Current Topics in Membranes and Transport, Volume 26 Na+ - H+ Exchange, Intracellular Ph, and Cell Function

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Current Topics in Membranes and Transport VOLUME 26

Na+-H+ Exchange, lntracellular pH, and Cell Function

Advisory Board

M . P . Bluustcin G . Blobel E . Curafoli J . S . Cook Sir H . L . Kornberg

D . Louvurd C . A . Pustcrnuk W . D . Stein W . Stocckcnius K . J . Ullrich

Current Topics in Membranes and Transport Edited by Arnost Kleinzeller

Felix Bronner

Depurtment of Physiology Universiry of Pennsylvcoiia School qf Medicine Philudelpliiii, Pennsylvania

Dcpcirtment of Oral Biologv University of Connecticut Hculth Center Furniington. Conncvticitt

VOLUME 26

Na+-H+ Exchange, lntracellular pH, and Cell Function Guest Editors Peter S. Aronson

Walter F. Boron

Departments of Medicine und Pliv.siology Yale University School of Medicine N e w Haven, Conneclicitt

1)c~purlmenlof

Physiology Yule University Scliool of Mrdicino Nenj Haven Connecticut I

Volume 26 is part ot the series (p. xiii) from the Yale Department of Physiology under the editorial supervision of:

Joseph F. Hoffman

Gerhard Giebisch

Department of Physiology Yale University School of Modic~inc~ N e w Huven, Connrcticitr

Ikpiirtincvi~of

Physiology Yule University School of Medicine Ncvir Hcrven, Cotineclicwl

1986

@) ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

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Contents

Preface, xi Yale Membrane Transport Processes Volumes, xiii

PART I.

CHAPTER

GENERAL ASPECTS OF INTRACELLULAR pH REGULATION AND Na+-H+ EXCHANGE

I.

lntracellular pH Regulation by Leech and Other Invertebrate Neurons R . C. THOMAS AND W. R . SCHLUE

1. Introduction, 3 11. Methods, 6 111. Results with Leech Neurons, 6 1V. Conclusion, 12 References, 12

CHAPTER

2.

Approaches for Studying lntracellular pH Regulation in Mammalian Renal Cells WALTER F. BORON

1. Introduction, I5

An Optical Absorbance Technique for Measuring lntracellular pH, 16 lntracellular pH Regulation in Mammalian Renal Cells. 22 IV. Conclusions, 31 References. 32 11. 111.

V

vi

CONTENTS

CHAPTER

3.

Aspects of pHi Regulation in Frog Skeletal Muscle ROBERT W. PUTNAM AND ALBERT ROOS

I. 11.

Ill. IV. V. VI. VII. VIII.

1x.

Introduction, 36 Methods, 36 Steady-State pH., 37 Response of pH, to Acid Loading, 42 Nature of pH,-Regulating Systems, 45 Properties of Na-H Exchange, 47 Factors Affecting the Rate of Na-H Exchange, 48 Comparison with Other Cells, 51 Summary, 52 References, 53

CHAPTER

4.

Molecular Properties and Physiological Roles of the Renal Na+- H Exchanger +

PETER S. ARONSON AND PETER IGARASHI 1. Introduction, 57 11. Kinetics of the Renal Nat-H+ Exchanger, 58 Ill. Biochemistry of the Renal Na+-H+ Exchanger, 65 IV. Role of the Renal Na+-Ht Exchanger in Facilitating Anion Transport, 70 References. 75

PART 11. CHAPTER

5.

Na+-H+ EXCHANGE AND CELL VOLUME REGULATION Volume-Sensitive Alkali Metal-H Transport in Amphiuma Red Blood Cells PETER M. CALA

I. Introduction, 79 11. Volume-Sensitive Fluxes in Amphiuma Red Blood Cells, 81 Ill. The Effects of DlDS upon Alkali Metal-H Exchange by Osmotically Swollen Cells: Activation versus Altered Selectivity, 88 IV. Activation of Alkali Metal-H Exchange: A Role for Diacylglycerol through Protein Kinase C, 90 V. The Relationship between Na-H and K-H Exchange in Amphiurnti Red Blood Cells, 95 VI. Summary, 96 References, 97

vii

CONTENTS CHAPTER

6.

Na-Proton Exchange in Dog Red Blood Cells JOHN C. PARKER

I. Introduction, 101 11. Anion Requirement, 102 111. Fixation of the Transport Mechanism by Glutaraldehyde, 103 IV. Protein Fluxes, 105

V. Lithium Effects, 108 References, 114

CHAPTER

7.

Activation of the Na+-H+ Antiport by Changes in Cell Volume and by Phorbol Esters; Possible Role of Protein Kinase S. GRINSTEIN, S. COHEN, J. D. GOETZ, A. ROTHSTEIN, A. MELLORS, AND E. W. GELFAND

I. Introduction, I IS Basic Properties of Na+-Ht Exchange in Lymphocytes. I I6 Na+-Hi Exchange in Volume Regulation, 120 1 v . Stimulation of Na+-H+ Exchange by Phorbol Esters, 122 V. Similarities of the Phorbol Ester and Volume-Induced Activation, 126 VI. Possible Involvement of a Protein Kinase in Volume Regulation, 128 v11. Osmotically Induced Changes in Phosphoinositide Turnover, 13 1 v111. Concluding Remarks, 133 References, 134 11. 111.

PART 111. CHAPTER

8.

Na+-H+ EXCHANGE AND CONTROL OF CELL GROWTH The Generation of Ionic Signals by Growth Factors W. H. MOOLENAAR, L. H. K. DEFIZE, P. T. VAN DER SAAG, AND S. W. DE LAAT

1. Introduction. 137 11. Ionic Signals i n Growth Factor Action, 138 111. Possible Physiological Role of Inlracellular Ionic Change\. 148 1V. A Monoclonal Antibody Approach to the Dissociation of Early Event\ in EGF Action. 150 V. Concluding Remarks, 153 References, 154

CONTENTS

viii CHAPTER

9.

Control of Mitogenic Activation of Na+-H+ Exchange D. CASSEL, P. ROTHENBERG, B. WHITELEY, D. MANCUSO, P. SCHLESSINGER, L. REUSS, E. J. CRAGOE, AND L. GLASER

I. Introduction, 157 11. The Activation of Na+-H+ Antiport in A431 Epidermoid Carcinoma Cells and

NR6 Fibroblasts, 158 References, 171

CHAPTER

10.

Mechanisms of Growth Factor Stimulation of Na+-H+ Exchange in Cultured Fibroblasts MITCHEL L. VILLEREAL, LESLIE L. MIX-MULDOON, LUCIA M. VICENTINI, GORDON A. JAMIESON, JR., AND NANCY E. OWEN

I . Introduction, 175 11. Stimulation of Na+ Influx in HSWP Cells by Serum and Peptide Mitogens, 176 111. Characterization of the Transport System Mediating Mitogen-Stimulated Na+

Influx, 177 1v. Involvement of Ca2+in the Mitogen Activation of the Na+-H+ Exchanger, 179 V. Possible Role of Protein Kinase C in the Activation of Na’ Exchange in HSWP Cells and Other Fibroblast Systems, 182 VI. Evidence for Phospholipase Involvement in the Mitogen Activation of Na+-H+ Exchange in HSWP Cells, 185 VII. Possible Involvement of Phospholipases in the Mobilization of Intracellular Ca”, 187 VIII. Which Phospholipase Is Activated by Mitogen Stimulation? 187 IX. Summary of the Proposed Mechanism for Activation of the Na+-H’ Exchanger by Mitogens, 189 References. 191

CHAPTER

I.

B Lymphocyte Differentiation: Role of Phosphoinositides, C Kinase, and Na+-H+ Exchange PHILIP M. ROSOFF AND LEWIS C. CANTLEY

I. Inti duction. 193 11. Effects of Lipopolysaccharide on Phosphatidylinositol Turnover and Cytosolic Free Ca2+,195 111. Effects of Phorbol Esters on Phosphatidylinositol Turnover and Cytosolic Free Ca2+, 196

IV. Conclusion, 197 References, 198

CONTENTS CHAPTER

12.

ix

Na+-H+ Exchange and Growth Control in Fibroblasts: A Genetic Approach JACQUES POUYSSEGUR. ARLETTE FRANCHI, MlCHlAKl KOHNO, GILLES L'ALLEMAIN, AND SONIA PARIS

1. Introduction, 202 11. Fibroblast Mutants Altered in the Na+-H' Antiport Activity, 203 111. Characterization and Properties of Two pH,-Regulating Systems in Fibroblasts, 205 IV. Growth Factor Activation of the Na+-H+ Antiporter, 208 V. pH, Controls Reinitiation of DNA Synthesis and Growth, 21 I v1. Toward a Molecular Identification of the Na+-H+ Antiport System, 215 VII. Conclusions, 217 References, 218

PART IV.

CHAPTER

13.

ROLE OF Na+-H+ EXCHANGE IN HORMONAL AND ADAPTIVE RESPONSES Hormonal Regulation of Renal Na+-H+ Exchange Activity BERTRAM SACKTOR AND JAMES L. KINSELLA

I. Introduction. 223 I I . Functions of Na+-H+ Exchange in the Proximal Tubule, 224 111. Measurement of Na+-Hi Exchange Activity in Renal Brush Border Membrane Vesicles, 224 IV. Extrinsic Effectors of Renal Na+-H' Exchange Activity. 228 V. Hierarchy of Hormonal Effect of Na+-H' Exchange, 240 References, 243

CHAPTER

14.

Adaptation of Na+-H+ Exchange in the Proximal Tubule: Studies in Microvillus Membrane Vesicles JULIAN L. SEIFTER AND RAYMOND C. HARRIS

I.

Introduction, 245 Experimental Approach, 247 Effects of Uninephrectomy and Dietary Protein on Na+-H+ Exchange, 249 IV. Effects of Potassium Depletion on Na+-H+ Exchange, 256 V. Other Models of Adaptation, 257 VI. Summary and Conclusions, 258 References, 259 11. 111.

CONTENTS

X

CHAPTER

IS.

The Role of lntracellular pH in Insulin Action and in Diabetes Mellitus RICHARD D. MOORE

I.

Introduction, 264 Mechanism of Insulin Effect upon pH,, 270 Effects of Insulin-Mediated Changes in pH, upon Glycolysis, 274 1V. Role of lntracellular pH in Insulin Action, 279 V. Model of Ionic Part of Mechanism of Insulin Action, 281 VI. Clinical Implications, 283 References. 288 11. 111.

CHAPTER

16.

The Proton as an Integrating Effector in Metabolic Activation WILLIAM B. BUSA

1. Introduction, 291 11. Four Systems Involving Regulatory pH, Changes, 292 111. Comparison of in Vitro pH/Activity Profiles with in Vivo Responses, 295 IV. Conclusion: Why Use pHi as a Metabolic Regulator'? 302 References, 304

Index, 307

Preface Since 1975, the Yale University Department of Physiology has sponsored annual symposia on topics concerned with membrane transport processes. The goal of the symposium held on December 13-15, 1984 was to describe the properties of the plasma membrane Nat -H exchanger, to discuss its role and that of other acid-base transport systems in the regulation of intracellular pH, and to relate Na+-H+ exchange and intracellular pH to other important aspects of cell function. For this purpose, a group of distinguished scientists from diverse disciplines was assembled, and they have contributed the chapters to this volume. The chapters have been organized into four major sections. The first deals with general aspects of intracellular pH regulation and Na'-H ' exchange: it covers methods for studying intracellular pH regulation, describes different acid-base transport systems involved in the regulation of intracellular pH, and details various kinetic and biochemical properties of the Na+-H+ exchanger in particular. Subsequent sections deal with the role of Na+-H+ exchange in specific aspects of cell function. Thus the second section describes the mechanisms by which the Na+-H+ exchanger is activated and contributes to the restoration of cell volume when cells are placed in anisotonic media. The third section describes the mechanisms by which the Na+-H+ exchanger is activated and plays a physiologic role in the cell response to mitogenic growth factors and promoters of differentiation. Finally, the last section examines the role of Na'-H+ exchange in mediating cell and organ responses to hormones and adaptive stimuli, which is seen to be part of a general theme of the proton as an integrating effector in metabolic activation. We gratefully acknowledge the invaluable assistance of Linda Brouard, our conference coordinator, whose efforts contributed so greatly to the success of the symposium. We wish also to acknowledge the generous financial support of the Kroc Foundation, the Burroughs Wellcome Co., Stuart Pharmaceuticals, McNeil Pharmaceutical, Hoechst Pharmaceuticals, Inc., Americas, Inc., Miles Laboratories, Merck Sharp and Dohme, and WPI, Inc. +

PETERS. ARONSON WALTERF. BORON xi

This Page Intentionally Left Blank

Yale Membrane Transport Processes Volumes Joseph F. Hoffman(ed.). (1978).“MembraneTransport 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.). (1 98 1). “Molecular Mechanisms of Photoreceptor Transduction”: Volume IS 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 111 (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, lntracellular pH, and Cell Function”: Volume 26 of Current Topics in Membranes and Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, Orlando. xiii


Part I

General Aspects of Int race1Iular pH Regulation and Na+-H+ Exchange


CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 26

Chapter 7

IntracelIular pH Regulation by Leech and Other Invertebrate Neurons R . C . THOMAS Department of Physiology Bristol University Brisrol, England AND

W . R . SCHLUE Institute for Zoologie I Diisseldorf University Diisseldorf, Federal Republic of Germany

I. Introduction. .......................... A. Acid Extrusion Mechanisms . . . . . . . . B . Leech Neurons.. . . . . . . . . .

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

11. 111.

IV .

3

A. Acid Extrusion in the P B. Effect of Amiloride and Removal of External N a . . ..................... C. Acid Extrusion in Bicarbonate-Free Solutions . . . . . . . . Conclusion. . . ..................................... References ............................................

1.

4 5

6 6 6

7 10

12 12

INTRODUCTION

Ignorance of the importance of the field of intracellular pH (pHi)regulation among scientists able to investigate it prevented significant progress until about 1976. The increasing variety of methods available in the years since then, however, has transformed the picture, although very few text3 Copyright 0 1986 hy Academic Press, Inc. All nghts of reproduction in any form reserved.

4

R. C. THOMAS AND W. R. SCHLUE

books today have even a paragraph about pHi regulation. It is already clear that pHi regulatory mechanisms vary considerably from preparation to preparation, and so far four different ion exchange systems for acid extrusion have been identified, as shown in Fig. 1 . A. Acid Extrusion Mechanisms

It is now well established that in most cells pHi is close to the external pH, too alkaline to be explained by a passive distribution of H+ or HC03 ions (for review see Roos and Boron, 1981; Thomas, 1984). Net passive fluxes would tend to acidify pH,, so an active acid extrusion mechanism is required to keep pHi constant. (There appears to be no special mechanism promoting acid uptake to accelerate pHi recovery from excessive alkalinization.) 1. SODIUM-HYDROGEN EXCHANGE

Johnson et al. (1976) were the first to propose an amiloride-sensitive Na-H exchange as the mechanism of the large pHi increase in sea urchin eggs after fertilization. What appears to be essentially the same system has since been described in a variety of vertebrate preparations (see Thomas, 1984), and it may well represent the principal vertebrate pH, regulating system. With invertebrate neurons, however, Na-H exchange has so far only been described in crayfish neurons (Moody, 1981), in which sensitivity to amiloride has not been established.

2. BICARBONATE-CHLORIDE EXCHANGE As red blood cells circulate round the body they gain bicarbonate and lose chloride in the tissues, with the reverse happening in the lungs. This process has been known for decades and is variously called the chloride shift or the Hamburger interchange. It is mediated by a carrier in the (AMILORIDEI

(SITS) Y

A

FIG.1. Diagram of four ion exchange mechanisms for intracellular pH regulation. Inhibitors are shown in parentheses.

1. INTRACELLULAR pH REGULATION BY NEURONS

5

membrane which is specifically blocked by SITS (4-acetamide-4’-isothiocyanatostilbene-2,2’-disulfonic acid). When pH, regulation in squid axons was found to require bicarbonate (Boron and deWeer, 1976b)and chloride ions and to be blocked by SITS (Russell and Boron, 1976), it was naturally concluded that squid axon pHi regulation involved Cl--HCOF exchange. Further investigation, however, showed that Na+ ions were required (see Boron and Russell, 1983). At present simple CI-HC03 exchange is not thought to play any role in pH, regulation in invertebrate cells.

3. SODIUM-DEPENDENT CHLORIDE-BICARBONATE EXCHANGE This mechanism is supposed to involve the influx of one Na+ and one HCO? ion in exchange for the efflux of one CI- and one H + ion. (The H+ ion may be replaced by a second HCO: ion.) It was first proposed to explain acid extrusion by snail neurons (Thomas, 1977) and has subsequently been found in crayfish neurons (Moody, 1981) and barnacle muscle (Boron et al., 1981). Like simple CI-HC03 exchange, it is blocked by SITS, but it is not affected by amiloride. It has not yet been described in a vertebrate preparation. 4. SODIUM CARBONATE (MONOVALENT)-CHLORIDE EXCHANGE

This mechanism has been shown to operate in red blood cells by Becker and Duhm (1978), who suggested that it might also account for pH; regulation in cells in which such regulation depended on Na+, HCO? , and Clions. If so, Li+ should be a good substitute for Na+, since it also forms ion pairs with the carbonate ion. In snail neurons Li+ is only a poor substitute for Na+ (Thomas, 1976b),but no detailed studies of the ion pair possibility have been undertaken. Boron et al. (1981) have ruled out the mechanism in barnacle muscle, but Boron (1985) has recently shown that it may explain pHi regulation in squid giant axons. 6. Leech Neurons

The leech nervous system is segmented into ganglia, most of which have the same layout and contain several cells large enough to be investigated with intracellular microelectrodes. Thus leech neurophysiology has been extensively investigated (see Muller et al., 19811, usually in preparations superfused with HCO: free solutions. Nothing is known about pH, regulation in leech neurons, so we have undertaken an investigation using neutral ligand pH-sensitive microelectrodes. A more detailed account of this investigation has been given elsewhere (Schlue and Thomas, 1985).

6

R. C.THOMAS AND W. R. SCHLUE

II. METHODS

Specimens of the medical leech, Hirudo medicinalis,were pinned to the bottom of a dish and cut open and parts of the ventral nerve cord removed. Two or three ganglia were then pinned to the base of a small chamber and continually superfused with leech saline at room temperature. Under visual observation selected neurons were then penetrated with a previously calibrated double-barreled pH-sensitive microelectrode. Most experiments were done on the largest cells in the ganglia, the Retzius cells. (For more details see Deitmer and Schlue, 1981; Schlue and Deitmer, 1980, 1984.) The bicarbonate-free saline contained 115 mM NaCl, 4 mM KCI, 1.8 mM CaCI2, 10 mM HEPES (pH 7.4), and 11 mM glucose. Bicarbonate-buffered solutions contained NaHC03 instead of HEPES and were equilibrated with 2% C 0 2 in oxygen. Sodium-free solutions contained N-methyl-D-glucamine instead of Na, and ammonium solutions had NH4Cl instead of part of the NaCl. Double-barreled pH-sensitive microelectrodes were made as described for K+-sensitive microelectrodes by Schlue and Deitmer (1980) except that the K+exchanger was replaced by a proton cocktail (Fluka, Switzerland). Each barrel of the electrode was connected to one input of a WPl F-223A differential electrometer (made in Connecticut!) with Ag-AgCI wires. The complete setup, including solutions and amplifier input probes, was enclosed in a Faraday cage. 111.

RESULTS WITH LEECH NEURONS

In preliminary experiments we found that leech neurons could be penetrated relatively easily with the double-barreled pH-sensitive microelectrodes, and, with care, stable recordings could be maintained for over an hour. The pHi was about 7.3 in both bicarbonate and bicarbonate-free solutions and was stable in both. The average membrane potential was about -40 mV, but ranged from -20 to -50 with no obvious correlation with pHi , except at the beginning of an experiment when a low Emcaused by damage was often associated with a fall in pHi. Recovery from such acidification was faster in the presence of bicarbonate. A. Acid Extrusion in the Presence of Bicarbonate

Apart from the accidental method of acid loading by damage, we have used three ways of decreasing pHi to allow us to study the mechanism of


7

its recovery. These methods of acid loading were exposure to propionic acid, exposure to C 0 2 , and loading with ammonium ions by exposure to NH4CI. With the weak acids, pH, recovery occurs in the presence of the added acid, but with NH: the acidification and recovery occurs only after the NH4CI is removed. For this reason the NH: technique has been widely used in pHI research since its introduction by Boron and De Weer (1976b). In the experiment shown in Fig. 2 we acid-loaded a leech neuron four times, once with C 0 2 and three times with NH4CI. Before the cell was penetrated, the pH electrode was calibrated by running pH 6.4 leech saline through the bath. Then two neurons were abortively penetrated before a reasonably stable recording was obtained. Then the superfusing solution was changed from a C02-freeone to one of the same pH (7.4) but buffered with 2% COz and 11 mM HCO?. The entry of C 0 2 caused a transient fall in pHi as it was converted to carbonic acid, followed by a pHi recovery beyond its earlier value to about 7.3 in 20 min. During the experiment the response to NH4CI was tested. The first exposure was for 2 min, and the second and third were for 1 min each. During the first NH4CI exposure it was possible to record the pH, decrease (after the initial large increase) caused by NH: entry. The shorter exposures allowed only the pHi increase to be seen; this is due to the rapid entry of NH3 molecules. Ammonia entry is fast and completely reversible-it has no effect on pHi lasting beyond its removal. On the other hand, NH: entry is slower and not readily reversible because of the negative Em.Thus most NH; leaves the cell as NH3 when external NH4CI is removed, the H + ions given up causing the fall in pH, (Boron and De Weer, 1976a; see also Thomas, 1984). B. Effect of Amiloride and Removal of External Na

After the final exposure to NH4CI, amiloride was added to the superfusate. This did not block pHi recovery, but did slow it by about half. If amiloride fails to inhibit completely because of lack of potency, it might work better in the absence of Na. Figure 3 shows that the removal of external Na did cause a complete blockage, although 3 mM amiloride caused only some slowing of pHi recovery. Either a single mechanism was blocked by amiloride in the absence of Na or there are two mechanisms, and the amiloride-insensitive one is blocked by Na removal. The next result (Fig. 4) suggests that, as expected, the amiloride-sensitive mechanism is also blocked by Na removal. The pH, recovery from acidification is completely blocked by the removal of external Na (re-

.

cop HCOi-free

64

coz-.

2 %cop , 11 rnM-HC05

HC0; -free

I

72 74

____

U

20 mM - NHqCI

U

20mM - NHqCl

U

20 mM- NHqCI 2 mM-amiloride

FIG.2. Pen recording of membrane potential (Em) and intracellular pH (pH,) of a leech neuron, preceded and followed by calibration of the pH microelectrode. The COIcontent of the superfusion solution is shown in the middle, and periods of application of NH,CI and amiloride are shown at the bottom.

9


5 rnin No-free

n

U

U

U

20rnM NHLCI

20rnM- NHbCl

20 mM NHhCl

-

-

3 mM - arntloride

FIG. 3. The effect of amiloride and Na removal on leech neuron pH, recovery from NH,CI-induced acidification. Superfusate contained I I mM bicarbonate. -_ -40

- 60

5 min 2 -1. c02 , I 1 rnM - HCO;

coz-,

HCOj-free

64

-- _ I

72 7.4

U 20 mM-NH&I

-

U

U

20mM-NHLCI

20mM-NH&I

U 20mM-NH&l

Na - free

FIG. 4. The effect of Na removal on leech neuron pH, recovery in the presence of bicarbonate.

10

R. C.THOMAS AND W. R. SCHLUE

placed by N-methyl-D-glucamine). This leaves unsettled the choice between (1) two separate mechanisms, both requiring Na, one blocked by amiloride, or (2) a single mechanism partially inhibited by amiloride. The second possibility is the less likely one in view of amiloride’s potency on other systems it inhibits. If there are two separate mechanisms, the amiloride-insensitive one may well require bicarbonate. C. Acid Extrusion in Bicarbonate-Free Solutions

The experiment shown in Fig. 5 illustrates the lack of any dramatic effect of bicarbonate removal. The preparation was twice acid-loaded and the pHi allowed to recover in 11 m M bicarbonate, then acid-loaded again and allowed to recover in the absence of bicarbonate. The recovery was perhaps a little slower, but not obviously so. But if allowance is made for the probable halving of intracellular buffering power in the C02-free solution (Thomas, 1976a; Roos and Boron, 1981),then the acid extrusion rate in the absence of bicarbonate is really much slower than it is in its presence. In other words, bicarbonate removal appears to reduce acid extrusion by roughly half.

O r -20 w

-

Em

_..

A

-

-60

5 min 2% c 0 2 , I I m M - HCO;

c02-. HCOj-free

c02:. HCOj-free

I

I,

7.0 7.2

7.6

U 20mM-NH&l

U 20mM-NHbCl

U ZOmM-NH&I

FIG.5 . The effect cf bicarbonate removal on leech neuron pH, recovery from NHdCIinduced acidification.

11


> E - 20 0 1

w

-40t

-60

-

5 min .-

I

6.8 7.2 7.4

U

U

20 mM - NHhCl

20 mM - NHbCl

I 2 rnM -arniloride

FIG. 6. The effect of amiloride in the absence of bicarbonate on leech neuron pH, recovery.

2 mM-arniloride

6.Lr 6.6 /I a

F"z..

70 12 14

+c" U

u

20mMNHbCl

2%C02, IlmM-HCO;

0 '4 u 2%C02, 1lmM-HCOj

FIG.7. The effect of bicarbonate application on leech neuron pH, recovery in the presence of amiloride.

12

R. C. THOMAS AND W. R. SCHLUE

All bicarbonate-dependent acid extrusion systems are blocked by SITS. With leech neurons we have also found (not illustrated) that SITS slows pHi recovery in the presence of bicarbonate but does not block it. The amiloride-sensitive mechanism is presumably insensitive to SITS. If bicarbonate removal blocks the amiloride-insensitive mechanism, then amiloride and bicarbonate removal together should completely block pHi recovery. This is shown in Fig. 6, based on an experiment in which there was no bicarbonate present. Amiloride inhibition of pHi recovery was readily overcome by bicarbonate application, as shown in Fig. 7. Presumably the amiloride-inhibited system was not affected by bicarbonate, which simply allowed the SITS-sensitive bicarbonate-dependent system to operate. IV. CONCLUSION

If acid extrusion by leech neurons must be explained by using only two of the four models shown in Fig. 1, then we conclude that in the absence of bicarbonate pHi is regulated by an amiloride-sensitive Na-H exchange. When bicarbonate is present, as it is in uiuo, there is presumably a dual mechanism of pHi regulation: Na-H exchange and a Na-dependent HCO3-C1 (presumably) exchange. We cannot distinguish between the snail neuron (Na-H-HC03-CI) and squid axon (NaCO3-CI) models. Thus pHi regulation in leech neurons seems similar to that described in crayfish neurons by Moody (1981). Why there should be a dual mechanism is unclear, unless it is related to the large changes likely in the blood CO;?and HCOi levels of both animals as they move between land and freshwater environments. ACKNOWLEDGMENTS We are grateful to both the Medical Research Council and the Deutche Forschungsgemeinschaft for money and to Ernst Friedrick of Dusseldorf for excellent technical help. REFERENCES Becker, B. F., and Duhm, J. (1978). J . Physiol. (London) 282, 149-168. Boron, W. F. (1985). J. Gen. Physiol. 85, 325-345. Boron, W. F., and De Weer, P. (1976a). J . Gen. Physiol. 61, 91-1 12. Boron, W. F., and De Weer, P. (1976b). Nature (London) 259, 240-241. Boron, W. F., and Russell, J. M. (1983). J. Gen. Physiol. 81, 373-399. Boron, W. F., McCormick, W. C., and Roos, A. (1981). Am. J . Physiol. 240, C80-89. Deitmer, J. W., and Schlue, W. R. (1981). J . Exp. Biol. 91, 87-101. Johnson, J. D., Epel, D., and Paul, M. (1976). Nature (London) 262, 661-664. Moody, W. J. (1981). J . Physiol. (London) 316, 293-308.


13

Muller, K . J., Nicholls, J. G., and Stent, G . (eds.) (1981). “Neurobiology of the Leech.” Cold Spring Harbor Lab., Cold Spring Harbor, New York. Roos, A., and Boron, W. F. (1981). Physiol. Rev. 61, 296-434. Russell, J. M . , and Boron, W . F. (1976). Nature (London) 264, 73-74. Schlue, W. R.,and Deitmer, J. W . (1980). J . Exp. B i d . 87, 23-43. Schlue, W. R.,and Deitmer, J . W. (1984). J . Neurophysiol. 51, 689-704. Schlue, W. R.,and Thomas, R. C. (1985). J . Physiol. 364, 327-338. Thomas, R. C. (1976a). J . Physiol. (LondonJ 255, 715735. Thomas, R. C. (1976b). J . Physiol. (London) 263, 212-213P. Thomas, R. C. (1977). J . Physiol. (London) 273, 317-338. Thomas, R. C. (1984). J . Physiol. (London) 354, 3-22P.


CURRENT TOPICS IN MEMBRANES A N D TRANSPORT, VOLUME 26

Chapter 2

Approaches Intrace1Iular WALTER F . BORON Department of Physiology Yale University School of Medicine New Haven, Connecticut

I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. An Optical Absorbance Technique for Measuring Intracellular pH

A. Optical Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Calibrating the Intracellular Dye. . . . . . E. Comparison of Microelectrode and Dy s ......................

A. Rabbit Proximal Straight Tubule. . ........... ........... B. Rabbit Cortical Collecting Tubule. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Single LLC-PK, Cells in Culture . ........... ............ IV. Conclusions ...... ............................................... References ....... ..............................................

1.

15 16 16 18 18 19

21 22 22 24 28 31

32

INTRODUCTION

There are two major reasons why it is important to study the regulation of intracellular pH (pH,) in epithelial cells. In the first place, because virtually all intracellular processes are expected to be pH sensitive, pH, regulation is likely to be of extreme importance for the normal functioning of the epithelial cell. In addition, controlled changes in pH, could play a role in modulating transepithelial solute transport, modifying biochemical or endocrinological function, or regulating cell growth and differentiation. The second major reason for studying pH, regulation in epithelial cells is 15 Copyright 0 19x6 by Academic Press, Inc All right5 of reproduction i n any form re\erved

16

WALTER F. BORON

that certain of these cells engage in the transepithelial transport of acidbase equivalents. Because the same ion transport systems involved in transepithelial acid-base transport are likely to be intimately involved in pHi regulation, an examination of transport mechanisms affecting pHi is likely to shed light on transepithelial transport processes that are of vital importance to the organism. In this chapter, I will summarize some of the recent work in which we and our collaborators have used an optical absorbance technique to study acid-base transport in mammalian renal cells. Our approach has been to introduce a relatively impermeant pH-sensitive dye into the cells by exposing them to a permeant ester derivative of the dye. Intracellular esterases then convert the colorless precursor to the dye, which is relatively trapped inside the cell. We measure the absorbance spectrum of intracellular dye by illuminating the cells with a small spot of light, focusing the transmitted light on a diffraction grating, and finally projecting the resulting intensity spectrum on a linear array of 1024 photodiodes. After computing the absorbance spectrum, we calculate pHi by comparing the shape of this spectrum to standard intracellular dye absorbance spectra, obtained under conditions of known pHi . As employed in our laboratory, this technique provides reasonably accurate pHi values (i.e., within 0.10) with excellent temporal resolution (-1 sec), pH resolution (-O.Ol), and spatial resolution (-10 pm). In this chapter, I discuss our use of this optical method in measuring pHi in two isolated perfused segments of the rabbit nephron, the proximal straight tubule (PST), and the cortical collecting tubule (CCT), as well as single cultured cells of the pig kidney line LLC-PKI . 11.

