and Rhoads, R. E. (1978). Translational recognition of the 5‘-terminal7-methylguanosine of globin messenger RNA as a function of ionic strength. Biochemistry 17, 2450-2455. Cochran, B. H., and Stiles, C. D. (1982). Molecular cloning of gene sequences regulated by platelet-derived growth factor (PDGF). J . Cell Biol. 95, 198a (Abstr.). Cress, A. E., Culver, P. S., Moon, T . E., and Gerner, E. W. (1982). Correlation between amounts of cellular membrane components and sensitivity to hyperthermia in a variety of mammalian cell lines in culture. Cancer Res. 42, 1716-1721. Dale, B. A., Haugen-Scofield, J. A., Hennings, H., Stanley, J. R., and Yuspa, S. H. (1983). Identification of filaggrin in cultured mouse keratinocytes and its regulation by calcium. J . Invest. Dermatol. 81, 90s-95s. Darnell, J. E., Jr. (1982). Variety in the level of gene control in eukaryotic cells. Nature (London) 297, 365-371. Das, M. (1980). Mitogenic hormone-induced intracellular message: Assay and partial characterization of an activator of DNA replication induced by epidermal growth factor. Proc. Natl. Acad. Sci. U . S . A . 77, 112-1 16. Dazord, A., Langlois-Gallet, D., and Saez, J. M. (1981). Mechanism of corticotropin action on adrenal protein synthesis. The effect of inhibitors of protein synthesis and calcium chelators. Horm. Res. 15, 270-282. DeCoursey, T. E., Chandy, K.G., Gupta, S., and Cahalan, M. D. (1984). Voltage-gated K + channels in human T lymphocytes: A role in mitogenesis? Nature (London) 307, 465468. De Laat, S. W., Boonstra, J., Moolenaar, W. H., Mummery, C. L., van der Saag, P. T., and van Zoelen, E. J. J. (1982). Cation transport and growth control in neuroblastoma cells in culture. In “Membranes in Growth and Development” ( G . Giebisch and A. J. Hoffman, eds.), pp. 211-236. Liss, New York. De Morales, M. M., Blat, C., and Harel, L. (1974). Changes in the phosphorylation of nonhistone chromosomal proteins in relationship to DNA and RNA synthesis in BHK-21 (C-13) cells. Exp. Cell. Res. 86, Ill-119.

252

KATHl GEERING

Dennis, W. H., and Yatvin, M. B. (1981). Correlation of hyperthermic sensitivity and membrane microviscosity in E. coli K1060. Int. J . Radiat. B i d . 39, 265-271. Dub& F., and Guemer, P. (1983). CaZ+influx and stimulation of protein synthesis in sea urchin eggs. Exp. Cell Res. 147, 209-215. Duesberg, P. H., and Vogt, P. K. (1979). Avian acute leukemia viruses MC29 and MH2 share specific RNA sequences: Evidence for a second class of transforming genes. Proc. Natl. Acad. Sci. U.S.A. 76, 1633-1637. Dulbecco, R., and Elkington, J. (1975). Introduction of growth in resting fibroblastic cell cultures by Ca++.Proc. Natl. Acad. Sci. U.S.A. 72, 1584-1588. Edelman, G. M. (1984). Cell adhesion and rnorphogenesis: The regulator hypothesis. Proc. Natl. Acad. Sci. U.S.A. 81, 1460-1464. Epel, D. (1980). Ionic triggers in the fertilization of sea urchin eggs. Ann. N.Y. Acad. Sci. 339,74-85. Epel, D., Steinhardt, R., Humphreys, T., and Mazia, D. (1974). An analysis of the partial metabolic derepression of sea urchin eggs by ammonia; the existence of independent pathways. Dev. Biol. 40,245-255. Farese, R. V. (1971). Calcium as a mediator of adrenocorticotrophic hormone action on adrenal protein synthesis. Science 173, 447-450. Fausto, N. (1984). Messenger RNA in regenerating liver: Implications for the understanding of regulated growth. Mol. Cell. Biochem. 59, 131-147. Garry, R. F., Bishop, J. M., Parker, S., Westbrook, K., Lewis, G., and Waite, M. R. F. (1979). Na+ and K+ concentrations and the regulation of protein synthesis in Sindbis virus-infected chick cells. Virology 96, 108-120. Gany, R. F., Ulug, E. T., and Bose, H. R., Jr. (1982). Membrane-mediated alterations of intracellular Na+ and K+ in lytic-virus-infected and retrovirus-transformed cells. Biosci. Rep. 2, 617-623. Geering, K., Girardet, M., Bron, C., Kraehenbuhl, J. P., and Rosier, B. C. (1982). Hormonal regulation of (Na+,K+)-ATPasebiosynthesis in the toad bladder. Effect of aldosterone and 3,5,3’-triiodo-~-thyronine.J. Biol. Chem. 257, 10338-10343. Gerson, D. F. (1982). The relation between intracellular pH and DNA synthesis rate in proliferating lymphocytes. In “Intracellular pH: Its Measurement, Regulation, and Utilization in Cellular Functions” (R. Nuccitelli and D. W. Deamer, eds.), pp. 375-383. Liss, New York. Gillies, R. J. (1981). Intracellular pH and growth control in eukaryotic cells. I n “The Transformed Cell” (I. L. Cameron and T. B. Pool, eds.), pp. 347-395. Academic Press, New York. Gillies, R. J. (1982). Intracellular pH and proliferation in yeast, tetrahymena and sea urchin eggs. In “Intracellular pH: Its Measurement, Regulation, and Utilization in Cellular Functions” (R. Nuccitelli and D. W. Deamer, eds.), pp. 341-359. Liss, New York. Godefroy-Colburn, T., and Thach, R. E. (1981). The role of mRNA competition in regulating translation. IV. Kinetic model. J . Biol. Chem. 256, 11762-1 1773. Gordon, J., Nielsen, P. J., Manchester, K. L., Towbin, H., Jimenez de Asua, L., and Thomas, G. (1982). Criteria for establishment of the biological significance of ribosomal protein phosphorylation. Curr. Top. Cell. Regul. 21, 89-99. Grainger, J. L., Winkler, M. M., Shen, S. S., and Steinhardt, R. A. (1979). Intracellular pH controls protein synthesis rate in the sea urchin egg and early embryo. Dev. B i d . 68, 396-406. Gray, M.A., Micklem, K. J., and Pasternak, C. A. (1983). Protein synthesis in cells infected with Semliki Forest virus is not controlled by intracellular cation changes. Eur. J . Biochem. 135. 299-302.

7. REGULATION OF DNA, RNA, AND PROTEIN SYNTHESIS

253

Gupta, N . K. (1982). Roles of eukaryotic initiation factor 2 ancillary factors in the regulation of eukaryotic protein synthesis initiation. Curr. T o p . Cell. Regul. 21, 1-33. Gurley, L.R . , Tobey, R. A., Walters, R. A., Hildebrand, C. E., Hohmann, P. G., D‘Anna, J. A., Barham, S. S., and Deaven, L. L. (1978). Histone phosphorylationand chromatin structure in synchronized mammalian cells. In “Cell Cycle Regulation” ( J . R. Jeter, Jr., I. L. Cameron, G. M. Padilla, and A. M. Zimmerman, eds.), pp. 37-60. Academic Press, New York. Hackstadt, T., and Mallavia, L. P. (1982). Sodium and potassium transport in herpes simplex virus-infected cells. J . Gen. Virol. 60, 199-207. Handler, J. S ., Preston, A. S . , Perkins, F. M., Matsumura, M., Johnson, J. P., and Watlington, C. 0. (1981). The effect of adrenal steroid hormones on epithelia formed in culture by A6 cells. Ann. N . Y. Acad. Sci. 372,442-454. Harper, J. F., Cheung, W. Y., Wallace, R. W., Huang, H. L.. Levine, S. N., and Steiner, A. L. (1980). Localization of calmodulin in rat tissues. Proc. Natl. Acud. Sci. U.S.A.77, 366-370. Heldin, C. H., and Westermark, B. (1984). Growth factors: Mechanism of action and relation to oncogenes. Cell 37, 9-20. Hennings, H., Michael, D., Cheng, C., Steinert. P., Holbrook, K.. and Yuspa, S. H. (1980). Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell 19, 245-254. Hennings, H., Holbrook, K. A., and Yuspa, S . H. (1983a). Potassium mediation of calciuminduced terminal differentiation of epidermal cells in culture. J. Invest. Dermurol. 81, 50s-55s.

Hennings, H., Holbrook, K. A., and Yuspa, S. H. (1983b). Factors influencing calciuminduced terminal differentiation in cultured mouse epidermal cells. J. Cell. Physiol. 116, 265-281. Hesketh, T. R.,Smith, G. A., Moore, J. P., Taylor, M. V.. and Metcalfe, J. C. (1983). Free cytoplasmic calcium concentration and the mitogenic stimulation of lymphocytes. J. Biol. Chem. 258, 4876-4882. Hickie, R . A , , Wei, J. W., Blyth, L. M., Wong, Y. W., and Klaassen, D. J. (1983). Cations and calmodulin in normal and neoplastic cell growth regulation. Can. J . Biochrm. Cell B i d . 61, 934-941. Hidaka, H., Sasaki, Y., Tanaka, T., Endo, T., Ohno, S., Fujii, Y., and Nagata, T. (1981). N (6-aminohexyl)-5-chloro-I-naphthalenesulfonamide, a calmodulin antagonist, inhibits cell proliferation. Proc. Natl. Acad. Sci. U . S . A . 78, 4354-4357. Hightower, K. R . . (1983). The influence of calcium on protein synthesis in the rabbit lens. Invest. Ophrhalmol. Visual Sci. 24, 1422- 1426. Holley, R. W., Armour, R.,Baldwin, J. H., and Greenfield, S. (1983). Preparation and properties of a growth inhibitor produced by kidney epithelial cells. Cell Biol. Inr. Rep. 7, 525-526. Hubbard, S . C., and Ivatt, R. J. (1981). Synthesis and processing of asparagine-linked oligosaccharides. Annu. Rev. Biochem. 50, 555-583. Jenkins, N. A., Kaumeyer, J. F., Young, E. M., and Raff, R. A. (1978). A test for masked message: The template activity of messenger ribonucleoprotein particles isolated from sea urchin eggs. Dev. Biol. 63, 279-298. Johnson, D. C . , and Schlesinger, M. J. (1980). Vesicular stomatitis virus and Sindbis virus glycoprotein transport to the cell surface is inhibited by ionophores. Virology 103,407424. Jgkgensen, P. L. (1980). Sodium and potassium ion pump in kidney tubules. Physiol. Rev. 60, 864-917.

254

KATHl GEERING

Jprgensen, P. L. (1982). Mechanism of the Na+,K+pump protein structure and conformations of the pure (Na+ + K + ) = l ATPase. Biochim. Biophys. Acra 694, 27-68. Jungmann, R. A,, and Kranias, E. G. (1977). Nuclear phosphoprotein kinases and the regulation of gene transcription. Int. J . Biochem. 8, 819-830. Kanungo, M. S., and Thakur, M. K. (1979). Phosphorylation and acetylation of nonhistone chromosomal proteins of the brain of rats of various ages and their modulation by calcium and estradiol. Biochem. Biophys. Res. Commun. 86, 14-19. Kaplan, E. and Richman, H. G. (1973). Calcium stimulation of uridine-6-3H incorporation into RNA of rat myocardium. Proc. SOC. Exp. Biol. Med. 142, 487-489. Katz, A. I. (1982). Renal Na-K-ATPase: Its role in tubular sodium and potassium transport. Am. J. Physiol. 242, F207-F219. Kaumeyer, J. F., Jenkins, N. A., and Raff, R. A. (1978). Messenger ribonucleoprotein particles in unfertilized sea urchin eggs. Deu. Biol. 63, 266-278. Kemper, B., and Stolarsky, L. (1977). Dependence on potassium concentration of the inhibition of the translation of messenger ribonucleic acid by 7-methylguanosine 5’phosphate. Biochemistry 16,5676-5680. Kisilevsky, R., Treloar, M. A., and Weiler, L. (1984). Ribosome conformational changes associated with protein S6 phosphorylation. J . Biol. Chem. 295, 1351-1356. Klein, G. (1983). Specific chromosomal translocations and the genesis of B-cell-derived tumors in mice and men. Cell 32, 311-315. Koch, K. S., and Leffert, H. L. (1979). Increased sodium ion influx is necessary to initiate rat hepatocyte proliferation. Cell 18, 153-163. Kozak, M. (1978). How do eucaryotic ribosomes select initiation regions in messenger RNA? Cell 15, 1109-1123. Kuchler, R. J. (1967). The role of sodium and potassium in regulating amino acid accumulation and protein synthesis in LM-strain mouse fibroblasts. Biochim. Bipohys. Acta l36, 473-483. Kypson, J., and Hait, G. (1971). The effects of ouabain, calcium and potassium on the transport and incorporation of some amino acids into rabbit atrial tissue in uitro. J. Pharmacol. Exp. Ther. 177, 398-408. L’Allemain, G., Franchi, A., Cragoe, E., Jr., and Pouysstgur, J. (1984a). Blockade of the Na+/H+antiport abolishes growth factor-induced DNA synthesis in fibroblasts. Structure-activity relationships in the amiloride series. J. Biol. Chem. 259, 4313-4319. L’Allemain, G., Paris, S., and Pouysskgur, J. (1984b).Growth factor action and intracellular pH regulation in fibroblasts. Evidence for a major role of the Na+/H+antiport. J . Biol. Chem. 259, 5809-5815. Langdon, R. C., Fleckman, P., and McGuire, J. (1984). Calcium stimulates ornithine decarboxylase activity in cultured mammalian epithelial cells. J. Cell. Physiol. 118, 39-44. Leader, D. P. (1980). The control of phosphorylation of ribosomal proteins. I n “Recently Discovered Systems of Enzyme Regulation by Reversible Phosphorylation; Molecular Aspects of Cellular Regulation” (P. Cohen, ed.), Vol. 1, pp. 203-233. Elsevier, Amsterdam. Ledbetter, M. L. S., and Lubin, M. (1977). Control of protein synthesis in human fibroblasts by intracellular potassium. Exp. Cell Res. 105, 223-236. Lennarz, W. J. (1983). Glycoprotein synthesis and embryonic development. CRC Crit. Rev. Biochem. 14,257-272. Levenson, R., Macara, I. G., Smith, R. L., Cantley, L., and Housman, D. (1982). Role of mitochondria1 membrane potential in the regulation of murine erythroleukemia cell differentiation. Cell 28, 855-863.

7. REGULATION OF DNA, RNA, AND PROTEIN SYNTHESIS

255

Levine, B. A., and Dalgarno, D. C. (1983). The dynamics and function of calcium-binding proteins. Biochim. Biophys. Acta 726, 187-204. Ling, L., and Cantley, L. (1984). The (Na,K)-ATPase of Friend erythroleukemia cells is phosphorylated near the ATP hydrolysis by an endogenous membrane-bound kinase. J. Biol. Chem. 259, 4089-4095. Linzer, D. I. H., and Nathans, D. (1983). Growth-related changes in specific mRNAs of cultured mouse cells. Proc. Natl. Acad. Sci. U.S.A. 80, 4271-4275. Lipton, P., and Heimbach, C. J. (1978). Mechanism of extracellular potassium stimulation of protein synthesis in the in uitro hippocampus. J . Neurochem. 31, 1299-1307. Lipton, P., and Robacker, K. (1983). Glycolysis and brain function: [K+], stimulation of protein synthesis and K+ uptake require glycolysis. Fed. Proc., Fed. Am. Soc. Exp. Biol. 42, 2875-2880. Lopez-Rivas, A., Adelberg, E. A., and Rozengurt, E. (1982). Intracellular K+ and the mitogenic response of 3T3 cells to peptide factors in serum-free medium. Proc. Natl. Acad. Sci. U . S . A . 79,6275-6279. Lubin, M. (1964). Cell potassium and the regulation of protein synthesis. In “The Cellular Functions of Membrane Transport” ( J . F. Hoffman, ed.), pp. 193-211. Prentice Hall, New York. Lubin, M. (1967). Intracellular potassium and macromolecular synthesis in mammalian cells. Nature (London) 213, 451-453. Lubin, M. (1980). Control of growth by intracellular potassium and sodium concentrations is relaxed in transformed 3T3 cells. Biochem. Biophys. Res. Commun. 97, 1060-1067. Lubin, M., Cahn, F., and Coutermarsh, B. A. (1982). Amiloride, protein synthesis, and activation of quiescent cells. J. Cell. Physiol. 113, 247-251. Mackenzie, C. G., Mackenzie, J. B., and Beck, P. (1961). The effect of pH on growth, protein synthesis, and lipid-rich particles of cultured mammalian cells. J . Biophys. Biochem. Cytol. 9, 141-156. McKnight, G. S., Hager, L., and Palmiter, R. D. (1980). Butyrate and related inhibitors of histone deacetylation block the induction of egg white genes by steroid hormones. Cell 22, 469-477. Martin-Perez, J . , Siegmann, M., and Thomas, G. (1984). EGF, PGF2, and insulin induce the phosphorylation of identical S6 peptides in Swiss mouse 3T3 cells: Effect of CAMPon early sites of phosphorylation. Cell 36, 287-294. Marver, D., and Kokko, J. P. (1983). Renal target sites and the mechanism of action of aldosterone. Mineral Electrolyte Metab. 9, 1-18. Matteson, D. R., and Deutsch, C. (1984). K channels in T lymphocytes: A patch clamp study using monoclonal antibody adhesion. Nature (London) 307, 468-471. Mazia, D., and Ruby, A. (1974). DNA synthesis turned on in unfertilized sea urchin eggs by treatment with NH40H. Exp. Cell Res. 85, 167-172. Means, A. R., Lagace, L., Guerriero, V., Jr., and Chafouleas, J. G. (1982). Calmodulin as a mediator of hormone action and cell regulation. J . Cell. Eiochem. 20, 317-330. Mendoza, S. A., Wigglesworth, N. M., Pohjanpelto, P., and Rozengurt, E. (1980). Na entry and Na-K pump activity in murine, hamster, and human cells. Effect of monensin, serum, platelet extract, and viral transformation. J. Cell. Physiol. 103, 17-27. Mix, L. L., Dinerstein, R. J., and Villereal, M. L. (1984). Mitogens and melittin stimulate an increase in intracellular free calcium concentration in human fibroblasts. Biochem. Biophys. Res. Commun. 119, 69-75. Moolenaar, W. H., Yarden, Y., de Laat, S. W., and Schlessinger, J. (1982). Epidermal growth factor induces electrically silent Na’ influx in human fibroblasts. J . Biol. Chem. 257. 8502-8506.

256

a T H l GEERING

Moore, R. E., Goldsworthy, T. L., and Pitot, H. C. (1980). Turnover of 3’-polyadenylatecontaining RNA in livers from aged, partially hepatectomized, neonatal, and Moms 5123C hepatoma-bearing rats. Cancer Res. 40, 1449-1457. Moms, J. D. H., Metcalfe, J. C., Smith, G. A., Hesketch, T. R., and Taylor, M. V. (1984). Some mitogens cause rapid increases in free calcium in fibroblasts. FEES Lett. 169, 189- 193. Mummery, C. L., van der Saag, P. T., and de Laat, S. W. (1983). Loss of EGF binding and cation transport response during differentiation of mouse neuroblastoma cells. J . Cell. Biochem. 21,63-75. Murty, C. N., Oliveros, B., and Sidransky, H. (1982). Effect of hypertonic sodium chloride on polyribosomes and protein synthesis of kidneys of rats. Proc. SOC.Exp. Biol. Med. 171,258-265. Norrie, D. H., Wolstenholme, J., Howcroft, H., and Stephen, J. (1982). Vaccinia virusinduced changes in “a+] and [K+]in HeLa cells. J . Gen. Virol. 62, 127-136. O’Donnell, M. E., Cragoe, E., Jr., and Villereal, M. L. (1983). Inhibition of Na+ influx and DNA synthesis in human fibroblasts and neuroblastoma-glioma hybrid cells by amiloride analogs. J . Pharmacol. Exp. Ther. 226, 368-372. Offenbacher, S., and Kline, E. S. (1975). Differential phosphorylation of rat liver nuclear non-histone proteins in vitro. Biochem. Biophys. Res. Commun. 66, 375-382. Oppermann, H., Levinson, A. D., Varmus, H. E., Levintow, L., and Bishop, J. M. (1979). Uninfected vertebrate cells contain a protein that is closely related to the product of the avian sarcoma virus transforming gene (src). Proc. Natl. Acad. Sci. U . S . A . 76, 18041808. Owen, N. E., and Villereal, M. L. (1983). Efflux of 4sCa2+from human fibroblasts in response to serum or growth factors. J. Cell. Physiol. 117, 23-39. Pardo, J. P., and Fernandez, F. (1982). Effect of calcium and calmodulin on RNA synthesis in isolated nuclei from rat liver cells. FEES Lett. 143, 157-160. Park, W., Jansing, R., Stein, J . , and Stein, G. (1977). Activation of histone gene transcription in quiesent WI-38 cells or mouse liver by a nonhistone chromosomal protein fraction from HeLa S3 cells. Biochemistry 16, 3713-3721. Parsons, P. G., Musk, P., Goss, P. D., and Leah, J. (1983). Effects of calcium depletion on human cells in vitro and the anomalous behavior of the human melanoma cell line MM170. Cancer Res. 43, 2081-2087. Petty, K. J., Kokko, J. P., and Marver, D. (1981). Secondary effect of aldosterone on Na-K ATPase activity in the rabbit cortical collecting tubule. J . Clin. Invest. 68, 1514-1521. Pool, T. B., Cameron, I. L., Smith, N. K. R., and Sparks, R. L. (1981). Intracellular sodium and growth control: A comparison of normal and transformed cells. In “The Transformed Cell” (1. L. Cameron and T. B. Pool, eds.), pp. 397-420. Academic Press, New York. Pouysstgur, J., Chambard, J. C., Franchi, A., Paris, S., and Van Obberghen-Schilling, E. (1982). Growth factor activation of an amiloride-sensitive Na+/H+exchange system in quiescent fibroblasts: Coupling to ribosomal protein S6 phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 79, 3935-3939. Pressman, B. C., and Fahim, M. (1982). Pharmacology and toxicology of the monovalent carboxylic ionophores. Annu. Rev. Pharmacol. Toxicol. 22, 465-490. Ranu, R. S. (1982). Regulation of protein synthesis in rabbit reticulocyte lysates: Effect of Ca2+,Mg2+,and Mn2+on self-phosphorylation and heterophosphorylation catalyzed by the heme-regulated and double-stranded-RNA-activated protein kinases. Biosci. Rep. 2, 813-817.

7. REGULATION OF DNA, RNA, AND PROTEIN SYNTHESIS

257

Reznik, V. M., Villela, J., and Mendoza, S. A. (1983). Serum stimulates Na entry and the Na-K pump in quiescent cultures ofepithelial cells (MDCK). J. Cell. Physiol. 117,21 I 214. Rhoads, R. E., Hellmann, G. M., Remy, P., and Ebel, J . P. (1983). Translational recognition of messenger ribonucleic acid caps as a function of pH. Biochemistry 22, 6084-6988. Ribeiro, S. M. F., and Armelin, H. A. (1984). Ca2+and Mg2+requirements for growth are not concomitantly reduced during cell transformation. Mol. Cell. Biochem. 59, 173181.

Richter, J. D., Smith, L. D., Anderson, D. M., and Davidson, E. H. (1984). Interspersed poly(A) RNAs of amphibian oocytes are not translatable. J. Mol. B i d . 173, 227-241. Robinson, J. K., Freinkel, R. K., and Gotschalk. R. (1984). Effect of retinoic acid and low calcium conditions on surface glycoconjugates defined by differential lectin labelling in mouse epidermal cell culture. Br. J . Dermafol. 110, 17-27. Rossow, P. W., Riddle, V. G., Pardee, A. B. (1979). Synthesis of labile, serum-dependent protein in early GI controls animal cell growth. Proc. Natl. Acad. Sci. U . S . A .76,44464450. Rozengurt, E. (1979). Early events in growth stimulation. I n “Surfaces of Normal and Malignant Cells” (R. 0. Hynes, ed.), pp. 323-353. Wiley, Chichester. Rozengurt, E . (1981). Stimulation of Na influx, Na-K pump activity and DNA synthesis in quiescent cultured cells. Adu. Enzyme Regul. 19, 61-85. Rozengurt, E., and Heppel, L. A. (1975). Serum rapidly stimulates ouabain-sensitive &Rb+ influx in quiescent 3T3 cells. Proc. Narl. Acad. Sci. U.S.A. 72, 4492-4495. Rubin, A. H., and Sanui, H. (1979). The coordinate response of cells to hormones and its mediation by the intracellular availability of magnesium. Cold Spring Harbor Conf. Cell P r o / f . 6, 74 1-750. Rubin, H., Vidair, C., and Sanui, H. (1981). Restoration of normal appearance, growth behavior, and calcium content to transformed 3T3 cells by magnesium deprivation. Proc. Natl. Acad. Sci. U . S . A . 78, 2350-2354. Saborio, J. L . , Pong, S. S., and Koch, G. (1974). Selective and reversible inhibition of initiation of protein synthesis in mammalian cells. J. Mol. Biol. 85, 195-21 I . Schatzman, R. C., Grifo, J. A., Merrick, W. C., and Kuo, J. F. (1983). Phospholipidsensitive Ca2+-dependentprotein kinase phosphorylates the p subunit of eukaryotic initiation factor 2 (eIF-2). FEES Lett. 159, 167-170. Schorderet-Slatkine, S . , Schorderet, M., and Baulieu, E. E. (1977). Progesterone-induced meiotic reinitiation in uitro in Xenopus laeuis oocytes. A role for the displacement of membrane-bound calcium. Differentiafion 9, 67-76. Schuldiner, S . , and Rozengurt, E. (1982). Na+/H+ antiport in Swiss 3T3 cells: Mitogenic 79,7778stimulation leads to cytoplasmic alkalinization. Proc. Nut/. Acad. Sci. U.S.A. 7782. Shainberg, A,, Yagil, G., and Yaffe, D. (1969). Control of myogenesis in uifro by Ca2+ concentration in nutritional medium. Exp. Cell Res. 58, 163-167. Shay, J. W. (1983). Cytoplasmic modification of nuclear gene expression. Mol. Cell. Biochem. 57, 17-26. Shipley, G. D., Childs, C. B., Volkenant, M. E., and Moses, H. L. (1984). Differential effects of epidermal growth factor, transforming growth factor, and insulin on DNA and protein synthesis and morphology in serum-free cultures of AKR-2B cells. Cancer Re.s. 44, 710-716. Shur, B. D. (1984). The receptor function of galactosyltransferase during cellular interactions. Mol. Cell. Biochem. 61, 143-158.

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Siekierka, J., Manne, V., and Ochoa, S. (1984). Mechanism of translational control by partial phosphorylation of the a subunit of eukaryotic initiation factor 2. Proc. Natl. Acad. Sci. U.S.A. 81, 352-356. Sikorska, M., MacManus, J. P., Walker, P. R., and Whitfield, J. F. (1980). The protein kinases of rat liver nuclei. Biochem. Biophys. Res. Commun. 93, 1196-1203. Smith, G. L. (1977). Increased ouabain-sensitive &Rubidium+uptake after mitogenic stimulation of quiescent chicken embryo fibroblasts with purified multiplication-stimulating activity. J . Cell Biol. 73, 761-767. Smith, J. B., and Rozengurt, E. (1978). Serum stimulates the Na+,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., Levenson, R., Housman, D., and Cantley, L. (1982). Evidence that a Na+/Ca2+antiport system regulates murine erythroleukemia cell differentiation. J . Biol. Chem. 257, 773-780. Sornlyo, A. P. (1984). Cellular site of calcium regulation. Nature (London) 309, 516-517. Sonenshein, G. E., and Brawerman, G. (1976). Differential translation of mouse myeloma messenger RNAs in a wheat germ cell-free system. Biochemistry 15, 5501-5506. Sorimachi, M. (1983). Inhibition of Na pumps enhances Ca-dependent release of vasopressin from the isolated neurohypophysis of the rat. Jpn. J. Physiol. 33, 1061-1066. Sparks, R. L., Pool, T. B., Smith, N. K. R., and Cameron, I. L. (1983). Effects of amiloride on tumor growth and intracellular element content of tumor cells in uiuo. Cancer Res. 43,73-77. Stanley, J. R., and Yuspa, S. H. (1983). Specific epidermal protein markers are modulated during calcium-induced terminal differentiation. J. Cell Biol. 96, 1809- 18 14. Steinhardt, R. A., and Epel, D. (1974). Activation of sea-urchin eggs by a calcium ionophore. Proc. Natl. Acad. Sci. U.S.A. 71, 1915-1919. Steinhardt, R., Zucker, R., and Schatten, G. (1977). Intracellular calcium release at fertilization in the sea urchin egg. Deu. Biol. 58, 185-196. Stevens, F. C. (1983). Calmodulin: An introduction. Can. J. Biochem. Cell Biol. 61, 906910. Stith, B. J., and Maller, J. L. (1984). The effect of insulin on intracellular pH and ribosomal protein S6 phosphorylation in oocytes of Xenopus laeuis. Deu. Biol. 102, 79-89. Suchard, S. J . , Lattanzio, F. A,, Jr., Rubin, R. W., and Pressman, B. C. (1982). Stimulation of catecholamine secretion from cultured chromaffin cells by an ionophore-mediated rise in intracellular sodium. J . Cell Biol. 94, 531-539. Tartakoff, A. M. (1982). The role of subcompartments of the Golgi complex in protein intracellular transport. Philos. Trans. R . SOC.Lond. Ser. B 300, 173-184. Tartakoff, A. M. (1983). Perturbation of the structure and function of the Golgi complex by monovalent carboxylic ionophores. I n “Methods in Enzymology” (S. Fleischer and B. Fleischer, eds.), Vol. 98, pp. 47-59. Academic Press, New York. Tartakoff, A., and Vassalli, P. (1978). Comparative studies of intracellular transport of secretory proteins. J . Cell Biol. 79, 694-707. Thomas, G., Thomas, G., and Luther, H. (1981). Transcriptional and translational control of cytoplasmic proteins after serum stimulation of quiescent Swiss 3T3 cells. Proc. Natl. Acad. Sci. U.S.A. 78, 5712-5716. Toback, F. G. (1980). Induction of growth in kidney epithelial cells in culture by Na+. Proc. Natl. Acad. Sci. U.S.A. 77, 6654-6656. Tolan, D. R., and Traut, R. R. (1981). Protein topography of the 40 S ribosomal subunit from rabbit reticulocytes shown by cross-linking with 2-iminothiolane. J . Biol. Chem. 256, 10129-10136. Traugh, J. A. (1981). Regulation of protein synthesis by phosphorylation. In “Biochemical

7. REGULATION OF DNA, RNA, AND PROTEIN SYNTHESIS

259

Actions of Hormones” ( G . Litwack, ed.), Vol. 8, pp. 167-208. Academic Press, New York. Truscello, A., Geering, K., Gaggeler, H. P., and Rossier, B. C. (1983). Effects of butyrate on histone deacetylation and aldosterone-dependent Na+ transport in the toad bladder. J . Biol. Chem. 258, 3388-3395. Tsurumi, T., Aoki, H., Nishiyama, Y., Shibata, M., Maeno, K., and Seo, H. (1983). Effect of high salt treatment on influenza B viral protein synthesis in MDCK cells. Microbiol. Immunol. 27, 519-529. Tupper, J. T., Del Rosso, M., Hazelton, B., and Zorgniotti, F. (1978). Serum-stimulated changes in calcium transport and distribution in mouse 3T3 cells and their modification by dibutyryl cyclic AMP. J. Cell. Physiol. 95, 71-84. Vidair, C., and Rubin, H. (1982). Mg*+-sensitive alterations in CaZ+regulation associated with cell transformation. J. Cell. Physiol. 113, 398-404. Walsh-Reitz, M. M., Toback, F. G., and Holley, R. W. (1984). Cell growth and net Na+ flux are inhibited by a protein produced by kidney epithelial cells in culture. Proc. Narl. Acad. Sci. U . S . A . 81, 793-796. Weber, L. A., Hickey, E. D., Nuss. D. L., and Baglioni, C. (1977). 5’-terminal 7-methylguanosine and mRNA function: Influence of potassium concentration on translation in vitro. Proc. Nail. Acad. Sci. U . S . A . 74, 3254-3258. White, B. A., Bauerle, L. R., and Bancroft, F. C. (1981). Calcium specifically stimulates prolactin synthesis and messenger RNA sequences in GH, cells. J . Biol. Chrm. 12, 5942-5945. Whitfield, J. F., MacManus, J. P., Rixon, R. H., Boynton, A. L., Youdale, T., and Swierenga, S. (1976). The positive control of cell proliferation by the interplay of calcium ions and cyclic nucleotides. A review. In V i m 12, 1-18. Whitfield, J. F., Boynton, A. L., MacManus, J. P., Sikorska, M., and Tsang, B. K. (1979). The regulation of cell proliferation by calcium and cyclic AMP. Mol. Cell. Biochem. 27, 155-179. Whitfield, J. F., MacManus, J. P., Boynton, A. L., Durkin, J., and Jones, A. (1982). Futures of calcium, calcium-binding proteins, cyclic AMP and protein kinases in the quest for an understanding of cell proliferation and cancer. I n “Functional Regulation at the Cellular and Molecular Levels” (R. A. Corradino, ed.), pp. 61-87. Elsevier, New York. Wilde, C. J., Hasan, H. R., White, D. A., and Mayer, R. J. (1981). The effect of calcium on synthesis and degradation of mammary cytosolic proteins and casein. Biochem. Biophys. Res. Commun. 103, 934-942. Williams, G. T., and Johnstone, A. P. (1983). ADP-ribosyl transferase, rearrangement of DNA, and cell differentiation. Biosci. Rep. 3, 815-830. Winkler, M. M. (1982). Regulation of protein synthesis in sea urchin eggs by intracellular pH. In “Intracellular pH: Its Measurement, Regulation, and Utilization in Cellular Functions” (R. Nuccitelli and D. W. Deamer, eds.), pp. 325-339. Liss, New York. Wolf€, D. J., Ross, J. M., Thompson, P. N., Brostrom, M. A., and Brostrom, C. 0. (1981). Interaction of calmodulin with histones. Alteration of histone dephosphorylation. J . Biol. Chem. 256, 1846-1860. Wu, F. S., Park, Y. C., Roufa, D., and Martonosi, A. (1981). Selective stimulation of the synthesis of an 80,000-dalton protein by calcium ionophores. J . Biol. Chem. 256,53095312. Youdale, T., and MacManus, J. P. (1975). Failure of tritiated thymidine incorporation into DNA to reflect the autoradiographically demonstrable calcium-induced increase in thymic lymphoblast DNA synthesis. J . Cell. Physiol. 86, 495-502.

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CURRENT TOPlCS 1N MEMBRANES AND TRANSPORT, VOLUME 27

Chapter 8

Energy Metabolism of Cellular Activation, Growth, and Transformation LAZAR0 J . MANDEL Department of Physiology Duke University Medical Center Durham, North Carolina 27710

I. Introduction.. . . . . . . . ............................................ 26 I 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 . 261 A. Energy Activation in Skeletal Muscle. ................................ 261 B. Brown Fat Thermogenesis.. . . . . . . 268 C. Activation of Energy Metabolism in 270 D. Energy Activation in Mononuclear Phagocytes and Polymorphonuclear Leukocytes ........................................................ 21 I IV. Energy Metabolism of Cells in Culture ........... . . . . . . . . . . . . . . . . . . . . . . . . . 272 A. Adaptation to Conditions in Culture . . . . . . 212 B. Adaptation of Cultured Cells to Hypoxia.. ............................ 215 C. Response of Energy Metabolism of Quiescent Cells to Growth Factors 216 and Specific Hormones. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Energy Metabolism of Malignant Cells 218 A. Aerobic Glycolysis in Tumors.. ...................................... 21x B. Oxidative Metabolism in Tumors 282 286 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.

INTRODUCTION

Biological cells have the ability to regulate their energy metabolism such that, in the steady state, energy production equals energy consumption. Many adult cell types, such as brain, liver, and kidney, normally 26 1 Copyright 0 19116 by Academic Pre%r.Inc. All rights of reproduction in m y form reserved.