AN OPTICAL ABSORBANCE TECHNIQUE FOR MEASURING INTRACELLULAR pH

A. Optical Apparatus

The methods, which are summarized in this section, are discussed in detail in a recent paper (Chaillet and Boron, 1985). We obtain the absorbance of intracellular dye by illuminating the cells with white light and then measuring the intensity of light transmitted through the cells over a range of wavelengths. At any wavelength, the absorbance A is given by -lOg(Z/Zo), (1) where ZO is the intensity of the incident light (i.e., that striking the cells) and Z is the intensity of the transmitted light. The central component of A

=

17

2. INTRACELLULAR pH REGULATION IN RENAL CELLS

power Supply

Tubule -

Diffraction Grating

---- Photodiode

Array

FIG. I . Optical system. The apparatus is built around a modified Leitz inverted microscope (Diavert). An isolated perfused tubule or a cultured cell is located on the lower coverslip, which forms the floor of the chamber. See the text for details. (From Chaillet and Boron, 1985. Reproduced by permission of the Rockefeller University Press.)

our optical system (see Fig. I) is a modified inverted microscope, the stage of which supports a chamber that holds an isolated tubule or cultured cells. The bottom and top of the chamber are formed by glass coverslips, and the space between these coverslips (-I mm) is filled with a Ringer solution. The light source is a 100-W quartz tungsten-halogen lamp placed in a light- and airtight, water-cooled housing. After passing through lenses and filters, some of the light is diverted by a beam-splitting cube to a photodiode, which is part of a feedback circuit that keeps the incident light intensity very stable. The remainder of the light is directed at two shutter-controlled pinholes at the level of the field diaphragm. Light passing through the pinholes is then focused on the plane of the biological preparation by a 32x objective that acts as a condenser. The tubule or cultured cells can be arranged so that light from one pinhole strikes the cells, whereas the light from the other passes through the chamber without ever striking a cell. Thus, the light intensity measured when only the former pinhole is open is I , whereas the intensity measured with only the latter open is l o .

18

WALTER F. BORON

Regardless of which pinhole is open, light passing through the chamber’s lower coverslip is collected by a lox objective and eventually focused on a diffraction grating. The resulting spectrum, in the wavelength range of 350 to 850 nm, is focused on a 1024-element linear array of photodiodes. Thus, diodes at one end of the array measure the intensity of light at the blue end of the spectrum, while those at the other, at the red end. The effective wavelength resolution is about 2 nm. The entire array can be scanned in -16 msec, though we generally record spectra once every 1 to 2 sec. The analog signal from the photodiodes is converted to a digital number with 14-bit precision, and the entire intensity spectrum is stored on a hard disk. The absorbance spectrum is calculated by using Eq. (1) from the Z and ZO spectra that are retrieved from the hard disk. B. Obtaining Absorbance Spectra of intracellular Dye

At any wavelength, the total measured absorbance is the sum of the absorbance of the cells per se and of the intracellular dye. We obtain an estimate of the absorbance due to intracellular dye alone in the following manner. At the outset of the experiment, before the cells are loaded with dye, an absorbance spectrum is obtained that represents the spectrum of the cells alone. This “cell absorbance” is later subtracted from the “total absorbance” of dyed cells to yield a rough estimate of the dye absorbance. The reason this estimate is rough is that the absorbance of the cells may change during the course of the experiment owing to changes in the composition of the Ringer or simply the passage of time. Thus, we apply a final correction in which we exploit the fact that dye absorbance is undetectable at wavelengths above 550 nm, whereas the absorbance spectra are reliable beyond wavelengths of 750 nm. Although the rough estimate of dye absorbance should be zero at wavelengths above 550, it is usually slightly positive or negative. Our approach (see Chaillet and Boron, 1985) has been to fit the rough estimate of dye absorbance between 600 and 750 nm to a straight line and then subtract this line from the entire estimated spectrum. We have verified that this correction procedure is valid when applied to undyed tubules, and we believe that it produces an excellent estimate of the true absorbance spectrum of intracellular dye. C. Introducing a pH-Sensitive Dye into Cells

The dye we have used in our experiments is 4’,5’-dimethyl-5(and 6)carboxyfluorescein (Me2CF), to which the cell is relatively impermeable. Me2CFcan be obtained as its colorless diacetate ester (Me2CFAc2),which

19


coon DIM €THY L CARBOXYFLUORESCEIN D IACETATE

coon DIMETHYL CARBOXYFLUORESCEIN (MeZCF)

(MeZCFAcz)

FIG.2. Hydrolysis of the colorless precursor 4’.5’-dimethyl-5(and6)-carboxytluorescein diacetate (Me2CF) to the pH-sensitive dye 4’,5’-dimethyl-5(and 6)-carboxyfluorescein (Me2CF). (From Chaillet and Boron, 1985. Reproduced by permission of the Rockefeller University Press.)

is relatively permeant, especially at low values of extracellular pH (pH,). As schematized in Fig. 2, Me$FAc2 enters the cell, where it hydrolyzed by native esterases, yielding MeZCF,which leaks out of the cell relatively slowly. This same approach was used by Thomas cr al. (1979) for introducing carboxyfluorescein into Ehrlich ascites cells. D. Calibrating the lntracellular Dye

We found that the spectral properties of intracellular Me2CFdiffer from those of the extracellular dye, possibly due to binding or metabolism inside the cell (Chaillet and Boron, 1985). In particular, the wavelength of maximal absorbance of the intracellular dye is red shifted (i.e., increased in wavelength) by -5 nm, its absorbance peak at comparable pH values is slightly broader, and its pKI, at comparable dye concentrations is increased by -0.3 pH units. For these reasons, one cannot calculate pH, values by comparing an intracellular dye spectrum to an extracellular “standard.” The ideal approach would be to obtain intracellular dye absorbance spectra after setting pHi to several predetermined values. AIthough there is no way for unambiguously fixing pH, at some predetermined value, the method of Thomas er al. (1979) appears to be reasonably valid. These authors used the K-H exchanger nigericin to approximately equalize the transmembrane K+and H+ gradient. If [K+], = [K+],, then it follows that [H+],= [H+],. Thus, in principle at least, pH, can be set to different values merely by manipulating pH,. The results of such a nigericin calibration of Me2CF in salamander proximal tubules is shown in Fig. 3A. It can be seen that the peak absorbance falls as pH, falls. The ratio of

A

-

u"z

- 8.10 - 7.81

2.0

U

m

a

zm U

1.0

0.0 ' 400

600

500

700

WAVELENGTH (nm)

c

1

Lo

.. -

2.0

-

'.O

I

-

L I

I

I

6.0

7.0

A (Peak) A (470) (no0)

1.0

J

I

I

8.0

9.0

PH

FIG. 3. Intracellular calibration of Me2CF. (A) Absorbance spectra of dye contained within the cells of an isolated perfused salamander proximal tubule. As described in detail in the text, the nigericin method was used to vary the apparent pHi between 5.74 and 8.10. Spectra, corrected for the absorbance of tubule, were scaled to have an absorbance of unity at 470 nm,the in uitro isosbestic wavelength. (B) I n uitro and in uiuo calibration curves for Me2CF.The ratio of the peak absorbance (505 nm for the in uitro dye, left ordinate; 509-51 1 nm for the in uiuo dye, right ordinate) to the absorbance at the in uitro isosbestic wavelength (470 nm) is plotted as a function of pH. The in uitro data were obtained with Me2CF contained in a cuvette at a concentration of 10 pM and have a fitted pK value of 6.90. Similar in virro data (not shown) obtained at a dye concentration of 3 mM had a fitted pK of 7.05. The in uiuo data were pooled from three separate tubules, having peak absorbances at wavelengths of 51 1, 510, and 509 nm. The intracellular dye concentrations were 2-4 mM. (From Chaillet and Boron, 1985. Reproduced by permission of the Rockefeller University

21


the absorbance at the peak (-510 nm) to that at 470 nm as a function of pHi (Fig. 3B) follows a standard pH titration curve. It is evident from Fig. 3B that, compared to an in v i m calibration curve, the intracellular curve is appreciably shifted toward alkaline pH values. There are two potential problems with this nigericin calibration method. First, one must accurately know the initial [K+]i.Second, even if [K+]i is known at the beginning of an experiment, subsequent changes of pH, must necessarily produce changes in [K+]i since nigericin exchanges K+ for H+. Nevertheless, comparing pHi values obtained with pH-sensitive microelectrodes to those calculated employing the nigericin calibration method, we have shown that dye-derived pHi values are accurate to within -0.1. This degree of accuracy is probably sufficient for most biological studies. E. Comparison of Microelectrode and Dye for Measuring Rapid pHI Changes

To determine whether it is possible to compute rapid pHi transients from MezCF absorbance spectra, we simultaneously measured the pHi of salamander proximal tubules with pH-sensitive microelectrodes and the Me2CF absorbance technique, Figure 4 illustrates the result of such an experiment in which the tubule was briefly exposed from both the luminal and basolateral surfaces to Ringer containing 20 mil4 total NHd (i.e., NH: LUMEN and BATH : *O

20 NH: +------I

r I

/

FIG.4. Comparison of pHi values simultaneously measured with MelCF (labeled dye) and a liquid-membrane pH-sensitive microelectrode. The tubule was simultaneously exposed to Ringer in which 20 mM Na+ was replaced mole for mole with NH: . The solutions were buffered to pH 7.5 with 13 mM HEPES at 22°C and were nominally HCO, free. The pH of the electrode tracing was raised by 0.10. (From Chaillet and Boron, 1985. Reproduced by permission of the Rockefeller University Press.)

22

WALTER F. BORON

and NH3) in the nominal absence of HCO? . The application of the NH: causes a rapid pHi increase as extracellular NH3 enters the cell and combines with H+ to form NH: . This is followed by a slower pHi decrease as NH: enters the cell and partially dissociates into NH3 and H+. Removal of the extracellular NH:-NH3 elicits a rapid pHi decrease as all intracellular NH: dissociates into NH3 (which exits the cell) and H+ (which remains behind). This pHi decline represents an intracellular acid load. Finally, the cell’s pHi-regulating mechanism (primarily Na-H exchange in this experiment) causes a slower recovery of pHi to the control level. As can be seen, the changes of pHi produced by the application and withdrawal of NH: were reported almost identically by the dye and microelectrode techniques (Fig. 4). Excellent agreement between the two methods was also obtained when the pHi perturbation was produced by lowering basolateral [HCO?] at constant pCOz. We conclude that pH, values calculated from MezCF absorbance spectra are sufficiently accurate for studying pHi regulation, both under steady-state conditions and during rapid pHi transients. 111.

INTRACELLULAR pH REGULATION IN MAMMALIAN RENAL CELLS

A. Rabblt Proximal Straight Tubule

When subjected to an NH$ pulse in nominally HCOT-free Ringer, the rabbit proximal straight tubule (PST) undergoes the same series of pHi changes as did the salamander PT of Fig. 4 (Chaillet and Boron, 1984). The pHi recovery from the NH:-induced acid load indicates that these rabbit PST cells have at least one HCOT-independent pHi-regulating mechanism. Evidence from membrane vesicle experiments indicates that there is a Na-H exchanger at the luminal membrane of rabbit proximal tubules (see Aronson, 1985). We found that when Na+ is removed from both the lumen and the bath (i.e., basolateral surface), the pHi of the PST falls by -0.6 (Nakhoul and Boron, 1985b). The rate of pHi decline is about four times faster when the Na+ is removed from the lumen only as compared to the bath only. Conversely, when Naf is reintroduced to the lumen only, the pHi recovery is about five times faster than when the Na+ is returned to the bath only. Although these observations are consistent with a luminal Na-H exchanger, we were somewhat concerned that, in the presence of 29 mM luminal Na+, 1 mM amiloride only inhibited the pHi recovery by about 25%.


23

Further experiments indicated that luminal Na-H exchange is not the only mechanism by which pHi can recover from an acid load. We found that when we remove luminal and basolateral Na' and maintain the Nafree state, pHi slowly recovers over a 5- to 10-min period, eventually rising 0.1-0.2 beyond the control value (Nakhoul and Boron, 1985b). This pHi overshoot is an intriguing phenomenon. Under Na-free conditions, acid extrusion via Na-H exchange should be blocked. If there were no comparable decrease in the steady-state rate of intracellular acid loading under these conditions, then one would predict that the steady-state pHi would have to be lower than normal. The overshoot implies either a disproportionate decrease in acid loading or a disproportionate increase in acid extrusion. Thus, it is possible that the mechanism responsible for the pHi recovery in the absence of Na' was relatively inactive under normal conditions but somehow was activated by the removal of Na+ or the accompanying fall of pHi. The alkalinization could be due to a change in cell metabolism. Alternatively, the Na-independent pHi recovery could be caused by the activation of a transporter at the luminal andlor basolatera1 cell membranes. Insertion of an H + pump into the luminal membrane has been demonstrated in turtle urinary bladders (Gluck et al., 1982) and is thought to be signaled by a decrease in pHi. Indeed, ATP-dependent H+ transport has been identified in membrane vesicles derived primarily from luminal membranes of rat proximal tubules (Kinne-Saffran et ul., 1982). Another unexpected observation was that a luminal Na-acetate cotransport system seems to make even a greater contribution to pHi recovery than does the Na-H exchanger, at least under the conditions of our Naremoval-Na-readdition protocol. When acetate-acetic acid, initially at a total concentration of 10 mM, is removed from both lumen and bath, pHi initially alkalinizes by 0.1 to 0.2, but then slowly declines to a level -0.4 less than the control value. Readdition of external acetate-acetic acid, on the other hand, causes a rapid transient acidification, followed by a slower pHi recovery to the control value. The initial, rapid alkalinization that follows removal of external Ac- is almost certainly caused by the efflux of acetic acid (HAc). The slower pHi decrease that follows cannot be due to acetate (Ac-) efflux per se inasmuch as almost all Ac- should have combined with H + to produce HAc, which exited from the cell during the first several seconds after external Ac- removal. Thus, the pHi decline probably reflects the reduced activity of an acid-extruding mechanism that is somehow Ac- dependent. Thus, if the cells' acid-loading rate remained approximately constant, the reduction of total acid extrusion would be expected to lower the steady-state pH;. The initial, rapid pH, decrease that follows the return of external Ac--HAc is due to the influx of HAc.

24

WALTER F. BORON

The slower pHi recovery, on the other hand, is probably due to the activation of an acid-extruding mechanism that is Ac- dependent. We had previously found that Na+ removal causes a sustained fall of pHi (see the earlier discussion). Under Na+-free conditions, removal of external Ac--HAc causes a rapid pHi increase that is not followed by a pHi decline, and returning external Ac--HAc caused a rapid pHi decrease that was not followed by a pHi recovery. Thus, the slower phases of pHi change that normally follow the removal-readdition of external Ac--HAc are Na+ dependent. Furthermore, in experiments conducted in the presence of Na+, we found that removal of Ac--HAc from only the lumen causes the usual biphasic pHi changes, whereas removal of Ac-HAc from only the bath causes a sustained intracellular alkalinization that is not followed by a pHi decline. Thus, the Na-dependent, acetate-dependent acid-extrusion mechanism most likely is located at the luminal membrane. Further support for the hypothesis of a luminal Na+- and Ac-dependent acid extruder comes from experiments in which tubules were subjected to bilateral Na+ removal. We found that the pHi recovery that follows the readdition of 145 mM Na+ to the lumen is inhibited by about two-thirds by the bilateral removal of Ac--HAc. Furthermore, the pHi recovery that follows that readdition of 29 mM luminal Na+, which is only -25% inhibited by 1 mM amiloride in the presence of acetate, is -60% inhibited by the amiloride in the absence of acetate. The Na+- and Ac-dependent acid-extruding mechanism could be a Na-Ac cotransport system acting in conjunction with nonionic diffusion of HAc. After entering at the luminal membrane, Ac- would combine with H+ to yield HAc, which could then diffuse from the cell across the luminal and/or basolatera1 membranes. The entry of Ac- per se would have little effect on pHi because of the large difference between pHi and the pK of Ac-; the intracellular alkalinization would result from the exit of HAc. The simplest interpretation of these data is that there are at least three mechanisms by which pHi can recover from an acid load in the nominal absence of HCO3 : (1) amiloride-sensitive Na-H exchange at the luminal membrane, (2) Na-Ac cotransport at the luminal membrane, and (3) a Naindependent mechanism. 8. Rabblt Cortical Collecting Tubule

The cortical collecting tubule (CCT) of the rabbit is composed of at least two distinct cell types (Kaissling and Kriz, 1979), the principal cells (PC), which account for about two-thirds of the total cells, and the intercalated cells (IC). The former are believed to play a crucial role in Na+ and K+ transport, and the latter in transepithelial acid-base transport. The iso-

25


lated, perfused CCT is known to engage in the net secretion of acid (i.e., from bath to lumen) when taken from animals fed a normal or acidic diet (McKinney and Burg, 1977, 1978a; Koeppen and Helman, 1982) but to secrete alkali when taken from animals fed an alkaline diet (McKinney and Burg, 1977, 1978b; Lombard el al., 1983). When a CCT is illuminated with a beam of light 10 pm in diameter, using the optical apparatus described earlier, two to six cells will be in the light path. Thus, the measured pH, will be a complicated average of the pHi values of all of these cells. Although in some cases only PCs would be in the light path, in most instances, both PCs and ICs would contribute to the measured pHi. The following experiments, described in detail elsewhere (Chaillet ef al., 1985), were undertaken to obtain a general description of pH, regulation in the cells of the CCT under nominally HCOy-free conditions. It is understood that this experimental approach does not allow one to distinguish between PCs and ICs. Figure 5 illustrates an experiment in which a CCT, incubated in pH 7.4 HEPES Ringer at 38"C, was twice acid-loaded by prepulsing with Ringer 20 NH:

LUMEN :

BATH :

''OF

u 20 NH; U

20 NH: I

0 No'

I

20 NH:

0 Na+

b

3

h 2 min H

FIG.5 . Recovery of the pH, of a rabbit cortical collecting tubule from an NH;-induced acid load in the presence and then in the absence of extracellular Na'. See the text for details. During the periods indicated by 0 Na+, Na' in the lumen or bath (i.e., basolateral solution) was replaced mole for mole with N-methyl-D-glucammonium. The solutions were 1985. Reproduced by buffered to pH 7.4 with 32 mM HEPES at 38°C. (From Chaillet et d., permission of the Rockefeller University Press.)

26

WALTER F. BORON

containing 20 mM NH:-NH3. As noted earlier, the initial alkalinization induced by the application of extracellular NH:-NH3 (segments ah and e f ) is due to the influx of NH3, whereas the slower acidification (hc and f g ) is probably due primarily to the influx of NH; . The removal of extracellular NH:-NH3 induces a rapid pHi fall (cd and g h ) as intracellular NH: dissociates into NH3 (which exits the cell) and H + (which remains inside). By the time most of the NH: has exited (points d and h ) , the cells are loaded with acid. This NH: prepulse is merely a trick for acid loading the cells and is formally equivalent to injecting them with acid. In the presence of extracellular Na+, pHi recovers rapidly and completely ( d e ) . However, when Na+ is removed from both the lumen and the bath, pHi recovers slowly and incompletely (hi).Thus, there are at least two mechanisms by which the measured pHi can recover from an acute acid load, one of which is rapid and Na+ dependent and the other slower and Na+ independent. To further examine the nature of the rapid, Na-dependent mechanism, we acid-loaded CCT cells in the absence of Na+ and monitored pHi while returning Na+ first to the lumen only and then to the bath only. As shown in Fig. 6, the readdition of Na+ to the lumen (bc) elicits a small increase in LUMEN:

1 Na+

BATH :

20 NH:

20 NH;

'

0

145

I

0

'

1

0 I

145

1

u

:I 7.95

PH i

INa+

645 I min

H

FIG.6. Dependence of the pHi recovery of a rabbit cortical collecting tubule on luminal versus basolateral Na+. The protocol was similar to that of Fig. 5, except that 145 m M Na' was first returned to the lumen (segment bc) and then to the bath ( d e ) .(From Chaillet et d., 1985. Reproduced by permission of the Rockefeller University Press.)

27


the rate of pH, recovery, whereas the readdition of Na+ to the bath ( d e ) causes a rapid pH, recovery. In a total of five similar experiments, the readdition of Na+ to the bath alone caused a pH, recovery that was -14 times faster than when Na+ was added to the lumen alone. Thus, the rapid, Na-dependent mechanism of pH, recovery is located primarily, if not exclusively, at the basolateral membrane. In other experiments (not shown), we quantitated the basolateral Na+ dependence of the rapid pH, recovery mechanism and found that the dependence of the pH, recovery rate on basolateral "a+] is adequately described by Michaelis-Menten kinetics, with an apparent K , of -27 mM. Because the Na-dependent mechanism functions in the nominal absence of HCOj, we thought that it was most likely to be a Na-H exchanger. To test this hypothesis, we performed the experiment of Fig. 7, in which the CCT cells were acid-loaded in the nominal absence of Na+ and then I5 mM Na+ was added back to the lumen in either the presence or the absence of 1 mM amiloride. The addition of Na' in the presence of the amiloride causes very little change in the rate of pH, recovery ( b c ) , whereas the addition of the same amount of Na+ in the absence of amiloride leads to a rapid pH, recovery ( d e ) . In three similar experiments at an amiloride level of 50 p M , the inhibition was -62%. These data thus indicate that the rabbit CCT has a rather potent Na-H exchanger located at the basolateral membrane. In addition, there appears to be a second, low-capacity mechanism that increases pH, in the total absence of Na+. It is possible that this slow, Na-independent pH, recovery is simply due to a leakage of buffer (i.e., HEPES) across the cell

'La+

20 NH;

LUMEN

2ONH;

BATH 8 00

0

1

I mMAm

'

I

15

1

0

15

'

1

145

7 50

PH, 7 00

6.50

FIG. 7. Effect of basolateral amiloride on the Na-dependent pH, recovery in a rabbit cortical collecting tubule. The protocol was similar to those of Figs. 5 and 6, except that the tubule was exposed to I mM amiloride hetween points h and ('. (From Chaillet 0 1 (//., 19x5. Reproduced by permission of the Rockefeller University Press.)

28

WALTER F. BORON

membrane. Alternatively, it may reflect the activity of a specialized transport mechanism, such as the electrogenic H+ pump believed to be present in the luminal membrane of ICs. As to the physiological roles of these two mechanisms, a definitive statement will be possible only after pHi regulation has been separately studied in PCs and ICs. However, circumstantial evidence suggests that the basolateral Na-H exchanger is present in at least the PCs. If so, it is likely that the major role of the Na-H exchanger is to regulate pHi in these cells and has little role in transepithelial acidbase transport. Indeed, it is known that both acid secretion (Koeppen and Helman, 1982) and alkali secretion (Schuster, 1985; Star et al., 1985) are not significantly affected by the total removal of Naf. If the basolateral Na-H exchanger played a significant role in either of these processes, they would be expected to be noticeably inhibited by Na+ removal. C. Slngle LLC-PK, Cells In Culture

The optical approach employed with renal tubules can also be applied to single cells in culture. The LLC-PKI cell line grows normally in culture, but can also be made to differentiate. Work by others on these cells has demonstrated an amiloride-sensitive 22Na influx (Cantiello et al., 1984; Haggerty et al., 1985) suggestive of an Na-H exchanger. We studied pHi regulation in a clone (clone 4) of LLC-PKI cells in order to verify the existence of a Na-H exchanger, as well as to determine whether HCO: transport plays a role in pHi ion these cells (Chaillet et al., 1986). Experiments were performed on both rapidly growing and quiescent cells, with the results being qualitatively the same in both. The first set of experiments (not shown) was designed to detect Na-H activity. In order to minimize the contribution of possible HCO? transport mechanisms on pHi , we conducted these experiments in the nominal absence of HCOy and employed a 32 mM HEPES buffer. As expected, when cells were acid-loaded by prepulsing with NH; (as in Figs. 4-7), pHi rapidly recovered, following an exponential time course. The recovery was very sensitive to reductions of “a+],. In six cells in which the pHi recovery rate was measured in 0 and 145 mM Na+ at the same pHi, the recovery was inhibited 88% by total removal of Na+. Reducing “a+], to only 29 mM Naf produced a 74% inhibition (n = 3). Amiloride (1 mM) completely blocked the pHi recovery in 29 mM Na+. These observations indicate that LLC-PK, cells can regulate their pHi by means of Na-H exchange. An interesting result that came out of the preceding experiments was a calculation of the intracellular buffering power of LLC-PKI cells. This

29


can be determined by applying a known acid load to the cell and observing the resulting pHi under conditions in which transport processes cannot modify pHi. In the preceding experiments, the acid load was applied by removing NH: from the external solution, and Na-H exchange was blocked by conducting the experiments in the absence of Na+. HCOy transport should have been minimized by the nominal absence of HCOy . The details of the calculation have been discussed previously (Boron, 1977). We found that the average non-COz buffering power of LLC-PK, was 6.2 mM (n = 12), to our knowledge the lowest value ever reported (see Roos and Boron, 1981). The second series of experiments was designed to detect possible HCO? transport mechanisms. Figure 8 illustrates an experiment in which we twice replaced all extracellular C1- with glucuronate. The first instance of C1- removal (segment ab) caused a rapid and reversible pHi increase of -0.3, as would be expected of a CI-HC03 exchanger. When C1- was subsequently removed in the presence of 50 p M DIDS ( c d ) , an inhibitor of HCO? transport in other systems, both the rate and extent of the pHi increase were reduced. In other experiments (not shown), we found that the initial rate of the pHi increase induced by C1- removal was reduced 91% ( n = 3) by conducting the maneuver in the nominal absence

0

c1-

0 c1-

DIDS

2 min

FIG.8. Effect of CI- removal on the pH, of a single, rapidly growing LLC-PK, cell incubated in a HCOT-containing Ringer's solution. See the text for details. The solutions were buffered to pH 7.4 with 25 mM HCO;/5% CO? at 37°C. (From Chaillet e? d.,1986. Reproduced by permission of the National Academy of Sciences.)

30

WALTER F. BORON

of HCO:. On the other hand, conducting the maneuver in the absence of Na+ failed to inhibit the pHi increase. The preceding results were consistent with the existence of a simple (i.e., Na-independent) CI-HC03 exchanger. If this CI-HC03 exchange hypothesis is correct, then the exchanger should produce pHi changes in response to alterations in [HCOy],. In the experiment of Fig. 9, [HCOy], was three times reduced from its normal value of 25 mM to 5 mM in solutions equilibrated with 5% C 0 2 (i.e., pH, was reduced from 7.4 to 6.7). As can be seen, the reduction in [HCOy], caused a rapid and reversible pHi decrease of -0.25. If these pHi changes were mediated by a CIHCO3 exchanger, then they should be reduced by removal of C1-. The nominal removal of C1- produced a large pHi increase ( c d ) , as in the experiment of Fig. 8, presumably due to HCOl uptake by a Cl-HCO3 exchanger. After pHi had nearly stabilized, [HCOy], was once again lowered. In the nominal absence of C1-, the rate and magnitude of the pHi changes were greatly reduced. In four experiments, the magnitude of the pHi change was reduced by an average of -79%. Returning C1- to the external solution caused pHi to return toward its initial value (fg), after which a reduction of [HCOy], once again elicited large and reversible pHi changes. In an experiment not shown, we found the pHi changes produced by reducing pH, from 7.4 to 6.7 in the nominal absence of HCOl to

0 c1pH 6.7

7.60

pH 6.7

pH 6.7

d

,,H(

7.40 .

7.20

-

7.00

6.80 2 min

FIG. 9. CI- dependence of the pHi response to lowering extracellular [HCO,], in a single, rapidly growing LLC-PK, cell. See the text for details. (From Chaillet e/ a / . . 1986. Reproduced by permission of the National Academy of Sciences.)


31

be only -16% as large as those in the presence of HCOT. The pH, changes induced by altering [HCO,],, were also reduced -82% by 50 pA4 DIDS (n = 2). The nominal removal of Na', however, failed to inhibit the pH, changes. The most straightforward interpretation of the preceding results is that LLC-PKI cells possess both a Na-H and a Na-independent CI-HC03 exchanger. An interesting observation is that the estimated maximal fluxes of acid-base equivalents, calculated as the product of total intracellular buffering power and the rate of pH, change, were much greater for the putative CI-HC03 exchanger than for the Na-H exchanger. For example, a typical acid extrusion rate for the Na-H exchanger was 0.7 mmol of acid extruded from each liter of cell water per minute. The equivalent fluxes of acid-base equivalents mediated by the putative CIHC03exchanger in the experiments of Figs. 8 and 9 were 5.1 and 6.4 mMl min, respectively. This comparison mmt be interpreted with caution inasmuch as the Na-H and CI-HC03 exchangers were assayed under different conditions and under conditions very different from those prevailing in a normal cell. In particular, one might argue that the Na-H exchange rate could be increased by a factor of two to four by greatly increasing the magnitude of the intracellular acid load or perhaps, as can be done in other cells, by stimulating the cell with growth factors (Moolenaar et ul., 1983) or a change in cell volume (Grinstein et ul., 1983). Nevertheless, our observations would indicate that the CI-HC03 exchanger is potentially at least as potent as the Na-H exchanger in terms of transporting acid-base equivalents across the cell membrane. IV. CONCLUSIONS

We have developed an optical absorbance technique that permits the measurement of rapid pH, changes in isolated perfused renal tubules and in single cultured cells. In isolated perfused salamander proximal tubules, the dye approach yields pH, values that agree to within 0.1 with those obtained with pH-sensitive microelectrodes, both in the steady state and during pHi transients. Applied to mammalian cells, the optical technique has permitted us to identify several acid-base transport systems: (1) a luminal Na-H exchanger, a Luminal Na-acetate cotransporter, and a possible Na-independent acid-extrusion mechanism in the rabbit proximal straight tubule; (2) a basolateral Na-H exchanger in one or both cell types of the rabbit cortical collecting tubule; and (3) independent Na-H and ClHC03 exchangers in cultured LLC-PK, cells. Inasmuch as this optical technique is relatively easy to use, it should be of value in further studies

32

WALTER F. BORON

of the transport systems already identified, as well as in characterizations of acid-base transport systems in other mammalian epithelia. REFERENCES Aronson, P. S . (1985). Kinetic properties of the plasma membrane Na+-H+ exchanger. Annu. Rev. Physiol. 47, 545-560. Boron, W. F. (1977). Intracellular pH transients in giant barnacle muscle fibers. Am. J. Physiol. 233, C61-C73. Cantiello, H., Thompson, I., and Rabito, C. (1984). Expression and modulation of different Na+ transport systems in a cell line (LLC-PK,) with characteristics of proximal tubular cells. Fed. Proc., Fed. Am. SOC.Exp. Biol. 43, 448 (Abstr.). Chaillet, J. R., and Boron, W. F. (1984). Intracellular pH regulation in rabbit proximal tubules studied with a pH-sensitive dye. Kidney Inr. 25, 273 (Abstr.). Chaillet, J. R., and Boron, W. F. (1985). Intracellular calibration of a pH-sensitive dye in isolated perfused salamander proximal tubules. J. Gen. Physiol. 86, 765-794. Chaillet, J. R . , Amsler, K., and Boron, W. F. (1986). Optical measurement of intracellular pH in single LLC-PK, cells: demonstration of CI-HC03 exchange. Proc. Natl. Acad. Sci. U.S.A. 83, 522-526. Chaillet, J . R., Lopes, A. G., and Boron, W. F. (1985). Basolateral Na-H exchange in the rabbit cortical collecting tubule. J . Gen. Physiol. 86, 795-812. 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. Nail. Acad. Sci. U.S.A. 79, 4327-4331. 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. Haggerty, J. G., Cragoe, E. J., Jr., Slayman, C. W., and Adelberg, E. A. (1985). Na+/H+ exchanger activity in the pig kidney epithelial cell line, LLC-PK, : Inhibition by amiloride and its derivatives. Uiochem. Uiophys. Res. Commun. 127, 759-767. Kaissling, B., and Kriz, W. (1979). Structural analysis of the rabbit kidney. Adv. Anal. Embryol. Cell Biol. 56, 1-123. Kinne-Saran, E., Beauwenw, R., and Kinne, R. (1982). An ATP-driven proton pump in brush-border membranes from rat renal cortex. J . Membr. Biol. 64, 67-96. Koeppen, B. M., and Helman, S. I. (1982). Acidification of luminal fluid by the rabbit cortica1 collecting tubule perfused in vitro. Am. J. Physiol. 242, F521-FS31. Lombard, W. E., Kokko, J. P., and Jacobson, H. R. (1983). Bicarbonate transport in cortical and outer medullary collecting tubules. Am. J . Physiol. 244, F289-F296. McKinney, T. D., and Burg, M. B. (1977). Bicarbonate transport by rabbit cortical collecting tubules: Effect of acid and alkali loads in vivo on transport in vitro. J . Clin. Invest. 60,766-768. McKinney, T. D., and Burg, M. B. (1978a). Bicarbonate absorption by rabbit cortical collecting tubules in vitro. Am. J . Physiol. 234, F141-Fl45. McKinney, T. D., and Burg, M. B. (1978b). Bicarbonate secretion by rabbit cortical collecting tubules in vitro. J . Clin. Invest. 61, 1421-1427. Moolenaar, W. H., Tsien, R. Y.,vander 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. Nakhoul, N., and Boron, W. F. (1985a). lntracellular pH regulation in rabbit proximal straight tubules: Basolateral HCOJ transport. Kidney Inr. 27, 286 (Abstr.).