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maintain a relatively constant metabolic rate, although these cells have developed adaptive mechanisms to deal with metabolic changes in the body. Other adult cells, such as those from skeletal muscle, brown fat, sperm, oocytes, and neutrophils, maintain themselves mostly in a lowactivity state but can be rapidly activated to accelerate their energy requirements. Cells placed in culture require different amounts of energy when they are growing as compared to the quiescent state. Growth activation of quiescent cells causes a rapid increase in metabolic rate and substantial changes in metabolic patterns to sustain the increased metabolic demand. Similarly, differentiation and malignant transformation also elicit fundamental alterations in metabolic rates and patterns within the affected cells. Most significantly, rapidly growing tumors demonstrate a rapid rate of aerobic glycolysis which is not present in most normal cells, Most of the metabolic alterations discussed in this chapter concern pathways in carbohydrate metabolism. The major pathways involved are shown in Fig. I (Morgan and Faik, 1981). Energy in the form of ATP can be obtained primarily through (1) oxidative metabolism, involving the tricarboxylic acid cycle and the oxidation of FADH and NADH in the mitochondria1 cytochrome chain (not shown), or (2) glycolytic metabolism from starch or glucose to lactate, which can occur either in the presence or absence of oxygen. The pentose cycle is dependent on glucose or other sugar metabolism and provides NADPH and a variety of anabolic intermediates required for cell growth (Reitzer et al., 1980). Most adult cells rely mainly on oxidative metabolism for their energy needs, while fetal cells, cells in culture, and tumor cells use a combination of glycolysis and oxidative metabolism (Pedersen, 1978; Morgan and Faik, 1981). This chapter provides examples of metabolic control in a variety of tissues to highlight the changes in energy metabolism that occur during activation, growth, and differentiation. First, a description of metabolic control in renal cells is offered as an example of this process in cells that normally maintain a relatively constant metabolic rate. The next section provides a variety of examples of energy activation used by adult cells that are quiescent most of the time but can be rapidly activated. Some of the examples selected describe the metabolic changes that are secondary to the alterations in ion transport discussed in other chapters of this volume. The last two sections describe changes in energy metabolism that occur when cells are placed in culture and in cells that undergo malignant transformation.

STARC"

1 I

SUCROSE

'-ern

MAD pT+

SUCCIUTE

F W A T E

2-~S?lC9GLVCfRATE

f

P W S w O t y K PVRWATE

GLUTAM1 N f

'"' d

UAD(P)

W

T

f

GDP

MAD(P)N

TmcmmxnK: ACIDC~CLE

o(- KfTOQLUTARATE

ISOCITRATI

-

-

CITRATE

NaD

FIG.1. Schematic diagram of major pathways involved in carbohydrate metabolism. Abbreviations: FDPase, fructose diphosphatase, EC 3.1.3.1 I ; GPI, glucose-phosphate isomerase, EC 5.3.1.9; GPK, 3-phosphoglycerate kinase, EC 2.7.2.3; HK, hexokinase, EC 2.7.1.1 ; PC,pyruvate carboxylase, EC 6.4. I. I; PEPCK, phosphoenolpyruvate carboxykinase, EC 4.1. I .32; PFK, phosphofructokinase, EC 2.7.1.11 ;PK, pyruvate kinase, EC 2.7.1.40; PRPP synthetase, phosphoribosylpyrophosphate synthetase, EC 2.7.6.1; RK, ribokinase, EC 2.7.1.15; TOK, triokinase, EC 2.7.1.28. [Reproduced with permission from Morgan and Faik (1981).]

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II. CONTROL OF ENERGY METABOLISM IN ADULT CELLS THAT MAINTAIN A RELATIVELY CONSTANT METABOLIC RATE

Many healthy tissues in an organism maintain a relatively constant metabolic rate which varies only slightly when external stimuli are imposed on the organism. For example, the oxygen consumption of the human kidney, brain, and abdominal organs are almost unchanged between rest, light physical work, and heavy physical work (Lehninger, 1975). Similarly, profound alterations in the acid-base status of mammals produce no change in the oxygen consumption of the kidney, despite dramatic changes in metabolic substrate utilization (Cohen and Kamm, 1976). Nevertheless, when experimentally stressed, the kidney displays a very tight coupling between energy supply and energy demand (Mandel and Balaban, 1981). This suggests that the relative constancy of the metabolic rate observed in these organs is due to a constant rate of work by these tissues rather than any metabolic limitation. This section will use experimental results obtained primarily in my laboratory with mammalian proximal renal tubules to illustrate the process by which the control of metabolic energy occurs when the energy demands on the cell are varied. The general principles learned are applicable to numerous other cells, such as liver and brain. The kidney derives most of its energy from oxidative metabolism (Cohen and Kamm, 1976; Mandel and Balaban, 1981). Therefore, to understand this control, it is important first to determine how much of the mitochondria1 respiratory capacity is used by the renal cell for its normal energy requirements. Measurement of the respiratory capacity of the in siru mitochondria provides the maximal rate at which the mitochondria consume oxygen coupled to the production of ATP. In isolated mitochondria, this maximal rate of oxygen consumption has been denoted as the “state 3 rate” by Chance and Williams (1956) and is obtained by the addition of a saturating concentration of ADP (Lardy and Wellman, 1952). Intact tubules are impermeant to ADP and thus, to perform the experiment, the plasma membranes must first be permeabilized to ADP. In a proximal tubule suspension from the rabbit kidney, this permeabilization was accomplished with the detergent digitonin, which binds preferentially to cholesterol and thus at low concentrations seems to be specific for plasma membranes (Harris et al., 1981). Without digitonin, ADP has no effect on respiration. Figure 2 shows various traces of experiments performed on a renal tubule suspension measuring its oxygen content vs. time, and the slopes, given by the numbers next to the traces, measure the rates of oxygen consumption (Qo2).Trace A measures the Qo2 of tubules placed in a KCI medium to approximate the intracellular ionic composi-

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Time

FIG. 2. Oxygen consumption traces of renal proximal tubules. The rate of consumption in nmol 02/min/mg protein is indicated by the numbers next to the traces. (A) Added digitonin (Dig), 0. I mg/mg protein; ADP, 0.38 mM. (B) Added nystatin (Nys), 0.026 mglmg protein; ouabain (Oub), 60 pM. ( C )Added ouabain, 60 F M ;nystatin. 0.018 mg/mg protein. [Adapted from Hams ct a/. (198l).]

tion. In the absence of Na, the initial Qo2 is low and plasma membrane permeabilization with digitonin does not affect the Qo, until ADP is added. ADP (375 pM)accelerates respiration by a factor of four or five to about 35-45 nmol 02/min - mg protein. This ADP-stimulated rate is termed the respiratory capacity of the mitochondria in the intact cell. This rate is identical to the state 3 rate of mitochondria isolated from proximal rabbit tubules when both respiratory rates are normalized to their cytochrome a content (Harris et al., 1981). This maximal rate was compared with oxygen consumption rates obtained in intact tubules bathed in a solution containing 150 mM Na, as well as other required salts and substrates, a situation close to that normally found in uiuo. Under these conditions, normal respiration is about 20-25 nmol/min * mg protein (trace B), which is 50-60% of maximal. Since most of the work of the kidney is devoted to active Na transport (Cohen and Kamm, 1976; Mandel and Balaban, 1981), we determined how much of the respiration can be modulated by this process. Two extreme conditions were examined: maximal activation of Na,K-ATPase activity (the enzyme involved in the active step for Na transport-see Mandel and

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Balaban, 1981) and total inhibition of the Na,K-ATPase. Maximal activation was obtained by the use of nystatin, a polyene antibiotic that makes the cells permeable to Na and K, thereby equilibrating intra- and extracellular Na and K. This treatment causes intracellular Na to increase from approximately 30 mM under normal conditions to 150 mM, which increases Na,K-ATPase activity, increasing the demand for ATP and causing a large increase in respiration, as shown in Fig. 2. Total inhibition of the Na,K-ATPase was obtained by use of ouabain. The effect of nystatin on Qo, occurs only through the Na,K-ATPase because the stimulation of Qo, by nystatin is fully inhibited by ouabain (trace C). If ouabain is added first, nystatin is without effect. Table I summarizes the results of a series of similar experiments comparing respiratory rate and capacity in rabbit proximal tubules. The respiratory capacity is 44 nmole 02/min mg protein. If we define this as 100% of the ATP generating capacity of the tubular mitochondria, we see that the spontaneous rate of respiration is 56% of maximal. Stimulation of Na,K-ATPase activity with nystatin increases respiration to the maximum capacity, and ouabain reduces respiration to 30% of capacity. Therefore, alterations in active transport could command up to 70% of the total respiratory capacity of the tubule (Harris et al., 1981). These changes in respiration are accomplished with little, if any, change in ATP content in the presence of adequate metabolic substrates. With nystatin, ATP declines by only 15% and with ouabain, measured in other experiments (not shown here), ATP content increases by about 6% (Balaban et al., 1980). Therefore, it appears that the cell adjusts its respiration to the energetic demands in that a constant ATP level is maintained. Normally operating at 56% of capacity, the proximal tubule would be expected to have a lot of flexibility in responding to metabolic demands. This flexibility is indeed seen when the rate of active transport is experimentally

-

TABLE I RESPIRATORY RATEAS COMPARED TO RESPIRATORY CAPACITY PROXIMAL TUBULES FROM T H E RABBITKIDNEY Experimental condition

(nmol/min.mg protein)

Respiratory capacity Spontaneous rate Nystatin Ouabain

4422 25 & 2 4422 14

QO2

Percentage of respiratory capacity 100 56 100

30

IN

ATP (nmol/mg protein) 9.8 8.5

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altered. Another example of such an adjustment is obtained by stimulation of gluconeogenesis, another energy-requiring process. The rate of glucose production can be increased 2-3-fold by the addition of the fatty acids butyrate and valerate, or by succinate. This stimulation is accomplished without affecting either the ATP content in the tubule suspension or fluid transport, measured in parallel experiments in isolated perfused rabbit proximal tubules (Gullans ef al., 1984). Taken together, these results demonstrate that the renal cells have sufficient metabolic reserve capacity to maintain active transport and provide energy for other energy-requiring processes, such as gluconeogenesis. No evidence is found for energy limitations that would require sharing of a limited supply of energy. Quite the contrary, when sufficient metabolic substrate is supplied to the proximal tubules, there is enough energy for all dissipative processes. Thus, the renal cells adjust their rate of respiration to maintain their ATP levels approximately constant. 111.

CONTROL OF ENERGY METABOLISM OF ADULT CELLS THAT CAN BE RAPIDLY ACTIVATED

Adult cells such as skeletal muscle, brown fat, sperm, oocytes, and neutrophils maintain themselves mostly in a low activity state and rely on a variety of mechanisms to increase their rate of energy production when they are activated. As is seen in each of these examples, activation of function also requires activation of energy metabolism. The examples selected provide an interesting cross section of the types of mechanisms used by cells to activate their energy metabolism. The description of these processes in sperm, eggs, and leukocytes is particularly relevant to other chapters in this volume which discuss the changes in ion transport that occur during activation in these and other cells. A. Energy Activation In Skeletal Muscle

In the human body, skeletal muscle can increase its metabolic rate 20fold from rest to heavy work (Lehninger, 1975). Measurements on frog sartorius muscles suggest that the ATP expenditure can increase nearly 1000-fold from rest to isometric tetanus (Kushmerick, 1983). During rest, 90% of the muscle’s energy requirements are met by oxidative metabolism (Kushmerick and Paul, 1976). Muscle contraction sets in motion a variety of biochemical changes that depend on the duration of the stimulus, the capacity for oxidative metabolism, and diffusional limitations for metabolic substrates and oxygen (i.e., capillary density). The interaction

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among these three variables determines the extent of high-energy depletion that occurs in the muscle during contraction and whether the energetic recovery after contraction is through anaerobic or oxidative metabolism. Muscles with high aerobic capacity and ample capillary networks, such as cardiac (Page, 1978) or insect flight muscle (Hansford, 1980), can maintain muscle activity for prolonged periods of time since the rate of aerobic energy production equals the rate of energy consumption. Muscles with a lesser mitochondrial respiratory capacity, such as the frog sartorius (the mitochondria1 volume fraction is only I%-Kushmerick, 1983), deplete their high-energy stores (mainly creatine phosphate) during contraction and replete them during a subsequent period of recovery. Experiments with well-oxygenated muscles in uitro demonstrate that this recovery is mainly through oxidative metabolism (Kushmerick and Paul, 1976). However, in the intact organism, muscle recovery may also involve anaerobic metabolism due to diffusional limitations of oxygen. This factor was highlighted by early experiments of Milliken (1939) showing a decrease in oxyhemoglobin saturation of blood in the capillaries of cat soleus muscle within 200 msec after tetanic activity. This response was interpreted as a rapid reduction of oxygen in the tissue. This reduction lasted about 10 seconds beyond the duration of the tetanic contraction, suggesting that the tissue was oxygen-limited during this time. Indeed, muscle contraction in uivo elicits an increase in muscle lactate production (Lehninger, 1975), signifying an increase in glycolysis. Most of the stimulation in glycolysis is probably due to anaerobiosis. The glycolytic rate is also enhanced by the activation of glycogen phosphorylase caused by the increase in cytoplasmic calcium activity during contraction (Danforth er al., 1962). These considerations demonstrate that skeletal muscle has a very large reserve capacity for both anaerobic and oxidative metabolism. Either one of these may be rapidly activated by the energetic needs of muscle contraction and/or recovery. However, only oxidative metabolism can supply energy at a sufficient rate to maintain muscle contraction over an extended time period. 6. Brown Fat Thermogenesis

The main function of brown fat is heat production (Himms-Hagen, 1976; Nicholls and Locke, 1984), and this tissue is found in all animals capable of non-shivering thermogenesis, including hibernators as well as newborn lambs, and harp-seal pups (Himms-Hagen, 1976). Brown fat is distributed in discrete masses which represent 1-5% of total body mass.

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The tissue contains fat globules and an extremely high mitochondrial content, which accounts for its brown color and the high oxygen consumption observed in the stimulated tissue. The thermogenic response of brown fat is catecholamine mediated, and oxygen consumption can be increased up to 40-fold in this tissue (HimmsHagen, 1976). The acute response can be mimicked in v i m by the addition of norepinephrine (NE), and this response has been studied in preparations of isolated adipocytes from brown fat (Nicholls and Locke, 1984). Within 1 minute of norepinephrine binding to the adipocyte plasma membrane, both the rate of lipolysis and respiration increases. The NE-induced lypolysis seems to occur through an increase in intracellular cyclic AMP and a cyclic AMP-mediated protein kinase which activates a lipase (Himms-Hagen, 1976). Associated with this rapid lipolytic rate is an increased activity of long-chain acyl CoA transferase, part of the specialized transport systems required for fatty acid entry into the mitochondria. This observation highlights the synchronous regulation of substrate supply and energy requirements by the cell. Studies with isolated brown fat mitochondria have demonstrated both a relatively low H+-ATP-synthase activity and a unique mechanism for graded uncoupling that permits respiration to proceed independently from ATP synthesis. In brown fat mitochondria, the ratio of H+-ATP-synthase to cytochrome oxidase activity is 10-20-fold lower than in other mammalian mitochondria (Houstek and Drahota, 1977). Furthermore, the maximal rate of ATP synthesis is only a small fraction of the maximal capacity of the respiratory chain under uncoupled conditions (Cannon et al., 1973). Thus, it is unlikely that an increase in coupled respiration contributes significantly to brown fat thermogenesis, but rather respiratory uncoupling appears to be the responsible mechanism. The work of Nicholls and co-workers (reviewed by Nicholls and Locke, 1984) has pioneered experiments which define the molecular steps involved in the regulation of uncoupling by brown fat mitochondria. Through their studies, these investigators not only have clarified the mechanism of brown fat thermogenesis but have also contributed significantly to the understanding of mitochondrial function in general. By simultaneously measuring the mitochondrial proton motive force and the respiratory rate, Nicholls and co-workers were able to assess directly the degree of coupling in their mitochondrial preparation (Nicholls, 1974, 1979). Isolated brown fat mitochondria were found to be coupled only in the absence of endogenous fatty acids and in the presence of purine nucleotide di- or triphosphates. Various investigators (Rafael et al., 1969; Nicholls, 1979) found that only in the presence of 2 mM ATP or GTP could respiratory control by ADP be demonstrated in this tissue. Mito-

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chondrial uncoupling is attained through a 32,000-Mr protein which has been isolated from the inner membrane of brown fat mitochondria. This protein modulates the hydroxyl ion permeability of that membrane (Heaton et al., 1978), which could alternately be considered a H+ leak pathway. The H+ leak is increased by the presence of fatty acids and decreased by purine nucleotide di- or triphosphates (Heaton et al., 1978; Nicholls, 1979). This explains how ATP and GTP promote respiratory control by decreasing the H+ leak. Regulation of the H+ leak in uiuo has been attributed to the actions of the fatty acids and fatty acyl-CoA esters that accumulate following activation of lipase by norepinephrine. The fatty acids (Nicholls and Locke, 1984) and fatty acyl-CoA esters (Strieleman and Shrago, 1985) appear to have a dual role of reversing the nucleotide inhibition of the 32,000-Mr protein and of increasing the oxygen consumption by supplying respiratory substrates. A steady-state concentration of fatty acids is reached which corresponds to a balance between lipolysis and oxidation. All these events reverse upon termination of lipolysis. In summary, brown fat thermogenesis seems to involve the synchronous mobilization of fatty acids to serve as respiratory fuel and mitochondrial uncoupling to increase the respiratory rate and, thus, increase heat production. The free fatty acids and fatty acyl-CoA esters themselves may modulate uncoupling in complex interaction with cytosolic ATP. C. Activation of Energy Metaboilsm in Sperm and Eggs

Both sperm and eggs of many organisms remain relatively quiescent most of the time until they prepare themselves for fertilization in the case of sperm and actual fertilization for the egg. The main activation of energy metabolism in sperm occurs through the “respiratory dilution effect.” For example, in sea urchins, when “dry” semen (>lolo cells/ml) is diluted in seawater, there is a dramatic increase in respiration (Gray, 1928). Most of this increase in metabolic rate is probably due to the concomitant activation of flagellar motility (Nishioka and Cross, 1978). This activation process has been studied extensively in intact and glycerol- or Tritonpermeabilized sperm preparations from various species of sea urchins. Activation appears to be a strong function of intracellular pH, as reviewed recently by Busa and Nuccitelli (1984). A role for Na-H exchange in activation is apparent from the rapid acidification of the medium that is normally observed but is prevented by dilution into Na-free seawater (Nishioka and Cross, 1978). Subsequent addition of Na causes the acid release and activation of flagellar motility (and, presumably, respiration).

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271

On the basis of these results, Nishioka and Cross (1978) suggested that dilution of sperm in seawater stimulated Na-H exchange, which resulted in a net release of acid to the medium and alkalinization of the intracellular compartment. The role of intracellular alkalinization in sperm activation has been directly assessed in permeabilized preparations. In the presence of added ATP, flagellar motility in sea urchin sperm displays a strong dependence on pH, being immotile below pH 7.4 and maximal at pH 8-8.3, depending on species (Gibbons and Gibbons, 1972; Goldstein, 1979). The axonemebound dynein ATPase from sea urchin sperm also displays a strong pH dependence, with no activity below pH 7 and increasing to a maximum value at pH 9.5 (Gibbons and Fronk, 1972). Thus, the activation of energy metabolism in sea urchin sperm seems to occur through increased demand for ATP, stimulated by the activation of flagellar motility due to the increased intracellular pH. It is presently unclear whether the increased intracellular pH also affects respiration directly and whether other intracellular messengers are also involved in the activation process (Busa and Nuccitelli, 1984). Similarly, eggs undergo an internal alkalinization upon fertilization that appears to depend on Na-H exchange (Busa and Nuccitelli, 1984). However, due to the large array of intracellular changes that occur with fertilization (see Epel, 1975), it has been difficult to determine how these are causally related to the observed increase in metabolic rate (Epel, 1969). The role of the increased intracellular pH in the activation process has also remained obscure (Busa and Nuccitelli, 1984). D. Energy Activation in Mononuclear Phagocytes and Polymorphonuclear Leukocytes Energy activation by these blood components provides a different type of example for an activation mechanism than the ones previously described. Upon activation, mononuclear phagocytes and polymorphonuclear leukocytes exhibit an increase in both oxygen consumption and glucose-CI oxidation resulting from increased activity of the hexose monophosphate shunt (Sbarra and Karnovsky, 1979). This increase in respiration leads to the formation of oxygen metabolites, such as superoxide (Babior et al., 1973) and peroxide (Iyer et al., 1961), which presumably are part of the antimicrobial mechanism of phagocytosis (Babior et al., 1973; Babior, 1978; Johnston et al., 1975). This activation of the hexose monophosphate shunt can be simulated by the addition of phorbol esters. In a macrophage-like cell line, Kiyotaki

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et al., (1984) observed a 20-fold stimulation of glucose-CI upon addition of phorbol myristate acetate (PMA). In contrast, the oxidation rate of glucose-C6 was extremely low and remained unchanged upon addition of PMA, suggesting that metabolic energy was not obtained through the oxidation of glucose. The oxidation of glucose through the hexose monophosphate shunt requires ATP for the phosphorylation of glucose to glucose 6-phosphate. It is presently unclear which metabolic pathways are used for the generation of this ATP, since inhibitors of mitochondria1 oxidative phosphorylation do not affect the ATP content of macrophages (Kiyotaki et al., 1984). Another interesting aspect of this activation process that has been recently studied in neutrophils is the attendant marked acidification of the surrounding medium (Borregaard et al., 1984; Van Zweiten et al., 1981). Stimulation of the hexose monophosphate shunt leads to a large enhancement in the rate of acid production (Lehninger, 1975). This acid load is rapidly exported out of the cells because there is very little change in the intracellular pH after activation (Grinstein et al., 1985). The responsible transport mechanism appears to be an amiloride-sensitive Na-H exchanger that has been characterized recently (Grinstein and Furuya, 1986). IV. ENERGY METABOLISM OF CELLS IN CULTURE

A. Adaptation to Conditions In Culture

When normal adult cells are separated from their original tissue and placed in culture, an adaptation to their new environment occurs. Such an adaptation may result in the expression of some but not other properties present in the original tissue. The literature is replete with reports of a variety of growing strategies designed to foment expression of specific properties (e.g., Paul, 1965; Levintow and Eagle, 1961; Bissel et al., 1972; Bissel and Guzelian, 1980). In terms of energy metabolism, it appears that most, if not all, growing conditions presently used result in a decline in oxidative metabolism and a great enhancement of aerobic glycolysis (Warburg, 1926; Aisenberg, 1961; Morgan and Faik, 1981). A typical example of such an adaptation is observed when liver cells are placed in culture (Gebhart et al., 1978). The liver normally relies almost entirely on oxidative metabolism for its energetic needs, utilizing mainly lactate and fatty acids as metabolic substrates (Berry, 1974). Furthermore, the liver is normally gluconeogenic, converting a variety of substrates into glucose (Krebs et al., 1974). Within 4 hours of being placed in culture, the initially

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spherical hepatocytes adhere to the collagen-coated bottom of a tissue culture flask and begin to flatten out and assume as cuboidal shape. After 24-30 hours, the cells form islets of epithelial structure with granular cytoplasm. As seen in Fig. 3, within hours of being placed in culture, lactate uptake ceases and instead the cells begin to produce lactate in large quantities. On the other hand, glucose production declines after the first day and, thereafter, net glucose uptake, which is glycolyzed to lactate, ensues. The activity of lactate dehydrogenase was also measured in these studies as a function of time in culture and was found to decline precipitously by more than 50% within the first 4 hours of culture. In this study by Gebhart et al., (1978) there were no measurements made of the activity of glycolytic enzymes as a function of time. However, numerous other studies in a variety of cell types in culture (see below) have found a direct correlation between the glycolytic rate and the activity of the key glycolytic enzymes hexokinase (HK), phosphofructokinase (PFK), and

A

:>A

0.2 O

- 0.2-

-0.h

/

release

O\

0

\

\O

-

o o -o .

- 0.6

lactate (A), and urea (0) by cultured FIG.3. Rates of release or uptake of glucose (0). hepatocytes as a function of culture age. Abscissa: culture time in days; ordinate: pmol/mg protehhour. [Reproduced with permission from Gebhart er d.(1978).]

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pyruvate kinase (PyKi). Therefore, it may be safely assumed that the activity of these enzymes increases in parallel with the increased rate of lactate production, as shown in Fig. 2. Similarly, Stanisz et al. (1983) studied the changes in energy metabolism that heart cells undergo when placed in culture. In the intact tissue, heart cells obtain most of their energy from fatty acid oxidation (Evans, 1964; Opie, 1972). When placed in culture, the cells reduced their reliance on fatty acids to 5-10% of their energetic requirements. Instead, glucose now provided 35-45% of the energy through aerobic glycolysis, and glutamine oxidation provided the remaining 45-55% of the energy. This type of adaptation to a combination of aerobic glycolysis and oxidative metabolism is clearly seen in a variety of established mammalian cell lines. For example, LLC-PKI cells, derived from the proximal kidney of the pig, rely on both aerobic glycolysis and oxidative metabolism for their energy supply, each contributing about one-half of the energy (Sanders et al., 1983). This mixture of glycolytic and oxidative metabolism allows the cultured cells more flexibility to survive in a medium that provides energy through only one of these metabolic pathways. Oxygen deprivation or inhibition of oxidative metabolism stimulates glycolysis sufficiently to maintain normal function (Morgan and Faik, 1981; Sanders et al., 1983). On the other hand, glucose deprivation under aerobic conditions abolishes glycolysis and leads to increased glutamine oxidation to maintain ATP levels (Zielke et al., 1984). The importance of each of these metabolic pathways for cell growth has been evaluated in considerable detail, and some interesting patterns have begun to emerge. (1) Most of the glucose is metabolized to lactate. In experiments with I4C-labeledglucose, most of the I4CO2evolved comes from C-1 carbons, indicating oxidation by the hexose monophosphate shunt (Morgan and Faik, 1981; Zielke et al., 1984). Hardly any oxidation of glucose carbons occurs in the TCA cycle in cultured cells (Morgan and Faik, 1981; Zielke et al., 1984). Because lactate oxidation is minimal (Zielke et al., 1984), pyruvate dehydrogenase may be inhibited in culture. (2) Glucose provides essential anabolic intermediates required for RNA synthesis through the hexose monophosphate shunt (Morgan and Faik, 1981; Zielke et al., 1984). Substitution of inosine, thymidine, and uridine for glucose in the growth medium allows a rate of growth comparable to that obtained with glucose (Zielke et al., 1976). Under these conditions, glutamine provides most of the metabolic energy. Similarly, work with glycolysis-deficient mutants (Pouyssegur et al., 1980; Morgan and Faik, 1980) has shown that cells can obtain their energy needs entirely from oxidative metabolism. In these cells, even though the enzyme glucosephosphate isomerase (see Fig. 1) is deficient, normal oxidation of

8. CELLULAR ACTIVATION, GROWTH, AND TRANSFORMATION

275

[I-'4C]glucose still occurs to provide essential anabolic intermediates through the hexose monophosphate shunt. (3) Glucose inhibits glutamine oxidation with a Ki of 95 pA4 in human diploid fibroblasts (Zielke et al., 1984), but ketone bodies and fatty acids do not affect glutamine oxidation. Conversely, glutamine does not affect the aerobic glycolytic rate (Zielke et al., 1984).The mechanisms responsible for the inhibition of glutamine oxidation by glucose have not been studied in detail. However, the inhibition of oxidative metabolism by glucose bears a strong similarity to the Crabtree effect described for Ehrlich ascites tumor cells (Koobs, 1972; Ibsen, 1961). This effect has been ascribed to the binding of intracellular phosphate by glucose and glycolytic intermediates so that intracellular phosphate becomes limiting for oxidative metabolism (Koobs, 1972). However, it is presently unclear whether the same mechanism is operative in cultured cells. (4) There is conflicting evidence as to whether the capacity for oxidative metabolism is inhibited in culture. On one hand, a recent study by Zuurveld et al. (1985) showed that many enzymes of oxidative metabolism have comparable activities in cultured human skeletal muscle as compared to biopsy material. However, a key enzyme that was inhibited 50% in culture was cytochrome c oxidase. Unfortunately, no oxygen consumption measurements were made to compare directly their respiratory capacities. Other studies (e.g., Sanders et al., 1983) suggested that in LLC-PK, cells, oxidative metabolism was limited in its ability to supply energy to maintain normal epithelial transport function. This observation is in sharp contrast to that seen in renal tissue, in which there is a large excess mitochondria1 respiratory capacity (Harris et al., 1981), as discussed in Section 11. How does the adaptation toward aerobic glycolysis occur when cells are placed in culture? The answer to this question remains elusive, despite the 60-year-old knowledge (Warburg, 1926) that tumor cells undergo this type of change (see Section V). It will be extremely interesting to attempt an elucidation of the nature of the signal(s) that triggers this response. Two types of studies that could help in that direction are (1) the adaptation of cultured cells to hypoxia and (2) the metabolic response of quiescent cells in culture to the addition of certain hormones and growth factors. These are discussed next. B. Adaptation of Cultured Cells to Hypoxia

Human lung fibroblasts (WI-38) and rat fibroblasts grown under hypoxic conditions for 96 hours markedly increased their content of glycolytic enzymes (Simon et al., 1981). Both PyKi and PFK more than dou-

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bled during this time period. Conversely, cytochrome oxidase and superoxide dismutase activities were significantly diminished. A variety of other cell types also showed similar behavior when exposed to low oxygen tensions in culture (Simon et al., 1977, 1978). A study of Robin et al. (1984) on mouse lung macrophages and L8 rat skeletal muscle cells in culture demonstrated that all 1 1 glycolytic enzymes increased in activity when the cells were exposed to hypoxic conditions. The increased activity was only observed in the glycolytic enzymes, because three cytosolic nonglycolytic enzymes did not change their activity and six enzymes of oxidative phosphorylation showed decreased activity. Using monoclonal antibodies, both Hance et al. (1980) and Ptashne et al. (1983, 1985) showed that the increased activity of PyKi and PFK was due to an increase in actual enzyme content. On the other hand, Murphy et al. (1984) showed a significant decrease in mitochondrial enzymes of these cells when subjected to hypoxia. It has been recently suggested by Cole et al. (1985) that cultured cells, even those grown on permeable supports, may be hypoxic even when aerated fluid is allowed to circulate under the support. When these investigators cultured LLC-PK, cells on a collagen substrate, there was little ammonia production from glutamine at low pH. In contrast, proximal renal tubules in uiuo produce large amounts of ammonia under these conditions (Pitts, 1974). Since ammonia production occurs in the mitochondria, Cole et al. (1985) hypothesized that the mitochondrial enzymes may not have been expressed in culture due to hypoxic growing conditions. Consequently, they cultured the same cells in a rocking flask in suspension culture in order to improve oxygenation. These cells did produce ammonia, and their rate of production was pH sensitive. Unfortunately, these investigators have not determined whether other mitochondrial enzymes or the respiratory rate also increase activity under these culturing conditions, and neither have they measured the activity of glycolytic enzymes. The experiments just described suggest that an important reason why most cells in culture become glycolytic may be that they are grown under hypoxic conditions. This idea is certainly intriguing and, thus, is worthy of further exploration. C. Response of Energy Metabolism of Quiescent Cells to Growth Factors and Specific Hormones

Addition of serum to quiescent (serum-deprived) 3T3 cells in culture causes a stimulation of DNA synthesis and renewed proliferation. Prior to

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the initiation of DNA synthesis, serum induces a series of “early events,” some of which are described in other chapters of this volume. Among these is a sharp increase in ATP turnover which can be observed within the first 5 minutes following addition of growth factors (Talha and Harel, 1983). This effect is not dependent upon protein synthesis, is unaffected by the presence of ouabain, and occurs whether oxidative metabolism is functioning or is inhibited (Talha and Harel, 1983). The increased ATP turnover is supplied by a striking increase in glycolysis (Diamond et al., 1978). This increase in glycolytic rate can also be obtained by addition of epidermal growth factor (EGF) or insulin. The glycolytic increase observed in 3T3 cells is not affected by exogenous cyclic nucleotides nor ouabain but appears to depend on the presence of extracellular calcium (Diamond el al., 1978). Addition of the calcium ionophore A23 187 (0.5 pM) produces nearly maximal stimulation of glycolysis, suggesting a role for intracellular free calcium in the signaling process. However, obscuring the role of calcium in this process are the observations that EGF and serum addition increase cytosolic free calcium in these cells, whereas insulin does not (Rozengurt and Mendoza, this volume). Measurements of glycolytic intermediates show a decrease in the concentrations of glucose 6-phosphate and fructose 6-phosphate and an increase in the concentration of fructose diphosphate 30 minutes after serum addition, following overnight incubation in serum-free medium (Fodge and Rubin, 1973). These changes are consistent with an increase in PFK activity, the rate-limiting enzyme in glycolysis. Direct measurement of increased PFK activity after growth factor addition bears this out. Schneider et al. (1978) found enhanced activity of PFK that appeared to be a specific response to growth-promoting factors and did not require new protein synthesis. This increased activity was preserved after cellular homogenization, demonstrating a persistent alteration in the enzyme. These findings were in contrast to the transient increases in PFK activity that could be obtained by inhibition of oxidative metabolism (by dinitrophenol and oligomycin addition), which did not survive homogenization. These results suggest that growth factors cause a more permanent form of activation that involves preexisting inactive molecules of PFK. It is even possible that a different form of PFK may be involved, as discussed in Section V. These results suggest that another important aspect of the promotion of aerobic glycolysis in cultured cells may be their inherent response to growth factors. These factors, possibly in conjunction with local hypoxia, may be important in predisposing cultured cells toward aerobic glycoly sis.

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V.

ENERGY METABOLISM OF MALIGNANT CELLS

Warburg (1926, 1956) proposed that the basis of malignancy could be found in the alteration in energy metabolism present in tumor cells. He observed that tumor cells displayed a large increase in aerobic glycolysis and had inhibited mitochondria1 function. On the basis of these observations, he proposed that carcinogens elicited an irreversible injury to the respiratory apparatus, causing glycolysis to be stimulated even under aerobic conditions to supply the needed ATP. Warburg (1956) speculated further that “Because of the morphological inferiority of fermentation energy, the highly differentiated body cells are converted by this into undifferentiated cells that grow wildly-the cancer cells. A very different focus for the increased glycolytic rate was provided by Racker (1976), who proposed that an inefficient Na,K-ATPase was responsible for the high rate of aerobic glycolysis in tumor cells. This investigator proposed that in tumor cells a lesion was present in this enzyme which caused it to hydrolyze ATP at high rates, thereby increasing cytosolic ADP levels which would maintain glycolysis in a stimulated state. However, direct measurements of potassium uptake in Ehrlich ascites cells by Balaban and Bader (1983) found the same K-ATP stoichiometry in these cells as in normal cells, demonstrating that the Na,K-ATPase was functioning normally in these tumor cells. The present day thinking of a number of investigators (e.g., Morgan and Faik, 1981; Pedersen, 1978; Weinhouse, 1966; Nakashima et al., 1984) is that respiratory impairment is not a necessary condition for neoplasia. Various types of evidence suggest that Warburg’s speculations on the metabolic basis of neoplastic transformation were oversimplified, although his observations were pioneering and accurate. During the past 25 years, numerous investigators (see Pedersen, 1978, for review) have found that high aerobic glycolysis is neither an underlying factor of neoplasia nor a universal characteristic of tumors. Rather, it appears to be one of the numerous altered characteristics that is expressed by malignant transformation in most, but not all, tumors. ”

A. Aerobic Glycolysis In Tumors

The alterations in energy metabolism that occur as a result of malignant transformation have been studied in detail in a number of cell types. A very general observation is a large increase in glycolytic rate (e.g., Dunaway and Smith, 1971; Singh et al., 1974a,b; Bustamante et al., 1981; Nakashima et al., 1984). Chick embryo cells transformed by infection

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8. CELLULAR ACTIVATION, GROWTH, AND TRANSFORMATION

with Rous sarcoma virus display a large increase in glucose entry (e.g., Weber, 1973) and a doubling of activity of hexokinase, PFK, PyKi, and glucose 6-P dehydrogenase (Singh et al., 1974a). Using a temperaturesensitive mutant, these investigators showed that infection by itself did not cause these changes. When the cells were infected and grown at the nonpermissive temperature (for transformation) of 42°C none of the changes were observed. Lowering the temperature to 36°C caused rapid initiation of transformation and a concomitant increase in glycolytic flux (see Fig. 4). Measurements of the intracellular levels of glycolytic intermediates confirmed that the glycolytic flux increased with transformation (Singh et al., 1974b). 500

-

400-

i? .300D 9,

F

8 200

-

1

I

I

4

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J

16

20

24

28

Hours after Shift to 36"

FIG.4. Time course of increases in glucose transport rate and glycolytic enzymes during transformation by Rous sarcoma virus. Cells infected with tsNY68 and held at 42°C were shifted to the permissive temperature (36°C). and at various times the rate of transport of 30-methylglucose (3-OMG) and the activity of hexokinase (HK)and lactate dehydrogenase (LDH)were measured. [Reproduced with permission from Weber et ul. (1984).]