33

Nakhoul, N., and Boron, W. F. (1985b). Intracellular-pH regulation in rabbit proximal straight tubules: Dependence on external sodium. Fed. Proc., Fed. A m . Soc. Exp. B i d . 44, 1898 (Abstr.). Roos, A., and Boron, W. F. (1981). Intracellular pH. Physiol. Rev.61, 296-434. Schuster, V. L. (1985). Cyclic-AMP-stimulated bicarbonate secretion (BiS) in rabbit cortical collecting tubules (CCT). Kidney I n t . 27, 288 (Abstr.). Star, R., Knepper, M., and Burg, B. (1985). Bicarbonate secretion by rabbit cortical collecting duct: Role of chloride/bicarbonate exchange. Kidney Inr. 27, 289 (Abstr.). Thomas, J. A., Buchsbaum, R. N., Zimniak, A., and Racker, E. (1979). Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 81, 2210-2218.


CURRENT TOPICS IN MEMHRANES A N D TRANSPORT. VOLLJMI; 26

Chapter 3

As ects of pHi Re ulation in rog Skeletal duscle

F

ROBERT W . PUTNAM' A N D ALBERT ROOS Department of Cell Biology and Physiologv und Deportment of Anesthesiology Washington University School of Medicine St. Louis, Missouri

II.. I11. I. 111. 111.

IV. IV.

VV.. VI. VI.

VII. VII.

. . . . . . . . . . . . . . . . . . 36 36 31 37 ...................... 38 . . . . . . . . . . . . . . . . 38 38 ...................... D. Depolarization ..................................... 39 ................... E. Insulin .......................... ............. 41 . . . . . . . . . . 42 ......................... 42 42 A. NHICl Prepulse.. . . . . . . . . . . . . . . . . . . . . . . . . . 42 B. C 0 2 Acidification . . . . . . . . . . , . . . . . . . . . 43 C. DMO Acidification . . . . . . . . . 44 D. Comparison of pH, Recovery ...................... . . . . . . . . . . .. . . . . . . . . . _ . . . . . . . . 45 Nature of pHi-Regulating Systems .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 45 A. Amiloride-Sensitive Transport.. . . . 45 46 B. SITS-Sensitive Transport . . . . . . ........................ ................................... ...... 41 Properties of Na-H Exchange . . . . . . . . . . . . . . . . . 41 48 B. pHi Dependence . . . . . . . . . C. "a], Dependenc 48 48 . . . . . . . . . . . . . . 48 48 A. Growth-Promoting Factors . . . . . . . . . . . . . . . . . . 49 . . . . . . . . . . . . . . . 50 D. Hypertonic Solutions ......................... 50 Comparison with Ot 51

Steady-State pH, . . . . . . . . .. . . . . . . . . . . . . .

I

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

VIII. VIII. IX. IX. Summary. . . . . . ........ . . . . . . . . . .. . . . . . . . . .

I Present address: Department of Physiology and Biophysics. Wright State University School of Medicine, Dayton, Ohio 45401.

35 Copyright C I986 by Academic Press. Inc. All rights of reproduction in any form reserved.

36

ROBERT W. PUTNAM AND ALBERT ROOS

1.

INTRODUCTION

Intracellular pH (pHJ in nearly all cells is higher than it would be if H ions were in electrochemical equilibrium. At steady state, this high pHi is maintained in the face of acidifying influences such as H influx, metabolic acid production, and HCO3 efflux, suggesting the presence of active transport systems which remove acid equivalents from the cell. The properties of these transport systems can best be studied by acid loading, to which most cells respond by returning pHi toward its initial steady-state value. We will refer to this response as pHi regulation. Regulation of pHi in response to an acid load has been studied in a variety of invertebrate cells including barnacle muscle, snail neuron, squid giant axon, crayfish neuron, and crab muscle (for a review see Roos and Boron, 1981). The pHi regulation has also been studied in some vertebrate cells. Two types of vertebrate skeletal muscle have been examined: mouse soleus muscle (Aickin and Thomas, 1977a,b) and frog skeletal muscle (Bolton and Vaughan-Jones, 1977;Abercrombie et al., 1983). In this chapter, we will describe the results of our studies on pHi regulation in frog skeletal muscle. We will first describe some changes which affect the steady state pHi. Next, we will examine the properties of the membrane transport systems responsible for pHi regulation and some factors that can modify this regulation. Finally, a comparison will be made between pHi regulation in frog muscle with that in other cells. II. METHODS

Details of our experimental procedures have been described previously (Abercrombie et al., 1983;Putnam, 1985).Briefly, a small bundle of about 30 fibers was dissected from the dorsal head of the semitendinosus muscle of Rana pipiens. The bundle was mounted in a small chamber (1.1 ml) and superfused with various solutions at 22°C. Membrane potential (V,) and pHi were recorded with conventional 3 M KC1-filled glass microelectrodes (10-20 Mil) and pH-sensitive recessed-tip glass microelectrodes (Thomas, 1974,1978),respectively, and displayed on separate channels of a strip chart recorder. In some experiments, pH-sensitive electrodes containing a neutral ion carrier were used (Ammann et al., 1981). These electrodes were constructed and calibrated as described by Reuss and Costantin (1984). The bicarbonate-free Ringer solution contained (in mM): I18 Na, 2.5 K, 118.5CI, 2 Mg, 4 Ca, 0. I EDTA. It was buffered with 10 mM N-2hydroxyethylpiperazine-N’-2-ethanesulfonicacid (HEPES) to pH, 7.35

3. pHi REGULATION IN FROG SKELETAL MUSCLE

37

and equilibrated with 100% 0 2 .The bicarbonate Ringer solution contained (in mM): 114 Na, 2.5 K,104.5 CI, 24 HCO3, 2 Mg, 4 Ca, 0.1 EDTA. It was equilibrated with 5% C02/95% 0 2 ; pH, was 7.35. For the purpose of depolarizing the fibers, 50 mM NaCl was replaced with 50 mM KCI. When the K x CI product was kept constant or in CI-free solutions, gluconate salts replaced chloride salts. When Na was reduced, N-methylD-glucammonium (NMDG) was used as substitute. For NH4Cl-containing solutions, NH4CI replaced NaCl on a mole for mole basis. When the divalent cation concentration was increased, these ions were added in the form of CI salts, and NaCl was reduced so that CI concentration remained unchanged. The tonicity of hypertonic solutions was doubled (2T) by adding 250 mM mannitol. We have employed three methods to acidify cells: ( I ) exposure to C 0 2 / HC03-containing solutions; (2) the NH4CI prepulse technique; or (3) exposure to solutions containing the weak acid dimethyl-2,4-oxazolidinedione (DMO, pK; 6.1). The mode of acidification with these techniques has been described previously (Boron and De Weer, 1976; Roos and Boron, 1981). Recovery rates were measured during the first 20 min of pHi recovery upon addition of C02/HC03,addition of DMO, or removal of NH4CI. In most fibers, the recovery was linear during this period. When the recovery was not linear during this period, the rate was estimated by the chord from lowest pHi to pHi 20 min later. Rates of pHi recovery are expressed per hour, ApHilhr. All values are reported as the mean f 1 standard error of the mean (SEM). 111.

STEADY-STATE pH1

Fibers superfused with HEPES-buffered solution (2.5mM K, nominally C 0 2 free) had a pHi of 7.23 k 0.02 (n = 26), comparable to pH,s measured previously in frog muscle (Bolton and Vaughan-Jones, 1977; Abercrombie et al., 1983). The equilibrium potential for H + (pH, 7.35) is -7 mV, far less than the actual V , of -92 2 I mV. Thus, in frog muscle, as in most cells (Roos and Boron, 1981), the H ions are not at equilibrium across the cell membrane; there is a net inward electrochemical gradient for protons. We studied the effect of several factors on steady-state pHi . A. Changes In pH,

Bolton and Vaughan-Jones (1977) showed that pHi fell by only 0.02 when the external pH (HEPES-buffered solution, nominal absence of

38


C 0 2 )was changed from 7.2 to 6.2. In fibers depolarized in 50 mM K to about -20 mV, we found a greater effect of extracellular acidification. When we lowered the pH of the superfusate from 7.35 (HEPES buffered) to 6.4 (PIPES buffered), pHi fell by 0.08. Raising external pH from 7.35 (HEPES buffered) to 8.3 (glycylglycine buffered), produced a more striking effect; pHi increased by 0.16. Intracellular pH changed slowly and attained a new steady state within 30 min. These changes were reversible. The mechanism underlying these small, slow changes in pHi is unexplained, as is the greater effect of extracellular alkalinization versus acidification. B. Na-Free Solutions

In eight fibers exposed to Na-free solution [Na replaced by N-methyl-Dglucammonium (NMDG), nominal absence of COz]for at least 2 hr, pHi was 7.20 -+ 0.04 (Vn, = -89 ? 2 mV) (Abercrombie et a / . , 1983), that is, nearly the same as in the presence of Na, 7.23. As will be shown in Section IV,B, removal of Na from the superfusate almost completely inhibits the acid-extruding mechanisms of frog muscle cells. Since steadystate pHi is determined by the balance between the rate of acid extrusion and that of acid loading (H influx, HC03 efflux, metabolic acid production), the absence of intracellular acidification observed in Na-free solutions indicates that the rate of acid loading is very slow in frog muscle. C. Hypertonic Solutions

Hypertonic solutions result in an increase in steady-state pHi of about 0,17 (Abercrombie and Roos, 1983). Thus, fibers superfused with a solution of twice normal tonicity (2T) made by the addition of mannitol had a steady-state pHi of 7.40 f 0.04, V , = -88 ? 1 mV ( n = 17). A similar alkaline shift in pHi due to hypertonic solutions was seen in rat diaphragm muscle (Adler et al., 1979, but a small acid shift was seen in mammalian cardiac muscle (Ellis and Thomas, 1976). A part of the alkaline shift can be explained by changes in the pK' of intracellular buffers. The cell shrinkage induced by hypertonic solutions increases intracellular ionic strength p, which for a weak monovalent base such as imidazole (the major buffer group of cellular proteins; Curtin and Woledge, 1978) would increase pK' according to pK'

=

pK

+ 0.5fi/(1 + fi)

(Bates, 1973), where pK is the limiting value at zero ionic strength. As-

3. pH, REGULATION IN FROG SKELETAL MUSCLE

39

suming that p is 0.22 for frog muscle under isotonic conditions (Abercrombie and Roos, 1983) and that it doubles in 2T solution, pK' (and thus pHi) should increase by 0.04 in hypertonic solution. This is only about a fourth of the observed change. [In Abercrombie and Roos (1983) the square roots in front of the p's were inadvertently omitted. The increase in pK' on doubling tonicity was erroneously given as 0.06.1 It is not clear what is responsible for the remainder of the increase in steady-state pHi. It is possible that some of the alkalinization in hypertonic solution is the consequence of the fiber's attempt to regulate its volume by increasing Na-H exchange (see Section V1I.D): the exchange of external Na for internal H will increase the internal osmotic pressure, thereby increasing cell volume. A similar alkalinization is seen during cell volume regulation in lymphocytes (Grinstein et al., 1983).

D. Depolarization Steady-state pH, is not significantly affected by depolarization in isotonic solutions. The pHi was 7.26 t 0.02 in fibers depolarized to V , = -22 ? 0.3 mV ( n = 79) in 50 mM K. However, a striking transient acidification can be seen upon depolarization (Abercrombie and Roos, 1983). Since depolarization leads to contracture, these experiments were done in 2T solutions, which largely block the mechanical response (Hodgkin and Horowicz, 1957; Gordon and Godt, 1970) but do not interfere with Ca release (Taylor et al., 1975). Upon depolarization, there was an abrupt acidification of about 0.3 to 0.5, followed by a rapid return toward initial pH, and then by a continued slow upward drift of pH, (Fig. IA). In Na-free solutions, a similar degree of acidification was observed, but the rapid recovery was incomplete, and the upward drift was eliminated (Fig. IB). Tetracaine (2 mM), which blocks Ca release (Almers and Best, 1976; Luttgau and Oetliker, 1968), prevented the transient acidification (Fig. IC). These data strongly suggest that the transient acidification is due to the Ca release induced by depolarization. In agreement with this, raising tonicity threefold, which supposedly blocks Ca release (Gordon and Godt, 1970). blocked the transient acidification. Caffeine-induced Ca release also produced marked acidification (Abercrombie and Roos, 1983). The recovery from this acidification is largely Na independent, but a portion seems to be due to a Na-requiring process (Fig. lB), possibly a Na-H exchange. This is shown by the findings that in the absence of Na, the rate of recovery is slower and is incomplete and that the extracellular acidification in the immediate vicinity of the fibers is less than in the presence of Na (Abercrombie and Roos, 1983).


40 Hypertonic ( 2 1 )

Hypertonic ( 2 7 )

Normtonic ( 1 T 1

20 min u

- -30 - -40 Vm - -50 - -60

pHiIf

No t 2mM Tetrocoino

7.7

No-free

7.2

l:b

FIG. I . Initial course of pH, and V,,,when the muscle was depolarized with 50 mM K. (A) In hypertonic (2T) solution. Note the prompt and transient acidification followed by a slow pHi rise to above control. (B) In hypertonic (2T) Na-free solution. Sodium was omitted both from the 2.5 m M K solution in which the muscle was presoaked for 40 min and from the 50 rnM K solution. Note prompt and transient acidification followed by a maintained undershoot. (C) In isotonic Na Ringer solution containing 2 mM tetracaine, applied 10 min before introduction of the electrodes. The drug depolarized the membrane, an effect that has previously been observed (Almers and Best, 1976). Tetracaine nearly abolished the early acidification. [From Abercrombie and Roos (1983) with permission.]

There are at least two explanations for the link between Ca release and transient acidification upon depolarization. First, each Ca ion released might be exchanged for two H ions (either through membrane transport or on protein buffers). Assuming a total myoplasmic Ca concentration of 1 mM in fibers depolarized in isotonic solutions containing high K (Somlyo et af., 1981), the concentration might well be double this amount in 2T solutions. Given a buffering power of 54 m M (Abercrombie and Roos, 1983), the pHi change due to H-Ca exchange would thus be 4/54 = 0.074, much less than the acidification actually observed (Fig. 1A). A second effect of Ca on pHi may be through activation of glycogenolysis (Ozawa et af., 1967; Cohen et af., 1980; Cohen, 1982), which would increase acid

41

3. pH1 REGULATION IN FROG SKELETAL MUSCLE

production and directly acidify the cell. The rapid Na-independent recovery, then, may correspond to removal of the acidic products by either metabolic conversion or diffusion out of the cell after Ca has been returned to the sarcoplasmic reticulum. At least a part of the extracellular acidification observed close to the fiber (Abercrombie and Roos, 1983) may be due to this diffusion. E. Insulin

Insulin has been shown to alkalinize frog muscle cells (Moore, 1979; Putnam, 1985). We have followed the time course of insulin-induced alkalinization (Fig. 2) and found the following: there is an initial lag of about 20 min after insulin application, followed by alkalinization amounting to 0.05 to 0.10 which requires about 1 hr for its full development, after which a new steady-state pHi is reached (Putnam, 1985). This alkalinization has been proposed as a necessary requirement for the activation of glycolysis by insulin (Fidelman et af.,1982), in the same way that cellular alkalinization has been found to be necessary for the action of growth factors on cells (Boonstra et al., 1983; L'Allemain et al., 1983).

1-100

- 7.6 A

B

4

1

C t

D t

-

-

, -__

7.4 PHI

7.2

IrnU/rnl INSULIN

2 5 K , OOt'ABSA

FIG.2. Effect of insulin on pH,. ( A ) lnsulin added to superfusate, V , = -96 mV, pH, = 7.38. (A to B) 1Prnin lag period before pH, began to rise. ( B to C) 54-min period during which the fiber alkalinized by 0.05 pH units. (C to D) N e w steady state, V , = -97 rnV, pH, = 7.42. [From Putnarn (1985) with permission.]

42


IV.

RESPONSE OF pH1 TO ACID LOADING

The pHi recovery rate in acid-loaded fibers was assessed using either an NH4CIprepulse or CO;! acidification. A few fibers were also acidified with the weak acid DMO. Both normally polarized fibers (V, about -90 mV, 2.5 mM K) and depolarized fibers ( V , about -20 mV, 50 mM K) were studied. A. NH&l Prepulse

We found that both polarized and depolarized fibers recovered at nearly the same rate from the intracellular acidification induced by removal of previously applied NH4Cl. Both removal of Na and amiloride were effective in nearly abolishing recovery (Putnam and Roos, 1983). ‘SITS hardly affected recovery. The NH4CI had to be applied for a longer period and often at higher concentrations in depolarized fibers than in normal fibers in order for the pHi undershoot after NH4C1removal to be comparable under the two conditions. Most likely, the reason is that the “plateau acidification” during NH4Cl application is due to the passive influx of NH: (Boron and De Weer, 1976) and thus is reduced in depolarized fibers. 8. C02 Acldlflcatlon

When normally polarized fibers were acidified to about 6.9 by exposure to 5% C02/24 mM HCO,, pHi recovery was slight, amounting to only 0.03 ApHilhr (Figs. 3A and 4). The slow recovery could be converted to an acidification by 1 mM amiloride or by complete substitution of external Na by NMDG (Fig. 4). In contrast to the small overshoot observed in normally polarized fibers upon removal of COZ,there was a pHi undershoot in the presence of amiloride or in the absence of Na, as expected (Abercrombie et al., 1983). In all cases, acidification with C 0 2 caused a depolarization of about 8 mV. In fibers depolarized in 50 mM K, constant C1, C 0 2 acidification resulted in brisk pHi recovery at a rate of 0.3 ApHilhr (Figs. 3B and 4). The membrane depolarized by 3 mV to -19 mV. When C02 was removed, there was a marked pHi overshoot (Fig. 3B). About half of the recovery in these depolarized fibers could be inhibited by amiloride and about half by SITS. When fibers were exposed for 20 min to SITS and then, after removal of SITS, exposed to amiloride, recovery was reduced by 70%. That recovery is not abolished under these conditions might be due to

43

3. pH, REGULATION IN FROG SKELETAL MUSCLE B

A

--50

-

--60vm

20 min

--70

--80

--90 --lo0

-

5% c

5% coz

___

2.5 K

a

f

17.6

I

__

50K. constant CI

FIG.3. pH, recovery in response to acidification induced by C 0 2 in normally polarized and depolarized fibers. pH, was 7.35 throughout. ( A ) A fiber in 2.5 mM K. pH, did not recover and returned to the initial value when CO: was removed. (B) A fiber depolarked in 50 mM K, constant CI. Recovery rate was 0.30 ApHJhr. Recovery occurred beyond the equilibrium pH,. When C 0 2 was removed, a considerable overshoot of pH, was observed. [From Abercrombie P I ul. (1983) with permission.]

partial reversibility of the SITS inhibition. In the absence of Na, recovery from CO? acidification was nearly abolished (Fig. 4) (Abercrombie r t d., 1983).

C. DMO Acidification

Depolarized fibers, acidified with 30 mM DMO (Na DMO substituted for an equal amount of NaCI) to about 6.8, recovered at a rate of 0.12 ApHi/hr (Abercrombie et al., 1983). The acidification also resulted in a 3 to 4 mV depolarization. Upon removal of DMO, there was a pHi overshoot.

44


. x 1

co2

0.4~

50 K, constant CI

2 5 K

1

1

0.3 RATE OF RECOVERY (ApHi/h)

1

o.2 0.I

-0IL

-

1

FIG.4. Ionic and drug sensitivities of pH, recovery in frog muscle fibers. The height of the bar is the mean; the vertical lines are 2 1 SEM. Fibers in 2.5 or 50 mM K (constant CI) and acidified with C 0 2 . Note that recovery rate is much higher in 50 than in 2.5 mM K. The slow recovery in 2.5 mM K could be converted into a slow acidification with either amiloride solutions or Na-free solutions. The brisk recovery in 50 mM K was 50% inhibitable with either amiloride or SITS. Na-free solutions nearly abolished recovery. [From Abercrombie er al. (1983) with permission.]

D. Comparison of pH1 Recovery Rates

In order to compare the rates of pHi recovery, we must take account of the total buffer content of the fibers, which differs under the various conditions. This content is the product of total buffering power and fiber water volume. Total buffering power includes both intrinsic buffering power (pi) and that due to the presence either of bicarbonate ( ~ H C O J or DMO (pDM0). We will use as reference the total buffer content of fibers in 2.5 mM K, acidified to 6.9 with 5% CO2. In these fibers, pi is about 26 mM is 2.3[HCO3]i = 19 (Abercrombie et al., 1983; Putnam, 19851, and PHCO~ mM. Thus, total buffer content is (26 + 19)V mmol, where V is water volume of the fiber in 2.5 mM K. To compare the rate of pHi recovery of fibers in 2.5 mM K and acidified after a NH&I pulse with that under the reference conditions, the observed rate must be multiplied by the ratio of total buffer contents, 26V/(26 + 19) V = 0.58. For fibers depolarized in 50 mM K, constant CI, fiber swelling must also be taken into account. Fiber water is increased to I S V , but intrinsic buffer content remains unchanged at 26V mmol (Abercrombie et al., 1983). Therefore, the observed rates of pHi recovery in depolarized fibers (constant CI) acidified with either C 0 2 or a NH4CI prepulse must be multiplied by (26 + 19 X 1.5) V/(26 + 19) V = 1.21 or 26V/(26 19) V = 0.58, respectively. Note that the latter value is

+

45

3. pH, REGULATION IN FROG SKELETAL MUSCLE TABLE I ADJUSTED pH, RECOVERY RATES(ApH,/hr)O 50 mM K b

2.5 mM K NH&I

Control 1 mM Amiloride 0.1 mM SITS Amiloride + SITS Na-free

0.15

* 0.02 -

-

-

COI 0.03 -0.06

?

2

-

NH4CI 0.01 0.02

-0.07 t 0.02

Cot

0.17 2 0.02 0 . 3 6 2 0.04 0.02 2 0.01 0.21 2 0.02 0.13 0.03 0.19 t 0.02 0.11 t- 0.04 0.00 t 0.01 0.04 0.02

*

*

DMO 0.14

-

a The adjusted rates, which allow comparison among the various conditions, were calculated by multiplying the observed rates and SEMs by factors that take into account the differences in fiber volume and total buffering power (see Section IV,D). 50 mM K , constant external CI.

the same as that for normally polarized fibers acidified with a NH4CI prepulse, as it should be. The recovery rate of depolarized fibers upon exposure to DMO must be multiplied by (26 + 18.4 x I .5) V/(26 + 19) V = 1.19, where 18.4 is PDMO.Table I lists the various recovery rates adjusted for differences in buffer content. V.

NATURE OF pH,-REGULATING SYSTEMS

On the basis of the data presented in the preceding section, we conclude that two independent pHi-regulating systems exist that may produce recovery from acidification. This is most clearly seen in the response of pHi to CO;! acidification in depolarized fibers (Table I). Only about half of the recovery under these conditions is inhibited by amiloride and half by SITS. When recovery is measured in the absence of Na, it is nearly abolished. These data indicate the presence of an amiloride-sensitive transporter and a SITS-sensitive transporter which requires Na. A. Amiloride-Sensitive Transport

This component of pHi recovery is probably a Na-H exchange. It might be expected that, at a particular pHi , the amiloride-sensitiverate of recovery is about the same whether C02/HCO3 is present or not (taking into account differences in buffer content; see Section IV,D and Table I). Indeed, the average, adjusted rate of pHi recovery after a NH4CIprepulse (nominal absence of C02/HC03)was comparable to that of the SITS-

46


insensitive recovery from C02 acidification both measured in depolarized fibers (Table I) and could be inhibited by amiloride. The pHi recovery both in depolarized fibers and in polarized fibers in the nominal absence of C 0 2 (NH4CI prepulse technique) is due to Na-H exchange. This agrees with our observation that the rate of recovery from DMO acidification (also in the nominal absence of C0JHC03) was comparable to that after a NH4CI prepulse (Table I). The rate of pH, recovery after a NHXI prepulse was the same in normally polarized fibers as in depolarized fibers (Table I). Normally polarized fibers acidified by C 0 2 showed only slight recovery (Table I), which in the presence of amiloride or in the absence of Na was converted into a slow acidification. We attribute this acidification to HC03 efflux. Assuming constant field, we calculated a HC03 permeability of 7.4 x lop8cm/sec (Abercrombie et al., 1983), which is comparable to the value reported previously (Woodbury, 1971). This acidifying HC03 efflux is only significant in normally polarized fibers, since in 50 mM K, V , = E H C OTherefore, ~. the actual recovery rate in normally polarized fibers must be taken as the sum of the observed slow recovery rate and the rate of acidification upon Na removal and is the same as the amiloride-sensitive recovery in depolarized fibers. The Na-H exchange accounts for the entire recovery from C02 acidification in normally polarized fibers, since amiloride and Na removal produce the same slow acidification. B. SITS-Sensitive Transport

The SITS-sensitive component of recovery could be demonstrated in depolarized fibers (50 mM K , constant CI) acidified with C 0 2 , but not in those acidified with a NH4CI prepulse. Thus, HC03 seems to be required for the SITS component. The rate of recovery in depolarized fibers acidified with C02 is reduced by a half by CI removal. The recovery remaining in CI-free solution is inhibited by amiloride, but is not affected by SITS. This indicates that the SITS-inhibitable component of recovery requires CI. Moreover, the complete inhibition of recovery in fibers deprived of Na indicates that Na is also required for this component of recovery. Thus, the ionic requirements of the SITS-sensitive transporter are similar to those of the pHi-regulating mechanism in many other cells (Thomas, 1977; Boron et al., 1981; Moody, 1981; Boron and Russell, 1983). In normally polarized fibers, Na-H exchange can account for all of the recovery from COz acidification. The appearance of a SITS-sensitive component of recovery in fibers depolarized in 50 mM K, constant C1, may well be due to the increase in intracellular C1 from about 2 mM to about 30 mM (Abercrombie and Roos, 1983) under these conditions. As

3. pHi REGULATION IN FROG SKELETAL MUSCLE

47

previously shown (Abercrombie eta/., 19831, fibers depolarized in 50 mM K, constant K x C1 product (which should not affect intracellular C1) showed a recovery rate (0.18 rt 0.04 ApHJhr, n = 7) that was significantly lower than the rate at constant C1. Thus, the SITS-sensitive component of pHi recovery in frog muscle is probably fully active only when intracellular C1 is elevated above the resting level. For the remainder of this chapter we will discuss the properties of the Na-H exchange only and factors that influence the rate of Na-H exchange. VI.

PROPERTIES OF Na-H EXCHANGE

To further characterize the properties of the Na-H exchanger, the pHi recovery from acidification after a NH4CI prepulse was studied in fibers depolarized in 50 mM K. Under these conditions, pHi recovery is almost entirely due to Na-H exchange (Table I) and is greater than if C 0 2 were used for acidification because of the lower buffering power. We studied the Na-H exchange to determine whether it is electroneutral and to characterize its pHi dependence and [NaIo dependence. A. Electroneutrality

It has been shown in several systems that the stoichiometry for Na-H exchange is 1 : 1. We have tested this under conditions in which only this exchange is operative, i.e., after a NH4CI prepulse, by examining the change in V , associated with inhibition of the exchange. The H efflux J H can be calculated from the measured rate of pH, recovery, ApHJAt, assuming the fiber to be a cylinder, by

JH = (ApH,/At) PT(d4) where PTis total intracellular buffering power and d is fiber diameter. For a particular fiber, ApHJAt after removal of NH&l was 0.55 ApHJhr, and PT(equal to p,) was 22 mM. A value of 127 p m for d was assumed (taking account of fiber swelling in 50 m M K, constant C1; see Abercrombie et al., 1983). This yields an H efflux of I I x lo-'? mol/cm2 sec. If this were all due to electrogenic Na-H exchange with an exchange ratio Na : H of, say, 1 :2, a current of (J/2)F = 5.1 x lo-' A/cm2 would be generated. This current, across a membrane resistance R , , of 4000 R cm2 (Fatt and Katz, 1951) would hyperpolarize the membrane by 2 mV. Thus, amiloride should depolarize the membrane by 2 mV. A potential change of 0.5 mV is easily detectable in our experiments. The fact that no measurable potential change was seen upon arniloride application strongly suggests that

48


Na-H exchange is electroneutral. A similar conclusion was reached for Na-H exchange in frog muscle fibers exposed to hypertonic medium (Abercrombie and Roos, 1983). B. pHI Dependence

Depolarized fibers were acidified to different degrees after exposure to either 20 or 40 mM NH&l for varying times. Recovery measured during the first 20 min linearly decreased as pHi was raised from between 6.5 to 7.2. For comparison, Na-H exchange in Necturus gallbladder similarly increases with decreasing pHi, reaching a maximal value at PHi -6.5 (Reuss and Petersen, 1985). In mammalian renal brush border membrane vesicles, the relationship between the amiloride-sensitive Na entry and intravesicular pH is nonlinear: the rate of change of Na entry increases as intravesicular pH decreases (Aronson et al., 1982). Kinsella and Sacktor (1984) showed that in this preparation amiloride-sensitiveNa uptake saturates at an intravesicular pH of about 5.5. Lung fibroblasts showed sigmoidal dependence of Na-H exchange on pHi (Paris and Pouyssegur, 1984), whereas a linear pHi dependence of recovery rate was seen in human neutrophils (Simchowitz and Roos, 1985), in thymic lymphocytes (Grinstein et al., 1984), and in barnacle muscle fibers (Boron et al., 1979). C. [NaIo Dependence

Recovery rate of depolarized fibers from acidification after a NH4C1 pulse increased with “a],; the data could be fit to a Michaelis-Menten curve. The K,,,(about 12 mM) is within the range of values (10-25 mM) observed in epithelial cells (Kinsella and Sacktor, 1984; Weinman and Reuss, 1982) and cultured fibroblasts (Paris and Pouyss6gur, 1984), but is less than K,’s in cultured carcinoma cells (Rothenberg et al., 1983) and in thymic lymphocytes (Grinstein et al., 1984), which are 42 and 59 mM, respectively. VII.