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The sequence of glycolytic changes occurring with transformation were described by Weber et al. (1984) in temperature-sensitive mutants infected with Rous sarcoma virus. As shown in Fig. 4, the increase in hexose transport appears with little apparent lag, whereas the increase in hexokinase activity may follow the change in transport rate by as much as a few hours. This increase in glucose flux may actually be a prerequisite for the induction of increased glycolytic enzyme activity because Bissell (1976) showed that inhibition of glucose transport in transformed cells caused a reversion to more normal glycolytic patterns. The mechanism for the increase in hexose transport appears to involve increased synthesis of transporters in the transformed cells (Weber er al., 1984). Three lines of evidence support this contention: ( 1 ) the increased transport rate seems to be due to a change in V,,, with no change in K , (Weber, 1973), (2) the change requires protein synthesis (Kawai and Hanafusa, 1971; Kletzien and Perdue, 1976), and (3) the number of glucose transporters increases in proportion to the increased transport rate, as measured by cytochalasin B binding (Salter and Weber, 1979) and monoclonal antibodies raised against the human erythrocyte glucose transporter (Weber et al., 1984). The increase in glucose transport rate greatly exceeds the requirements for growth when compared to the growth-related uptakes of other substances, such as potassium and various amino acids (Weber et al., 1984). Thus, only glucose transport increased in a transformation-specific manner, that is, cells transformed by Rous sarcoma virus transported hexose sugars at rates five-fold higher than normal cells proliferating at the same rate (Weber et al., 1984). According to Pedersen and co-workers (Pedersen, 1978; Bustamante and Pedersen, 1977; Bustamante et al., 1981; Parry and Pedersen, 1983), the localization, characteristics, and high activity of tumor hexokinase predispose these cells to high aerobic glycolytic rates. These investigators found that in a variety of tumor cells, at least 50% (and in some cases up to 70%) of the hexokinase was associated with the outer mitochondria1 membrane. In contrast, isolated liver mitochondria displayed minimal hexokinase activity and even this amount appeared to be due to microsomal contamination (Parry and Pedersen, 1983). The close functional association of hexokinase with tumor mitochondria was demonstrated by the ability to stimulate respiration when glucose was added to isolated H91 hepatoma mitochondria but not to isolated liver mitochondria following an ADP-induced respiratory burst (Bustamante and Pedersen, 1977). This result should be obtained only if the hexokinase reaction glucose

+ ATP =glucose 6-phosphate + ADP

8. CELLULAR ACTIVATION, GROWTH, AND TRANSFORMATION

28 1

occurs at the expense of mitochondrially synthesized ATP. The glucosestimulated respiration was inhibited by oligomycin and stimulated by respiratory uncouplers, suggesting coupling of the hexokinase reaction to oxidative phosphorylation. In addition, the glucose-stimulated respiration was not subject to end-product inhibition by 0.6 mM glucose &phosphate, in contrast to normal hexokinase. Some extremely interesting experiments from this laboratory were performed by combining mitochondrial with cytosolic extracts of normal and Ehrlich ascites tumor cells (Bustamante et al., 1981). As shown in Fig. 5 , addition of isolated tumor mitochondria to tumor cytosol increased the rate of lactate production to that found in the original tumor cell. In contrast, addition of isolated liver mitochondria to tumor cytosol inhibited lactate production to almost the same low level seen in the liver cytosol (Fig. 5). In another series of experiments, starting with liver cytosol, addition of liver mitochondria elicited no change in the rate of lactate production, whereas tumor mitochondria1 addition caused a fourfold stimulation of this process. This stimulatory effect of tumor mitochondria could be mimicked on an activity basis by hexokinase specifically solubilized from tumor mitochondria. These results from Bustamante et al. (198 1) strongly suggest that the mitochondrially associated hexokinase plays a key role in the high rate of aerobic glycolysis in tumors. In further support of this hypothesis, they found a good correlation between the rate of tumor growth, the rate of aerobic glycolysis, and the specific activity of

.-c

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0-• +Tumor Mitochondria

------------1 1

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Mitochondriol Protein Added (mgl

FIG.5 . Effect of tumor and liver mitochondria on the glycolytic rate of tumor cytosol. Aliquots of fresh tumor cytosol(O.8 mg of protein) were incubated with indicated amounts of fresh tumor (L@) or liver (-0) mitochondria. [Reproduced with permission from Bustamante e t a / . (1981).]

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mitochondrial hexokinase in 12 tumor cell lines. These investigators found that only the tumor cells lines with high aerobic glycolysis had detectable mitochondrial hexokinase, whereas none of this hexokinase was detectable in slow-growing tumors which displayed a low rate of aerobic glycolysis (see below). Finally, Bustamante et al. (1981)argue that this hexokinase, by producing large quantities of ADP, could take the place of the loosely coupled ATPase postulated by Racker (1976). The presence of this special form of hexokinase in tumor cells has prompted the investigation of possible therapeutic interventions aimed at the specific inhibition of this enzyme. Two reports from Floridi el al. (1981a,b)describe that Lonidamine [ 1-(2,4-dichlorobenzyl)-lH-indazol-3carboxylic acid, a powerful antispermatogenic agent] increases lactate production in normal cells but inhibits it in neoplastic cells, suggesting that it preferentially inhibits the mitochondrially bound hexokinase. However, this drug also inhibits respiration at higher concentrations. The rates of aerobic glycolysis found in tumor cells cover a very wide range, as shown in Fig. 6. In slow and intermediate growth rate hepatomas, the glycolytic rate is low and comparable to that present in the original liver tissue. Rapidly growing hepatomas display an intermediate rate, whereas very rapidly growing tumors have extremely high glycolytic rates. Interestingly, most tumors display a Pasteur effect, although some exceptions have been noted (Simon et al., 1981). Most normal tissues show low levels of aerobic glycolysis (Fig. 6),with the exception of a few tissues such as the retina (Fig. 6),vascular smooth muscle (Paul, 1983), and cornea (Thies and Mandel, 1985).In uiuo measurements by Cori and Cori (1925), Warburg (19261,and Gullino et al. (1967)found that rapidly growing tumors produced large amounts of lactate, as shown in Fig. 6, whereas normal tissues did not. This tabulation clearly demonstrates that neoplastic transformation expresses itself to varying degrees in the glycolytic metabolism of cells. High aerobic glycolysis is not a requisite property of tumorigenesis; it just happens that the easiest tumors to study and, consequently, the ones studied the most, are the very rapidly growing tumors, which display this property. A similar range of expression is found in the cellular mitochondrial content of tumors, as described next. 6. Oxidative Metabolism in Tumors

In an excellent review, Pedersen (1978)compared the mitochondrial content of tumors relative to their respective normal tissue of origin. As seen in Table I1 (reproduced from this review), most tumor cell lines demonstrated a reduction in mitochondrial content of 50% or more. Two

-

A Kidney

4A

Aerobic DAnerobic - Normal tissues

iA

16 7794A 7793 5123C H-35

tissues Intermediate growth rate hepatomas

7288 C 3924A

-

3683

Novikoff

Liver

Rapidly growing hepatomas Lactate arterial-venous difference, mg/lOO mi

B

S

H-91 Jensen

Bu Very rapidly growing tumors

JA

Rous

A

Ehrlich

A ~

I

I

1

I

I

700 100 200 300 400 500 600 Approximate lactic acid production, ClmOlelg wet tissuelh

800

FIG.6 . Lactic acid production of normal tissues and tumors (in uirro and in uiuo). [Reproducedfrom Pedersen (1978).]

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TABLE I1 MITOCHONDRIAL CONTENT OF TUMORS RELATIVE TO NORMAL TISSUE^ Tumor or rapidly growing tissue

Animal

Hepatoma (dimethylazobenzene induced) Hepatoma (Novikoff) Hepatocellular carcinoma (HC-252) Hepatoma 98/15 Hepatoma C-57 Hepatoma 3924A Mammary adenocarcinoma Hepatoma 16 Hepatoma 7800 Fetal liver Regenerating liver (2 days)

Mouse Rat Rat Mouse Mouse Rat Mouse Rat Rat Rat Rat

Mitochondria1 content (% of normal) 22-33 25 25 35-50 40 40

50 91 100

50-60 85, 66

Reproduced from Pedersen (1978) with permission.

notable exceptions in this list were the Morris hepatomas 16 and 7800, which are relatively slow growing, highly differentiated tumors that have a normal or only a slightly elevated glycolytic capacity (Aisenberg and Morris, 1963; Morris, 1965; LaNoue et al., 1974).Interestingly, fetal liver displays a large reduction in mitochondrial content, but in regenerating liver the drop in mitochondrial content is only moderate. This observation suggests that rapid growth rate by itself does not cause the decrease in mitochondrial content but, rather, this decrease is part of the neoplastic transition. (Similarly, little mitochondrial hexokinase is found in regenerating liver-Bustamante et al., 1981.) On the other hand, the decrease in mitochondrial content exhibited by these and many other tumors (Warburg, 1926, 1956; Aisenberg, 1961; Pedersen, 1978) may indicate that the normal-to-neoplastic transformation in those tumors constitutes a reversion to a fetal (less differentiated) state with low mitochondrial content and high aerobic glycolysis. Isolated tumor mitochondria have been studied by a number of investigators (for reviews, see Aisenberg, 1961; Weinhouse, 1966; Pedersen, 1978). After overcoming some initial problems related to their isolation, most investigators have found that tumor mitochondria are coupled and capable of producing ATP. Mitochondria from slowly growing tumors appear normal, but those from rapidly growing tumors have morphological and compositional abnormalities (Pedersen, 1978). One of the most striking differences appears to be the cholesterol enrichment (up to 4-fold)

8. CELLULAR ACTIVATION, GROWTH, AND TRANSFORMATION

285

of the inner mitochondrial membrane found in a number of hepatomas (see Wallach, 1975). It is believed that this might be due to loss of cholesterol-feedback biosynthetic control in tumor cells. Nevertheless, tumor mitochondria appear to be coupled. However, their substrate requirements may be different from those of normal mitochondria; for example, it is possible that tumor mitochondria do not readily oxidize pyruvate (Pedersen, 1978). In addition, it appears that, as tumor cells become more malignant, their capacity to utilize fatty acids as respiratory substrates declines (Stanisz et a1., 1983). These considerations suggest that the reduced respiratory rate observed in highly glycolyzing tumors may be in part due to the decrease in mitochondrial content, which in turn decreases their respiratory capacity. The proportion of respiratory capacity used for ATP production may depend on the type of tumor and the metabolic substrates present. In the presence of both glucose and respiratory substrates, H-91 cells do not respire maximally, because addition of uncouplers markedly stimulates respiration (Bustamante and Pedersen, 1977). On the other hand, various considerations suggest that AS-30D cells respire at their maximal rate (Nakashima et al., 1984). The proportion of energy obtained from oxidative phosphorylation or glycolysis also varies with tumor type and the substrates present. The slowly growing tumors derive more than 90% of their energy from oxidative metabolism (Pedersen, 1978). In highly glycolyzing tumors, when glucose and respiratory substrates are present, 40-60% of the ATP is still obtained from oxidative metabolism (Balaban and Bader, 1984; Nakashima et al., 1984; Ikehara et al., 1984). It is interesting that either oxidative metabolism or glycolysis alone can provide the normal cellular needs for ATP. In particular, Ehrlich and HeLa cells have been grown in galactose or fructose instead of glucose. Under these conditions, respiratory substrates such as glutamine can supply all the needed energy to the cell without the production of lactate (Pedersen, 1978; Reitzer et al., 1979). As with nontransformed cells in culture (see the previous section), sugars are mainly utilized to provide anabolic intermediates through the hexose monophosphate shunt (Reitzer et a/., 1980). An interesting phenomenon observed most clearly in highly glycolyzing cells is the inhibition of respiration by the addition of glucose-the Crabtree effect (Koobs, 1972; Ibsen, 1961). In tumor cells, the close association of hexokinase with the mitochondria may explain this effect. The rapid phosphorylation of glucose by mitochondrial ATP may cause a reduction of phosphate in the vicinity of the site of oxidative phosphorylation (Koobs, 1972) by the following reactions:

286

LAZAR0 J. MANDEL

ADP

+ Pi

oxidative phosphorylation hexokinase

+

Net

-

(mitochondrially bound;

glucose

+ Pi

ATP

+ HOH

glucose 6-phosphate

glucose 6-phosphate

+ ADP

+ HOH

In addition, other glycolytic intermediates can also bind phosphate, limiting its availability for oxidative phosphorylation. In summary, it appears that cancer cell lines exhibit a broad spectrum of aerobic glycolytic and oxidative activities. Slowly growing, highly differentiated tumors rely mostly on oxidative metabolism and metabolically resemble normal cells. Fast growing tumors display a high rate of aerobic glycolysis and inhibited mitochondria1 content. The aerobic glycolysis may be due to a mitochondrially bound form of hexokinase that permits rapid phosphorylation and subsequent metabolism of glucose. At the same time, the phosphorylation of glycolytic intermediates may limit the availability of phosphate for oxidative phosphorylation, causing its inhibition. Although glycolysis is of primary importance in providing ATP for fast-growing tumor cells, about 40-60% of ATP is obtained through oxidative metabolism. These results suggest that any attempts to inhibit tumor cell growth through metabolic interference must realize that these cells have developed the ability to produce energy simultaneously from both oxidative and glycolytic sources. ACKNOWLEDGMENTS

I would like to thank Ms. Elizabeth Holmes for typing the manuscript. This work was supported in part by Grant AM26816 from the National Institutes of Health. REFERENCES Aisenberg, A. C. (1961). The Glycolysis and Respiration of Tumours.” Academic Press, London. Aisenberg, A. C., and Moms, H. P. (1963). Energy pathways of hepatomas H-35 and 7800. Cancer Res. 23,566-568. Babior, B. M. (1978). Oxygen-dependent microbial killing by phagocytes. N.Engl. J . Med. 298,659. Babior, B. M., Kipnes, R. S., and Curnutte, J. I. (1973). Biological defence mechanisms. The production by Leukocytes of superoxide, a potential bactericidal agent. J . Clin. Invest. 52, 741, 1973. Balaban, R. S., and Bader, J. P. (1983). The efficiency of (Na+ + K+)-ATPase in tumorigenic cells. Biochim. Biophys. Acta 730,271-275. Balaban, R. S., and Bader, J. P. (1984). Studies on the relationship between glycolysis and (Na+ + K+)-ATPase in cultured cells. Biochim. Biophys. Acta 804,419-426. Balaban, R. S., Mandel, L. J., Soltoff, S., and Storey, J. M. (1980). Coupling of Na-KATPase activity to aerobic respiratory rate in isolated cortical tubules from tbe rabbit kidney. Proc. Narl. Acad. Sci. U.S.A. 77,447-451.

8. CELLULAR ACTIVATION, GROWTH, AND TRANSFORMATION

287

Berry, M. N. (1974). Energy demand of endogenous metabolism and gluconeogenesis in liver cells from normal and hyperthyroid rats. I n “Regulation of Hepatic Metabolism” (F. Lundquist and N. Tygstrup, eds.), pp. 568-578. Academic Press, New York. Bissell, M. H. (1976). Transport as a rate limiting step in glucose metabolism in virustransformed cells: Studies with cytochalasin B. J. Cell. Physiol. 89, 701-710. Bissell, D. M. and Guzelian, P. S. (1980). Phenotypic stability of adult rat hepatocytes in primary monolayer culture. Ann. N . Y. Acad. Sci. 349, 85-98. Bissell, M. J., Hatie, C., and Rubin, H. (1972).Patterns ofglucose metabolism in normal and virus-transformed chick cells in tissue culture. J. Nail. Cancer Inst. 49, 555-565. H., and Tauber, A. I. (1984). Proton secretion by stimulated Borregaard, N., Schwartz, .I. neutrophils. Significance of hexose monophosphate shunt as source of electrons and protons for the respiratory burst. J. Clin. Invest. 74, 455-459. Busa, W. B., and Nuccitelli, R. (1984). Metabolic regulation via intracellular pH. Am. J . Physiol. 246, R409-R438. Bustamante, E., and Pedersen, P. L. (1977). High aerobic glycolysis of rat hepatoma cells in culture: Role of mitochondrial hexokinase. Proc. Natl. Acad. Sci. U . S . A . 74, 37353739. Bustamante, E., Moms, H. P., and Pedersen, P. L. (1981). Energy metabolism of tumor cells: Requirement for a form of hexokinase with a propensity for mitochondrial binding. J. Biol. Chem. 256, 8699-8704. Cannon, B., Nicholls, D. G., and Lindberg, 0 . (1973). In Mechanisms in Bioenergetics” (G. F. Azzone, S. Papa, E. Quagliariello, and N. Siliprandi, eds.), pp. 357-363. Academic Press, New York. Chance, B., and Williams, C. M. (1956). The respiratory chain and oxidative phosphorylation. Adv. Enzymol. Relat. Areas Mol. Biol. 17, 65-134. Cohen, J. J., and Kamm, D. E. (1976). Renal metabolism: Relation to renal function. In “The Kidney” (B. M. Brenner and F. C. Rector, eds.), pp. 126-200. Saunders, Philadelphia. Cole, L. A., Scheid, J., and Tannen, R. L. (1985). LLC-PK, cultured renal tubule cells: Direct exposure to oxygen permits a direct and adaptive effect of pH on ammoniagenesis. Kidney Int. 27, 271. Con, C. F., and Con, G . T. (1925). The carbohydrate metabolism of tumors. 11. Changes in the sugar, lactic acid, and CO2-cornbiningpowers of blood passing through a tumor. J. Biol. Chem. 65, 397-405. Danforth, W. H., Helmreich, E., and Con, C. F. (1962). The effect of contraction and of epinephrine on the phosphorylase activity of frog sartorious muscle. Proc. Natl. Acad. Sci. U . S . A . 48, 1191-1 199. Diamond, I., Legg, A., Schneider, J. A., and Rozengurt, E. (1978). Glycolysis in quiescent cultures of 3T3 cells: Stimulation by serum, epidermal growth factor, and insulin in intact cells and persistence of the stimulation after cell homogenization. J. Biol. Chem. 253, 866-871. Dunaway, G. A,, Jr., and Smith, E. C. (1971). A comparative study of some of the enzymes involved in glucose metabolism of human diploid and SV4O-transformedhuman diploid cells. Cancer Res. 31, 1418-1421. Epel, D. (1969). Does ADP regulate respiration following fertilization of sea urchin eggs? Exp. Cell Res. 58, 312-319. Epel, D. (1975). The program of and mechanisms of fertilization in the echinoderm egg. Am. 2001.15, 507-522. Evans, J. R. (1964). Importance of fatty acid in myocardial metabolism. Circ. Res. 14, 15 (SUPPI.2), 96-106. Floridi, A., Paggi, M. G., D’Atri, S., De Martino, C., Marcante, M. L., Silvestrini, B., and

288

LAZAR0 J. MANDEL

Caputo, A. (1981a). Effect of lonidamine on the energy metabolism of Ehrlich ascites tumor cells. Cancer Res. 41,4661-4666. Floridi, A., Paggi, M. G., Marcante, M.L., Silvestrini, B., Caputo, A., and De Martino, C. (1981b). Lonidamine, a selective inhibitor of aerobic glycolysis of murine tumor cells. J . Natl. Cancer Inst. 66, 497-499. Fodge, D. W., and Rubin, H. (1973). Activation of phosphofructokinase by stimulants of cell multiplication. Nature (London) New Biol. 246, 181-183. Gebhardt, R., Bellemann, P., and Mecke, D. (1978). Metabolic and enzymatic characteristics of adult rat liver parenchymal cells in non-proliferatingprimary monolayer cultures. Exp. Cell Res. 1l2, 431-441. Gibbons, B. H., and Gibbons, I. R. (1972). Flagellar movement and adenosine triphosphatase activity in sea urchin sperm extracted with Trition X-100.J . Cell Biol. 54,365-381. Gibbons, I. R., and Fronk, E. (1972). Some properties of bound and soluble dynein from sea urchin sperm flagella. J. Cell B i d . 54, 365-381. Goldstein, S.F. (1979). Starting transients in sea urchin sperm flagella. J. Cell Biol. 80, 6168.