FACTORS AFFECTING THE RATE OF Na-H EXCHANGE

A. Growth-Promoting Factors

The alkalinization induced by insulin in frog muscle (see Section III,E) is mediated by activation of Na-H exchange (Moore, 1981; Putnam,


49

1985). Other growth-promoting factors have been shown to activate Na-H exchange. These include epidermal growth factor (EGF) in carcinoma cells (Rothenberg et a/., 1983), thrombin and insulin in lung fibroblasts (Paris and Pouyssegur, 1984), and EGF and nerve growth factor in pheochromocytoma cells (Boonstra et af., 1983). The mechanism of this stimulation of Na-H exchange is not fully understood. Whatever mediates the effect of growth factors, Paris and Pouyssegur (1984) showed that these factors can increase the affinity of the Na-H exchanger for internal H ions by 0.3 units, without affecting either its affinity for external Na or the maximal rate of exchange. 6. lntracellular Ca and Depolarlzation-Induced Ca Release

Increased intracellular Ca has been shown to increase the activity of alkali metal-H exchange in cultured fibroblasts (Mix et al., 1984) and red blood cells (Cala, 1983). In frog muscle, we studied whether the Na-H exchanger might be affected by Ca release induced by depolarization in high-K solutions. It is known that in response to high K, cytoplasmic Ca transiently rises; the rise subsides within a few minutes (Blinks er al., 1978). Despite this transient nature of the Ca increase, membrane transport processes such as 3-0-methylglucose uptake can remain elevated for hours (Holloszy and Narahara, 1965; Valant and Erlij, 1983). Thus, although the Ca transient may be short-lived, its effect on transport processes could be prolonged. We measured the effect on pH, recovery of caffeine, a drug which induces Ca release. When caffeine (4 mM) was added to fibers in 15 mM K, constant CI (V, about -50 mV) for at least an hour, the recovery rate from CO2-induced acidification was similar to that in fibers in 15 mM K not exposed to caffeine. Caffeine also did not affect recovery in normally polarized fibers in 2T medium (Abercrombie and Roos, 1983). When tetracaine, which blocks Ca release, was applied prior to depolarization in 50 mM K and maintained during depolarization, the pH, recovery from COz acidification was significantly less than without tetracaine. However, further experimentation has made it clear that this inhibition is not related to the effect of tetracaine on Ca release. The recovery rates from C02 acidification were studied in fibers first depolarized in 50 mM K, constant K x C1 product, and again after repolarization of these fibers in normal frog Ringer (2.5 mM K). During the depolarization, the recovery rate was increased, whereas the rate in the repolarized fibers was much lower and similar to that in fibers that never had been depolarized. Clearly, the history of Ca release in these fibers had

50


no effect on subsequent pHi recovery. However, this does not eliminate the possibility of a Ca effect on pHi recovery if the recovery could have been measured during the transient elevation of intracellular Ca. C. External Divalent Cations

External divalent cations seem to inhibit amiloride-sensitive Na uptake in cultured fibroblasts (Villereal, 1982) and chick heart cells (PiwnicaWorms et al., 1985). This suggests an inhibition of Na-H exchange by divalent cations. We investigated the effect of external divalent cations on pHi recovery from COz acidification in frog muscle fibers depolarized in 50 mM K (constant CI) to about -20 mV (Putnam and Roos, 1985). Increasing Ca above our “standard” value (4 mM) significantly decreased recovery rate, an effect which was found to be due to inhibition of the Na-H exchange. The SITS-sensitive component of recovery was not significantly affected by the increased Ca concentration. We also tested the ability of other divalent cations to inhibit Na-H exchange during COz acidification. Magnesium and strontium were only slightly effective, whereas even low concentrations of nickel or cadmium distinctly inhibited recovery. When a NH&l prepulse was used for acidification, in which case nearly all the recovery is due to Na-H exchange, these results could be confirmed. These observations suggest an inhibitory divalent cation binding site on the Na-H exchanger, with binding order Cd > Ni > Ca > Sr 2 Mg. It is of interest that an external modifier site on the Na-H exchanger of renal tubule vesicles has been described (Ives et al., 1984). This site is inhibitory, binds amiloride and Li, and is distinct from the Na transport site. Also, a positive modulatory binding site for internal H has been demonstrated for the Na-H exchanger (Aronson et al., 1982). D. Hypertonic Solutions

The pHi recovery in 2T solutions differs markedly from that in isotonic solutions (Abercrombie and Roos, 1983). In 2T, normally polarized fibers exhibited slow recovery in response to C02-induced acidification (Fig. S ) , as did fibers in isotonic medium (Fig. 4). When the fibers were depolarized in SO mM K (2T medium), the brisk recovery was nearly completely abolished by amiloride and Na-free solutions, but unaffected by SITS (Fig. 5 ) . Thus, in contrast to depolarized fibers in isotonic medium, recovery is entirely due to Na-H exchange. In 2T medium (in the presence of SITS), depolarized fibers exhibited an adjusted rate of Na-H exchange of 0.34 ApHilhr (using a factor of 1.07; see Section IV,D). Under similar

51

3. pH, REGULATION IN FROG SKELETAL MUSCLE 50 K, constant C I , 2 T

0 . 5 - 2.5 K, 2T

I r

I

0.5m M SITS

0.4I

(9)

(4)

T]

1

RATE OF 0.3RECOVERY IN 5% C02 (ApHiIh)

o,2 -

0.1 -

(3)

Na-

ImM Amiloride

Free

(5)

(5)

1

1 1

I 'T 1 I 1 1

FIG.5 . The recovery rate from COz acidification of fibers in 2.S or 50 mM K (constant CI) in hypertonic (2T) solutions. The height of the bar is the mean; the vertical lines are ? I SEM. Note that recovery is much higher in depolarized fibers (50 mM K ) than in normally polarir.ed fibers (7.5 rnM K). The recovery i n 5 0 niM K is nearly aholished by amiloridr and Na-free solutionh. hut unaffected hy SITS. IFi-om Ahercrombie and Koos (1983) with pel-mission. I

isotonic conditions, the adjusted rate of Na-H exchange was only 0. Is) ApH,lhr (Table 1). The mechanism by which, under hypertonic conditions, the rate of Na-H exchange is increased and the rate of the SITSsensitive component is inhibited is unknown. VIII.

COMPARISON WITH OTHER CELLS

The pH, recovery mechanisms in frog skeletal muscle most closely resemble those of crayfish neurons (Moody, 1981). I n both cell types, two independent mechanisms are present: a Na-H exchange and a SITSsensitive component which requires HCO?, C1, and Na. MOUWsoleus fibers also have two independent recovery mechanisms, but in these cells Na-H exchange predominates and the SITS-sensitive component does not appear to require Na (Aickin and Thomas, 1977b). In three inverte1979, brate preparations, the barnacle muscle (Boron, 1977; Boron et d., 1981),snail giant neuron (Thomas, 19771, and squid giant axon (Boron and Russell, 1983), the only mechanism for pH, recovery is one that exchanges external Na and H C 0 3 for internal CI. It is SITS sensitive. Sea urchin eggs have a Na-H exchange which is amiloride sensitive (Johnson

52


et al., 1976). In a variety of vertebrate cells, an amiloride-sensitive Na-H exchange has been demonstrated (Deitmer and Ellis, 1980; Weinman and Reuss, 1982; Rothenberg et al., 1983; Boron and Boulpaep, 1983a; Simchowitz and Roos, 1985).In some of these cells, a CI-HC03 exchange has also been found, in which CI enters and HC03 leaves either to allow pHi recovery in response to an alkaline load (Vaughan-Jones, 1982; Simchowitz and Roos, 1985) or to allow CI entry in NaCl transporting epithelia (Reuss and Costantin, 1984). In salamander proximal tubules, a SITSsensitive transporter results in a C1-independent Na and HC03 efflux which is electrogenic (Boron and Boulpaep, 1983b). A unifying principle for this variety of pHi-regulating mechanisms has yet to be proposed. The properties of the Na-H exchanger in frog muscle are similar to those of the exchanger in other cells. Its stoichiometry seems to be 1 : 1 (see Section VI,A), its rate is increased as pHi is reduced (see Section VI,B), and its K , for external Na is in the range of 10 to 20 mM (see Section V1,C). Whether the inhibition of Na-H exchange by certain external divalent cations in frog muscle (see Section VJ1,C) is a property shared by the exchanger in other cells remains to be seen. IX. SUMMARY

Frog skeletal muscle fibers, like nearly all other cells, maintain a pHi which is more alkaline than if H were at equilibrium across the cell membrane. The steady-state pHi of frog fibers (in 2.5 mM K and in the nominal absence of C02) is 7.23. Over a period of hours, it is not affected by depolarization to -20 mV (in 50 mM K) or by inhibiting the pHi-regulating mechanisms in Na-free medium. In the nominal absence of C 0 2 , changes in pH, affected steady-state pHi only slightly, with external alkalinization having a somewhat larger effect than acidification. Both insulin and hypertonic solutions caused an alkaline shift in steady-state pHi . Frog muscle fibers have two membrane transport systems responsible for pHi recovery from an acid load. One, a Na-H exchange, can be inhibited by amiloride or by removal of Na from the external medium. The other, which requires HC03, C1, and Na, can be inhibited by SITS. The Na-H exchanger seems to be equally active in normally polarized and depolarized fibers, whether they are acidified by COz or by an NH4Cl prepulse. The SITS-sensitive component manifests itself when the fibers are acidified with C 0 2and depolarized in 50 mM K, constant C1 (to about -20 mV). The resulting elevation of intracellular C1 may be necessary for the appearance of this component of recovery. When normally polarized cells are acidified with CO;! , the pHi recovery (which is only due to Na-H exchange) is largely masked by an acidifying HCO3 efflux.


53

The Na-H exchange in frog muscle is electroneutral, increases in a linear way as pHi is reduced, and has a K , for “a], of about 12 mM. Insulin increases the activity of Na-H exchange. External divalent cations inhibit it, perhaps by binding to a modifier site on the exchanger with binding order Cd > Ni > Ca > Sr IMg. Hypertonic solutions (2T) increase the activity of the Na-H exchanger and, in addition, inhibit the SITS-sensitive component of recovery. The Na-H exchanger in frog muscle is comparable to that in a variety of other vertebrate cells, while the SITS-sensitive component may be more similar to that found in invertebrate nerve and muscle cells. ACKNOWLEDGMENTS We thank T. Wilding for performing some of the experiments. Drs. R. Abercrombie and L. Reuss critically read the manuscript; this led to a number of improvements. S . Eads and C. Krah are gratefully acknowledged for typing the manuscript. This work was supported by National Institutes of Health grant 00082 and Research Career Award HR-19608 to A. R. REFERENCES Abercrombie, R. F., and Roos, A. (1983). The intracellular pH of frog muscle: its regulation in hypertonic solutions. J. P h y . ~ i d (London) . 345, 189-204. Abercrombie, R. F., Putnam, R. W.. and Roos, A. (1983). The intracellular pH of frog skeletal muscle: its regulation in isotonic solutions. J. Physiol. (London) 345, 175-187. Adler, S., Anderson, B., and Zett, B. (1975). Effect of osmolarity on intracellular pH of rat diaphragm muscle. Am. J. Physiol. 228, 725-729. Aickin, C. C., and Thomas, R. C. (1977a). Micro-electrode measurement of the intracellular pH and buffering power of mouse soleus muscle fibres. J. Physiol. (London)267, 791810.

Aickin, C. C., and Thomas, R. C. (1977b). An investigation of the ionic mechanism of intracellular pH regulation in mouse soleus muscle fibres. J . Physiol. (London) 273, 295-3 16. Almers, W., and Best, P. M. (1976). Effects of tetracaine on displacement currents and contraction of frog skeletal muscle. J. Physiol. (London) 262, 583-61 1 . Ammann, D., Lanter, F., Steiner, R., Schulthess, P., Shijo, Y . , and Simon, W. (1981). Neutral carrier based hydrogen ion selective microelectrode for extra- and intracellular studies. Anal. Chem. 53, 2267-2269. Aronson, P. S., Nee, J., and Suhm. M. (1982). Modifier role of internal H+ in activating the Na+-H+exchanger in renal microvillus membrane vesicles. Noture (London)299, 16 I163. Bates, R. G . (1973). “Determination of pH: Theory and Practice: (2nd Ed.). Wiley, New York. Blinks, J. R., Riidel, R., and Taylor, S. R. (1978). Calcium transients in isolated amphibian skeletal muscle fibres: Detection with aequorin. J. Physiol. (London)277, 291-323. Bolton. T.. and Vaughan-Jones, R. (19771. Continuous direct measurement of intracellular chloride and pH in frog skeletal muscle. J. Physiol. (London) 270, 801-833. 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 (PC 12) cells. J. Cell B i d . 97, 92-98.

54


Boron, W. F. (1977). Intracellular pH transients in giant barnacle fibers. A m . J . Physiol. (Cell Physiol. 2 ) 233, C61-C73. Boron, W. F., and Boulpaep, E. L. (1983a). lntracellular pH regulation in the renal proximal tubule of the salamander. Na-H exchange. J . Gen. Physiol. 81, 29-52. Boron, W. F., and Boulpaep, E. L. (1983b). Intracellular pH regulation in the renal proximal tubule of the salamander. Basolateral HCOj transport. J . Gen. Physiol. 81, 53-94. Boron, W. F., and De Weer, P. (1976). Intracellular pH transients in squid giant axons caused by CO?, NH3, and metabolic inhibitors. J . Gen. Physiol. 67, 9 1-1 12. Boron, W. F., and Russell, J. M. (1983). Stoichiometry and ion dependencies of the intracelMar pH-regulating mechanism in squid giant axon. J . Gen. Physiol. 81, 373-399. Boron, W. F., McCormick, W. C., and Roos. A. (1979). pH regulation in barnacle muscle fibers: Dependence on intracellular and extracellular pH. Am. 1.Phy.sio1. (Cell Phv.siol. 6) 237, C185-CI93. Boron, W. F., McCormick, W. C., and Roos, A. (1981). pH regulation in barnacle muscle fibers: Dependence on extracellular sodium and bicarbonate. A m . 1. Physiol. (Cell Physiol. 9) 240, C80-C89. Cala, P. M. (1983). Cell volume regulation by Amphiitmu red blood cells. The role of Ca2- as a modulator of alkali metal/H+ exchange. J . Gen. Physiol. 82, 761-784. Cohen, P. (1982). The role of protein phosphorylation in neural and hormonal control of cellular activity. Nuture (London) 296, 613-620. Cohen, P., Klee, C. B., Picton, C., and Shenolikar, S. (1980). Calcium control of muscle phosphorylase kinase through the combined action of calmodulin and troponin. I n “Calmodulin and Cell Function” (D. M. Watterson and F. F. Vincenzi, eds.). Ann. N . Y . Acad. Sci. 356, 151-161. Curtin, N. A., and Woledge, R. C. (1978). Energy changes and muscular contraction. Physiol. Rev. 58, 690-761. Deitmer, J. W., and Ellis, D. (1980). Interactions between the regulation of the intracellular pH and sodium activity of sheep cardiac Purkinje fibres. J . Physiol. (London) 304,471488. Ellis, D., and Thomas, R. C . (1976). Direct measurement of the intracellular pH of mammalian cardiac muscle. J . Physiol. (London) 262, 755-771, Fatt, P., and Katz, B. (1951). An analysis of the end-plate potential recorded with an intracellular electrode. J . Physiol. (London) 115, 320-370. Fidelman, M. L., Seeholzer, S. H., Walsh, K. B., and Moore, R. D. (1982). Intracellular pH mediates action of insulin on glycolysis in frog skeletal muscle. A m . J . Physiol. (Cell Physiol. 11) 242, C87-C93. Gordon, A. M., and Godt, R. E. (1970). Some effects of hypertonic solutions on contraction and excitation-contraction coupling in frog skeletal muscles. J . Gen. Physiol. 55, 254275. Grinstein, S . , Clarke, 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 Na+/H+antiport. J . C e n . Physiol. 83, 341-369. Hodgkin, A . L., and Horowicz, P. (1957). The differential action of hypertonic solutions on the twitch and action potential of a muscle fibre. J . Physiol. (Londonj 136, 17P. Holloszy, J. O., and Narahara, H. T. (1965). Studies of tissue permeability. X . Changes in permeability to 3-methylglucose associated with contraction of isolated frog muscle. J . B i d . Cbem. 240, 3493-3500. Ives, H. E., Mircheff, A. K., Yee, V. J . , and Warnock, D. G. (1984). Distribution and


55

regulation of the Na*/H+ antiporter in the renal proximal tubule epithelial cell. I I I "Hydrogen Ion Transport in Epithelia" ( J . F. Forte, D. G . Warnock. and F. C. Rector. Jr.. eds.), pp. 117-126. Wiley. New York. Johnson, J . D., Epel. D., and Paul. M . (1976). Intracellular pH and activation of sea urchin eggs after fertilisation. NUIWP(London) 262, 661-664. Kinsella, J . L.. and Sacktor, B. (1984). Na--H- exchange in isolated renal brush border membrane vesicles: Regulation by metabolic acidosis and glucocorticoids. In "Hydrogen Ion Transport in Epithelia" (1. F. Forte. D. G. Warnock, and F. C. Rector. Jr.. eds.). pp. 127-137. Wiley. New York. L'Allemain, G., Paris, S . . and Pouyssegur. J . (19x4). 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. Liittgau. H. C., and Oetliker. H. (1968). The action of caffeine on the activation of the contractile mechanism in striated muscle fibres. J . Pliysiol. (London) 194, 5 1-74, Mix. L. L., Dinerstein. R. J., and Villereal. M. L. (1984).Mitogens increase intracellular pH and Ca?' in human fibroblasts. Biophys. J . 45, 86:i. Moody. W. J.. Jr. (1981).The ionic mechanism of intracellular pH regulation in crayfish neurones. J . Physiol. (Londoii) 316, 293-308. Moore, R. D. (1979). Elevation of intracellular pH by insulin in frog skeletal muscle. Biochem. Biophys. Re.\, Commctn. 91, 900-904. Moore, R. D. (1981). Stimulation of Na: H cxchmge by insulin. Biop/iy.\. J. 33, 203210. Ozawa, E.. Hosoi, K.. and Ebashi. S. (1967). Reversible stimulation of muscle phosphorylase h kinase by low concentrations of calcium ions. J . Bioc./w/n.( T o k y ) )61, 53 1-533. Paris, S.. and PouyssCgur. J . (1984). Growth factors activate the Na-lH+ antiporter in quiescent fibroblasts by increasing its affinity for intracellular H'. J . B i d . C'hem. 259, 10989-1 0994. Piwnica-Worms. D.. Jacobs, R.. Horres, C. R.. and Lieberman. M. (1985). NalH exchange in cultured chick heart cells. pH, regulation. J . Gen. P/iv.siol. 85, 43-64. Putnam. R. W. (1985). Effect of insulin on intracellular pH in frog skeletal muscle fibers. ~ mJ . . ~ / l y . s i o / .(cPi/~ k y s i o i I. 7) 248, c m - c 3 3 6 . Putnani, R . W . . and Roos, A . (1983). N a : H exchange in frog skeletal muscle: Its role in recovery of intracellular pH (pHi) from acid loading. A m . Z o o / . 23. 996a. Putnam, R. W., and Roos. A. (1985). External divalent cations inhibit NaiH exchange in frog skeletal muscle. Biopliys. J . 49, 48%. Ruess. L., and Costantin. J . L. (1984). CI / H C 0 3 exchange at the apical membrane of Necttrrcts gallbladder. J . GeIi. Phy.siol. 83, 801-818. Reuss. L.. and Petersen. K . - U . (1985). Cyclic AMP inhibits Na+/H+exchange at the apical membrane of Nectrrrcts gallbladder epithelium. J . (;en. P/iy,!io/.85, 409-429. Roos, A., and Boron, W . F. (1981). Intracellular pH. PIrysiol. Rev. 61, 296-434. Rothenberg, P.. Glaser. L.. Schlesinger. P.. and Cassel, D. (1983). Activation of Na-/H ' exchange by epidermal growth factor elevates intracellular pH in A431 cells. J . B i d . Chem. 258, 12644-12653. Simchowitz, L.. and Roos. A. (1985). Regulation of intracellular pH in human neutrophilh. J . Gen. Pliysiol. 85, 443-470. Somlyo. A. V . , Gonzalez-Serratos, H., Shuman. H.. McClellan. G . . and Somlyo. A. P. (1981 ). Calcium release and ionic changes in the sarcoplasmic reticulum of tetanized muscle. J . Cell Biol. 90, 577-594. Taylor. S . R . , Riidel, R., and Blinks, I . R. (1975). Calcium transients in amphihian miisclc. Fed. Proc., Fed. A m . Soc. EX^. Riol. 34, 137'9-138 I .

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Thomas, R. C. (1974). Intracellular pH of snail neurones measured with a new pH-sensitive glass micro-electrode. J . Physiol. (London) 238, 159-180. Thomas, R. C. (1977). The role of bicarbonate, chloride and sodium ions in the regulation of intracellular pH in snail neurones. J . Physiol. (London) 273, 3 17-338. Thomas, R. C. (1978). “Ion-Sensitive Intracellular Microelectrodes. How to Make and Use Them.” Academic Press, San Francisco. Valant, P., and Erlij, D. (1983). K+-stimulated sugar uptake in skeletal muscle: role of cytoplasmic Ca2+.Amer. J . Physiol. (Cell Physiol. 14) 245, C12S-CI32. Vaughan-Jones, R. D. (1982). Chloride-bicarbonate exchange in the sheep cardiac Purkinje fibre. I n “Intracellular pH: Its Measurement, Regulation, and Utilization in Cellular Functions” (R. Nuccitelli and D. W. Deamer, eds.), pp. 239-252. Liss, New York. Villereal, M. L. (1982). Inhibition of the serum-dependent, amiloride-sensitive sodium transport pathway in human fibroblasts by extracellular divalent cations. J . Cell Physiol. 111, 163- 170. Weinman, S. A., and Reuss, L. (1982). Na+-H+ exchange at the apical membrane of NecINTUS gallbladder. Extracellular and intracellular pH studies. J . Cen. Physiol. 80, 299321. Woodbury, J. W. (1971). Fluxes of H+ and HCOi across frog skeletal muscle cell membranes. In “Ion Homeostasis in the Brain” (B. K. Siesjo and S. C. Sorensen, eds.), pp. 270-289. Munksgaard, Copenhagen.


Chapter 4

Molecular Properties and Ph siolo ical Roles of the enal a+-H+ Exchanger

K

a

PETER S. ARONSON AND PETER IGARASHI Departments of Medicine and Physiology Yale University School of Medicine New Haven. Haven, Connecticul

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction.

11. Kinetics of o f the t Renal Na+-H+ Exchanger . . . . . . . . . . .

.......... A. Stoichiometry . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. .. ......... . . B. Specificity and Modes.. . . . . ........... .......... C. Interaction with H+ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... D. Inhibitors. .. . . . . . . . ........... 111. Biochemistry of the Renal er......... A. Effect of of Histidine Histidine M Modification . . . . . . . .......... B. Effect of Carboxyl M C. Labeling of the Tran .......... IV. Role of the Renal Na+-H A. HCOy Reabsorption . . . . . . ..... . . . . ....... . . . . . . . . .......................... ........... B. Organic Anion Reabsorption . . . . . . . . . . , . , . . . . . . . . . . . . . . . . . . . . .. . ............ C. CI- Reabsorption.. . . . . ............. . . . . . . ...........,. . . . . . . .............. . . . . , . . . . . . . . . D. Anion Transport and Intracellular H . . . . . . , . . . . . . . . . . . . . . . . . . . . .. . . . .. .. Intracellular p H References. . . . . . . . . . . . . . , . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

57 58 58 59 62 64 65 65 67 68 70 70 71 72 74

I. INTRODUCTION

Although the concept that acidification of the urine occurs by a process of Na+-for-H+exchange is at least four decades old (Pitts, 1948), the actual existence of a directly coupled Na+-H+ exchange process in the luminal (microvillus, brush border) membrane of renal proximal tubule cells was not demonstrated until relatively recently (Murer ef al., 1976). Microvillus membrane vesicles isolated from renal cortex have subsequently 57 Copyright 8 1986 by Academlc Press. Inc All nshtb of reprduction in any form reserved

58

PETER S. ARONSON AND PETER IGARASHI

served as a useful model system for characterizing the kinetic and biochemical features of a Na+-Hi exchanger whose properties are largely representative of plasma membrane Na'-H+ exchangers in general (Aronson, 1985). The principal purpose of this chapter is to review studies conducted in our laboratory on the molecular properties of the renal microvillus membrane Na+-H+ exchanger. We will also review studies indicating that additional transport systems for the transfer of acids or bases exist in parallel with the Na+-H+ exchanger on the luminal membrane of proximal tubule cells since these studies have important implications concerning the physiologic role of the renal Na+-H+ exchanger in facilitating anion transport.

11.

KINETICS OF THE RENAL Na+-H+ EXCHANGER

A. Stoichiometry

An in-to-out Hf gradient drives uphill Na+ uptake into rabbit renal microvillus membrane vesicles, and an out-to-in Na+ gradient drives uphill Ht extrusion, consistent with the presence of a Naf-H+ exchange process (Kinsella and Aronson, 1980). Three sets of observations argue that the stoichiometry of this exchange is 1 : 1. First, the membrane potential does not act as a driving force for Nat uptake by Na+-H+ exchange (Kinsella and Aronson, 1980). For example, in the presence of an in-to-out H+ gradient, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), an H+ ionophore, causes the membrane potential to become more inside-negative, Nevertheless, FCCP has no effect on Na+ accumulation via the Na+-H+ exchanger. Similarly, in the presence of an in-to-out K+ gradient, the K + ionophore valinomycin causes the membrane potential to become more inside-negative but does not affect Na+ uptake. Altering the membrane potential by imposing outto-in gradients of anions of varying conductance also does not influence Na+ influx. These findings imply that the process of Nat-Ht exchange is electroneutral with a Na+ : H + coupling ratio of 1.0. It should be noted that changing the membrane potential has no effect on Nat uptake whether the internal pH of the vesicles is 5.9 or 7.5, suggesting that internal pH does not modify the stoichiometry of exchange. Another set of observations (Kinsella and Aronson, f982) indicating that the Na+ : H + coupling ratio is 1.0 is that the Nat-H+ exchanger comes to equilibrium and mediates no net fluxes of Nat or H C whenever the transmembrane Na+ gradient is balanced by a H+ gradient of the same

4. THE RENAL Na+-H- EXCHANGER

59

magnitude and direction (i.e,, whenever [Na'],,/[Na+]i is equal to [H+],,/ [H+]i). For example, if vesicles preloaded with Na' and having internal pH 6.5 are diluted I : 10 into media of varying external pH, there is net efflux of Na+ when the pH of the external medium is 6.5, net influx of Na+ when external pH is 8.5, but no initial net flux of Na' when external pH is 7.5. Thus, the Na+-H+ exchanger comes to equilibrium and mediates no net ion flux when the I0-fold in-to-out Nat gradient is just balanced by a l0-fold in-to-out H + gradient. If the coupling ratio of the Na+-H+ exchanger were not 1 .O, the transport system would not have been at equilibrium when similarly directed Na' and H' gradients of equal magnitude were imposed (Aronson, 1981). For instance, if the Na+ : H' coupling ratio were 2 : I , bringing the Na'-H' exchanger to equilibrium would require imposing a 100-fold in-to-out H ' gradient in order to balance a 10fold in-to-out Na+ gradient. A Na' : H coupling ratio of I .O is consistent not only with a I : I stoichiometry. but also with such other possible stoichiometries as 2 : 2 or 3 : 3 . However, the rate of Na+ influx via N a + - H t exchange is a simple, saturable, Michaelis-Menten function of lNa+I,,, consistent with the presence of only a single external transport site for Na' (Kinsella and Aronson, 1981a). Moreover, raising [H+], inhibits Na+ influx by protonation of a single external site (Aronson et NI., 1983). Binding of external H' at this site is mutually exclusive of the binding of external Na'. Together, these findings suggest that Na' and H t compete for binding to a single, saturable, external transport site, arguing that the actual stoichiometry of the Nat-Ht exchanger is 1 : I .

B. Specificity and Modes An out-to-in Na+ gradient drives uphill H + efflux from renal microvillus membrane vesicles (Kinsella and Aronson, 1980, 1981a), and an in-to-out Na+ gradient drives uphill Hi influx (Seifter et ul., 1984).Thus, the transport system is readily reversible and can mediate Na+-H' exchange in either direction. Moreover, "Na efflux is accelerated by the presence of external Na+ (Kinsella and Aronson, 1981a; Aronson et al., 1983), and 22Nainflux is accelerated by the presence of internal Na+ (Aronson ct d., 1982), indicating the existence of a Na' -Nat exchange mode. Along with Na+ and H + , the renal Na+-H' exchanger ha5 affinity for Lit and NH; and can function in additional exchange modes involving pairs of these four cations. For example, external Lit is a single competitive inhibitor of Na+ influx (Kinsella and Aronson, 1981a; Mahnensmith and Aronson, 19851, suggesting that binding of Li+ to the external trans-

60

PETER S.ARONSON AND PETER IGARASHI

port site occurs and is mutually exclusive of the binding of Na+. Furthermore, an out-to-in Li+ gradient drives uphill H+ extrusion (Kinsella and Aronson, 1980, 1981a), indicating the existence of a Lit-H+ exchange mode. Similarly, external NH: competitively inhibits Na+ influx, and an out-to-in NH: gradient generates an inside-alkaline pH gradient (Kinsella and Aronson, 1981a). However, the extent to which the latter finding results from entry of NH; via nonionic diffusion of NH3 rather than from carrier-mediated NH:-H+ exchange is not clear. Nevertheless, transport of NH; by the Na+-H+ exchanger is suggested by the finding that external NH: accelerates 22Naefflux (Kinsella and Aronson, 1981a), consistent with the existence of a Na+-NH: exchange mode. These properties-a 1 : 1 stoichiometry, affinity for Lit and NH: in addition to Na+ and H + , and multiple exchange modes involving these four cations-can be easily explained by the simple model of a transport protein that can bind a Na+, H+, Li+, or NH: on one side of the membrane and exchange it for one or another of these same cations on the opposite side of the membrane. This model of the transport system as a cation exchanger is illustrated by the kinetic scheme in Fig. 1. If A+ represents Na+, then net exchange of external Na+ for internal H+ can occur via the cycle 1 + 2 + 3 + 4 + 5 + 6 as follows. First, A: binds to the outwardly facing, unloaded form of the transporter To (step 1). The transporter then undergoes a conformational change (step 2) from the outwardly facing, loaded form AtTo to the inwardly facing, loaded form A+Ti. Release of A+ to the inside solution (step 3) is then followed by binding of H: (step 4) to the inwardly facing, unloaded form Ti. After a conformational change of TH: to TH: (step 5 ) , H: is released (step 6), thereby regenerating To to start the cycle again. Similarly, the alternative

A+T,

B+T,

0

A+T~

B*Ti

FIG. 1. Kinetic model of the renal Na+-H+ exchanger.

61

4. THE RENAL Na+-H+ EXCHANGER

- --

substrate B: (e.g., external Li') can exchange with internal H+ via the cycle 1' 2' + 3' 4 + 5 ---* 6. Exchange of A: for A: (e.g., Na+-Nat exchange) can occur via the cycle I 2 + 3 -+ 3 2 + I , and exchange of B: for A: (e.g., NH:-Na+ exchange) can occur through the cycle 1' + 2' 3' 3 2 + 1. This kinetic scheme clearly can account for the multiple exchange modes involving H+ and other cations sharing the exchanger. It should be emphasized, however, that this particular kinetic scheme, although possessing the important attribute of simplicity, is not unique for explaining the modes of the plasma membrane Na+-H+ exchanger. Alternative kinetic schemes, some allowing simultaneous binding of H+ and A+ or B + , are also possible, as are models of the Na+-H+ exchanger as a cation-hydroxyl cotransport system (Aronson, 1985). Indeed, while we will continue the convention of referring to this transport system as a cation-proton exchanger, the reader should realize that interpretations based on models of cation-hydroxyl cotransport are equally plausible. Based on measurements at external pH 7.5 of the K, for Na+ influx (613 mM) and the K , values for inhibition of Na+ influx by NH: (4-1 1 mM) and Li+ (1-2 mM), it appears that the selectivity sequence for the binding of substrates to the external transport site of the renal Na+-H+ exchanger is Li+ > NH: 2 Na+ , whereas there is no detectable affinity for K + , Rb+, Cs+,or choline (Kinsella and Aronson, 1980, 1981a; Aronson rt al., 1983; Mahnensmith and Aronson, 1985). Interestingly, there is a different selectivity sequence for the relative rates of transport of these cations once they are bound. For example, external Na+ accelerates the rate of 22Na efflux more than does a saturating level of external H' (Aronson et al., 1983). This implies that the rate-limiting step for entry of bound Na+, either translocation inward (e.g., step 2 in Fig. 1 ) or release from the inwardly facing form of the transporter (e.g., step 3 in Fig. l ) , must be faster than the corresponding rate-limiting step for entry of bound H+ (e.g., step 5 or 4 in Fig. 1). External NH: accelerates the efflux of ??Naas much as does external Na+ (Kinsella and Aronson, 1981a), suggesting that the rate-limiting step for entry of bound NHimust also be faster than the rate-limiting step for entry of bound H+. In contrast, external Li+ reduces the rate of 22Naefflux under conditions in which H+ is the only other transportable cation in the external medium, indicating that the ratelimiting step for entry of bound Li+ must be slower than that for entry of H+. Together, these findings suggest that the rate constants for the ratelimiting step for entry of externally bound cations are in the sequence Na+ = NH: > H+ Lit. Consistent with this sequence is the observation by Ives et al. (1983) that the V,,, for exchange of internal H+ with external Lit is lower than that for exchange of internal H+ with external Na'.

-

-

62


C. Interaction with H+

The kinetics of interaction of external H+ with the renal Na+-H' exchanger has been examined in some detail (Aronson ct a / . , 1983).The rate of Na+ uptake into vesicles with internal pH 6.0 is progressively inhibited as external pH is lowered from 8.5 to 6.0, the data conforming to a simple titration curve with apparent pK 7.3-7.5. A Dixon plot (Lee, l / V versus [ H + ] , )of the data yields a straight line, consistent with the idea that inhibition of Na+ influx by external H+ arises from protonation of a single site with apparent KH 35 nM. Moreover, reducing external pH elevates both the K , for Na+ influx and the Ki for inhibition of Na+ influx by Lit or NH;, with virtually no effect on either the V,,, for Na+ influx or the slopes of the Dixon plots for inhibition of Na+ influx by external Lit and NH:. These observations indicate that the binding of external H+ is mutually exclusive of the binding of external Nat, Li', and NH; to the renal Na+-H+ exchanger. Whether the external binding site for H+ is actually identical to, or physically within, the external binding site for Na+, NH;, and Li+ is not known. Nevertheless, in the absence of evidence to the contrary, the simplest concept is that external H + , Na', Li+, and NH; mutually compete for binding at a common external transport site, as schematically illustrated in Fig. 2. Whereas, as just described, the kinetic effects of external Hf can be ascribed to simple titration of the external cation transport site, the kinetic effects of internal H + are more complex (Aronson Pt d.,1982). First, when the rate of Na+ influx is measured as a function of internal pH, a greater than first-order dependence of the rate on the internal H + concentration is observed. This is not consistent with any reaction mechanism, such as the one illustrated in Fig. I , that involves the binding of internal H + to a single binding site on a single conformation of the transporter. One possibility is that there exists more than one binding site for OUTSIDE

INSIDE

Na'. NH:,

Lit, or Ht

FIG. 2. Schematic diagram summarizing the kinetic properties of the renal Na+-H+ exchanger.


63

internal H + . Given the arguments presented earlier that the stoichiometry of the Na+-H exchanger is I : I , any additional site(s) for internal H i must permit binding without transport. Thus, these results suggest that internal H', independent of its role as a transportable substrate, can serve as an allosteric activator of the Na' -Hi exchanger. Consistent with this concept is the finding that the rate of "Na flux via an exchange mode not involving H+ transport, namely, Na+-Na+ exchange, is also activated by raising internal H i . The primacy of kinetic factors over thermodynamic factors in regulating the rate of Na'-H+ exchange is well illustrated by the activating effect of internal H + on Na' efflux. When membrane vesicles are preloaded with Na' at internal pH 7.5 or 6.9 and then diluted 1 : 100 into Na+-free media at external pH 7.5, Na' efflux via the Na+-H+ exchanger is threefold faster from vesicles with internal pH 6.9 than from vesicles with internal pH 7.5. In this experiment, the rate of Na+-H+ exchange is actually greater under the condition in which its thermodynamic driving force is smaller. In the case of vesicles with internal pH 7.5, the net thermodynamic driving force for exchange of internal Na+ with external H' is the 100: I in-to-out Na' gradient unopposed by any H' gradient: in the case of vesicles with internal pH 6.9, the same in-to-out Na' gradient is opposed by a 4 : 1 in-to-out H' gradient. No laws of thermodynamics are violated, of course, because even at internal pH 6.9 the net thermodynamic driving force for exchange of internal Na' with external H+ is still positive. These results clearly show the important role of internal Hi as a potent kinetic activator of the Na'-Hi exchanger. The most straightforward and probable explanation for the activating effect of internal H' is that there exist one or more inwardly facing, titratable groups, protonation of which causes activation of the transport system. However, the presence of a modifier site that is separate and distinct from the internal H' transport site is not the only possible explanation for the anomalous kinetic effects of internal H + . As discussed in more detail elsewhere (Aronson, 1985), kinetic schemes, such as random-order reaction mechanisms, that allow for binding of H' to a single internal transport site on two or more conformations of the transporter can also give rise to such phenomena as greater than linear dependence of Na+ influx on internal [H+],stimulation of Na+-Na' exchange by internal H + , and stimulation of net Na+ efflux by internal H t . Despite uncertainty concerning the underlying molecular mechanism, it is clear that there is an important asymmetry in the interactions of H+ with the renal microvillus membrane Na+-H' exchanger, with the net result that internal H 'serves as an apparent kinetic activator of the transport system, as schematically illustrated in Fig. 2.


D. Inhibitors

The effects of several potential inhibitors on the renal Na+-H+ exchanger have been examined. Acetazolamide ( M),furosemide M ) , and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid M ) have no direct effects on this transport system (Kin(SITS, 3 x sella and Aronson, 1980).The renal Na+-H+ exchanger is inhibited by the diuretic amiloride (Kinsella and Aronson, 1981b). The Dixon plot for amiloride inhibition of the Naf uptake rate is linear and indicates the presence of a single inhibitory site having a K ; value of 7 to 30 pM, depending on the experimental conditions (Kinsella and Aronson, 1981b; Aronson et al., 1983; Mahnensmith and Aronson, 1985). Amiloride inhibition is rapid in onset and quickly reversible (Kinsella and Aronson, 1981b), implying that its inhibitory site is on the external face of the transporter. In contrast to the results of Ives el al. (1983), we find that amiloride increases the K, for Na+ with no effect on the V,, for Na+ uptake (Kinsella and Aronson, 1981b; Mahnensmith and Aronson, 1985), indicating that amiloride is a simple, competitive inhibitor whose binding is mutually exclusive of the binding of Naf. Raising the external H+ concentration (Aronson et al., 1983) or adding Lit to the external medium (Mahnensmith and Aronson, 1985) increases the K i value with no significant effect on the slope of the Dixon plot for amiloride inhibition of Na+ influx. These findings indicate that the binding of amiloride is also mutually exclusive of the binding of external H+ or Lit. The binding of substances to a protein can be mutually exclusive by virtue of allosteric effects rather than simply as the result of competition for a single physical site. Thus, the number of physically distinct sites actually involved in the mutually exclusive binding of H+, Na+, Lit, and amiloride to the renal Na+-H+ exchanger is uncertain. Nevertheless, the simplest concept to explain these results is that amiloride, Na+, H+, and Li+ all mutually compete for binding at the external transport site of the renal Na+-H+ exchanger, as schematically shown in Fig. 2. The kinetics of inhibition of the renal Na+-H+ exchanger by quinidine differs from that by amiloride (Mahnensmith and Aronson, 1985). Quinidine is a mixed-type inhibitor; it not only increases the K , , but also reduces the V,,, for Na+ influx, indicating that binding of quinidine is not completely mutually exclusive of the binding of Na+. Similarly, quinidine not only increases the Ki values, but also increases the slopes of the Dixon plots for inhibition of Na+ influx by Lit and amiloride, indicating that binding of quinidine is not completely mutually exclusive of the binding of Lit and amiloride. Together, these data signify that quinidine must interact with the renal Na+-Ht exchanger at a minimum of one site that is


65

distinct from the external transport site where Na+, H + , NH; , Lit, and amiloride mutually compete with one another. In contrast to the linear Dixon plots for inhibition of Na+-H+ exchange by external H+, Lit, and amiloride (Aronson et al., 1983; Mahnensmith and Aronson, 1985), the Dixon plot for quinidine inhibition is curvilinear with a Hill coefficient >1.0. One interpretation of this finding is that quinidine interacts at more t h m a single site on the Na+-H+ exchanger. If one such site were the external transport site, it would explain the effect of quinidine to increase the K,,, for Na+ and to increase the K , values for inhibition by amiloride and Li+. This possibility, namely, that quinidine interacts both at the external transport site and at a site distinct from it, is schematically illustrated in Fig. 2. However, a nonlinear Dixon plot with Hill coefficient >1.0 does not necessarily require the presence of more than one binding site for an inhibitor (Segel, 1975). Greater than linear dependence on inhibitor concentration can also arise when an inhibitor binds to a single site on two different conformations of an enzyme or a transporter. Accordingly, the binding of quinidine to a single site on both the unloaded (e.g., To in Fig. 1) and substrate-loaded (e.g., A+Toin Fig. I ) conformations of the Na+-H+ exchanger could alternatively explain its nonlinear, mixed-type pattern of inhibition. Thus, although our data require the presence of at least one quinidine binding site that is separate from the external transport site of the Na+-H+ exchanger, the possible interaction of quinidine at the external transport site itself is uncertain.

111.

BIOCHEMISTRY OF THE RENAL Na+-H+ EXCHANGER

A. Effect of Histidine Modification

As reviewed earlier, external H+ interacts with the renal Na+-H+ exchanger by titrating a single site with an apparent pK value of 7.3 to 7.5 (Aronson et al., 1983). Based on this finding and the fact that the imidazo h m ring of histidine is the principal ionic group in proteins that is titratable at near-neutral pH values, we have evaluated the effect of histidinespecific reagents on the activity of the renal microvillus membrane Na+-H+ exchanger (Grill0 and Aronson, 1986). Photooxidation in the presence of Rose Bengal, a procedure widely used to modify histidine residues albeit with only moderate specificity (Lundblad and Noyes, 1984), progressively and irreversibly inhibits the initial rate of Na+ influx via Na+-H+ exchange. However, under conditions that cause 50% or greater inactivation of transport, Rose Bengal-catalyzed photooxidation

66


significantly disrupts vesicle integrity, rendering more detailed kinetic studies unfeasible. Diethylpyrocarbonate (DEPC), the most selective reagent currently available for covalent modification of histidine residues (Miles, 1977; Lundblad and Noyes, 1984), causes greater than 90% inactivation of Na'-H+ exchange with no significant effect on vesicle integrity. The time course of inactivation by DEPC follows pseudo-first-order kinetics down to below 10% residual activity, suggesting that modification of a single class of histidine residues results in complete loss of transport function. Consistent with this concept, the loss of transport activity resulting from pretreatment of vesicles with DEPC is due entirely to a reduction in V,, with no effect on the K , for Na' influx. Inactivation of Nat-Ht exchange by DEPC is reversed by hydroxylamine, confirming that inactivation results from modification of a histidine residue rather than a sulfhydryl group, another moiety that may possess a pK value in the neutral range. Diethylpyrocarbonate is known to react with imidazolium groups in their unprotonated form (Miles, 1977). Inactivation of the renal Na+-H+ exchanger by DEPC is accelerated when the pH of the external medium is progressively increased from pH 6 to pH 8, but is unaffected when internal pH is similarly varied. This finding indicates that inactivation by DEPC must result from modification of a histidine residue that is present on the external face of the Nat-H+ exchanger. Indeed, the effect of Na' on the process of inactivation by DEPC suggests that this external DEPCsensitive histidine residue may actually be the group that binds H+ at the external transport site. It should be recalled that kinetic studies indicate that the binding of Na+ and the binding of H+ at the external transport site are mutually exclusive. Thus, during pretreatment of vesicles with DEPC at pH 7, when the external transport site is partially protonated, the presence of Na+ enhances inactivation, presumably by displacing H' from its binding site. During pretreatment with DEPC at pH 8, when the external transport site is unprotonated, the presence of Na+ has little effect on inactivation. Additionally consistent with the presence of the DEPC-sensitive histidine residue at or near the external transport site of the Nat-Ht exchanger is the observation that amiloride protects against inactivation by DEPC, presumably as the result of steric hindrance. Of course, alternative explanations have not been excluded. For example, it is possible that the observed effects of pH, Na+, and amiloride on the process of inactivation by DEPC result from long-range allosteric interactions among physically separate sites for Na', H + , and amiloride rather than from direct interactions at a single site. Nevertheless, the hypothesis that an imidazolium ring is the titratable group that binds H t at the external transport site of the renal Nat-H+ exchanger is the simplest explanation for our data, and this possibility is shown schematically in Fig. 3.

67

4. THE RENAL Na+-H- EXCHANGER

FIG. 3. Model of the external transport site of the renal Na*-H+ exchanger

6. Effect of Carboxyl Modification

Although, as just discussed, the titratable group that binds H' at the external transport site of the renal Na+-H+ exchanger appears to be the imidazolium ring of a histidine residue. one would not expect such a group to avidly bind Na+. Anionic groups, such as carboxyls, are more likely candidates for this role. Indeed, Burnham v/ NI. (1982) reported that the carboxyl-activating reagent N-ethoxycarbonyl-2-et hoxy- I ,2-dihydroquinoline (EEDQ) inhibits Na+-H exchange in renal microvillus vesicles. Carbodiimides have been widely used as carboxyl-specific reagents (Lundblad and Noyes, 1984), and we therefore have evaluated the effects of these agents on the activity of the renal microvillus membrane Na+-H' exchanger (Igarashi and Aronson, 1985. 1986).Pretreatment of membrane vesicles with the hydrophobic carbodiimide N,N'-dicyclohexylcarbodiimide (DCCD) causes greater than 80% inactivation of Nal-H' exchange before significant disruption of vesicle integrity begins to occur. The time course of DCCD inactivation down to 20% residual activity follows pseudo-first-order kinetics, suggesting that modification of a single class of carboxyl groups results in irreversible inhibition of transport function. The hydrophilic carbodiimidcs I-ethyl-3-(3-dimethylaminopropyl)carbodiimide and I-cyclohexyl-3-(2-morpholinoethyl)carbodiimidemetho-ptoluenesulfonate are much less potent in causing inactivation than DCCD, implying that the carbodiimide-sensitive carboxyl group is located in a relatively hydrophobic microenvironment. lnactivation of Na+-H' exchange by DCCD proceeds much more rapidly at pH 6 than at pH 7.5. consistent with its reacting with a carboxyl group rather than with an amino, imidazolium, or aliphatic hydroxyl group. Although exogenous nucleophiles such a s glycine methyl ester have no effect, amiloride can completely protect and Lit can partially protect against inactivation by DCCD. These findings suggest that the DCCD-sensitive carboxyl group is located at or near the external transport site of the Na+-H + exchanger, as schematically illustrated in Fig. 3. Of course. the possibility cannot be excluded that protection by umiloride and L i t results from more long+

68


range, allosteric interactions and thus that the DCCD-sensitive carboxyl group is physically remote from the external transport site. The model shown in Fig. 3 can explain the known kinetic and biochemical properties of the Na+-H+ exchanger. According to this scheme, an essential carboxyl group is located at the external transport site and is involved in the binding of cations such as Na+ that are substrates for the transport system. The group that binds H+ at the external transport site is the imidazolium ring of a histidine residue. What then accounts for the competition for binding between H+ and Na+ (or other cations) as observed in kinetic studies? If the external transport site is actually located in a hydrophobic microenvironment, as suggested by the selectivity for inactivation by DCCD compared to hydrophilic carbodiimides, then the presence of unbalanced charge at this site is highly unfavorable thermodynamically. Binding of H+ to the imidazolium ring is stabilized by formation of a hydrogen bond between the protonated imidazolium group and the unprotonated carboxyl group. This in turn tends to preclude the binding of Naf to the site. Dissociation of Hf allows binding of Na+ to the carboxyl group, resulting in another relatively stable, uncharged configuration. Because titration of the imidazolium group would now introduce an unbalanced charge into the hydrophobic microenvironment, such an event is highly unfavorable thermodynamically. Thus, according to this model, the binding of H+ and Naf to the external transport site is mutually exclusive. It should be noted, however, that this model does allow the possibility for simultaneous occupancy of the external transport site by Na+ and H+. If the microenvironment of the site is sufficiently hydrophobic, such simultaneous occupancy cannot be detected at near-physiologic concentrations of Na+ and H + , as in the case of the renal Naf-H+ exchanger. Interestingly, recent evidence suggests that the binding of Na+ and H+ to the external transport site of the Na+-Hf exchanger may not be completely mutually exclusive in the case of lymphocytes (Grinstein et al., 1984) and cultured skeletal muscle cells (Vigne et al., 1982). In these cells the microenvironment of the external transport site may be sufficiently hydrophilic to allow simultaneous occupancy by Na+ and Hf to occur under physiological conditions. C. Labeling of the Transport Protein

The observations that DCCD inactivates the renal Na+-H+ exchanger irreversibly and that amiloride protects against this inactivation suggested a strategy for covalently labeling the transport protein or one of its subunits. Illustrated in Fig. 4 are autoradiograms resulting from an experi-


69

FIG.4. Autoradiogram illustrating labeling of the renal Na+-H+ exchanger. Microvillus membrane vesicles were incubated with 0.2 mM [I4C]DCCDfor 8 hr at 0°C in the absence (left lane) or presence (middle lane) of I mM amiloride, then washed and subjected to SDSpolyacrylamide gel electrophoresis. Molecular weight standards were run in the right lane.

70


ment in which renal microvillus membrane vesicles were incubated with [14C]DCCDin the absence or presence of amiloride, then washed and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Molecular weight standards are shown in the right lane. In the absence of amiloride, exposure to [I4C]DCCDresults in the labeling of multiple polypeptide bands (left lane). In the presence of amiloride, exposure to [i4C]DCCDalso results in the labeling of multiple bands, but the labeling of a single polypeptide having an apparent molecular weight of 100,000 is markedly reduced (middle lane). The labeling of no other polypeptide in the molecular weight range from 12,000 to 250,000 is significantly affected by amiloride (data not shown). Coomassie blue staining of the gel reveals the 100-kDa protein as a faint band comprising less than I % of total membrane protein. These findings tentatively identify the 100-kDa protein as the Na+-H+ exchanger or a subunit thereof. IV. ROLE OF THE RENAL Na+-H+ EXCHANGER IN FACILITATING ANION TRANSPORT A. HCO; Reabsorption

Along the mammalian proximal tubule, over 90% of the bicarbonate load presented by glomerular filtration is reabsorbed and a lumen-acid transtubular pH gradient is generated. As reviewed in detail elsewhere (Aronson, 1983), current evidence suggests that at least 80% of active H+ secretion and HCO, reabsorption across the luminal membrane of the proximal tubule cell takes place via the Na+-H+ exchanger. This process is illustrated in Fig. 5A. Primary active extrusion of Nat across the basolateral surface of the cell generates an out-to-in electrochemical gradient for Na+ across the luminal membrane. This Na+ gradient then serves as the driving force for secondary active H' secretion via the Na+-H+ exchanger. Under physiologic conditions, luminal HCO; serves as the principal acceptor for secreted H+, with its eventual disappearance from the lumen after carbonic anhydrase-catalyzed conversion of HzC03 to CO1 and H20. The intracellular OH- generated by H + secretion combines with C 0 2 to form intracellular HCO.7 through another reaction catalyzed by carbonic anhydrase. Thus, the net effect of active H+ secretion by the Na+-H+ exchanger is the reabsorption of HCO, across the luminal membrane against its electrochemical gradient. Downhill exit of HCO; across the basolateral membrane than completes the process of transtubular reabsorption.

71


A

Cell

Lumen Na*+

Lumen

Cell

.at+

Na'

Lumen

Cell

Not

\ H*

HA

O:-+oH-

Lumen

Cell

D

Lumen

E Na+

A-

Cell

.&

Na+

Fic;. 5 . Role of the lurninal membrane N a ' - t i ' exchanger in facilitating the absorption of HCOl (A). organic anions ( B and C), and C1 ( D and E). See the text for details (c.a.:

carbonic anhydrase.)

B. Organic Anion Reabsorption

There are at least two mechanisms by which Na+-H+ exchange can promote the reabsorption of organic anions across the luminal membrane of the proximal tubule cell. First, the transmembrane H + gradient generated by active H+ secretion via Na+-H+ exchange can drive the net absorption of lipid-soluble weak acids that cross cell membranes predominantly by nonionic diffusion (Fig. SB). For example, an out-to-in H' gradient drives the accumulation of formate by rabbit renal microvillus membrane vesicles (Karniski and Aronson, 1985). In contrast to the carrier-mediated anion transport processes described later, H + gradientstimulated formate uptake is not sensitive to inhibition by 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS). An out-to-in Na+ gradient can also drive the accumulation of formate by renal microvillus vesicles, but Na' stimulation of weak acid uptake is inhibited by addition of ionophores to

72


prevent the generation of a transmembrane pH gradient. These data indicate that Na+ stimulates the uptake of formate as the indirect result of Na+-H+ exchange rather than as the direct result of cotransport with Na+. A second mechanism for organic anion transport indirectly coupled to Na+-H+ exchange is via the carrier-mediated process of OH--anion exchange, as illustrated in Fig. 5C. For example, an out-to-in H+ gradient stimulates uphill urate accumulation in dog renal microvillus membrane vesicles by a process that is saturable and sensitive to inhibitors such as SITS, DIDS, probenecid, and furosemide (Blomstedt and Aronson, 1980). These findings are consistent with carrier-mediated H+-anion cotransport or OH--anion exchange but not with a process of simple, nonmediated, nonionic diffusion. In addition to urate, the same transport system has affinity for C1- (Kahn and Aronson, 1983), and probably all organic anions that have a single anionic group, are at least three carbon atoms in length, and have at least one unsubstituted carbon atom (e.g., PAH, lactate, P-hydroxybutyrate, the monovalent forms of succinate and maleate) (Guggino et al., 1983). This transport system can function in multiple exchange modes involving these anions including, for example, exchange of OH- (or cotransport of H+)with PAH, lactate, or succinate; exchange of C1- with urate or PAH; and exchange of urate with PAH, lactate, or succinate (Blomstedt and Aronson, 1980; Kahn and Aronson, 1983; Guggino et al., 1983). Although many of the organic anions sharing this transport system (e.g., lactate, P-hydroxybutyrate, succinate) are principally reabsorbed by Na+ cotransport systems that are present on the luminal membrane of proximal tubule cells (Wright, 1985), urate is not a substrate for cotransport with Na+ (Kahn and Aronson, 1983) and it is probably reabsorbed at least in part via OH--urate exchange (or H+urate cotransport) indirectly coupled to Na+-H' exchange, as shown in Fig. 5C. C. CI- Reabsorptlon

Two processes have been described by which the active uptake of C1across the luminal membrane of proximal tubule cells can be indirectly coupled to Na+-H+ exchange. One process, illustrated in Fig. 5D, involves Cl--HCO: exchange. As already discussed, the net effect of secondary active H+ secretion by Na+-H+ exchange in the presence of a COJHCO? buffer system is to maintain intracellular HCO: concentration above the level at which HCO: would be in electrochemical equilibrium across the luminal membrane. The resulting in-to-out electrochemical gra-


73

dient for HCO? can then serve as a driving force for tertiary active CIk uptake across the luminal membrane if a Cl--HCO? exchange system is present. In fact, in microvillus membrane vesicles isolated from Necturus kidney, CI- influx is stimulated by an in-to-out HCO: gradient and C1efflux is stimulated by an out-to-in HCOi gradient, consistent with the presence of a CIk-HCO? exchanger (Seifter and Aronson, 1984). Transport of C1- via this pathway is sensitive to inhibitors of red cell CltHCO? exchange such as DIDS, SITS, and furosemide. Direct cotransport of Na+ with C1- cannot be demonstrated in Necturus renal microvillus vesicles, but amiloride-sensitive Na+-H+ exchange is present. Thus, the active, electroneutral, Na+-coupled uptake of C1- that occurs across the luminal membrane of the Necturus proximal tubule cell in situ (Spring and Kimura, 1978) is probably due to CI--HCO7 exchange indirectly coupled to Na+-H+ exchange, as illustrated in Fig. 5D. An interesting property of the Necturus renal microvillus membrane anion exchanger is that in the physiologic range of pH values it can mediate CI--HCO? exchange but not Cl--OH- exchange (or H+-Clk cotransport) (Seifter and Aronson, 1984). This raises the possibility that generation of HCOi from intracellular OH- may under certain conditions be rate limiting for Na+-coupled C1- transport occurring by CIF-HCOT exchange indirectly coupled to Na+-H+ exchange. Although not examined in Necturus renal microvillus membrane vesicles, the stimulation of C1- uptake by an out-to-in Naf gradient in rabbit ileal microvillus membrane vesicles, which also contain Na+-H+ and Cl--HCOy exchangers, is dependent on COz and sensitive to inhibition of carbonic anhydrase with acetazolamide (Knickelbein et al., 1985), findings consistent with the idea that intracellular HCO7 generation can be rate limiting for Na+-coupled CI- transport by this mechanism. Recent evidence suggests that a major component of C1- reabsorption in the rabbit proximal tubule also takes place through an electroneutral, transcellular, Na+-coupled process (Baum and Berry, 1984), but we have been unable to demonstrate appreciable Na+-CI- cotransport or exchange of C1- with OH- or HCOl in microvillus membrane vesicles isolated from rabbit kidney (Seifter et al., 1984). However, an in-to-out formate gradient drives uphill CI- uptake into these vesicles, and an in-toout C1- gradient drives uphill formate accumulation, indicating the presence of a formate-CI- exchange process (Karniski and Aronson, 1985). In contrast to the nonionic diffusion of formic acid that also occurs in these membranes (see preceding discussion), C1--formate exchange is sensitive to inhibition by DIDS. Whereas in the absence of formate an out-to-in H+ gradient only minimally stimulates the uptake of R2Brused as tracer for C1-, the same H+ gradient in the presence of a physiologic formate

74


concentration (i.e., 0.2 mM) markedly stimulates **Brinflux and induces its uphill accumulation. The probable explanation for this observation is that the out-to-in H+ gradient drives uptake of formate by nonionic diffusion of formic acid, and then the resulting in-to-out formate gradient drives Br- influx. In essence, the presence of formate enables a transmembrane H+ gradient to serve as a driving force for CI- uptake. Thus, these data support a model of CI- absorption via CIF-formate exchange indirectly coupled to Na+-H+ exchange, as shown in Fig. 5E. According to this scheme, secondary active H+ secretion by Na+-H+ exchange acts to drive formic acid entry into the cell by nonionic diffusion, and the resulting in-to-out electrochemical gradient for formate then serves as a driving force for tertiary active CI- uptake across the luminal membrane via CIF-formate exchange. D. Anion Transport and lntracellular pH

The various absorptive processes for anions discussed in the preceding section involve the coupling of anion uptake to the inward flux of H+ or the outward flux of OH- or HCO? across the luminal membrane. Thus, each of these transport processes tends to load the cell with acid. As described in detail elsewhere in this volume (see Section I), the plasma membrane Na+-H+ exchanger plays a major role in regulating intracellular pH in diverse tissues. In particular, the Nat-H+ exchanger is a major pathway for extruding intracellular acid loads and maintaining intracellular pH at a significantly higher level than would be the case if HS were in equilibrium with the membrane potential. In this regard, the most important kinetic property of the N a t - H t exchanger is its greater than firstorder dependence on intracellular [H'], likely reflecting the presence of one or more internal modifier sites for H + . The high sensitivity of the rate of Na+-Hf exchange to changes in intracellular [H+]undoubtedly serves to mitigate the impact of anion exchange processes on intracellular pH in the proximal tubule and similar epithelia and enables steady-state rates of Na+-coupled anion transport to proceed at values of intracellular pH compatible with normal cell function.

ACKNOWLEDGMENT The careful typing of the manuscript by Carol Robinson is gratefully acknowledged. This work was supported by NIH grants AM-33793 and AM-17433 and an Established Investigatorship to P. S. Aronson from the American Heart Association.