Gray, J. (1928). The effect of dilution on the activity of spermatozoa. J . Exp. Biol. 5 , 337344. Grinstein, S.,and Furuya, W. (1986). Characterization of the amiloride-sensitive Na+/H+ antiport of human neutrophils. Am. J. Physiol.: Cell Physiol. 250, C283-C291. Grinstein, W., Elder, B., and Furuya, W. (1985). Phorbol ester-induced changes in cytoplasmic pH in neutrophils. Role of exocytosis in activation of Na/H exchange. Am. J . Physiol. 248, C379-C386. Gullans, S. R., Brazy, P. C., Dennis, V. W., and Mandel, L. J. (1984). Interactions between gluconeogenesis and sodium transport in rabbit proximal tubule. Am. J . Physiol. 246, F859-F869. Gullino, P. M., Grantham, F. H., Courtney, A. H., and Losonczy, I. (1967). Relationship between oxygen and glucose consumption by transplanted tumors in uivo. Cancer Res. 27, 1041-1052. Hance, A. J., Robin, E. D., Simon, L. M., Alexander, S., Herzenberg, L. A., and Theodore, J. (1980). Regulation of glycolytic enzyme activity during chronic hypoxia by changes in rate-limiting enzyme content. J. Clin. Invest. 66, 1258-1264. Hansford, R. G. (1980). Control of mitochondria1 substrate oxidation. Curr. Top. Bioenerg. 10, 217-278. Hams, S. I., Balaban, R. S., Barrett, L., and Mandel, L. J. (1981). Mitochondria1 respiratory capacity and Na+- and K+-dependent adenosine triphosphatase-mediated ion transport in the intact renal cell. J. Biol. Chem. 256, 10319-10328. Heaton, G . M., Wagenvoord, R. J., Kemp, A., and Nicholls, D. G. (1978). Brown-adiposetissue mitochondria: Photoafflnity labelling of the regulatory site of energy dissipation. Eur. J. Biochem. 82, 515-521. Himms-Hagen, J. (1976). Cellular thermogenesis. Annu. Rev. Physiol. 38, 315-351. Houstek, J., and Drahota, Z. (1977). Purification and properties of mitochondria1adenosine triphosphatase of hamster brown adipose tissue. Biochim. Biophys. Acta 484, 127-139. Ibsen, K. H. (1961). The Crabtree effect: A review. Cancer Res. 21, 829-841. Ikehara, T., Yamaguchi, H., Hosokawa, K., Sakai, T., and Miyamoto, H. (1984). Rb+ influx in response to changes in energy generation: Effect of the regulation of the ATP content of HeLa cells. J . Cell. Physiol. 119, 273-282. Iyer, G. Y. N., Islam, M. F., and Questel, J. H. (1961). Biochemical aspects of phagocytosis. Nature (London) 192, 535.

8. CELLULAR ACTIVATION, GROWTH, AND TRANSFORMATION

289

Johnston, R. B., Jr., Keele, B. B., Jr., Misra. H. P., Lehmeyer. J . E., Webb, L. S.. Bachner, R. L., and Rajagopalan. K. V. ( 1975).The role of superoxide anion generation in phagocytic bactericidal activity. Studies with normal and chronic granulomatous disease leukocytes. J . Clin. Invest. 55, 1357-1372. Kawai, A., and Hanafusa, H . (1971). The effects of reciprocal changes in temperature on the transformed state of cells infected with a RSV mutant Rous sarcoma virus. Virology 46, 470-479. Kiyotaki, C., Peisach, J., and Bloom, B. R. (1984). Oxygen metabolism in cloned macrophage cell lines: Glucose dependence of superoxide production, metabolic and spectral analysis. J . Immunol. 132, 857-866. Kletzien, R. F., and Perdue, J. F. (1976). Regulation of sugar transport in chick embryo fibroblasts and in fibroblasts transformed by a temperature-sensitive mutant of the Rous sarcoma virus. J . Cell. Physiol. 89, 723-728. Koobs, D. H. (1972). Phosphate mediation of the Crabtree and Pasteur effects. Science 178, 127-133. Krebs, H . A., Cornell, N. W., Lund, P., and Hems, R. (1974). Some aspects of hepatic energy metabolism. I n “Regulation of Hepatic Metabolism” (F. Lundquist and N. Tygstrup, eds.), pp. 549-564. Academic Press, New York. Kushmerick, M. J. (1983). Energetics of muscle contraction. I n “Handbook of Physiology. Skeletal Muscle” pp. 189-236. Am. Physiol. SOC.,Bethesda, MD. Kushmerick, M. J., and Paul, R. J. (1976). Aerobic recovery metabolism following a single isometric tetanus in frog sartorius muscle at 0°C. J . Physiol. (London) 254, 693-709. La Noue, K. F., Hemington. J . G., Ohuishi. T., Morris, H. P., and Williamson, J. R. (1974). Defects in anion and electron transport in Morris hepatoma mitochondria. I n “Hormones and Cancer” (K. W. McKerus, ed.), pp. 131-167. Academic Press, New York. Lardy. H. A., and Wellman. H. (1952). Oxidative phosphorylations: Role of inorganic phosphate and acceptor systems in control of metabolic rates. J. B i d . Chem. 195,215224. Lehninger, A. L. (1975). “Biochemistry: The Molecular Basis of Cell Structure and Function,” 2nd Ed. Worth, New York. Levintow, L., and Eagle, H. (1961). Biochemistry of cultured memmalian cells. Annu. Rev. Biochem. 30, 605-640. Mandel, L. J., and Balaban, R. S. (1981). Stoichiometry and coupling of active transport to oxidative metabolism in epithelial tissues. A m . J . Physiol. 240, F357-F371. Millikan, G. A. (1939). Experiments on muscle hemoglobin in uiuo: the instantaneous measurement of muscle metabolism. Proc. R. Soc. London Ser. B 123, 218-241. Morris. H. P. (1965). Studies in the development, biochemistry, and biology of experimental hepatomas. Adu. Cancer Res. 9, 227-302. Morgan, M. J . , and Faik, P. (1981). Carbohydrate metabolism in cultured animal cells. Biosci. Rep. 1, 669-686. Murphy, B. J., Robin, E. D., Tapper, D. P., Wong, R. J., and Clayton, D. A. (1984). Hypoxic coordinate regulation of mitochondria1 enzymes in mammalian cells. Science 223, 707-709. Nakashima, R. A., Paggi, M. G., and Pedersen. P. L . (1984). Contributions ofglycolysis and oxidative phosphorylation to adenosine 5’-triphosphate production in AS-30D hepatoma cells. Cancer Res. 44, 5702-5706. Nicholls, D. G. (1974). Hamster brown-adipose-tissue mitochondria. The control of respiration and the proton electrochemical potential gradient by possible physiological effectors of the proton conductance of the inner membrane. Eur. J . Biochem. 49, 573-583.

290

LAZAR0 J. MANDEL

Nicholls, D. G. (1979). Brown adipose tissue mitochondria. Biochim. Biophys. Acra 549, 129. Nicholls, D. G . . and Locke, R. M. (1984). Thermogenic mechanisms in brown fat. Physiol. Rev. 64, 1-64. Nishioka, D., and Cross, N. (1978). The role of external sodium in sea urchin fertilization. I n “Cell Reproduction: In Honor of Daniel Mazia” (E. R. Dirkson, D. M. Prescott, and C. F. Fox, eds.), pp. 403-413. Academic Press, New York. Opie, L. H. (1972). Metabolism of the heart in health and disease. Am. Heart J . 76,685-698. Page, E. (1978). Quantitative ultrastructural analysis in cardiac membrane physiology. A m . J. Physiol. 235, C147-CI58. Parry, D. M.,and Pedersen, P. L. (1983). Intracellular localization and properties of particulate hexokinase in the Novikoff ascites tumor: Evidence for an outer mitochondria1 membrane location. J . Biol. Chem. 258, 10904-10912. Paul, J. (1965). Carbohydrate and energy metabolism. I n “Cells and Tissues in Culture” (E. N. Willmer, ed.), pp. 239-276. Academic Press, London. Paul, R. J. (1983). Functional compartmentation of oxidative and glycolytic metabolism in vascular smooth muscle. A m . J . Physiol. 244, C399-CW. Pedersen, P. L. (1978). Tumor mitochondria and the bioenergetics of cancer cells. Prog. Exp. Tumor Res. 22, 190-274. Pitts, R. F. (1974). “Physiology of the Kidney and Body Fluids,” 3rd Ed. Year Book Medical Publishers, Chicago. Pouyssegur, J., Franchi, A., Salomon, J. L., and Silvestre, P. (1980). Proc. Nart. Acad. Sci. U.S.A. 77,2698-2701. Ptashne, K. A., Theodore, J., and Robin, E. D. (1983). Increased phosphofructokinase content during chronic hypoxia in cultured skeletal muscle (L8) cells. Biochim. Eiphys. Acta 763, 169-174. Ptashne, K., Morin, M., Hance, A., and Robin, E. D. (1985). Increased biosynthesis of pyruvate kinase under hypoxic conditions in mammalian cells. Biochim. Biophys. Acra 844, 19-23. Racker, E. (1976). Why do tumor cells have a high aerobic glycolysis? J. Cell. Physiol. 89, 697-700. Raphael, J., Ludolph,H.J., and Hohorst, H. J. (1%9). Mitochondrien aus braunem fettgewebe: Eutkopplung der atmungsketten phosphorylierung durch langkettige fettsauren und rekopplung durch guanosin triphosphat. Hoppe-Seyler’s Z. Physiol. Chem. 350, 1121-1 131. Reitzer, L. J., Wice, B. M., and Kennel], D. (1979). Evidence that glutamine, not sugar, is the major energy source for cultured HeLa cells. J. Biol. Chem. 254, 2669-2676. Reitzer, L. J., Wice, B. M., and Kennel], D. (1980). The pentose cycle: Control and essential function in HeLa cell nucleic acid synthesis. J . Biol. Chem. 255, 5616-5626. Robin, E. D., Murphy, B. J., and Theodore, J. (1984). Coordinate regulation of glycolysis by hypoxia in mammalian cells. J. Cell. Physiol. 118, 287-290. Salter, D. W., and Weber, M. J. (1979). Glucose-specificcytochalasin B binding is increased in chicken embryo fibroblasts transformed by Rous sarcoma virus. J. Biol. Chem. 254, 3554-3561. Sanders, M. J., Simon, L. M., and Misfeldt, D. S. (1983). Transepithelial transport in cell culture: Bioenergetics of Na-, D-glucose-coupled transport. J. Cell. Physiol. 114, 263266. Sbarra, A. J., and Karnovsky, M. L. (1979). The biochemical basis of phagocytosis. 1. Metabolic changes during the ingestion of particles by polymorphonuclear leukocytes. J. Biol. Chem. 234, 1355.

8. CELLULAR ACTIVATION, GROWTH, AND TRANSFORMATION

291

Schneider, J. A., Diamond, I., and Rozengurt, E. (1978). Glycolysis in quiescent cultures of 3T3 cells: Addition of serum, epidermal growth factor, and insulin increases the activity of phosphofructokinase in a protein synthesis-independent manner. J. Biol. Chem. 253, 872-877. Simon, L. M., Robin, E. D., Phillips, J. R., Acevedo, J., Axline, S. G., and Theodore, J. (1977). Enzymatic basis for bioenergetic differences of alveolar versus peritoneal macrophages and enzyme regulation by molecular 0 2 J. . Clin. Invest. 59, 443-448. Simon, L. M., Robin, E. D., Raffin, T. A., Douglas, W. H., and Theodore, J. (1978). Bioenergetic pattern of Type I1 pneumocytes in air and during hypoxia. J . Clin. Invest. 61, 1232-1239. Simon, L. M., Robin, E. D., and Theodore, J. (1981). Differences in oxygen-dependent regulation of enzymes between tumor and normal cell systems in culture. J . Cell. Physiol. 108, 393-400. Singh, M., Singh, V. N., August, J. T., and Horecker, B. L. (1974a). Alterations in glucose metabolism in chick embryo cells transformed by Rous sarcoma virus. Transformationspecific changes in the activities of key enzymes of the glycolytic and hexose monophosphate shunt pathways. Arch. Biochem. Biophys. 165, 240-246. Singh, V. N., Singh, M., August, J. T., and Horecker, B. L. (1974b). Alterations in glucose metabolism in chick-embryo cells transformed by Rous sarcoma virus: Intracellular levels of glycolytic intermediates. Proc. Null. Acud. Sci. U.S.A. 71, 4129-4132. Stanisz, J., Wice, B. M., and Kennell, D. E . (1983). Comparative energy metabolism in cultured heart muscle and HeLa cells. J . Cell. Physiol. 115, 320-330. Strieleman, P. J., and Shrago, E. (1985). Specific interaction of fatty acyl-CoA esters with brown adipose tissue mitochondria. A m . J . Physiol. 248, E699-E705. Talha, S., and Harel, L. (1983). Early effect of growth factors (EGF + insulin) upon ATP turnover in 3T3 cells. Exp. Cell Res. 149, 471-481. Thies, R. S., and Mandel, L. J. (1985). The role of glucose in corneal metabolism. A m . J . Physiol. 249, C409-C416. Van Sweiten, R., Wever, R., Hamers, M. N., Weening, R.,and Roos, D. (1981). Extracellular proton release by stimulated neutrophils. J . Clin. Invest. 68, 310-313. Wallach, D. F. H. (1975). “Membrane Molecular Biology of Neoplastic Cells.” Elsevier, Amsterdam. Warburg, 0. (1926). “Uber den Stoffwechsel der Tumoren.” Springer-Verlag, Berlin. Warburg, 0. (1956). On the origin of cancer cells. Science 123, 309-314. Weber, M. J. (1973). Hexose transport in normal and Rous sarcoma virus-transformed cells. J . Biol. Chem. 248, 2978-2983. Weber, M. J., Evans, P. K., Johnson, M. A., McNair, T. F., Nakamura, K. D., and Salter, D. W. (1984). Transport of potassium, amino acids, and glucose in cells transformed by Rous sarcoma virus. Fed. Proc., Fed. A m . Soc. Exp. Biol. 43, 107-112. Weinhouse, S. (1966). Glycolysis, respiration, and enzyme deletions in slow-growing hepatic tumors. Gann Monogr. 1, 99-115. Zielke, H . R., Ozand, P. T., Tildon, J. T., Sevdalian, D. A., and Cornblath, M. (1976). Growth of human diploid fibroblasts in the absence of glucose utilization. Proc. Natl. Acad. Sci. U.S.A. 73, 4110-4114. Zielke, H. R., Zielke, C. L., and Ozand, P. T. (1984). Glutamine: A major energy source for cultured mammalian cells. Fed. Proc., Fed. Am. Soc. Exp. Biol. 43, 121-125. Zuurveld, J. G. E. M., Oosterhof, A., Veerkamp, J. H.,and van Moerkerk, H. T. B. (1985). Oxidative metabolism of cultured human skeletal muscle cells in comparison with biopsy material. Biochim. Biophys. Acra 844, 1-8.