75

REFERENCES Aronson. P. S . (1981). A m . J . Phvsiol. 240, FI-FII. Aronson. P. S . (1983). A m . J . Phvsiol. 245, F647-F6.59. Aronson. P. S . (1985). Annu. Rru. Pky.siol. 47. 545-560. Aronson, P. S . . Nee, J.. and Suhm, M. A. (1982). Narnw (London) 299, 161-163. Aronson. P. S . , Suhm. M. A., and Nee, J . (1983). J . B i d . Chrm. 258. 6767-6771. Baum, M., and Berry, C. A. ( 1984). J . Clin. /nur.st. 74, 205-21 1. Blomstedt, J . W., and Aronson, P. S. (1980). J . C h i . Inursr. 65, 931-934. Burnham, C.. Munzesheimer, C.. Rabon. E.. and Sachs, G. (1982). Eiochini. Biophy.c.Actrr 685, 260-212. Grillo, F. G . , and Aronson. P. S. (1986). J . B;o/. 2000

300

>2000

50

100

W NH

C

H

2

0(Benzamil)

I50

80

100.0

-

-

The table summarizes data From three different laboratories that describe the effectiveness of a series of amiloride analogs as inhibitors of Na+-H+ exchange. Only a selected group of inhibitors is listed, and the original publications should be consulted for a more extensive list. Also shown in the last two columns are data on the toxicity, i.e., inhibition of functions other than Na+-H' exchange, of a few selected analogs. Note that toxicity and effectiveness as an inhibitor of Na+-H' exchange show poor correlation and that the effectiveness of the various amiloride analogs as inhibitors of Na+-Hi exchange varies by a factor of greater than 10'. Data from Zhuang ef al. (1984). Data from L'Allemain (1984a). Data from Vigne ef a/.(1984). Q

9. CONTROL OF MITOGENIC ACTIVATION OF Na+-H+ EXCHANGE

167

Insights into possible control mechanisms come from a systematic study of the effect of phorbol esters on the mitogenic response. Protein kinase C is a ubiquitous threonine/serine-specific protein kinase. Work in a number of laboratories has shown that this Ca2+-dependent protein requires diacylglycerol for activity. Phorbol esters are potent tumor promoters which have been shown by Nishizuka and co-workers to act as highly potent analogs of diacylglycerols and thereby activate protein kinase C (Nishizuka, 1983). The receptor for phorbol esters detected in binding studies appears to be protein kinase C. Phorbol esters also increase the level of tyrosine phosphorylation of cellular proteins (Gilmore and Martin, 1983; Bishop et al., 1983; Cooper et al., 1984; Grunberger et al., 1984), but this effect must be indirect, since protein kinase C does not have tyrosine kinase activity. Addition of phorbol esters that can activate protein kinase (for example, PMA) at relatively high concentration, lO-’M to A431 cells, activates Na+-H+ exchange, as measured by amiloride-sensitivechanges in pHi as well as by measurement of Na+ uptake (Whiteley et al., 1984). At low concentration, 10-9-10-8 M, PMA does not activate Na+-H+ exchange but dramatically blocks the mitogen-dependent (EGF, serum, or vanadate) activation of Na+-H+ exchange. This is illustrated in Fig. 5 by measurements of pHi. By contrast, PMA does not affect the activation of Na+-H+ exchange by hypertonicity, indicating that PMA does not directly affect the activity of the Na+-H+ antiport. Only phorbol esters that activate protein kinase C show this activity; for example, phorbol deacetate, which does not activate protein kinase C, also does not block the mitogen-dependent activation of Na+-H+ exchange (Whiteley et al., 1984). The effect is not restricted to A431 cells. PMA also blocks PDGF or serum-induced activation of Na+-H+ exchange in NR6 cells (Whiteley et al., 1985). The fact that this inhibition appears to be general, i.e., occurs in many cells and is not restricted to a single mitogen, strongly suggests that protein kinase C acts as a negative modulator for a broad range of mitogen receptors. A possible explanation for these observations comes from a series of reports (Cochet er al., 1984; lwashita and Fox, 1984; Hunter et al., 1984, Friedman er al., 1984) that indicate that protein kinase C can phosphorylate the EGF receptors on threonine residues and that this phosphorylation results in a loss of the EGF-dependent tyrosine kinase activity of this molecule. Taken together, these two sets of observations suggest that protein kinase C acts to modulate (inhibit) the activity of a variety of mitogen receptors and that the tyrosine kinase activity of these receptors is re-

168

D. CASSEL ET AL.

G--pM ;:)f=K;:: vanadr IOOpM C

0.6

+ PMA

-

-

II:_

E. Ca*'-free

100ng/rnl I%+FCSEGF

-

-1

IOrnin

FIG.5 . Effect of phorbol-12-myristate-13 acetate (PMA) on mitogen-induced cytoplasmic alkalinization. Each panel shows the effect of the addition indicated on pHi either added directly to A431 cells or following preincubation of cells with 10 mM PMA for 60 min. Note that preincubation with PMA abolishes changes in pHi due to serum, EGF, or vanadate, but not the change due to hypertonicity (Whiteley et al., 1984).

quired for their activation of the Na+-H+ exchange molecule. A diagrammatic summary of all of these observations is shown in Fig. 6. We can speculate on the regulatory significance of these observations by noting that a variety of hormones and mitogenic polypeptides have been shown to activate phosphatidylinositol breakdown via a pathway that yields triphosphoinositol and diacylglycerol (for review see Berridge and Irvine, 1984). The former results in the release of Ca2+into the cytoplasm from intracellular stores, probably the endoplasmic reticulum, and an elevation in intracellular Ca2+.A simultaneous increase in the concentrations of Ca2+should activate protein kinase C. As shown earlier, activation of protein kinase C inactivates the EGF receptor as a tyrosine kinase. EGF has been shown to activate phosphatidylinositol breakdown in A431 cells (Sawyer and Cohen, 1981), and PDGF to have this effect in 3T3 fibroblasts (Berridge et al., 1984; Brown et al., 1984). Thus each of these mitogens activates an enzyme which can render the mitogen receptor nonfunctional. The net activation of Na+-H+ exchange that will be

9. CONTROL OF MITOGENIC ACTIVATION OF Na*-H+ EXCHANGE

EZI=

pt,;

169

EGF phosphotyrosinr

P,,= photphothrroninr

d = phorbol esters or diocylglycerol

FIG.6. Diagrammatic representation of the activation of Na+-H+ exchange by mitogens and control by PMA. Reaction I is the binding of EGF to its receptor. I t results in a conformational change on the cytoplasmic side of the membrane receptor, which activates the tyrosine kinase activity of this molecule. Reaction 2 is hypothetical: it indicaks the phosphorylation by the kinase of a control molecule which blocks the function olthe Na+-H antiport but is removed from this molecule by phosphorylation. Na+-H+ exchange can now take place. This regulatory molecule is the substrate of a phosphatase which is blocked by vanadate; thus addition of vanadate. like activation of a tyrosine kinase. will activate Na+-H' exchange. The increased intracellular Na' is removed from the cell by the Na', K+-A'rPase (reaction 4). thereby increasing intracellular K'. PMA, an analog of diacylglycerol, binds to protein kinase C. This kiniise now attaches to the cytoplasmic membrane and phosphorylates the EGF receptor o n threonine. The consequence of this modification of the EGF receptor is that the tyrosine kinase activity is attenuated. thus limiting the ability of EGF to stimu1;ite Na'-Hi exchange. +

observed in this system will represent the steady-state level of the active form EGF receptor (or other mitogen receptors) under these conditions. The overshoot in pHi that is often seen when mitogens are added to cells could represent the difference in time required for the mitogenic stimulus to activate Na+-H+ exchange versus the time required to activate protein kinase C. A diagram that illustrates this speculation is shown in Fig. 7 . The notion that mitogens activate Na+-H+ exchange by first activating protein kinase C is made unlikely by these observations. Since PMA

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D. CASSEL ET AL.

H+

FIG. 7. The self-limiting nature of the mitogen-dependent activation of Na+-H+ exchange. The diagram illustrates the speculation that if mitogenic polypeptides stimulate formation of diacylglycerol in cells as well as an increase in cytoplasmic Ca2+,then the concomitant activation of protein kinase C by these endogenous activators will limit the mitogenic response, as illustrated in Fig. 6 and discussed in the text. The magnitude of the response (in this example activation of Na+-H+ exchange) will be a measure of that fraction of the mitogen receptor which is occupied by mitogen and has not been inactivated by phosphorylation or protein kinase C. Reaction 1 is the binding of mitogen to receptor and activation of tyrosine kinase of the receptor. Reaction 2 is the activation of Na+-H+ antiport by the mitogen receptor. Reaction 3 is the activation of “turnover” of phosphatidylinositol (PI) which results in Caz+release from the endoplasmic reticulum by inositol triphosphate (IP,) and formation of diacylglycerol (DAG), both of which activate (reaction 4) protein kinase C, which binds to the cytoplasmic membrane and by phosphorylation inactivates the mitogen receptor (reaction 5 ) .

inactivates the EGF receptor at lower concentrations than it activates Na+-H+ exchange, one would have to conclude that the EGF receptor is the preferred substrate for the protein kinase C and thus that this receptor would be inactivated well before activation of Na+-H+ exchange would be turned on. The activation of Na+-H+ exchange by EGF takes place in the absence of external Ca2+(Rothenberg et al., 1983a) and so the inacti-

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vation of the EGF response by low doses of PMA (Whiteley et al., 1985). In contrast, activation of Na+-H+ exchange by high concentrations of PMA only occurs in the presence of extracellular Ca2+,again suggesting that the activation of Na+-Hf exchange by EGF is not mediated via protein kinase C. Phorbol esters act as mitogens in a number of cell types, but with other cell types inhibit the mitogenic response. For example (C. K. Osborne et al., 1981; Chandler et al., 1980), we would speculate that the former effect is due to activation of one or more tyrosine kinases while the latter effect is due to the inactivation of protein kinase C of the mitogen receptor. The data presented have, for simplicity, mainly described experiments using A431 cells and EGF as the mitogen. Work in our own, as well as other, laboratories suggests that the general features of the regulation of Nat-Hf exchange observed in these cells apply equally to other cell types, such as fibroblasts of various origins, and to other mitogens, such as PDGF. It is likely that this regulatory mechanism will also apply to a variety of cell surface hormone receptors, and work in other laboratories has shown that activation of protein kinase C “desensitizes” the p-adrenergic receptor (Sibley et al., 1984; Kelleher et al., 1984) and the glucagon-stimulated adenylcyclase (Hayworth er al., 1984).This generalization is potentially important because A431 cells are malignant cells which do not require EGF for cell growth. The descriptive aspects of the activation of Na+-H+ exchange by mitogens are rapidly coming to an end; the next phase will require an understanding of these events at a molecular level. The recent availability of a mutant cell lacking the Naf-H+ antiport represents a major advance in this direction (See Chapter 12 by Pouyseggur er al., in this volume). ACKNOWLEDGMENTS The work presented in this chapter was supported by grant GM 18405 from the NIH and by grants from Monsanto Chemical Company. P. Rothenberg was supported by grant GM 02016; B . Whiteley was supported by grant OM 07067; D. Mancuso by grant CA 091 18. REFERENCES Aronson, P. S . (1983). Am. J . Physiol. 245, F647-659. Aronson. P. S . , Nee, J . , and Suhm, M. A. (1982). Nature (London) 299, 161-163. B e r r i d g . 1 . J., and Irvine, R. F. (1984). Nature (London) 312, 315-321. Berridge, M. J . , Heslop, J . P., Irvine, R. F . , and Brown, K. D. (1984).Biochern. J . 222, 195201.

Bishop, B., Martinez, R . , Nakamura, K . D., and Weber, M. J. (1983). Biochem. Biophys. Res. Commun. 115, 536-543. Brown, K. D., Blay, J . , Irvine, R. F., Heslop. J . P., and Berridge, M. J . (1984). Biochem. Biophys. Res. Commun. 123,377-384.

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Carpenter, G. (1984). Cell 37, 357-358. Cassel, D., Rothenberg, F., Zhuang, Y. X., Deuel, T. F., and Glaser, L. (1983). Proc. Natl. Acad. Sci. U . S . A . 80, 6224-6228. Cassel, D., Whiteley, B., Zhuang, Y. X., and Glaser, L. (1985). J . Cell. Physiol. l22, 178186. Chandler, C. E., and Henchman, H. R. (1980). J . Cell. Physiol. 105, 275-285. Cochet, C., Gill, G. W., Meisenhelder, J., Cooper, J . A., and Hunter, T. (1984). J . Biol. Chem. 259, 2553-2558. Cooper, J. A., Sefton, B. M., and Hunter, T. (1984). Mol. Cell. Biol. 4, 30-37. Friedman, B. A., Frackelton, A. R., Ross, A. H., Connors, J. M., Fujiki, H., Sugimura, T., and Rosner, M. R. (1984). Proc. Natl. Acad. Sci. U . S . A . 81, 3034-3038. Gilmore, T., and Martin, G . S. (1983). Nature (London) 308, 487-490. Grunberger, G., Zick, Y., Taylor, S. I., and Gorden, P. (1984). Proc. Nut/. Acad. Sci. U.S.A. 81, 2762-2766. Hayworth, C. M., Whitton, A. D., Kinsella, A . R., and Houslay, N. D. (1984). FEES Lett. 170, 38-42. Heldin, C . H., and Westermark, B. (1984). Cell 37, 9-20. Hunter, T., Ling, N., and Cooper, J. A. (1984). Nuture (London) 311, 480-483. Iwashita, S., and Fox, C. F. (1984). J . B i d . Chem. 259, 2559-2567. Kelleher, D. J., Pessin, J. E., Ruoko, A. E., and Johnson, G. L. (1984). Proc. Nutl. Acud. Sci. U . S . A . 81, 4316-4320. Kent Osborne, C., Hamilton, B.. Noves, M., and Ziegler, J. (1981). J . Clin. Invest. 67,943951. L’Allemain, G . , Franchi, A., Cragoe, E., Jr., and Pouyssegur, J. (1984a). J . Biol. Chem. 259,4313-4319. L’Allemain, G . , Paris, S., and Pouyssegur, J. (1984b). J . Biol. Chern. 259, 5809-5815. Mancuso, D., and Glaser, L. (1985). J . Cell. Physiol. 123, 297-304. Moolenaar, W. H., DeLaat, S. W., Mummery, C. L., and Van der Saag, P. T. (1982). 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. Nishizuka, Y.(1983). Nature (London) 308, 693-698. Okada, C. Y . , and Rechsteiner, M. (1982). Cell 29, 33-41. Okhuma, S., and Poole, B. (1978). Proc. Nutl. Acad. Sci. U . S . A . 75, 3327-3331. Pardee, A. B., Dubrow, R., Hamlin, J. C., and Kletzien, R. F. (1978). Annu. Rev. Biochem. 47, 715-750. Paris, S., and Pouyssegur, J. (1984). J . B i d . Chern. 259, 10989-10994. Pruss, R. M . , and Henchman, H. R. (1977). Proc. Natl. Acud. Sci. U . S . A . 74, 3918-3921. Reuss, L., Cassel, D., Rothenberg, B., Whiteley, B., Mancuso, D., and Glaser, L. (1986). Curr. Top. Membr. Tramp., in press. Rothenberg, P., Reuss, L., and Glaser, L. (1982). Proc. Nutl. Acad. Sci. U.S.A. 79,77837787. Rothenberg, P., Glaser, L., Schlesinger, P., and Cassel, D. (1983a). J . Biol. Chem. 258, 4883-4889. Rothenberg, P., Glaser, L., Schlesinger, P., and Cassel, D. (1983b). J . Biol. Chem. 258, 12644-12653. Sawyer, S . T., and Cohen, S. (1982). Biochemistry 20, 6280-6286. Schuldiner, S., and Rozengurt, E. (1982). Proc. Nutl. Acad. Sci. U.S.A. 79, 7778-7782. Sibley, D. R., Nambi, P.. Peters, J . R., and Lefkowitz, R . J. (1984). Biochem. Biophys. Rrs. Commun. 121,973-979. Smith, J. B., and Rozengurt, E. (1978). Proc. Nut/. Acud. Sci. U . S . A . 75, 5560-5564.

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Swarup, G., Cohen, S . , and Garbers. D. L. (1982). Biochem. Eiophys. Res. Comrnun. 107, 1104-1 109. Vigne, P., Frelin, C . , Cargoe, E. J . . Jr., and Lazdunski, M. (1984). Mol. Pharmrccd. 25, I3 1-136. Whiteley, B., Cassel, D., Zhuang. Y . X . , and Glaser, L. (1984). J . CelIEiol. 99, 1162- 1166. Whiteley, B . , Deuel, T., and Glaser, L. (1985). Eiochern. Eiophys. Res. Comm. 129, 854861. Zhuang, Y. X . , Cragoe, E. J . , Jr., Shaikewitz. T., Glaser, L., and Cassel, D. (1984). Biochemistry 23, 4481-4488.


CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 26

Chapter 10 Mechanisms of Growth Factor Stimulation of Na+-H+ Exchange in Cultured Fibroblasts MITCHEL L . VILLEREAL,' LESLIE L . MIX-MULDOON,* LUCIA M . VICENTINl,* GORDON A . JAMIESON, JR.,' A N D NANCY E. OWEN? *Department of Pharmacological and Physiological Sciences University of Chicago Chicago, Illinois and tDepartmeni of Biological Chemistry and Structure Chicago Medical School North Chicago, Illinois

I . Introduction ................

. . . . . . . . . . . 175

11. Stimulation of Na+ Influx in H 111. Characterization of the Transport System Mediating Mitogen-Stimulated

Na+ Influx . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Involvement of Ca2+in the Mitogen Activation of the Na+-H+ Exch V. Possible Role of Protein Kinase C in the Activation of Na+ Exchange in HSWP Cells and Other Fibroblast Systems.. .......................... VI . Evidence for Phospholipase Involvement in the Mitogen Activation of Na+-H+ Exchange in HSWP Cells ........................ VII. of Intracellular Ca2+ ................ ............... VIII. IX . Summary of the Proposed Mechanism for Activation of the Na+-H+ Exchanger by Mitogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.

182 185

187 187 189 191

INTRODUCTION

When nontransformed fibroblasts are grown in culture, there is an absolute requirement for the presence of growth factors in addition to the 175 Copyright 0 1986 by Academic Press. Inc. All nghts of reproduction in any form reserved.

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MITCHEL L. VILLEREAL ET AL.

normal complement of salts, amino acids, and vitamins present in the culture medium. If these growth factors are removed, the cells become arrested in the Go phase of the cell cycle. If growth factors are then added to these quiescent cells, a defined sequence of events occurs which leads eventually to cell division. Over the past four years our laboratory has been interested in defining the sequence of events which occurs after the binding of growth factors to their surface receptors. We decided that if we could select an early event initiated by growth factors and investigate the mechanism by which growth factors stimulate this early event, then we might obtain some valuable information concerning the action of growth factors. The early growth factor-induced event that we chose to investigate is the activation of the Na+-H+ exchange system which is present in the plasma membrane of a number of different cell types. The cell system that we chose to work with is a cultured human fibroblast strain (HSWP cells) which has normal growth characteristics in cell culture. In this chapter we will describe the activation of Na+ influx by growth factors in HSWP cells, discuss evidence that the Na+ influx is mediated by a Na+H+ exchange system, and describe our current hypothesis for how this transport system is activated in HSWP cells. In addition, we will discuss evidence in other fibroblast lines which suggests that there may be different mechanisms for regulating this transport system in different cell types. II. STIMULATION OF Na+ INFLUX IN HSWP CELLS BY SERUM AND PEPTIDE MITOGENS

Smith and Rozengurt (1978a) demonstrated that the addition of serum to cultured mouse fibroblasts (3T3 cells) would stimulate the influx of Na+ as measured by either **Na+influx or net Na+ influx. They subsequently demonstrated that serum would also stimulate Lit uptake by this pathway and that the serum-dependent Li+ uptake was inhibited by the Na+ transport inhibitor amiloride (Smith and Rozengurt, 1978b). Subsequent work from our laboratory demonstrated that amiloride had no effect on basal Na+ influx into HSWP cells, while it totally blocked the serum-sensitive component of Na+ influx, suggesting that serum stimulates a Na+ transport system that is virtually inactive under basal (growth factor-free) conditions (Table I). The serum activation of the amiloride-sensitive Na+ transport system is rapid since it appears to be fully stimulated as early as 30 sec (Villereal, 1981) after addition of serum. In addition to serum, several purified peptide mitogens are capable of activating the Na+ transport system in HSWP cells. Na+ influx can be stimulated by epidermal growth factor (EGF), vasopressin, and bradykinin (Owen and Villereal,

10. GROWTH FACTOR REGULATION OF Na+-H+ EXCHANGE

177

TABLE I EFFECTOF SERUM A N D AMILORIDE ON Na+ INFLUXI N HSWP CELLS"

Assay conditions

Na' influx (pmol/g protein-min)

Serum free Serum free + amiloride 10% Serum 10% Serum + amiloride A23 I87 A23187 + amiloride

5.0 2 0.2 4.9 C 0.3 26.1 C 1.2 5.5 C 0.8 23.2 2 1.4 5.2 t 0.5

Cells were serum deprived in HEPES-buffered Eagle's minimum essential medium plus 0.1%' fetal bovine serum. They were then assayed in either serum-free medium, medium containing 10% serum, or medium containing 5 pglml A23187. Net Na' uptake was assayed over a 5-min period in the presence of the Na+-K+ pump inhibitor digitoxin. Values for fluxes in the presence of amiloride were obtained from kinetic analyses by extrapolating to infinite amiloride concentration.

1983a). Although these individual peptides do not stimulate Na+ influx as effectively as serum, the combination of these three peptides can give as large a stimulation as seen with serum. All three of these peptides are able to produce a mitogenic response in HSWP cells, and the combination of the three stimulates the synthesis of DNA in these cells as effectively as serum (Owen and Villereal, 1983a). The Na+ influx stimulated by these peptide factors seems to be mediated by the same pathway as that responsible for serum-stimulated Na+ influx since the influx of Na+ induced by these factors is totally blocked by amiloride. 111.

CHARACTERIZATION OF THE TRANSPORT SYSTEM MED1AT1NG MITOGEN-STIMULATED Na INFLUX +

The mitogen-stimulated N a t influx appears to be mediated by a Na+H+ exchange system in HSWP cells. Although the initial observation that the serum-stimulated Na+ flux is amiloride sensitive suggested involvement of Na+-H+ exchange, it must be kept in mind that there are at least two other Na+ transport systems which are amiloride sensitive, namely, the Na' channel in tight epithelial cells and the Na+-Ca2+ exchanger. Thus, to determine whether a Na+-H+ exchanger was mediating the se-

178


rum-induced Na+ flux, we investigated the effects of pH on Na+ transport as well as the effect of activating the serum-sensitive Na+ influx pathway on intracellular pH. In early studies we observed that lowering the external pH would inhibit the influx of Na+ in the presence of serum, which is consistent with the competition of protons for the Na+ binding site on a Na+-H+ exchanger. Recent studies utilizing a pH-sensitive intracellular fluorescent probe (dimethylcarboxyfluorescein) demonstrated that the stimulation of HSWP cells by mitogens would produce an intracellular alkalinization (Fig. 1A) which was dependent on the presence of

40

30 20

B 40_ltmiloride

Wsh 150 Na

A23187

J.

\

20 10

'

I

1

I

I

I

I

2

4

6

8

10

12

Time, minutes FIG. I . Effect of growth factors and A23187 on intracellular pH in HSWP cells. Cells grown on glass coverslips were serum deprived for 4 hr and then loaded with the pHsensitive fluorescence probe dimethylcarboxyfluorescein. Cells washed free of extracellular probe were monitored for their fluorescence in a photon-counting microspectrofluorometer. (A) Cells were first given growth factors (EGF, 100 ng/ml + vasopressin, 100 ng/ml + bradykinin, 100 ng/ml + insulin, 1 &ml) in the presence of 150 mM ChCl (Na free). The ChCl medium was then replaced by medium containing 150 mM NaCl and growth factors added a second time. (B) Cells were incubated in the presence of 3 mM amiloride and 0.2 WLg/ ml A23187 was added. The amiloride and ionophore were then washed away and A23187 added a second time. (For details, see Muldoon et al., 1985).


179

external Na+ and could be blocked by the presence of amiloride (Muldoon et al., 1985). These observations confirmed in HSWP cells observations in a number of other cell systems that the Na' influx in response to various stimuli is the result of stimulation of Na+-H+ exchange (Johnson et al., 1979; Cassel et al., 1983; Moolenaar et al., 1983). In addition to the intracellular pH data indicating that the amiloridesensitive pathway which is activated is the Na+-H+ exchange system, there is also pharmacological and transport evidence which supports this hypothesis. Studies from a number of laboratories including our own have indicated that certain analogs of amiloride have very high affinity for inhibition of Na+ flux in fibroblast systems (O'Donnell et al., 1983; L'AIlemain et al., 1984; Lazdunski et al., 1983). The modification of amiloride which gives a higher affinity for inhibition of Na+ flux in these cells is known to reduce the affinity of these analogs for inhibition of the Na+ channel in tight epithelia. The fact that these agents inhibit mitogen-stimulated Na+ flux argues against involvement of epithelial-like amiloride-sensitive Na+ channels in this process. Furthermore, the Na+ flux in HSWP cells does not appear to be mediated by a Na+-Ca2+ exchanger either, because the Na+ influx is approximately an order of magnitude larger than the corresponding efflux of Ca2+and there is no observable Na' stimulation of Ca2+efflux in HSWP cells (Owen and Villereal, 1983b). Thus, it seems highly unlikely that either an epithelial-type Na+ channel or a Na+Ca2+ exchanger is involved in the mitogen-stimulated Na+ influx in HSWP cells. IV. INVOLVEMENT OF Ca2+ IN THE MITOGEN ACTIVATION OF THE Na+-H+ EXCHANGER

Now that the pathway which mediates the mitogen-induced Na+ influx has been described, it is time to ask how the Na+-H+ exchanger is activated in response to binding of these peptides to their surface receptors. Several years ago we began to investigate the possibility that Ca2+may serve as a second messenger for this activation process. There are several criteria which must be met in order to demonstrate that Ca2+ serves a second messenger function in this process: (1) an artificially induced elevation of intracellular Ca2+ should activate the Na+-H' exchanger; (2) mitogens should induce an elevation of the intracellular Ca2+activity; (3) an agent which can block the mitogen-induced rise in intracellular Ca2+ activity should block the activation of the Na+-H+ exchanger by mitogens; (4) there should be some logical mechanism by which an elevation of intracellular Ca2+ could activate the Na+-H+ exchanger. We will

180


briefly describe the data which satisfy these four criteria and thereby support the involvement of intracellular Ca2+in the process for activation of the Na+-H+ exchanger. The first attempt to implicate intracellular Ca2+as a second messenger in the activation of the Na+-H+ exchanger in HSWP cells utilized the Ca2+ionophore A23 I87 to artificially elevate the intracellular Ca2+concentration. It was shown that a concentration of A23187 which would stimulate a 5 x increase in Ca2+influx (Villereal, 1981) in the absence of serum would dramatically stimulate the influx of Na+ (Table I). The Na+ influx stimulated by A23 187 is totally inhibited by amiloride. Subsequent studies showed that influx could also be stimulated by the Ca2+ionophore ionomycin, which as we have shown stimulates a rise in intracellular Ca2+ activity in HSWP cells (Mix et al., 1984). Recent measurements of intracellular pH demonstrate that the addition of A23 187 will stimulate a dramatic alkalinization of the intracellular pH (Fig. IB). This alkalinization is for the most part dependent on the presence of extracellular Na+ (Muldoon et af., 1985), although there is a slight alkalinization in the absence of Na+ which is presumably due to A23187-mediated exchange of extracellular Ca2+for intracellular protons. The alkalinization of the intracellular pH in response to A23187 can be blocked by the addition of amiloride (Fig. IB) or by the removal of extracellular Ca2+(Muldoon et al., 1985), although long-term incubation with high doses of ionophore, which should mobilize intracellular stores of Ca2+,will stimulate the Na+-H+ exchanger and thereby lead to intracellular alkalinization. Thus, when taken together, these data suggest that the first criterion for involvement of intracellular Ca2+as a second messenger has been met. It has been stated earlier that if Ca2+is a second messenger in the activation process, then we should be able to measure a mitogen-induced rise in intracellular Ca2+activity. To investigate this possibility we used the Ca2+-sensitiveintracellular fluorescence probe quin 2, first described by Tsien (Tsien et al., 1981), to monitor intracellular Ca2+ activity in HSWP cells. It was shown that the addition of mitogens to quin 2-loaded cells would induce a transient rise in cellular fluorescence which was indicative of a rise in intracellular Ca2+activity (Mix et af., 1984). Since the intracellular pH studies indicate that mitogens can stimulate Na+-H+ exchange in the absence of extracellular Ca2+(Muldoon et a l . , 1985), it appeared that the Ca2+for the activation process is mobilized from intracellular stores. To test this possibility, we determined the effect of the intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) on the mitogen-induced rise in intracellular Ca2+activity. This compound has been proposed to block the mobilization of intracellular Ca2+in platelets (Rittenhouse-Simmons and Deykin,

181


1978) and smooth muscle (Maligodi and Chiou, 1974). We found that the addition of TMB-8 totally blocked the mitogen-induced rise in intracellular Ca2+activity (Fig. 2). The contention that TMB-8 acts by blocking the mobilization of intracellular Ca2+was supported by another series of experiments during which the effect of this compound on Ca2+efflux was determined. These studies showed that the addition of mitogens to 45Ca2+loaded cells induced a stimulation of 4SCa2tefflux which is consistent with a mobilization of intracellular Ca2+and the addition of TMB-8 blocked the mitogen-induced efflux of intracellular Ca2+(Owen and Villereal, 1983). The third criterion for Ca2+ involvement as a second messenger, namely, that blocking the rise in Ca2+activity should block the activation of Na+-H+ exchange, was tested by determining the effect of TMB-8 on the mitogen-induced activation of Na+-H+ exchange. It was found that the addition of TMB-8 blocked the serum stimulation of Na+ influx with a Ki of approximately 10 pM.The effect of TMB-8 appeared to be the result of its action on Ca2+rather than some nonspecific effect at the level of the membrane or the transport system, because TMB-8 had no effect on the stimulation of Na+ influx by A23187 (Owen and Villereal, 1982a). Thus, since blocking the mitogen-induced rise in Ca2+activity does block the activation of Na+-H+ exchange, the third criterion has been met. To test for a possible mechanism by which a rise in intracellular Ca2+ activity could activate the Naf-H+ exchanger, we investigated the effects

I

t

2

4

6

8

10

12

Time, minutes FIG. 2. Effect of TMB-8 on growth factor-induced increase in intracellular Ca2+ in HSWP cells. Cells grown on glass coverslips were serum deprived for 4 hr and then loaded with the Ca2+-sensitivefluorescence probe quin 2. Cells washed free of extracellular quin 2 were monitored for their fluorescence in a photon-counting microspectrofluorometer. At the first arrow, TMB-8 was injected into the flowthrough chamber to a final concentration of SO p M . A cocktail of growth factors (EGF, 100 ngiml: vasopressin, 100 ng/ml; bradykinin, 100 ng/ml; insulin, I pg/ml) was injected at the second arrow. The cells were then washed free of both drug and growth factors and growth factors were added again at the fourth arrow. (From Mix ef al., 1984.)

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of calmodulin inhibitors to determine whether Ca2+ could act via this Ca2+-dependent regulatory protein. It was found that a series of six known calmodulin inhibitors, the prototype of which was trifluoperazine (TFP), would inhibit both the serum-stimulated and the A23 187-stimulated influx of Na+ while having no effect on the basal Na+ influx (Owen and Villereal, 1982b). There was excellent agreement between the concentration of each agent needed to bind to calmodulin and the concentration needed to block the activation of Na+ influx. Subsequent studies showed that the naphthalene sulfonamide calmodulin inhibitor W13 was a potent inhibitor of the activation of the Na+-H+ exchanger while the much less potent dechlorinated analog W 12 was reasonably ineffective in blocking the activation of this transport system (Owen and Villereal, 1982a). These data suggest that the effect of Ca2+on the activation of the Na+-H+ exchanger may be mediated by calmodulin.

V.

POSSIBLE ROLE OF PROTEIN KINASE C IN THE ACTIVATION OF Na+ EXCHANGE IN HSWP CELLS AND OTHER FIBROBLAST SYSTEMS

We recently began to investigate the possible involvement of protein kinase C in the mitogen-induced activation of Na+-H+ exchange in HSWP cells. there were two reasons for suspecting the involvement of this kinase in the activation process. First, recent studies have demonstrated that many of the calmodulin antagonists that we used in earlier studies also inhibit the function of protein kinase C, suggesting that this enzyme, rather than calmodulin, may be the important one in the activation sequence. Second, studies from Rozengurt’s laboratory demonstrated that the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) would stimulate Na+ influx in 3T3 cells (Dicker and Rozengurt, 1981). Although this study predated the knowledge that the primary target for TPA is protein kinase C, in light of this new information the observation suggests a role for protein kinase C in the regulation of Na+ transport. Therefore, we investigated the effects of TPA on Na+ influx in HSWP cells. Our initial studies with TPA involved treating serum-deprived cells with various doses of TPA in the absence of mitogens to determine whether the addition of this compound alone could activate Na+ influx in HSWP cells. The outcomes of numerous experiments were negative. The dose range tried was extensive (1 ng/ml to 1 Fg/ml) and the cells were


183

preincubated with TPA for times between 1 min and 1, hr. The experiments were performed with cells at low and high densities that had been serum deprived for the normal time period used in our studies (4 hr) and with cells that had been arrested following chronic periods of serum deprivation (4 days). Under no circumstances was Na+ influx stimulated by the addition of TPA alone (Vicentini and Villereal, 1985). However, we also investigated the possibility that TPA might synergize with submaximal doses of A23187. Cells were preincubated with TPA for 2 min and then a dose of A23187 which gave a small stimulation of Na+ influx was added, There was a dramatic synergism between A23187 and TPA (Table 11). We were also interested in whether TPA could synergize with submaximal doses of mitogens. Thus, we preincubated cells with TPA for 2 min and then added low doses of growth factors. We found that instead of synergizing with growth factors, TPA actually inhibited the growth factor stimulation of Na+ influx (Table 11).At present it is too early to provide a detailed explanation for these data, but we will offer a couple of suggestions for how TPA could be working in the HSWP cell system. First, it is clear that TPA alone is not a sufficient signal to activate Na+-H+ exchange in HSWP cells. The question is whether protein kinase C is activated by TPA alone or whether there is a need for intracellular Ca2+to be elevated

TABLE I1 EFFECTOF TPA ON Na+ INFLUX HSWP CELLS~'

Assay conditions Serum free Growth factors TPA A23 I87 A23187 f TPA Growth factors

+ TPA

IN

Na' influx (pmol/g protein-min) 4.5 t 0.2 23.8 t 1 . 1 4.9 t 0.5 9.8 f 0.4 19.3 1.5 8.9 0.7

* *

Net Na' influx was measured as described in Table I . Cells were stimulated with a growth factor cocktail containing 100 ng/rnl epidermal growth factor, 100 ng/ml vasopressin, 100 n/ml bradykinin, and 1 pg/ml insulin. TPA was present at 100 ng/ml and A23187 was present at 1 pg/ml. Cells were preincubated with TPA for 2 rnin prior to initiation of Na+ influx.