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Index

A

Acidification, A 431 cells by permeant anion, 41 Alkali metal-chloride cotransport, in Amphiuma red blood cells, 207-208 Alkali metal-hydrogen exchange in Amphiuma red blood cells vs. alkali metal41 cotransport, 209210 and CI-HC03 exchange, parallel net transport in, 201-202 and CI-HCO, net transport, 207 electroneutral, 198-20 1 protein kinase C and, 213-215 volume-dependent sensitivity of, 210213 role in cell regulatory processes, 194I95 Alkali metal-hydrogen exchanger, and C1HCO, exchanger, parallel net transport in Amphiuma red blood cells, 20 1-202 Alkali metal ions, in Amphiuma red blood cells Ca2+-dependentflux, 202-205 C1 ions and buffer power of cells and medium, 207-209 Alkalinization epidermal growth factor-dependent, modulation by phorbol myristic acetate, 36 growth factor-induced in NR6 cells, 66 intracellular fertilization-evoked, 142- 143 induction by ionophore A23187 in cultured human fibroblasts, 73-74

293

mitogen-stimulated in human fibroblasts, 66 pH with 5,5-dimethyloxazolidine-2,4dione, 65 platelet-derived growth factor-induced in NR6 cells, amiloride effect, 30 Amiloride analogs, pharmacological interaction with Na+ transport systems, 80-81 effect on platelet-derived growth factorinduced alkalinization in NR6 cells, 30 inhibition of Na+-Ca2+ exchange, 80 Na+ channel in tight epithelia cells, 78-79 Na+-H+ exchange, 63, 79-80 nonspecific effect, 81 Amphipatic molecules, distribution and intracellular pH measurement, 41 Amphiuma red blood cells alkali metal-CI cotransport, 207-208 alkali metal-hydrogen exchange activation and control of, 210-215 vs. alkali metal-C1 cotransport, 209210 and CI-HC03 net transport, role of buffers, 207 electroneutral, 198-20 I protein kinase C and , 213-215 volume-dependent Ca2+sensitivity, 210-213 Ca2+-dependentalkali metal ion flux in, 202-205 ion and water content as function of time after suspension in hypotonic and hypertonic media, 199

INDEX

membranes with high alkali metal and H conductance, 208-209 net Na flux by, in hyperosmotic media, 205-206 net transport by alkali metal-H and C1HCOJ exchangers, 201-202 regulatory volume decrease in, 17 regulatory volume increase in, 17-18 volume regulatory ion flux pathways, 20 1 volume-sensitive ion fluxes in, 198-202 Amphotericin B increased Na+ and, 166 stimulation of Na+-K+ pump activity, 166, 168 Angiotension, stimulation of Na+ influx in cultured smooth muscle cells, 71 Anions Na+ cotransport with, 7 permeant, acidification of A 431 cells by, 47 replacement, identification of electroneutral alkali-metal ion transport with, 210 Anisosmotic activation, of C1-dependent cation cotransport, 92-93 0

Benzamil inhibition of Na+-Ca*+ exchange, 80 Na+-H+ exchange, 78 Bindin, and sperm-egg interaction, 138 Bombesin effect on ion fluxes and DNA synthesis in quiescent Swiss 3T3 cell cultures, 173-174 stimulation of Na+-K' pump activity, 166, 168 Bradykinin-stimulated Na+ influx, in human foreskin and lung fibroblasts, 167 Brown fat, thermogenesis, 268-270 Buffers, role in alkali metal-H and C1HCO, net transport, 207 C

Calcium-calmodulin complex, and chromosomal protein phosphorylation, 235-236

Calcium ion buffering and sequestration, 13-14 changes in DNA synthesis and, 225-226 protein synthesis and, 237-240 RNA synthesis induction and, 233-234 -dependent alkali-metal ion flux in Amphiuma red blood cells, 202-205 effect on thymidylate synthase induction, 23 1-232 eftlux Ca2+pump, 15 Na+-Ca2+ exchange, 14-15 stimulation by vasopressin, 180 expression of latent plasma membrane proteins and, 248-249 fluxes in quiescent fibroblastic cell lines, 180-182 influx, 13 Na+ countertransport and, 7-8 regulation of growth and differentiation in murine keratinocytes, 239 role in activation of Na+-H+ exchange, 73-75 sensitivity, volume-dependent, of alkali metal-H exchange in Amphiuma red blood cells, 210-213 transport by mitochondria, 13-14 transport pathways in plasma membrane. characteristics, 16 uptake by sarcoplasmic reticulum membranes, 14 Calcium ionophore A23187, see Ionophore A23 187 Calcium pump, 15 Calcium-sodium exchange, see Sodiumcalcium exchange Carbohydrate metabolism, see also Energy metabolism major pathways in, 263 Cation fluxes, monovalent, generality in quiescent cells after stimulated proliferation, 171 Cation transport, chloride-dependent, see Chloride-dependent cation cotransport Cell cultures energy metabolism in, 272-277 adaptation to culture conditions, 272-275 hypoxia, 275-276 response to growth factors and hormones. 276-277

INDEX

ionic transport systems, activation by serum and growth factor, 143I50 Na+ channel, 147-149 Na+-H+ exchange, 143-145 serum-stimulated electrical events, 145-147 murine keratinocytes, as model system for extracellular Ca2+regulation of growth and differentiation, 239 pituitary cells, ion transport and hormonal secretion in, 151-155 Cell growth, ion fluxes and, 176-180 cyclic AMP and, 176-177 protein kinase C and, 177-180 Cell lines A 431 cells acidification by permeant anion, 47 equilibrated with extracellular Na+, intracellular pH changes, 46 Na+-H+ exchange, phorbol myristic acetate effect, 35 plasma membrane vesicles, Na+-H+ exchange in, 39 BSC-I cells, effect of epidermal growth factor and serum addition, 24 MDCK cells, CI-dependent cation transport in, 108-111 NR6 cells growth factor-induced alkalinization, 66 Na+ influx in, effect of mitogens, 27 pH, effect of platelet-derived growth factor and fetal calf serum, 28 platelet-derived growth factor-induced alkalinization, amiloride effect, 30 Swiss 3T3 cells, ionic responses to growth factors effect of mitogens on Na+-H+ antiport, 169170 mitogens on Na+ entry into, 167168 Na+ entry on Na+-K+ pump activity, 166 Na+ cycle of, 170 Na+-H+ antiport system in cultured cells, 168-169 Na+-K+ pump activity, 165-166 tumor cells, see Tumors

295 Cell membrane permeability, spenn-induced changes in, 141-142 Cell volume, maintenance and regulation of, 15-18 regulatory volume decrease, 17 regulatory volume increase, 17-18 Chloride-dependent cation cotransport anisosmotic activation of, 92-93 chemical modifications, 95-96 in differentiated cells epithelial cells, 108-1 1 1 major modes, triggers, and cell types, 94 during differentiation, nonepithelial cells as models for 112-115 in Ehrlich ascites tumor cells, 108-111 in erythrocytes chemically modified K-CI transport, 104-105

general aspects, 100-103 K-CI transport, volume-stimulated, 103-104 Na-CI transport, 105-108 Na-dependent K-CI transport, 101 Na-K-2CI transport, 105-108 Na or Na-K transport, 102 functional aspects, 99-100 hormone activation of, 93-95 inhibitors, 97-98 K-CI cotransport, 94 kinetics and ionic interdependence, %97 major modes, triggers, and differentiated cell types, 94 in MDCK cells, 108-1 I I Na-CI cotransport, 94 Na-K-2CI cotransport, 94 in nonepithelial cells during differentiation, 112-115 pharmacologic effectors, 94 role of cellular metabolism, 98-99 thermodynamics, 91 Chloride-hydrogen carbonate, in Amphiu m a red blood cells and alkali metal-H net transport, 207 exchange, 12 exchanger, and alkali metal-H exchanger, parallel net transport, 201202 inhibition of, and changes in pH, 209 Chloride ion, alkali ions and buffer power

296

INDEX

of cells and medium in Amphiuma red blood cells, 207-209 Chromosomes, protein phosphorylation, Ca-calmodulin complex and, 235-236 Concanavalin A-induced mitogenesis, in peripheral blood lymphocytes, 149 Concentration of permeant ions, membrane potential changes and, 22-23 Conductance, membranes with high alkali metal and H, 208-209 Conductive transport, alkali metal ions, 197- I98 Cotransport CI-dependent. see Chloride-dependent cation cotransport HCOS, Na+-coupled, 1 I Countertransport systems Na+-Ca2+exchange, see Sodium-calcium exchange Na+-H+ exchange, see Sodium-hydrogen exchange Cyclic AMP activation of CI-dependent cation cotransport, 94 ion fluxes and, 176-177 stimulation of NaHCO,-HCI exchange, 12 D

Diazosul fonic acid, inhibition of Na+ transport by, 147-148 Differentiation Dictyostelium discoideum, Na+-H+ exchanger role, 72 epithelial cells, CI-dependent cation cotransport during, 108-1 I I erythrocytes CI-dependent Na or Na-K transport, 102 K-CI cotransport chemically modified, 104-105 volume-stimulated, 103-104 Na-CI or Na-K-2CI transport, 105I08 Na-independent K-CI transport, 101 keratinocytes (murine), Ca2+-regulated, 239 nonepithelial cells Cl-dependent cation cotransport during, 112-115

as models for cotransport during, 112115 ruminant hemopoietic cells, K-CI COtransport and, 106 702/3 cells, lipopolysaccaride-induced, 72-73 Diffusion potentials, membrane, 19-21 5,5-Dimethyloxazolidine-2,4-dione, alkalinization of intracellular pH with, 65 DNA polymerase activity, pH and, 232 DNA synthesis effect of ions on, 231-232 DNA polymerase activity, 232 thymidylate synthase induction, 23 1232 in growth factor-stimulated 3T3 cells as function of K+ internal concentration, 230 ionic changes and, 224-231 Ca2+and Mg2+,225-226 Na+ and K+,229-231 pH, 227-229 in quiescent Swiss 3T3 cells increased ion fluxes and, 173-176 inhibition of ion fluxes and, 172173 Dyes, for intracellular pH measurement cellular location of, 43-47 leakage rate and, 42

E Eggs, sea urchin activation of energy metabolism in, 27027 1 fertilization -evoked intracellular alkalinization, 142-143 potential, 138-140 resting potential, 133-135 sperm-egg interaction, 135-138 sperm-gated channels, 140-141 sperm-induced permeability changes, mechanism, 141- 142 timing of early events after, 132 Na+-H+ exchange in, 63-64 eIF-2, see Initiation factor (eIF-2) Electrodiffusion, H+ and HCO;, 10-1 1 Electrogenic pumps, 21-22 changes in transport rates of, 23

INDEX

Electroneutral transport, alkali metal ions, 197- 198 Endoplasmic reticulum, induction of Ca2+ release by inositol triphosphate, 14 Energy metabolism, see also Carbohydrate metabolism control in adult cells rapidly activated brown fat thermogenesis, 268-270 in mononuclear phagocytes, 271212 in polymorphonuclear leukocytes, 27 1-272 in skeletal muscle, 267-268 in sperm and eggs, 270-271 with relatively constant metabolic rate, 264-261 of cultured cells, 272-277 adaptation to conditions in culture, 272-275 hypoxia, 215-216 response to growth factors and hormones, 276-277 of malignant cells, 278-286 aerobic glycolysis in tumors, 278-282 oxidative metabolism in, 282-286 Enzymes, glycolytic, and glycolysis rate, correlation, 273-274 Epidermal growth factor -dependent alkalinization, modulation by phorbol myristic acetate, 36 effect on quiescent BSC-I cells, 24 energy metabolism response of quiescent cells in culture, 276-277 stimulation of Na+ influx in hepatocytes, 167 in human foreskin and lung fibroblasts, 167 and Na+-K+ pump activity in neonatal foreskin cells, 167 in rat pheochromocytoma cells, 7071 Epithelial cells differentiated, C1-dependent cation cotransport, 108-1 1 1 Na-independent K-CI transport, I I I Na or Na-K transport, 109 kidney, Na+-H+ antiport system, 168 MDCK, serum-stimulated Na+ uptake and Na+-K+ pump activity, 167

297 Erythrocytes Amphiuma activation and control of 210-215 alkali metal-C1 cotransport, 207-208 alkali metal-H exchange vs. alkali metal41 cotransport in 209-210 and C1-HC03 net transport, role of buffers, 201 volume dependent Ca2+sensitivity Of, 210-213 Ca2+-dependentalkali metal ion flux in, 202-205 ion and water content as function of time after suspension in hypotonic and hypertonic medium, 199 membranes with high alkali metal and H conductance, 208-209 protein kinase C and, 213-215 regulatory volume decrease in, I7 regulatory volume increase in, 17-18 volume regulatory ion flux pathways, 20 I volume-sensitive ion fluxes in, 198202 electroneutral alkali metal-H exchange, 198-201 net transport by parallel alkali metal-H and HCO, exchangers, 20 1-202 C1-dependent cation transfer during differentiation, 100-103 K-CI transport chemically modified, 104-105 volume-stimulated, 103- 104 Na-CI or Na-K-2CI transport, 105I08 Na-independent K-CI transport, 101 Na or Na-K transport, 102 N-Ethylmaleimide, effect on C1-dependent cation cotransport, 95 F

Fertilization, sea urchin egg intracellular alkalinization by, 142143 ionic responses to, 132-143 potential, 138-140 resting potential, 133-135

INDEX

sperm-egg interaction, 135-138 sperm-gated channels, 140-141 sperm-induced permeability changes, mechanism, 141-142 timing of early events after, 132 Fibroblasts, cultured Chinese hamster, Na+-H+ antiport system in, 168 human Ca2+role in Na+-H+ exchange, 73-75 intracellular alkalinization with ionophore A23187, 73-74 mitogen-stimulated, 66 Na+-H+ antiport system in, 168 Na+ influx stimulation with ionophore A 23187,73 differentiated, C1-dependent Na or Na-K transport, 113 Na+-H+ exchange in, 64-66 proliferative response, roles of cyclic AMP and ion fluxes, 176-177 protein kinase C and ion fluxes, 177180 quiescent cell lines, Ca2+ fluxes and, 180-182 Swiss 3T3 cells, ionic responses to growth factors effect of mitogens on Na+-H+ antiport, 169170 mitogens on Na+ entry into, 167168 Na+ entry on Na+-K+ pump activity, 166 Na+ cycle of, 170 Na+-H+ antiport system in cultured cells, 168-169 Na+-K+ pump activity, 165-166 Fick's law, 1% Fluorescein-dextran introduction to cells, method, 44 Force-flow coupling, in alkali metal ion fluxes, 195-197 Formyl-methionyl-leucyl-phenylalanine, stimulation of polymorphonuclear leukocytes with, 67-68 Friend erythroleukemic cells, Na+-Ca2+ exchange in, 58-59 Furosemide, diuretic effect in Cl-dependent cotransport systems, 97

G

Glucagon-stimulated Na+ influx, in hepatocytes, 167 Glucose, uptakehelease by cultured hepatocytes as function of age, 273 Glycolysis aerobic, in tumors, 278-282 rate and glycolytic enzymes, correlation, 273-274 of tumor cytosol, effect of tumor and liver mitochondria on, 281 Goldman-Hodgkin-Katz equation, 20 Gramicidin, increased Na+ and, 166 Growth factors action of, and changes in membrane voltage, 23-25 activation of ionic transport systems with lectin-induced mitogenesis in lymphocytes, 149-150 Na+ channel, 147-149 Na+-H+ exchange, 143-145 serum-stimulated electrical events, 145-147 cellular responses to, model, 131 epidermal, see Epidermal growth factor energy metabolism response of quiescent cells in culture, 276-277 -induced alkalinization in NR6 cells, 66 ionic responses in quiescent Swiss 3T3 cells effect of mitogens on Na+ entry into, 167-168 mitogens on Na+-H+ antiport, 169170 Na+ entry on Na+-K+ pump activity, 166 ion fluxes increase in, and DNA synthesis, 173-176 inhibition effect on DNA synthesis, 172-173 Na+ cycle, 170 Na+-H+ antiport system in cultured cells, 168-169 Na+-K+ pump activity, 165-166 platelet-debived, see Platelet-derived growth factor

299

INDEX

stimulation of ion fluxes in quiescent cells, 182 Growth hormone-secreting cells, see Pituitary cells H

Hemopoietic cells, ruminant, K-CI cotransport and cellular differentiation in, 106 Hepatocytes Na+-H+ exchange in, 66-67 Na+ influx stimulation by insulin, glucagon, and epidermal growth factor, 167 release or uptake of glucose, lactate, and urea as function of culture age, 273 Hexokinase, glycolytic rate and, 273 Hormones peptide, activation of cellular responses to, model, 131 stimulation of hormone secretion and ion transport in pituitary cells, 151-155 Hydrogen carbonate electrodiffusion, 10-11 and Hi, Na+-coupled transport, 11 Hydrogen carbonate-chloride exchange, see Chloride-hydrogen carbonate Hydrogen chloride-sodium hydrogen carbonate exchange, see Sodium hydrogen carbonate-hydrogen chloride exchange Hydrogen ion active transport CI -HC03 exchange, 12 H+ pump, 12-13 NaHC03-HCL exchange, 12 Nat-H+ exchange, 11-12 passive transport electrodiffusion of H+and HCOl , 10II HCO; cotransport, I I pump, 12-13 Hydrogen ion pump, 12-13 Hydrogen-sodium exchange, see Sodiumhydrogen exchange, 15 Hyperosmotic media, net Na flux by Amphiuma red blood cells in, 205-206 Hypoxia, adaptation of cultured cells to, 275-276

I Initiation factor (eIF-2) phosphorylation, Ca2+and, 246-247 lnositol 1,4,5-triphosphate, stimulation of Ca2+release in leaky Swiss 3T3 cells. 181 lnositol triphosphate-induced Ca2+release from endoplasmic reticulum, 14 Insulin energy metabolism response of quiescent cells in culture, 276-277 -stimulated Na+ influx in hepatocytes, 167 Intracellular pH, see pH, intracellular Ion fluxes alkali metal Ca2+-dependent,in Amphiuma red blood cells, 202-205 force-Row coupling, 195-197 forces driving conductive and electroneutral transport, 197-198 volume and/or pH sensitive, and identification of electroneutral alkali metal ion transport, 210 Ca2+fluxes in quiescent fibroblastic cell lines, 180-182 and initiation of DNA synthesis in quiescent cells, 170-176 generality of increased monovalent cation fluxes after stimulation, 171 increase effect on, 173-176 inhibition effect on, 172-173 net, mechanisms for, 19 net Na, by Amphiuma red blood cells in hyperosmotic media, 205-206 protein kinases and, 176-180 cyclic AMP and cell growth, 176-177 protein kinase C and cell growth, 177180 stimulation by growth factors in quiescent cells, 182 transmembrane by facilitated diffusion, 19 by simple diffusion, 19 volume regulatory pathways, in Amphiuma red blood cells, 201 volume-sensitive, in Amphiuma red blood cells, 198-202

300 electromechanical alkali-metal-H exchange, 198-201 net transport by parallel alkali metalH and CI-HC03 exchangers, 201202 Ionic responses to fertilization in sea urchin eggs fertilization potential, 138-140 intracellular alkalinization, 142-143 resting potential, 133-135 sperm-egg interaction, 135-138 sperm-gated channels, 140-141 sperm-induced permeability changes, mechanism, I41-I42 to growth factors in quiescent cells ion fluxes increase in, and DNA synthesis, 173-176 inhibition effect on D N A synthesis, 172-173 monovalent ion transport in Swiss 3T3 cells effect of Na+ entry on Na+-K+ pump activity, 166 Na+ cycle of, 170 Na+ entry into, effect of mitogens, 167-168 Na+-H+ antiport system, effect of mitogens, 168- 170 Na+ pump activity, 165-166 Ionic transport systems, see also Ion transport and hormone secretion, hormonal stimulation of, 150-155 serum and growth factor activation of lectin-induced mitogenesis in lymphocytes, 149-150 Na+ channel, 147-149 Na+-H+ exchange, 143-145 serum-stimulated electrical events, 145-147 Ionophore A23187 energy metabolism response of quiescent cells in culture, 276-277 and hypertonic medium, effect on cellular Na content of osmotically shrunken Amphiuma cells, 21 1 induction of intracellular alkalinization in cultured human fibroblasts, 73-74 -stimulation of

INDEX

Na+ influx in clutured human fibroblasts, 73 &Rb+efflux from lymphocytes, 149 Ion transport systems, see Ionic transport systems thermodynamics of alkali metal ion fluxes, 195-198 force-flow coupling, 195-197 forces driving conductive and electroneutral transport, 197-198 transport proteins, 5-6 carriers, 5-6 channels, 5 pumps, 6 Isoproterenol, activation of C1-dependent cation cotransport, 94 K

Kidney epithelial cells, Na+-H+ antiport system, 168 proximal tubules O2consumption traces, 265 respiratory capacity, respiratory rate compared to, 266 Kinetics CI-dependent cation transport, 96-97 Na+-H+ exchange system, 62-63 cultured fibroblasts, 64-66 cultured neural cells, 70-71 cultured smooth muscle cells, 71 hepatocytes, 66-67 lymphocytes, 68-70 neutrophils, 67-68 platelets, 67 sea urchin eggs, 63-64 1

Lactate, uptakehelease by cultured hepatocytes as function of age, 273 Lactic acid, production in normal tissues and tumors, 283 Lectin-induced mitogenesis in lymphocytes, 149-150 Leukocytes, polymorphonuclear energy activation in, 271-272

INDEX

301

stimulation with formyl-methionylleucyl-phenylalanine, 67-68 Lipopolysaccaride, differentiation induction in 70213 cells, 72-73 Liver mitochondria, effect on glycolytic rate of tumor cytosol, 281 Loop diuretics, and cation cotransport, 97-98 Lymphocytes differentiating pre-B cell line, Na+-H’ exchange in, 72-73 ionic transport systems, activation by serum and growth factor, 143-150 Na+ channel, 147-149 Na+-H+ exchange, 143-145 serum-stimulated electrical events, 145- 147 Na+-H+ exchange in, 68-70 peripheral blood, lectin-induced mitogenesis, 149-150 spleen, intracellular pH changes and [3H]thymidineincorporation after mitogenic stimulation, 227-229

M

Magnesium ion changes, and DNA synthesis, 225-226 Melittin effect on ion fluxes and DNA synthesis, in quiescent Swiss 3T3 cell cultures, 174- 175 stimulation of Na+ influx, 77 Na+-K+ pump, 166 Membrane potential, see also Membrane voltage BSC-1 cells, effect of epidermal growth factor and serum addition, 24 change of, mechanisms, 22-23 concentration, 22-23 permeability, 23 transport rates of electrogenic pumps, 23 diffusion potentials, 19-21 electrogenic pumps, 21-22 Membrane voltage, see also Membrane potential changes in, and growth factors, 23-25

Messenger RNA cap structure recognition, effect of K + , temperature, concentration, and pH, 244-245 competition between, K+ effect on, 244 posttranslational modification, ionic changes and, 236 Metabolism carbohydrate, see Carbohydrate metabolism cellular, role in C1-dependent cation cotransport, 98-99 energy, see Energy metabolism oxidative, in tumors, 282 -286 Methylmethane thiosulfonate, modification of K-CI cotransport, 104 Microelectrodes, intracellular pH measurement, 40 Mitochondria Caz+ transport by, 13-14 tumor and liver, effect on glycolytic rate of tumor cytosol, 281 Mitogenesis, in lymphocytes, induction by lectin, 149-150 Mitogenic response, modulation by protein kinase C, 33-37 Mitogens alteration of intracellular pH, 27-32 changes in intracellular pH induced by, 31 effect on Na+ entry into Swiss 3T3 cells, 167168 Na+-H+ antiport in fibroblasts, 169170 Na+-H+ exchange, 27-32 Na+ influx in NR6 cells, 27 pH, 169-170 Monensin, increased Na+ entry and, 166 Mononuclear phagocytes, energy activation in, 271-272 Muscle skeletal, energy activation in, 267-268 smooth, Na+-H+ exchange in, 71 N

Nersnt-Planck equation, 196 Nerve growth factor-stimulated Na+ influx in rat pheochromocytoma cells, 70-71

302

INDEX

Neural cells, cultured, Na+-H+ exchange in 70-71 Neuroblas toma Na+-H+ antiport system in, 168 NB2A and NIE, serum-stimulated Na+ influx, 71 NIE-1 15, serum-stimulated Na+ influx, 70 NG108-15 neuroblastoma-glioma, serumstimulated Na+ influx, 70 Neuromuscular tissues, C1-dependent Na or Na-K transport in, 113 Neutrophils, Na+-H+ exchange in, 67-68 Norepinephrine, activation of Cl-dependent cation cotransport, 94 Nuclear magnetic resonance, intracellular pH measurement, 41

0

I-Oleyl-2-acetylglycerol,as mitogen for Swiss 3T3 cells, 178

P

Pancreatic p cells, Na+-C3+ exchange in, 59-60 Parathyroid hormone, effect on bone resorption, role of Na+-Caz+ exchanger, 60 Peptide hormones, cellular response activation, model, 131 Permeability cell membrane egg, sperm-induced changes in, 141I42 potential changes, 23 modulators, effect on ion fluxes and DNA synthesis in quiescent Swiss 3T3 cell cultures, 174-176 PH changes in DNA synthesis and, 227-229 posttranslational mRNA modifications and, 236 protein synthesis and, 240 effect of mitogens on, 169-170

platelet-derived growth factor and fetal calf serum in NR6 cells, 28 effect on DNA polymerase activity, 232 recognition of mRNA cap structure, 244-245 inhibition of CI-HC03 exchange transport in Amphiuma red blood cells and, 209 intracellular alkalinization with 5,S-dimethyloxazolidine-2,4-dione, 65 alteration by mitogens, 27-32 changes after equilibration with extracellular Na+, 46 changes induced by mitogens, 31 effect on NaHC03-HCI exchange, 12 measurement distribution of amphipatic molecules, 41 microelectrodes for, 40 nuclear magnetic resonance, 41 optical methods, 41-47 cellular location of dye, 43-47 dye leakage rate, 42 regulation in nonactivated cells, role of Na+-H+ exchange, 60-61 role in Sa protein phosphorylation, 245246 sensitive alkali metal ion fluxes in Amphiuma red blood cells, and identification of electroneutral alkali metal ion transport, 210 Phagocytes, mononuclear, energy activation in, 271-272 Pharmacology definitions of Na+-H+ and Na+-Caz+ exchange systems, 78-81 interaction of amiloride analogs with Na+ transport systems, 80-8 I Phorbol esters activation of Na+-H+ exchange with, 75-77. 145 effect on ion fluxes and DNA synthesis in quiescent Swiss 3T3 cell cultures, 173-174 stimulation of Na+-K+ pump activity in Swiss 3T3 cells, 165, 168 Phorbol myristic acetate effect on A 431-cell Na+-H+ exchange, 35

INDEX

modulation of epidermal growth factordependent alkalinization, 36 Phosphofructokinase activity, and energy metabolism response of quiescent cells to growth factors, 277 glycolytic rate and, 273 Phospholipase, role in activation of Na+H+exchange, 77-78 Phosphorylation eIF-2, CaZ+and, 246-247 S6 protein, role of intracellular pH, 245246 Phytohemagglutinin-induced mitogenesis, in peripheral blood lymphocytes, 149 Pituitary cells, cultured, hormonal stimulation of ion transport and hormone secretion in, 151-155 Placenta, human, CI-dependent Na or NaK transport in, I 1 3 Plasma membrane Ca2+transport pathways, characteristics, 16 ions and expression of latent proteins, 248-249 Na+ transport pathways, characteristics of, 9 vesicles from A 431 cells, Na+-Hi exchange in, 39 Platelet-derived growth factor alkalinization in NR6 cells, amiloride effect, 30 effect on pH in NR6 cells, 28 stimulation of Caz+eftlux, 180 Na+-H+ antiport in cultured cells, 169 Na+ in cultured smooth muscle cells, 71 Na+-K+ pump activity in Swiss 3T3 cells, 165 synergistic action with vasopressin, 173 Platelets, Na+-H+ exchange in, 67 Polymorphonuclear leukocytes energy activation in, 271-272 stimulation with formyl-methionylleucyl-phenylalanine, 67-68 Posttranslational protein modifications expressions of latent plasma membrane proteins, Na+-Caz+ and, 248-249 intracellualr protein traflic, Na+-K+ levels and, 247-248

303 Potassium-chloride cotransport and cellular differentiation in ruminant hemopoietic cells, 106 in erythrocytes chemically modified, 104-105 volume-stimulated, 103-104 major modes, triggers, and cell types, 94 Na-independent, in differentiated epithelial and nonepithelial cells, I I I Potassium ion changes in DNA synthesis and, 229-231 protein synthesis and, 240-243 cotransport KCI, 10 NaKCl2, 8-10 effect on mRNA competition, 244 recognition of mRNA cap structure, 244-245 and Na+ levels, intracellular protein traffic and, 247-248 transport, 8-10 Prolactin, secretion by GH3 pituitary cells, 151 Protein kinase C and alkali metal-H exchange in Amphiuma red blood cells, 213-215 ion fluxes, and, 177-180 mitogenic response modulation by, 3337 role in activation of Na+-H+ exchange, 75-77 Proteins intracellular traflic and cell surface expression, posttranslational events affecting, 247-249 latent plasma membrane, ions and expression of, 248-249 transport, classification carriers, 5-6 channels, 5 pumps, 6 Protein synthesis effect of ions on, 243-247 eIF-2 phosphorylation, 246-247 mRNA competition, 244 recognition of mRNA cap structure, 244-245 S6 phosphorylation, 245-246 ionic changes and, 237-247

INDEX

Ca2+.237-240 K+ and Na+, 240-243 PH, 240 Pyruvate kinase, glycolytic rate and, 274 Q

Quin-2, monitoring Ca2+intracellular activity with, 74 R

Red blood cells, see Erythrocytes Respiration, renal proximal tubules, rate compared to capacity of, 266 Resting potential, sea urchin eggs, 133-135 RNA, messenger, see Messenger RNA RNA synthesis effect of ions on, 234-236 chromosomal protein phosphorylation, 235-236 posttranslational modification of mRNA, 236 ionic changes and, 232-236 Calf, 233-234 Na+, 234 S

Sarcoplasmic reticulum membranes, Ca2+ uptake by, 14 Sea urchin eggs, see Eggs, sea urchin Serum activation of ionic transport systems electrical events, 145-147 lectin-induced mitogenesis in lymphocytes, 149-150 Na+ channel, 147-149 Na+-H+ exchange, 143-145 effect on quiescent BSC-I cells, 24 energy metabolism response of quiescent cells in culture, 276-277 fetal bovine, stimulation of Na+ in cultured smooth muscle cells, 71 fetal calf, effect on pH in NR6 cells, 28 stimulation of Na+ influx in human foreskin and lung- fibroblasts, 167

in murine neuroblastoma cells, 70 and Na+-K+ pump activity in MDCK cells, 167 in NG108-15 neuroblastoma-glioma cell line, 70 in NIE and NB2A cells, 71 Skeletal muscle, energy activation in, 267268 Smooth muscle, Na+-H+ exchange in, 71 Sodium-calcium exchange, 14-15 in activated or differentiating cells Friend erythroleukemic cells, 58-59 pancreatic /3 cells, 59-60 parathyroid hormone and bone resorption, 60 amiloride inhibition of, 80 inhibition with benzamil, 80 in nonactivated cells, general properties, 57-58 Sodium-calcium exchanger, general properties, 57-58 Sodium channels serum and growth factor activation of, 147-149 tight epithelia cells, amiloride inhibition of, 78-79 Sodium-chloride cotransport major modes, triggers, and cell types, 94 Na-independent, in erythrocytes, 101 or Na-K-2Cl transport, in erythrocytes, 105- 108 Sodium cycle, 170 Sodium-hydrogen antiport system in cultured cells, 168-169 effect of mitogens on, 169-170 Sodium hydrogen carbonate-hydrogen chloride exchange CAMP stimulation of, 12 internal ATP requirement, 12 pH effect on, 12 Sodium-hydrogen exchange, 11-12 in A 431-cells phorbol myristic acetate effect, 35 in plasma membrane vesicles, 39 activation diagram of, 40 mechanism, 32-33 with TPA, 75-77 in activated cells fibroblasts, 64-66 hepatocytes, 66-67

INDEX

lymphocytes, 68-70 neural cells, 70-71 neutrophils, 67-68 platelets, 67 sea urchin eggs, 63-64 smooth muscle cells, 71 stimulation mechanism, see under stimulation mechanism in activated cells amiloride inhibition of, 79-80 in differentiating cells Dictyostelium discoideum, 72 pre-B lymphocyte cell line, 72-73 electroneutral, fertilization-activated, 142 increase with mitogens, 27-32 inhibition by amiloride, 62 with benzarnil, 78 Na+-H+ exchanger, properties in nonactivated cells, 62-63 regulation of intracellular pH in nonactivated cells, 60-61 serum and growth factor activation of, 143-145 stimulation mechanism in activated cells Ca2+ role, 73-75 phospholipase activity and, 77-78 protein kinase C role, 75-77 vanadate stimulation of, 32-33 Sodium-hydrogen exchanger, properties in nonactivated cells, 62-63 Sodium ion and Ca2+countertransport, 7-8 changes in DNA synthesis and, 229-231 protein synthesis and, 240-243 RNA synthesis induction and, 234 Cl-dependent transport in differentiated epithelial cells, 109 cotransport with anions, 7 with organic solutes, 7 -coupled transport of HCO; , I I efflux, 8 entry into Swiss 3T3 cells effect of mitogens on, 167-168 effect on Na+-K+ pump activity, 166 and expression of latent plasma membrane proteins, 248-249 extracellular, intracellular pH changes in cells equilibrated with, 46

and H+countertransport, see Sodiumhydrogen exchange -independent K-CI transport in differentiated epithelial and nonepithelial cells, I 1 1 in erythrocytes, 101 influx, 6-8 activation with TPA, 75-77 carriers, 7-8 channels, 7 ionophore A23187-stimulated in cultured human fibroblasts, 73 nerve growth factor- and epidermal growth factor-stimulated in rat pheochromocytoma cells, 70-7 I in NR6 cells, effect of mitogens on, 27 serum-stimulated in C6 glioma cells, 71 in murine neuroblastoma cells, 70 in NG108-15 neuroblastoma-glioma cell line, 70 in NIE and NB2A cells, 71 tetrodotoxin-sensitive, in nonexcitable cells, 7 and K+ levels, intracellular protein traffic and, 247-248 net flux by Amphiuma red blood cells in hyperosmotic media, 205-206 transport, chloride-dependent, in erythrocytes, 102 transport pathways in plasma membrane, characteristics, 9 Sodium-potassium-chloride cotransport, major modes, triggers, and cell types, 94 Sodium-potassium pump, in quiescent Swiss 3T3 cells, 165-166 effect of mitogens of Na+ entry into, 167-168 mitogens on Na+-H+ antiport, 169- 170 Na+ entry on activity of, 166 ionic responses to growth factors, 165176 Na+ cycle of, 170 Na+-H+ antiport system, 168-169 Sodium-potassium transport, CI-dependent in differentiated epithelial cells, 109 in erythrocytes, 102 Sodium transport systems, and amiloride analogs, pharmacological interaction, 80-8 I

306

INDEX

Sperm activation of energy metabolism in, 27027 1 -egg interaction, in sea urchins, 135-138 -gated channels, and fertilization potential, 140-141 -induced permeability changes in egg cell membrane, I4 1 -142 T

Temperature, effect on recognition of mRNA cap structure, 244-245 Tetrodotoxin-sensitive Na+ influx in nonexcitable cells, 7 Thermodynamics, C1-dependent cation cotransport, 91 Thermogenesis, brown fat, 268-270 Thymidine, 3H-labeled, intracellular pH changes after incorporation into splenic lymphocytes, 227-229 Thymidylate synthase induction, CaZ+ effects, 231-232 Thyrotropin-releasing hormone, secretion by GH3 pituitary cells, 151-155 Tissues, neuromuscular, C1-dependent Na or Na-K transport in, 113 Transport proteins, classification carriers, 5-6 channels, 5 pumps, 6 Tumors aerobic glycolysis in, 278-282 c6glioma, serum-stimulated Na+ influx, 71 Ehrlich ascites, CI-dependent cation transport in, 108-1 I 1 Friend erythroleukemic cells, Na+-Caz+ exchange in, 58-59 human leukemic cell line, Na+-H+ antiport system in, 168 lactic acid production in normal tissues and, 283 mitochondria, effect on glycolytic rate of tumor cytosol, 281 mitochondria1 content relative to normal tissue, 284

neuroblastoma N1E-115 cells, serum-stimulated Na+ influx, 70 NG108- 15 neuroblastoma-glioma, serum-stimulated Na+ influx, 70 NIE and NBZA, serum-stimulated Na+ influx, 71 oxidative metabolism in, 282-286 pheochromocytoma (PC-12) cells, nerve growth factor- and epidermal growth factor-stimulated Na+ influx in, 70-71 70213 cell line, lipopolysaccarideinduced differentiation, 72-73

U

Urea, uptakehelease by cultured hepatocytes as function of age, 273

V

Vanadate, stimulation of Na+-H+ exchange, 33 Vasopressin effect on ion fluxes and DNA synthesis in quiescent Swiss 3T3 cell cultures, 173-174 stimulation of Caz+efflux, mediation by pressor-type receptor, 180 Na+ influx in human foreskin and lung fibroblasts, I67 Na+-K' pump activity in Swiss 3T3 cells, 165 Volume maintenance, 15 Volume regulation regulatory volume decrease, 17 K-CI cotransport effect, 92 mechanisms of activation, 18 regulatory volume increase, 17-18 in Amphiuma erythrocytes, 17-18 mechanisms of activation, 18 Na-CI or Na-K-2CI effects, 93