184


to produce an activation of this kinase. In general it is thought that TPA can activate protein kinase C in most tissues in the absence of a rise in intracellular Ca2+ activity by increasing the affinity of this enzyme for Ca2+.In addition, the effect of TPA preincubation on the growth factor response suggests that the addition of TPA alone is producing some effect which is presumably mediated by protein kinase C. However, if A23187 and TPA are not synergizing at the level of the protein kinase C, at what level are they cooperating? One possibility is that the Na+-H+ exchanger is regulated by two phosphorylation events in HSWP cells, one of which is mediated by calmodulin in response to a rise in the intracellular Ca2+ activity and another which is mediated by protein kinase C in response to TPA or its physiological equivalent diacylglycerol. The regulation of the sarcoplasmic reticulum protein phospholamban by Caz+-dependent and CAMP-dependent kinases provides a precedent for regulation of the function of a protein by multiple phosphorylations. A question that arose from the work in the HSWP cell is why 3T3 cells seem to respond to TPA with an activation of Na+ influx while HSWP cells do not. Thus, we felt that it was important to try to duplicate these results in 3T3 cells. We found that the addition of TPA alone produced a significant stimulation of Na+ influx in 3T3 cells although maximal doses produced only about 50% of the stimulation seen with serum (Vicentini and Villereal, 1985). The synergistic effects between A23187 and TPA seen in HSWP cells were not observed in 3T3 cells nor were the inhibitory effects of TPA on the mitogen stimulation of Na+ influx seen. Since investigations in two cell systems produced quite different results in terms of the regulatory mechanism for activation of Na+ influx, it seemed important to test other cell lines to determine the generality of either or both mechanisms. To date we have performed similar experiments in two other cell systems and we found one, another human foreskin fibroblast, which behaves identically to HSWP cells and another, a human lung fibroblast (WI-38), which behaves like the 3T3 cell system (Vicentini and Villereal, 1985). Investigations are currently under way in other fibroblastic lines to determine into which category they can be classified. At present all that can be concluded is that there appears to be some diversity between cell lines concerning their mechanism for regulating the Na+-Hf exchange system in response to TPA. Whether that necessarily means that there is diversity between these cells in their mechanism for regulating Na+-H+ exchange in response to mitogens remains to be determined. It should be pointed out that since the doses of TPA necessary to activate Na+ flux are quite high in comparison to the Kd for specific TPA binding to its receptor site, it is possible that the activation of Naf-H+ exchange is via a mechanism other than protein kinase C-induced phosphorylation.


VI.

185

EVIDENCE FOR PHOSPHOLIPASE INVOLVEMENT IN THE MITOGEN ACTIVATION OF Na+-H+ EXCHANGE IN HSWP CELLS

There were initially two reasons for expecting that phospholipases may be involved in the mitogen activation of the Na+-H+ exchanger. First, our laboratory demonstrated that bradykinin, a peptide known to stimulate phospholipase activity in fibroblasts (Hong and Deykin, 1981), would stimulate both Na+ influx and cell growth in HSWP cells. Second, the work of Rozengurt et al. (1981) demonstrated that melittin, a compound which stimulates phospholipase activity, would stimulate Na+ influx in 3T3 cells, although these authors suggested that the effects of melittin were the result of nonspecific leaks created in the 3T3 cell membrane. On the basis of these two observations, we began to investigate the involvement of phospholipase activity in the regulation of Na+-H+ exchange in HSWP cells. The initial experiments to test for phospholipase involvement in the activation sequence utilized the known inhibitors of phospholipase activity, mepacrine and the Upjohn drug U-1002, to determine whether inhibition of phospholipase activity would block the mitogen activation of the Na+-H+ exchange system. It was found that both mepacrine and U-1002 would totally block the serum-stimulated Na+ influx in HSWP cells while having no effect on the basal Na+ influx (Vicentini et al., 1984). The absence of any effect on basal flux suggests that these agents are not having nonspecific effects at the membrane level. There was a good correlation between the dose response curves for inhibition of serum-stimulated phospholipase activity and the inhibition of Na' flux activation by these two agents. In addition to inhibiting the serum-induced Na+ influx, both of these agents blocked the mitogen-induced alkalinization of the intracellular pH (Muldoon et al., 1985). It was mentioned earlier that melittin stimulates Na' influx into 3T3 cells but that the authors of that paper felt that these effects were due to a nonspecific leak. We sought to test the alternative explanation that melittin was activating the Na+-H+ exchange system. It was found that in HSWP cells the addition of melittin to cells in the absence of serum induced a large increase in Na+ influx (Fig. 3) similar to that seen in 3T3 cells. However, these studies demonstrated that virtually all of the melittin-induced Na+ influx could be inhibited by amiloride, suggesting that the flux was being mediated by the same transport pathway which is activated by mitogens. The doses required to stimulate Na+ flux are in good agreement with the doses required to stimulate phospholipase activity, as measured by the release of arachidonic acid (Vicentini et d., 1984). Stronger

186


1

0

2

4

6

1

Time, min

I

200

I

I

400

600

I

800

I

1000

[Melittin] nqlml

FIG. 3. Melittin stimulation of Na+-H+ exchange in HSWP cells. Cells plated on 60-mm culture dishes were serum deprived for 4 hr and then assayed for net Na+ influx following the addition of the Na+-K+ pump inhibitor digitoxin. Points are means 5 SE of five replicate determinations in serum-free medium containing various concentrations of melittin. 0, control; 0, 2 mM amiloride; @, 100 p M mepacrine. Inset: Effect of melittin on intracellular pH. Intracellular pH was monitored as described in the legend of Fig. 1. Cells were incubated with 3 mMamiloride. Melittin was injected at the arrow to give a final concentration of 500 ng/ml. Then the cells were washed free of drugs with 150 mM Na+ medium and melittin was reinjected at the last arrow. For details on the Na+ flux measurements see Vicentini et a/. (1984) or on the pH measurements see Muldoon ef al. (1985).

evidence for the activation of the Na+-H+ exchange pathway by melittin was provided by our studies on the effect of melittin on the intracellular pH of HSWP cells (Muldoon et al., 1985). The addition of melittin to cells loaded with the pH-sensitive fluorescent probe dimethylcarboxyfluorescein in the absence of mitogens induces a dramatic intracellular alkalinization (Fig. 3 , inset). The alkalinization is dependent on the presence of Na+ in the extracellular medium (Muldoon et al., 1985) and can be blocked by the addition of amiloride (Fig. 3, inset).


187

VII. POSSIBLE INVOLVEMENT OF PHOSPHOLIPASES IN THE MOBILIZATION OF INTRACELLULAR Ca2+

Since there is substantial data that a mobilization of intracellular Ca2+is involved in the activation of the Na+-H+ exchanger in HSWP cells, it seemed important to ask whether the phospholipase activity might be involved in this mobilization process. The first step in investigating this possibility was to determine whether the phospholipase activation was upstream or downstream from the Ca2+mobilization. This was tested by determining whether the phospholipase inhibitors would block the A23187 stimulation of Na+-H+ exchange, with the thought that if the phopholipase effect were downstream from Ca2+ mobilization, or on a parallel pathway, then the inhibitors should still block the A23 187 stimulation of transport. However, we found that the addition of these inhibitors had no significant effect on the A23187-stimulated flux while they inhibited approximately 90% of the serum-stimulated flux in this same set of experiments (Vicentini et al., 1984). This observation suggests that the phospholipase activation occurs upstream from the Ca2+mobilization and also suggests the possibility that the phospholipase activity could be causative for Ca2+mobilization. The causal relationship between phospholipase activity and Ca2+mobilization was tested in two ways. First in quin 2-loaded cells the effect of phospholipase inhibitors on the mitogen-induced rise in Ca2+activity was tested. The addition of the phospholipase inhibitor U-1002 was quite effective in blocking the mitogen-induced rise in intracellular Ca2+activity (Muldoon et al., 1985). Second, the effect of the phospholipase activator melittin on intracellular Ca2+activity was tested. Melittin was quite effective in stimulating a rise in intracellular Ca2+activity in HSWP cells (Mix et al., 1984) and its effects were blocked by the addition of phospholipase inhibitor (Muldoon et al., 1985). The melittin-induced rise in intracellular Ca2+activity was also blocked by the intracellular Ca2+antagonist TMB8, suggesting that it raises intracellular Ca2+by a similar mechanism as that used by mitogens. VIII.

WHICH PHOSPHOLIPASE IS ACTIVATED BY MITOGEN STIMULATION?

In our early experiments with phospholipase stimulation and inhibition, the assay for level of activity was release of arachidonic acid from cells prelabeled with 3H-arachidonic acid. However, arachidonic acid can be released in response to stimulation of phospholipase A2 or by stimulation

188


of phospholipase C, which results in diacylglycerol production. Diacylglycerol can be broken down by the action of diacylglycerol lipase to give arachidonic acid. Based on studies in secretory cells (Berridge et al., 1983; Streb et a1 ., 1983) suggesting that phospholipase C action on polyphosphorylated phosphatidylinositol pools may play a role in Ca2+mobilization, we investigated whether the mitogen-stimulated activity that we had observed was mediated by phospholipase C. In this series of studies cells were labeled for 48 hr in the presence of 3H-inositol and then washed free of inositol. Cells were incubated for varying periods of time either in the presence or absence of mitogens and the cells extracted with trichloroacetic acid (TCA). The TCA extract was ether-washed, loaded on an anion exchange column, and then the various phosphorylated forms of inositol eluted with formate buffers. Initial studies demonstrated that serum, bradykinin, and vasopressin stimulate release of inositol phosphates (Vicentini and Villereal, 1984). Subsequent fractionation studies demonstrated that stimulation with mitogens elevates the inositol trisphosphate content of cells within 5 sec and that the content of this compound peaks at 15 sec at approximately threefold the basal level (Jamieson and Villereal, 1986). Inositol bisphosphate levels are elevated with a slower time course than for inositol trisphosphate, but the content increases to a higher level. The level of inositol monophosphate stays constant for approximately 30 sec before rising to a peak level at 2.5 min. This release pattern is consistent with that seen for serotonin stimulation of release of inositol phosphates in secretory cells (Berridge et al., 1983), where it was suggested that the hormonal coupled pool was the phosphatidylinositol 4'3'-bisphosphate pool (PIP2), which releases inositol trisphosphate upon action by phospholipase C. Since data in pancreatic cells and in hepatocytes have demonstrated that inositol trisphosphate is probably the second messenger which couples receptor binding to the mobilization of intracellular Ca2+(Streb et al., 1983; Burgess et al., 1984; Joseph er al., 1984),we investigated the effects of inositol trisphosphate on the mobilization of Ca2+in HSWP cells. The plasma membranes of HSWP cells were permeabilized by the addition of digitonin. Digitonin has relatively little effect on the permeability of internal cell membranes because of their low cholesterol content. The addition of 4sCa2+to permeabilized cells results in the uptake of 45Ca2+into intracellular compartments. This uptake is stimulated two- to fourfold by the addition of ATP to the system (Table 111). At a concentration of Ca2+of 150 nM, the uptake of 45Ca2+is predominantly into nonmitochondrial pools which appear to be membrane enclosed since Ca2+is released by the addition of Ca2+ionophore (Muldoon and Villereal, 1985). The addition of purified inositol trisphosphate to cells which have accumulated


189

TABLE I11 4sCa2+UPTAKEINTO PERMEABILIZED HSWP CELLS: EFFECTOF ATP A N D INOSITOL TRISPHOSPHATE~I

Assay conditions

Ca2+content (nmol/g protein)

Control ATP ATP + inositol trisphosphate

0.50 I .74 0.70

Cells were permeabilized with 5 pg/ml digitonin in a buffer containing 150 mM K', I mM Mg, 10 mM Na', and 150 nMCaZ+,with the Ca concentration maintained by the presence of 1 mM EGTA. Uptake of 45Ca2+was measured over a 5-min period for the control and plus-ATP conditions. For the inositol trisphosphate experiment the cells took up 4sCaZ+for 5 min and then inositol trisphosphate was added for an additional minute while the cells were still in the presence of ATP. Concentrations of inositol trisphosphate and ATP were I pM and I mM. respectively.

45CaZ+ into these intracellular pools in the presence of ATP results in the rapid release of 90% of the sequestered Ca2+(Table 111). These observations suggest that inositol trisphosphate may play an important role in the mobilization of intracellular Ca2+in cultured fibroblasts and that the phospholipase C which liberates inositol trisphosphate is most likely the phospholipase of importance in the mobilization of intracellular Ca2+and the activation of Na+-H+ exchange in HSWP cells. IX. SUMMARY OF THE PROPOSED MECHANISM FOR ACTIVATION OF THE Na+-H+ EXCHANGER BY MITOGENS

The data presented in this paper suggest that we have identified several key steps in the sequence of events occurring between binding of growth factors to their surface receptors and the activation of the Na+-H+ exchange system. Our current working model is shown in Fig. 4. The initial step after binding of peptide rnitogens appears to be the release of inositol trisphosphate from membrane pools of phosphatidylinositol 4',5'-bisphosphate (PIP2).This inositol trisphosphate then interacts with an internal Ca2+storage site to mobilize intracellular CaZ+thereby elevating the intracellular CaZ+activity. An elevation of Ca2+activity then acts through a Ca2+-dependentregulatory protein which somehow leads to the activa-

190

MITCHEL L. VILLEREAL ET AL. Melitt i n

I

Mepacri ne

PLase C Mitogens-

1i

Nail H*

Ins-P, + Diacylglycerol

Receptor

\

I/

Kinase

Ca2+CaM

+ca2+pool

[caz+li

7 1 2 1 1 8 7

\ TMB-8

C kinase4

FIG.4. Speculative model for sequence of events leading from receptor binding to activation of Na+ exchange.

tion of the Na+-H+ exchanger. The current data are most consistent with calmodulin being the Ca2+-dependentregulatory protein involved in the activation process; however, the synergism between A23 187 and TPA suggests that protein kinase C may also play some role in this process, although the lack of effect of TPA alone suggests that activation of protein kinase C is not a sufficient stimulus for activation of the Na+-H+ exchanger. The current data on phospholipase involvement are most readily explained on the basis of a mitogen activation of phospholipase C activity which acts to release inositol trisphosphate, which in turn mobilizes intracellular Ca2+.However, it should be pointed out that in general the compounds (mepacrine and melittin) that we have used to perturb phospholipase activity have been traditionally thought of as interacting with phospholipase A2 instead of phospholipase C. However, recent results in our laboratory indicate that mepacrine will inhibit the mitogen-induced release of inositol trisphosphate and that melittin will stimulate its release in the absence of mitogens (Jamieson and Villereal, in preparation), suggesting either that these compounds do interact with phospholipase C or that there is some regulation of phospholipase C activity by the breakdown products of phoshpolipase A2 activity. A report from Majerus’s laboratory indicating that mepacrine directly inhibits phospholipase C


191

activity suggests that effects of mepacrine in the HSWP cell are probably at the level of phospholipase C. In the summary scheme (Fig. 4), the possibility that the release of inositol trisphosphate could be potentiated via the stimulation of a kinase activity which would increase the substrate (PIP2) available to phospholipase C is presented. This possibility is suggested by the observation that certain oncogene products with kinase activity can phosphorylate phosphatidylinositol to the polyphosphorylated forms (Sugimoto et a / ., 1984; Macara et al., 1984). REFERENCES Berridge, M. J., Dawson, R. M., Downes, C. P., Heslop, T. P., and Irvine, R. F. (1983). Biochem. J . 212,473-482. Burgess, G. M., Godfrey, P. P., McKinney, J . S . , Berridge, M. J., Irvine, R. F., and Putney, J. W . , Jr. (1984). Nature (London) 309, 63-66. Cassel, D., Rothenberg, P., Zuang, Y., Deuel, T. F. and Glaser, L. (1983). Proc. Narl. Acad. Sci. U . S . A . 80, 6224-6228. Dicker, P., and Rozengurt, E. (1981). Biochem. Biophys. Res. Comrnrrn. 100, 433-436. Hong, S. L., and Deykin, D. (1981). J. Biol. Chem. 256, 5215-5219. Jamieson. G . , Jr., and Villereal, M. L. (1986). Arch. Biochem. Biophys.. submitted. Johnson, J . D., Epel, D., and Paul, M. (1976). Nature (London) 262, 661-664. Joseph, S. K., Thomas, A. P., Williams, R. J., Irvine, R. F., and Williamson, J. R. (1984).J . Biol. Chem. 259, 3077-3081. L'Allemain, G., Paris, S., and Pouyssegur, J. (1984). J. B i d . Chem. 295, 5809-5815. Macara, I. G . , Marinetti, G. V., and Balduzzi, P. C. (1984). Proc. Natl. Acad. Sci. U.S.A. 81, 2728-2732. Malagodi, M. H., and Chiou, C. Y. (1974). Eur. J . Pharmacol. 27, 25-33. Mix, L. L., Dinerstein, R. J., and Villereal, M. L. (1984).Biochem. Biophys. Res. Commun. 119, 69-75. Moolenaar, W. H., Tsien, R. Y., van der Saag, P. T., and de Laat, S. W. (1983). Narure (London) 304,645-648. Muldoon, L. L.. and Villereal, M. L. (1985). Fed.Proc., Fed. Am. Soc. Exp. B i d . 44, 1594. Muldoon, L. L., Dinerstein, R. J., and Villereal, M. L. (1985). Am. J . Physiol. 249, C140. O'Donnell, M. E., Becker, J. H., Cragoe, E. J., Jr., and Villereal. M. L. (1984). Fed.Proc.. Fed. A m . Soc. E x p . Biol. 43, 520a. Owen, N. E., and Villereal, M. L. (1982a).Biochem. Biophys. Res. Commun. 109,762-768. Owen, N. E., and Villereal, M. L. (1982b). Proc. Narl. Acad. Sci. U . S . A . 79, 3537-3541. Owen, N. E., and Villereal, M. L. (1983a). Cell 32, 979-985. Owen, N. E., and Villereal, M. L. (1983b). J . CelI. Physiol. 117, 23-29. Rittenhouse-Simmons, S . , and Deykin, D. (1978). Biochim. Biophys. Acta 543, 409-422. Rozengurt, E., Gelehrter, T. D., Legg, A,. and Pettican, P. (1981). Cell 23, 781-788. Smith, J . B., and Rozengurt, E. (1978a). Proc. Null. Acad. Sci. U . S . A . 75, 5560-5564. Smith, J. B., and Rozengurt, E . (1978b). J . Cell. Physiol. 97, 441-449. Streb, H., Irvine, R. F., Berridge, M. J., and Schultz, 1. (1983). Na/ure (London) 306, 67-69. Sugimoto, Y., Whitman, M., Cantley, L. C., and Erikson, R. L. (1984). Proc. Narl. Acad. Sci. U.S.A. 81, 21 17-2121.

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Tsien, R. Y. (1981). Nature (London) 290, 527-528. Vicentini, L., Miller, R. J. and Villereal, M. L. (1984). J . Biol. Chem. 259, 6912-6919. Vicentini, L. M., and Villereal, M. L. (1984). Biochem. Biophys. Res. Commun. U3,663670. Vicentini, L. M., and Villereal, M. L. (1985). Proc. Null. Acad. Sci. U.S.A. 82,8053-8056. Vigne, P., Frelin, C., Cragoe, E. J . , Jr., and Lazdunski, M. (1983). Biochem. Biophys. Res. Commun. 116, 86-90.

CURRENT TOPICS IN MEMBRANES A N D TRANSPORT. VOLUME 26

Chapter I I B Lymphocyte Differentiation: Role of Phosphoinositides, C Kinase, and Na+-H+ Exchange PHILIP M . ROSOFF Department of Pediatrics (Hematolugy-Onco[ogy.y)and Physiology New England Medical Center Tufts University School of Medicine Boston, Massachusetts and

LEWIS C . CANTLEY Department of Physiology TUBSUniversity School of Medicine Boston, Massachusetts

1. Introduction ...................... 11. Effects of Lipopolysacc and Cytosolic Free Ca2+. . ...................... 111. Effects of Phorbol Esters o and Cytosolic Free Ca?+.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. References ..................................................

1.

193 195

1% 197 198

INTRODUCTION

The initiation of proliferation or differentiation in resting or immature cells appears to require the activation of a Na+-H+ antiport system, leading to an increase in both cytosolic "a+] and pH (Rosoff and Cantley, 1985; Boron, 1984). Based on the initial observations of Burns and Rozengurt (1983), we and others have shown that enhanced activity of the antiport is apparently preceded by stimulation of protein kinase C, the phospholipid-dependent, Ca2+-activatedprotein kinase (Rosoff et al., 193 Copyright C 1986 by ALddemiC Presr. Inc All rights of icproduction in m y form r e w v e d

194

PHILIP M. ROSOFF AND LEWIS C. CANTLEY

1984; Besterman and Cuatrecacas, 1984; Moolenaar et al., 1984). In these studies, direct in uiuo activation of C kinase was accomplished via treatment of cells with the potent tumor promoter TPA (12-0-tetradecanoylphorbol-l3-acetate), which substitutes for endogenous diacylglycerol (DG) to specifically activate this enzyme (Castagna et al., 1982; Nishizuka, 1984; Berridge and Irvine, 1984; Berridge, 1984). In the cell, DG is produced via the phospholipase C (phosphatidylinositol-4,5-bisphosphatase)-catalyzed hydrolysis of phosphatidylinositol 4,S-bisphosphate (PIP2). This reaction also yields inositol 1,4,5-trisphosphate (IPJ), which has recently been shown to effect release of Ca2+from a nonmitochondrial storage pool (Streb et al., 1983; Berridge et al., 1984; Berridge and Irvine, 1984; Jospeph et al., 1984; Suematsu et al., 1984). A schematic outline of the phosphatidylinositol (PI) turnover pathway is shown in Fig. 1. We have been studying the chemically transformed murine pre-B lymphocyte cell line 70Z/3 as a model system for the analysis of the initiation of cellular differentiation. We have found that this process bears many similarities to growth factor induction of proliferation in quiescent, resting cells. 70Z/3 cells proliferate at a maximal rate in culture as surface IgMpre-B cells. Upon exposure to the B cell mitogen, lipopolysaccharide (LPS), 70Z/3 cells differentiate to a surface IgM+ B cell within 24 hr (Paige ef al., 1978). This process appears to require transcriptional activation of an already-rearranged K light chain gene as well as enhanced processing and translation of membrane p heavy chain mRNA (Perry and Kelley, 1979). Expression of surface IgM can also be induced by exposure of these

ATp\

I .- glucose-6-phosphate

FIG. I . The phosphatidylinositol turnover pathway. Abbreviations are PI, phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate; PIP*, phosphatidylinositol 4,5,-bisphosphate; IP, inositol 1-phosphate; IP2, inositol 1.4-bisphosphate; lP3, inositol I ,4,5-trisphosphate; DG, diacylglycerol; PA, phosphatidic acid; CTP, cytosine triphosphate.

11. B LYMPHOCYTE DIFFERENTIATION

195

cells to nanomolar concentrations of TPA, but not the biologically inactive phorbol ester PDD (Rosoff et al., 1984). The rate-limiting step in the induction of 70Z/3 cells is the activation of Na+-H+ exchange (Rosoff and Cantley, 1983; Rosoff et al., 1984). Indeed, reduction of external Na+ of addition of amiloride completely blocks both LPS and TPA induction of surface IgM expression. However, the amiloride block of LPS- or TPAinduced differentiation can be overcome by treatment with the Na+-H+ ionophore monensin (Rosoff and Cantley, 1983; Rosoff ef al., 1984; Rosoff and Cantley, 1984). Thus, an activated Na+-H+ exchanger is vital for the successful induction of differentiation in these cells. II. EFFECTS OF LIPOPOLYSACCHARIDE ON PHOSPHATIDYLINOSITOL TURNOVER AND CYTOSOLIC FREE Ca2+

Because of the close relationship between stimulus-coupled increases in phosphatidylinositol turnover and activation of protein kinase C by the consequent liberation of DG, we wished to examine the role of this process in the induction of differentiation in 70Z/3 cells treated with either LPS or TPA. The effect of LPS on phosphatidylinositol turnover was studied by labeling 70Z/3 cells with either [3H]glycerolor [3H]inositoluntil isotopic equilibrium was reached and then measuring the relative amounts of DG, PIP (phosphatidylinositol 4-phosphate), PIP2, and IP3 produced over time. After labeling, the cells were suspended in fresh medium at 37"C, LPS (10 pg/ml) was added at t = 0 and aliquots of cells were removed for analysis at 0, 1, 3, and 5 min. The lipids were extracted in 1 : I : 1 CHC13: CH30H : (1N)HCI and chromatographed on silica gel 60 thin-layer chromatography plates in either 7 : 3 benzene : ethyl acetate (for DG) or 45 : 35 : 8.5 : I .5 CHC13: CH30H: H 2 0: NH40H (for PIP and PIPJ. Water-soluble IP3 was measured by 15% trichloroacetic acid precipitation of [3H]inositol-labeled 70Z/3 cells. The TCA was removed by washing with diethyl ether, and the IP3 was separated by ion exchange chromatography of the formate form of Dowex 1x8-100 (Streb et al., 1984; Rosoff and Cantley, 1985). Radioactivity in all samples was quantitated by liquid scintillation counting of spots scraped off the TLC plates or of the IP3 eluant from the Dowex columns. Within I min of exposure to LPS, there was a 30% increase in DG production and a 55% increase in IP3 production. These increased levels were maintained for at least 5 min. At the same time, there was a 20% decrease in the amounts of PIP and PIP2 produced. These data would suggest that there was a stimulation of phospholipase C-catalyzed hydrol-

196

PHILIP M. ROSOFF AND LEWIS C. CANTLEY

ysis of PIP2 resulting in enhanced liberation of DG and IP3. The DG thus produced could then go on to activate protein kinase C, eventually leading to increased Na+-H+ exchange and differentiation. The elevated IP3 should enhance the release of Ca2+from an intracellular storage site (Berridge and Irvine, 1984; Berridge, 1984). We therefore measured changes in the concentration of cytosolic free Ca2+([Ca2+]i)in these cells in response to LPS. 70Z/3 cells were loaded with the fluorescent intracellular Cazt-chelator dye quin 2/AM (Tsien et al., 1982) and [Ca2+]idetermined with time after exposure to lO-pg/ml LPS. There was a very rapid increase in [Ca2+]i within 30 sec of treatment. The basal [Ca2+]iin 70213 cells is approximately 180 nM as measured by the method of Tsien et al. (1982). After LPS treatment the [Ca2+]irose to 230 nM and decayed gradually over 10 min to a point still well above control levels. In order to determine whether this increase in [Ca2+]iwas due to influx from an extracellular source or release from an intracellular site, quin 2-loaded 70Z/3 cells were treated with LPS in a Ca2+-freemedium containing 3 mM EGTA. This had no effect on the ability of LPS to stimulate a rise in [Ca2+Ii.In addition, incubation of the cells in a Na+-freebuffer did not block the LPS effect, suggesting that the increase in [Ca2+]ioccurs prior to stimulation of Na+-H+ exchange and the consequent increase in cell “a+], thus excluding a major role for Na+-Ca2+ exchange. These data are thus consistent with our results showing an increase in PI turnover in response to LPS. Enhanced production of DG and IP3 leads to stimulation of protein kinase C and cytosolic Ca2+release. 111. EFFECTS OF PHORBOL ESTERS ON PHOSPHATIDYLINOSITOL TURNOVER AND CYTOSOLIC FREE Ca2+

We next investigated the effects of phorbol esters and exogenously added diacylglycerol (OAG: l-oleoyl-2-acetylglycerol)on PI turnover and intracellular Ca2+mobilization in 70Z/3 cells. If enhanced PI turnover in response to growth factor or mitogen is required to stimulate protein kinase C, then direct activation of the enzyme via TPA or OAG would presumably bypass the PI turnover system entirely. In addition, one might predict that a specific feedback mechanism exists such that activated protein kinase C could attenuate its own stimulatory pathway by inhibiting further production of DG in the cell. This feedback inhibition could conceivably exist at several levels, the most obvious being at the growth factor receptor and at phospholipase C. Evidence for the former

11. 6 LYMPHOCYTE DIFFERENTIATION

197

has accumulated for the epidermal growth factor receptor in A431 human epidermoid carcinoma cells treated with TPA (Cochet et al., 1984; Iwashita and Fox, 1984; McCaffrey et al., 1984; Whitely er al., 1984). 70Z/3 cells were labeled to isotopic equilibrium with either [3H]glycerol or [3H]inositol and the inositol lipids, IP3, and DG isolated as described earlier after treatment with either TPA, PDD, or OAG. There was at least a 50% decrease in the levels of cellular DG within I min of exposure to OAG or TPA. Associated with this was a 30-40% fall in the amount of cellular IP3. Conversely, there was a significant rise in the levels of both PIP and PIP2. The biologically inactive phorbol ester PDD had no effect of PI turnover. These data are consistent with the hypothesis that direct activation of protein kinase C leads to a decrease in PI turnover via an apparent inhibition of phospholipase C. We also determined the concentration of cytosolic free Ca2+ in response to these agents. A fall in the level of IP3 might be expected to produce a decrease in the steady-state [Ca2+],.Therefore, 70213 cells were loaded with quin 2lAM as before and treated with 50 nM TPA, 50 nM PDD, or 25 pglml (68.8 puM) OAG. There was a very rapid fall in [Ca2+], detectable within 30 sec after exposure to either TPA or OAG. The [Ca2+], in TPA-treated cells slowly approached control levels over 10 min, but still remained below baseline. As in the case of the experiments on PI turnover, the inactive phorbol PDD had no effect on [Ca?’], . These data complement the results obtained for the effect of these drugs on PI turnover and suggest that the production of IP3 and changes in the steadystate concentration of Caf’ are closely related. IV.

CONCLUSION

In this paper we have presented data showing that mitogen-stimulated PI turnover in 70Z/3 pre-B lymphocytes is accompanied by release of Ca2’ from intracellular stores. This is probably accomplished via an increased production of IP3, a putative intracellular second messenger for Ca2+ release. In these cells, the rate-limiting step for LPS- or TPA-induced differentiation is activation of Na’-H+ exchange. The increase in [Ca2+Iimediated by IP3 is apparently not required for antiport stimulation in this cell system since phorbol esters also produce enhanced Na’-H’ exchange while at the same time causing an inhibition of intracellular Ca2+ release and an actual transient fall in [Ca2+]i.Our data do not exclude the possibility that, in the “normal” sequence of events following exposure to LPS, the small elevation in [Ca2+licomplements protein kinase C activation of the antiporter. The model shown in Fig. 2 schematically portrays the interrelationships among these various systems.

198

PHILIP

.

7 @

~

/

M. ROSOFF AND LEWIS C. CANTLEY

- - --- .

TPA. OAG

/ -

-

, /

0

LPS -D

x’0

Receptor

I ’

, ’

P I --+ PIP-PIp2

FIG. 2. Model of growth factor stimulation of 702/3 cells. Stimulatory pathways are indicated by pluses, while inhibitory events are indicated by minuses.

ACKNOWLEDGMENTS This work was supported by NIH grant GM28538 and Public Health Service grant I-KO8 CA00935-01 awarded by NCUDHHS to PMR. LCC is an Established Investigator of the American Heart Association. REFERENCES Berridge, M. J. (1984). Biochem. J . 220, 345-360. Berridge, M. J., and Irvine, R. F. (1984). Nature (London) 312, 315-321. Berridge, M. J . , Heslop, J. P., Irvine, R. F., and Brown, K. D. (1984).Biochem. J . 222, 195201. Besterman, J. M., and Cuatrecacas, P. (1984). J . Cell Biol. 99, 340-343. Boron, W. F. (1984). Nature (London) 312, 312. Bums, C. P., and Rozengurt, E. (1983). Biochem. Biophys. Res. Commun. 116,931-938. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. (1982). J . Biol. Chem. 257, 7847-7851. Cochet, C., Gill, G. N., Meisenhelder, J., Cooper, J. A,, and Hunter, T. (1984). J . Biol. Chem. 259,2553-2558. Hunter, T., Ling, N., and Cooper, J. A. (1984). Nature (London) 311,480-483. Iwashita, S., and Fox, C. F. (1984). J . Biol. Chem. 259, 2559-2567. Joseph, S. K., Williams, R. J., Corkey, B. E., Matchinsky, F. M., and Williamson, J. R. (1984). J . Biol. Chem. 259, 12952-12955. McCaffrey, P. G., Friedman, B., and Rosner, M. R. (1984). J . Biol. Chem. 259, 1250212507. Moolenaar, W. H., Tertoolen, L. G. J., and de Laat, S. W. (1984a). Nature (London) 312, 371-374. Moolenaar, W. H., Tertoolen, L. G. J., and de Laat, S. W. (1984b). Nature (London) 312, 371-373. Nishizuka, Y. (1984). Nature (Londo) 308, 693-698. Paige, C. J., Kincade, P. W., and Ralph, P. (1978). J . Immunol. 121, 641-647. Perry, R. P., and Kelley, D. E. (1979). Cell 18, 1333-1339. Rosoff, P. M., and Cantley, L. C. (1983). Proc. Natl. Acad. Sci. U.S.A. 80, 7547-7550. Rosoff, P. M., and Cantley, L. C. (1984). In “Regulation and Development of Membrane Transport Processes” (J. S. Graves, ed.). Wiley, New York. Rosoff, P. M., and Cantley, L. C. (1985). In “Stem Cell Physiology (J. Palek, ed.). Liss, New York, in press. Rosoff, P. M., and Cantley, L. C. (1985). J. Biol. Chem. 260, 9209-9215.

11. B LYMPHOCYTE DIFFERENTIATION

199

Rosoff, P. M . , Stein, L. F., and Cantley, L. C. (1984). J . B i d . Chem. 259, 7056-7060. Streb, H . , Irvine, R. F., Berridge, M . J . , and Schulz, I . (1983). Nutiire (London)306,67-69. Suematsu, E., Hiarta, M . , Hashimoto, T., and Kuiyama, H. (1984). Eiorhem. Biophys. Res. Commun. 120, 481-485. Tsien, R.Y., Pozzan, T., and Rink, T . J. (1982). Nuturr (London) 295,68-71. Whiteley, B., Cassel, D . , Zhuang, Y-X., and Glaser, L . (1984). J . Cell B i d . 99, 1162-1 166.


CURRENT TOPICS IN MEMBRANES A N D TRANSPORT, VOLUME 2h

Chapter 12 Na+-H+ Exchange and Growth Control in Fibroblasts: A Genetic Approach JACQUES POU YSSEGUR, ARLETTE FRANCHI, MICHIAKI KOHNO, GILLES L'ALLEMAIN, A N D SONIA PARIS Centre de Biochirnie CNRS, UniuersitP de Nice Nice, France

I. Introduction . . . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 11. Fibroblast Mutants Altered in the Na+-Hi Antiport Activity . . . . . . . . . . . . , . . 203 A. Mutants Lacking the Amiloride-Sensitive Na+-H+ Antiport Activity . . . . 203 111.

1V.

V.

VI. VII.

B. Mutants with an Altered Amiloride Binding Site and Increased Antiport Activity . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . Characterization and Properties of Two pH,-Regulating Systems in Fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Na+-H+ Antiporter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. A Na+-Dependent CIF-HCO, Antiporter.. . . . . . . . . . . . . . . Growth Factor Activation of the Na'-H' Antiporter.. . . . . . . . . . . . . . . . . . . . . . A. Cytoplasmic Alkalinization: An Early and Common Response of Quiescent Cells to Mitogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . B. Mechanism of Activation , ....................... pH, Controls Reinitiation of DN wth . . . . . . . . . . . . . . . . . . . A. Specific Inhibition of the Na+-H' Antiporter with Amiloride Analogs Blocks Reinitiation of DNA Synthesis.. . . . . , , . , B. Reinitiation of DNA Synthesis and Growth of Mutants Lacking Na+-H+ Antiport Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , Toward a Molecular Identification of the Na+-H' Antiport System ..................................... Conclusions . . . References . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2(M 205 205 207 208 208 210 211 212 212 215 217 218

201 Copyright 0 1986 by Academic Press, Inc. All right3 of reproduction In any form reserved.

202

JACQUES POUYSSEGUR ET AL.

I. INTRODUCTION

The recent progress in the chemical definition of external signals required to set in motion the Gn/GI+ S transition and to ensure continuous proliferation of normal cells in vitro is a first step toward the understanding of growth control at a molecular level. Polypeptide growth factors and hormones are essential regulators of animal cell proliferation. Their initial action is to bind to specific cell surface receptors. Although much has been learned about the structure and the biochemical characterization of the growth factor-hormone receptor interactions, little is known about how these external signals transduce their message. The early and common biochemical events stimulated by mitogens (ionic fluxes, phosphatidylinositol turnover, protein phosphorylation) have, therefore, drawn much attention. Among these events, stimulation of an amiloride-sensitive Na+ influx, first reported in sea urchin eggs after fertilization (Johnson et al., 1976), is one of the earliest and “universal” responses of quiescent cells to growth-promoting agents (for review, see Rozengurt, 1981). More recently, it has been clearly established that growth factor-stimulated Na+ influx results from activation of a membrane-bound Na+-H+ antiporter (Moolenaar et al., 1982; PouyssCgur et al., 1982; Owen and Villereal, 1983; Rothenberg et al., 1983; Paris and PouyssCgur, 1983; Frelin et al., 1983) leading to cytoplasmic alkalinization (Schuldiner and Rozengurt, 1982; Moolenaar et al., 1983; Cassel et al., 1983; L’Allemain et al., 1984b; Grinstein et al., 1984). Therefore, although no direct proof has been provided, intracellular pH (pHi) has been postulated as a possible mitogenic signal. To test this hypothesis directly we have used a line of Chinese hamster lung fibroblasts (CCL39) capable of entering a reversible Go/GI growtharrested state and amenable to genetic analysis (PouyssCgur ef al., 1980a,b, 1984). In this chapter we will present genetic and biochemical evidence establishing that the Na+-H+ antiporter is a major pHi-regulating system in fibroblasts, that growth factors activate the antiporter by increasing its pHi sensitivity, and that growth factor-induced cytoplasmic alkalinization (0.2-0.3 pH units) is essential for reinitiation of DNA synthesis and growth at neutral and acidic pH,. In addition, we will present specific selection procedures which have led to the isolation of three classes of mutants of the Na+-H+ antiport system: class I, mutants partially or totally defective; class 11, mutants with altered Na+ or amiloride binding sites; class 111, mutants overproducing the antiporter.

12. Na+-H+ EXCHANGE AND GROWTH CONTROL

203

II. FIBROBLAST MUTANTS ALTERED IN THE Na+-H+ ANTIPORT ACTIVITY

Early studies indicating a link between stimulation of Na+ influx and DNA synthesis were based on the following observations: (1) mitogenstimulated Na+ influx rates and DNA synthesis initiation rates follow the same dose-response curves (Pouyssegur et al., 1982; Leffert and Koch, 1982); (2) amiloride, which blocks Na+ influx, also blocks DNA synthesis (Koch and Leffert, 1979). However, since it was found that amiloride could rapidly permeate and concentrate in various cells and directly inhibit protein synthesis (Lubin and Cahn, 1981; Leffert et ul., 1982), it became clear that in order to assess the role of stimulated Na+ influx in mitogen action, another approach was necessary. Therefore, we decided first to analyze the biochemical features of the amiloride-sensitive Naf pathway of CCL39 cells and second to apply this knowledge in setting up methods for selecting mutants impaired in this system. A. Mutants Lacking the Amiloride-Sensitive Na+-H+ Antiport Activity

From studies based on net Hf fluxes across the plasma membrane of CCL39 cells, we have established that these cells possess a reversible, electroneutral, and amiloride-sensitive Na+-H+ antiporter with two distinct and mutually exclusive binding sites for Na+ and Hi' (Paris and Pouyssegur, 1983). Na+-depleted cells release Ht upon addition of external Na+. Conversely, Na+- or Li+-loaded cells take up H+ from the medium when shifted to a Na+- and Li+-free medium. This reversible Na+ (or Li+)-dependent H+ flux is inhibited by amiloride. The technique for isolation of mutants lacking Na+-H+ antiport activity is based on the reversibility of the Na+-H+ antiport and on the fact that Li+ is as efficient as Na+ for the transstimulation of H+ movements. The details of this technique are available (Pouyssdgur et ul., 1984). Briefly, and as shown schematically in Fig. IA, we have used the Na+H+ antiporter as a H+ vector killing device. First, the cells were loaded with Li+. Second, by incubating the cells at pH, 5.5, in a Nat- and Litfree choline CI saline solution, we created two chemical gradients of opposite direction: an inward-directed H+ gradient and an outward-directed Li+ gradient. Under such conditions, cell viability dropped dramatically within a few minutes as a consequence of massive and rapid H f entry; pHi was found to drop from 7.1 to below 5 after 5 min. This H'

204


A - MUTANTS DEFICIENT

BMJAMTS N I T H INCREASED ACTIVITY AND/OR LTERED NA*

n

AND WILORIDE BINDING SITES -:

iLI'

-DEPENDENT

INHIBITIONOF NA+-DEPENDENT

h

PS120 WT ARltO AR300 (RESISTANT) (KILED) (RESISTANT)

(KILLED)

I

h

NA*/H*

EXCHANGE ACTIVITY (RELATIVE VALUES)

100

300 APPARENT Kn FOR

30

20

900

N A (MM) ~ 12.5

K l FOR MILORIDE ANALOG WA(pM) 0.05

0.5

1,2

s

0

2 4 TIYE (min)

FIG. I . Principles of selection for mutants of the Na+-H+ antiport system and specific activity of three CCL39-mutant derivatives. (A) Principle of selection for mutants deficient in Na+-H+ antiport activity (PS120). Cells were loaded with LiCl as described and incubated in a Na+- and Li+-free medium at pH, 5.5 (Pouysstgur ct a/., 1984). (B) Principle of selection for mutants with an increased activity in Na+-H+ exchange and/or alteration in Na+ and amiloride binding sites (AR40 and AR300). (C) Na+-H+ exchange activity in CCL39 (WT) and mutant derivatives (PS120, AR40, AR300). Cells were loaded for 30 min with 50 m M N H F I and assayed after NH: removal for arniloride-sensitive Na+ influx as described (Pouysstgur et al., 1984).

suicide is indeed specifically generated by the operation of the Na+-H+ antiport, since addition of amiloride protects the cells by slowing down the rate of cytoplasmic acidification (PouyssCgur et al., 1984). When mutagenized CCL39 cells were submitted twice to this H+-suicide test, 90% of the resistant clones were found to be defective in Na+/H+ exchange activity, either partially (30-50% residual activity) or totally. One such mutant, PS120, has no detectable amiloride-sensitive Na+ influx when measured either under Li+-or H+-loadingconditions (Fig. 1C). This proton-suicide method is efficient, specific, and general for isolation of mammalian cells lacking Na+-H+ exchange activity. Its specificity is reflected by the fact that the mutant analyzed, PS120, is not impaired in other membrane transport systems: Na+,K+-ATPase, tetrodotoxin-sensitive Na+ channels or Na+-K+-CI- cotransporter. Finally, the same

6


205

method applied to mouse LTK- cells generated mutants with -50% of the Naf-Hf antiport activity and in a second mutagenized step mutants with no detectable activity (A. Franchi and J. Pouyssegur, unpublished results). The physiological and biochemical properties of PS 120 will be described in the next sections. B. Mutants with an Altered Amiloride Binding Site and Increased Antiport Activity

Another direction that we are currently developing is the selection of mutants overproducing the antiporter system. The common method of selecting “overproducers” is to use inhibitors which kill cells, or arrest their growth, and then to select for resistant mutants. Although several different mechanisms can mediate resistance, substantial elevation of the activity or amount of a protein, the target of the inhibitor, is often observed. Now, more than 20 examples of mammalian cells resistant to drugs are known to result from overproduction of a protein, and in at least 10 cases, this overproduction results from gene amplification (for review see Stark and Wahl, 1984; Schimke, 1984). The principle of selection we employed was just the opposite of that used for the isolation of Na+-H+antiport deficient cells. As illustrated in Fig. IB, we loaded the cells with protons in such a way that the operation of the Na+-H+ antiporter was rendered absolutely essential for protection of the cells against the lethal acid load. If the antiporter was partially inhibited with amiloride, the cells would not survive unless they overproduced the antiporter and/or were endowed with a mutation decreasing the affinity of the antiporter for amiloride. Briefly, selections are conducted as follows. First, CCL39 cells are acid loaded using the NH:-prepulse technique described by Boron and De Veer (1976). Cells are incubated 60 min with 50 mM NHdCI. Removal of external NH: induces a rapid and lethal intracellular acidification when the Na+-H+ antiporter is inhibited with amiloride for 60 min. A half-lethal dose is obtained with 0.3 p M of the potent arniloride analog 5-N-propenyl-N-rnethylamiloride(PMA) (L’Allemain et al., 1984a). Acidic selection was initiated with 0.3 p M PMA and repeated daily with progressively increasing concentrations of the inhibitor. Following this protocol two stable variants have been selected, AR40 and AR300, resistant, respectively, to 40 and 300 p M of PMA in the acidic test. Both display an increased Na+-H+ antiport activity: threefold in AR40 and ninefold in AR300 under conditions in which the internal sites are saturated with H t (Fig. IC). Also, both express a reduced affinity for the amiloride analog PMA of 10- and 20-fold, respectively (Franchi and Pouyssegur, 1984, and unpublished results). It is interesting that concomi-

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JACQUES POUYSS~GURET AL.

tant with the decrease in affinity for the amiloride binding site, AR300 has increased by 2.5-fold its affinity for the substrates Na+ and Li+. These preliminary results suggest that AR300 is overproducing a structurally modified antiporter molecule. 111.

CHARACTERIZATION AND PROPERTIES OF TWO pH,-REGULATING SYSTEMS IN FIBROBLASTS

A. The Na+-H+ Antiporter

Kinetic studies of the Na+-H+ antiporter identified in CCL39 cells (Paris and PouyssCgur, 1983) have shown ( I ) that external Na+ or Li+ stimulates H+ release and inhibits H+ uptake in a competitive manner (Ki = 2-3 mM) and (2) that amiloride is a competitive inhibitor for Na+ (K, = 3 p M ) . It was therefore expected that if the antiporter is a major cellular pHi-regulating system, then in response to an acid load, we should observe a rapid pHi recovery in presence of external Na+. Also, pHi recovery is expected to be faster in mutant cells expressing an increased Na+-H+ antiport activity. Conversely, this recovery should be very slow in the presence of amiloride, or in a Na+-free medium, or in a mutant lacking the Na+-H+ antiport activity. These predictions were all verified (L’Allemain et al., 1984b) (Fig. 2). We have seen in Section II,B that when cells are loaded with NH: and then shifted to a NHd-free medium, the rapid diffusion of the uncharged form NH3 out of the cell provokes a rapid cytoplasmic acidification (Boron and De Veer, 1976). Figure 2A shows that this acidification is strongly pronounced in PS120 lacking Naf-H+ antiport activity; pHi drops from 6.9 to 5.95 and persists below 6 for 30 min. In contrast, cells expressing Na+-H+ antiport activity (Fig. 2B,C,D) display a rapid and complete pHi recovery. In addition, we observed a positive correlation between the level of expression of Naf-H+ exchange activity and the rate of pHi recovery. The H+-extruding system is so efficient in AR300 that we cannot detect the intracellular acidification imposed by the NH:-prepulse technique (Fig. 2D) unless the Na+-H+ antiporter is blocked with amiloride in Na+-free medium. The fact that the Na+-H+ antiporter plays an important role in pH, regulation has been established for a variety of vertebrate cells (Schuldiner and Rozengurt, 1982; Moolenaar et ul., 1983; L’Allemain et al., 1984b; Rothenberg et al., 1983; Vigne et al., 1984; Grinstein et al., 1984; for review see Boron, 1983). Our results bring independent and additional support to this concept.

207


7.0

5.- 6.5

-

6.0

C

I

D

FIG. 2. pH, recovery in CCL39 cells and Na+-H’ antiport mutant derivatives in response to an acid load. Cells were loaded for 60 min with 50 mM NH,CI and, at the time indicated by the arrow NH; was removed and pH, measured with I4C-benzoic acid as reported by L’Allemain et al. (1984b3. (A) PSI20 lacking Na+-Ht exchange activity; (B) wild-type cells; (C) ARM; (D) AR300. (For specific activity of the Na+-H’ exchange in the four cell lines, see Fig. 1 .)

6. A Na+-Dependent CI--HCO;

Antiporter

Two observations led us to search for an additional pH,-regulating system in CCL39 cells. In bicarbonate-containing culture medium, the concentration of 5-N,N-dimethylamiloride (30 p M ) , which at "a+], = 35 mM inhibits more than 98% of the Na+-H' antiport activity, does not prevent or even affect the rate of clonal growth. In contrast, the same inhibitor abolishes growth in bicarbonate-free medium. Accordingly, PS120, lacking Na+-H+ antiport activity, is severely restricted for its growth only in HC0;-free medium at neutral and acidic pHs (Pouyssegur et al., 1984; Section V). In relation to these observations, we recently demonstrated the existence of a stilbene derivative (SITS)-sensitive C1-HCO; exchange in CCL39 cells (L'Allemain ~t al., 1985). Evidence for this system is based on 36C1- influx studies and on pH, measurements in PS120 mutant cells. 36C1- influx rate is a saturable function of [CI-1, with an apparent K , 7 mM; it is competitively inhibited by external HCO; ( K i = 3 mM) and stilbene derivatives (K, = 20 p M for SITS). We have %

208


seen (Fig. 1A) that pHi recovery following an acid load in PS120 is very slow. However, addition of external HCO; at pH, constant quickly restores the initial pHi. This acid-extruding mechanism is inhibited by SITS and requires external Na+. Since 22Na+influx is stimulated upon addition of HCO; to acid-loaded cells and this effect is completely abolished by SITS, we conclude that Na+ is cotransported with HCO; in exchange for intracellular C1-. Thus, in many respects, this system closely resembles the HCO;, Na+-CI- (H+) exchange described in various invertebrate cells (Thomas, 1977; Boron et al., 1981; Boron and Russel, 1983). In conclusion, Chinese hamster lung fibroblasts possess two pH,-regulating mechanisms: an amiloride-sensitive Na+-H+ antiport and a SITSsensitive HCO;, Na+-CI- exchange. These data are in agreement with the commonly accepted view (see Roos and Boron, 1981; Boron, 1983, for reviews) that, in mammalian cells, two distinct systems have replaced the unique Na+-dependent HCO;-CI- transport system of invertebrate cells. IV. GROWTH FACTOR ACTIVATION OF THE Na+-H+ ANTIPORTER A. Cytoplasmic Alkalinization: An Early and Common Response of Quiescent Cells to Mltogens

CCL39 cells can be arrested in Go/GIby serum growth factor deprivation (Pouyssdgur et al., 1980a) and stimulated to reinitiate DNA synthesis by addition of purified growth factors, in particular, a-thrombin and insulin (PouyssCgur et al., 1983; Van Obberghen-Schilling et al., 1983). One of the earliest responses of Go-arrested CCL39 cells to a-thrombin is the stimulation of an amiloride-sensitive Na+ influx. The magnitude of this early ionic response correlates well with the percentage of cells that reinitiate DNA synthesis. Insulin, which alone is not mitogenic for CCL39 cells, has very little effect on Na+ influx; however, when added together with a-thrombin, a potentiation of Na+ influx and DNA synthesis stimulation is observed (PouyssCgur et al., 1982). In Fig. 3 we show that a-thrombin and insulin stimulate a rapid cytoplasmic alkalinization in CCL39 cells. After a transient acidification, presumably due to a nonspecific increase in membrane permeability, a new resting pHi 0.2 to 0.3 pH units more alkaline is obtained in 5 to 8 min and persists for more than 60 min (Fig. 4). Again, insulin, which alone has very little effect on pHi, potentiates the a-thrombin-induced pHi rise (L’Allemain et al., 1984b). The nonmitogenic phenylmethyl sulfonyl conjugate of a-thrombin does not induce pHi rise, whereas a weak mitogen for CCL39, like epidermal

209


7.5

-

+TH INS

7.4 .

7.3 PHi 7.2. 7.1

7.0

-

1

1

0

.

I

.

I

.

4 8 12 TIME ( min)

I

.

~

~

16

FIG.3. Effect of growth factors on pH, in G,,-arrested wild-type and Na+-H' antiportdeficient cells. CCL39 cells (WT) and PSI20 mutant cells grown on glass coverslips were arrested in G,,/Gl by serum deprivation and loaded with 2',7'-bis(carboxyethyl)-5,6-~arboxyfluorescein (Moolenaar ef n l . , 1983). At time 0, a-thrombin ( I Uiml) and insulin (10 pglml) were added together to the cells preincubated at 30°C. pH,, 7.35.

growth factor, induces an intermediate intracellular alkalinization (0.10.15 pH units). Finally, we demonstrated that growth factor-induced cytoplasmic alkalinization is Na+ (or Li') dependent, is prevented by amiloride analogs, and is not detected in the mutant PS120 (Fig. 3) (L' Allemain et al., 1984b). This early biochemical response of quiescent cells is therefore entirely mediated by growth factor activation of the Na+-H+ antiporter.

Ot

-a+ , 0

lo

20 TIME(mi")

FIG.4. Comparative effect of a-thrombin and TPA on cytoplasmic alkalinization of arrested CCL39 cells. pH, was measured with 14C-benzoicacid as reported by L'Allemain rr a / . (1984b).

210


The phorbol ester TPA either alone or in combination with insulin is a weak mitogen for CCL39 cells arrested by 24 hr of serum deprivation (?Na+uptake is plotted against pH, values determined on parallel dishes (A), in the absence (0)or presence of athrombin, 1 U/ml, and insulin, 10 pg/ml ( 0 ) Growth . factors were added 15 sec before the 30-sec pulse of **Na+,pH, 7.4, 1 mM [Na-1, (Paris and PouyssCgur, 1984).

to additive effects on the amiloride-sensitive cytoplasmic alkalinization. Likewise, the addition of a-thrombin to cells treated for 30 min with maximally stimulating concentrations of TPA induced further stimulation of the Na+-H+ exchanger (S. Paris, unpublished data). These results may suggest that, besides activation of phospholipid-dependent protein kinase C, a pathway common to TPA (Castagna et al., 1982) and thrombin through polyphosphoinositide breakdown (Nishisuka, 1984; our unpublished results), thrombin may induce Na+-H+ antiporter activation via an additional pathway. V. pH1 CONTROLS REINITIATION OF DNA SYNTHESIS AND GROWTH

There is now increasing but still indirect evidence that cytoplasmic pH may play an important role in the metabolic activation of a variety of resting cells and in growth control (for a review see Nuccitelli and Deamer, 1982; Busa and Nuccitelli, 1984; Gillies, 1981).The development of very potent inhibitors of the Na+-H+ antiporter (L'Allemain et al., 1984a; Vigne el al., 1984a) and of mutants specifically defective in this pHi-regulating system allowed us to directly answer the question: Could a small change in pHi control the rate of DNA synthesis?

212


A. Specific Inhibition of the Na+-H+ Antiporter with Amiloride Analogs Blocks Reinitiation of DNA Synthesis

We have analyzed the potency of 28 analogs of amiloride for inhibition of the Na+-Ht antiporter (L’Allemain et al., 1984a). Our findings revealed that substitution within the guanidino group reduced the activity 20-1000-fold. On the contrary, substitution of the proton(s) of the 5amino group of amiloride increases potency up to 100-fold [5-N-propenylN-methylamiloride (PMA) has a Ki of 4 x lo-* MI.In HC0;-free medium and at low “a+], (50 mM) to reduce competition with amiloride, we found that growth factor-stimulated DNA synthesis of Go-arrested CCL39 cells is inhibited by amiloride and its analogs with the same rank order as that for Nat-Ht antiporter inhibition (L’Allemain et al., 1984a). To substantiate the conclusion that inhibition of DNA synthesis reflects the primary action of amiloride analogs on the Na+-H+ antiporter, we analyzed AR40, a mutant, with an altered Na+-H+ antiporter molecule (Franchi and Pouyssegur, 1984, 1985). AR40 has a modified amiloride binding site, its Ki for PMA being 10-fold increased (Fig. 6A). Interestingly, this specific alteration of the Nat-Ht antiporter is reflected in the inhibition of DNA synthesis since PMA was found to be 10 times less active in AR40 cells (Fig. 6B). These findings converge to establish that the functioning of the Na+-H+ exchange system is required for growth factor-induced DNA synthesis in a HC0;-free medium. We have seen that CCL39 cells possess a HC0;-CI- exchange system involved in pHi regulation. It is interesting to note that in presence of HCO;, 100% inhibition of the Na+-H+ antiport by amiloride analogs does not prevent reinitiation of DNA synthesis. Inhibition is only observed at high concentrations of amiloride analogs at which they are known to have secondary inhibitory effects on protein synthesis (Lubin and Cahn, 1981; L’Allemain et al., 1984a). 8. Reinitiation of DNA Synthesis and Growth of Mutants Lacking Na+-H+ Antiport Activity

The mutant PS120, in which growth factors fail to stimulate the amiloride-sensitive Na+ influx and cytoplasmic alkalinization, was useful in directly evaluating the role of increased pHi in mitogen action. Wild-type and mutant cells were arrested in Go and then stimulated with thrombin and insulin at various external pH values. Figure 7A shows that pH, dependence for thymidine incorporation is shifted in PS120 by 0.4 pH


213

[AMILORIDE ANALOG] p M

FIG.6. Inhibition of Na+-H+ antiport activity and growth factor-induced DNA synthesis by an amiloride analog (PMA). Dose-response curves in CCL39 cells and a mutant derivative AR40. (A) Na+-H+ antiport activity measured by amiloride-sensitive initial rates of 22Na+uptake (pH, 7.4 and 130 mM choline C1) in Li+-preloaded wild-type CCL39 cells (A) and the PMA-resistant mutant derivative AR40 (0). (B)Growth factor-induced DNA cells at external "a'] = 50 m M . Results synthesis in quiescent CCL39 (A)and AR40 (0) are expressed as percentage inhibition of the control *"a- influx ( A ) or [?H]thymidine (TdR) incorporation (B), measured in the absence of PMA.

units toward alkaline values. Up to pH, of 7.2, mutant cells fail to reinitiate DNA synthesis, whereas at this pH value, 60% of the maximal response has occurred in wild-type cells. Intracellular pH at the early stage of reinitiation was measured for each pH, (L'Allemain et a l . , 1984b) and pH, values were plotted against thymidine incorporation. Figure 7B clearly shows (1) that pH, dependence for reinitiation of DNA synthesis is extremely sensitive (90%of the response is obtained within a variation of 0.2 pH units), (2) that the pH, dependence is identical for mutant and wildtype cells, and (3) that below pH, of 7.2 (threshold value), growth factors fail to reinitiate DNA synthesis. Now, if reinitiation of DNA synthesis is carried out in the presence of HC07-COZ, very little, if any, difference is observed in the pH, dependence for DNA synthesis in wild-type and mutant cells (not shown). We found that in the presence of HCOY the

214


PHo

PHI

FIG.7. Growth factor-induced DNA synthesis and pHi dependence in wild-type and

were arrested in Na+-H+ antiport-deficient cells. Wild-type (A) and PSI20 mutant cells (0) Go/GIby serum deprivation for 24 hr. DNA synthesis was reinitiated by addition of IU/ml of a-thrombin and insulin (10 pg/ml) in HC0;-free medium, buffered with 30 mM of either MOPS (pH, 6.6-7.4) or EPPS (pH 7.4-8.2). [3H]thymidine incorporated during 24 hr in response to growth factors is plotted against either pH, (A) or pHi ( 8 ) .Intracellular pH was determi-d 10 min after growth factor addition, subsequent to a 60-min pre-equilibration at the various pH, values.

resting pHi of PS120 is “reverted” back to the wild-type value (L’Allemain et al., 1985),presumably by the operation of the SITS-sensitive C1-HCO; antiporter. Clonal growth of wild-type CCL39 cells in HC0;-free medium buffered with PIPES, MOPS, or HEPES is observed over a pHo range of 6.6 to 8.3, with an optimum value of around pH 7.4. In contrast, clonal growth of PS120 is precluded at pHo 7.2 and below. However, above this pH limit, clonal growth appears normal with an optimal pHo for growth shifted to alkaline values 8-8.3 (Pouysskgur et al., 1984). Here again, the pH-conditional growth of mutants lacking Na+-H+ antiport is restored to normal in bicarbonate-buffered medium. All these results on pHo-dependent growth are in good agreement with the DNA reinitiation synthesis data. Finally, to substantiate the idea that pHi controls the rate of DNA synthesis, we selected a variant, AS7, capable of growing rapidly at pHo 6.4, a nonpermissive pH for the parental cells (Table I). This variant also reinitiates DNA synthesis at a more acidic pHo (A. Franchi and J. PouyssCgur, unpublished results). Therefore, we expected that AS7 would maintain its pHi at a more alkaline value than that observed in the parent

215


TABLE 1 COMPARISON OF RESTING pH, A N D GROWTHRATEOF c c L 3 9 A N D ITS DERIVATIVE AS7“ PHO 6.4 6.8 7.2 7.4

Resting pH, AS7 CL39(WT) 6.75 (10) 6.94 (10) 7.07 (IS) 7.33 (8) 7.28 (14) 7.59 (8) ND ND

Growth Rate CCL39 (WT) 1.02 ND ND 38.6

AS7 11.4 ND ND 39.7

pH, was measured 15 min after growth factor stimulation of quiescent cells with thrombin and insulin (L’Allemain et al., 1984b).pH, values are the mean of the number of determinations indicated in parentheses. Growth rate is expressed as the ratio between cell numbers from day 4 to day 1 . N D means no data.

cells. This was indeed the case (Table I). Over the range of pH, 6.4 to 7.2, pHi of AS7 is 0.2-0.3 pH units higher than in the wild-type cells. Altogether, these independent results strongly support the idea that pHi exerts a tight control on the rates of DNA synthesis and growth. VI.

TOWARD A MOLECULAR IDENTIFICATION OF THE Na+-H+ ANTIPORT SYSTEM

Even though rapid progress over the past four years has advanced our understanding of the physiology and mechanistic aspects of the Na+-H+ antiporter, no information is available concerning the molecular structure of the antiport protein(s). It is now possible with the availability of [3H]labeled high-affinity amiloride analogs to “titrate” the Na+-H+ antiporter in solubilized membranes (Vigne et a l . , 1984~).This is a first step toward its purification. An alternative approach that we have chosen is the comparison of plasma membrane proteins and phosphoproteins in the parent cells CCL39 and two classes of mutants: the class lacking the Na+-H+ antiport activity and that overexpressing it and, it is hoped, overproducing the protein. The latter class of mutants, like AR300 and others that we have not described here, may undergo discrete amino acid changes in the antiporter molecule(s) as they express decreased affinity for amiloride andlor increased affinity for Na+ (Fig. IB). These changes might be detected by a charge shift in two-dimensional gel electrophoresis. Preliminary experiments are depicted in Fig. 8. Plasma membrane proteins of

FIG.8. Protein and phosphorylation pattern of purified plasma membrane of CCL39 cells and of the Na+-H+ antiport mutant derivatives PS120 and AR300. Plasma membranes were purified as reported by Courtneidge et a / . (1980) and proteins were separated in a 7.515% SDS-gradient gel electrophoresis. (A) Plasma membrane proteins stained with Coomassie blue corresponding to CCL39 (WT), PS120 (PS), and AR300 (AR). (B) Plasma membrane proteins after in v i m phosphorylation (incubation at 0°C in presence of [-y-”P]ATP without exogenous kinases).


217

CCL39, PS120, and AR300 cells, purified after centrifugation at the 2013.5% sucrose interface (Courteneidge et al., 1980), do not show major changes (Fig. 8A). However, marked differences can be detected after in vitro phosphorylation of these plasma membranes: First, AR300 shows an increased level of two phosphoproteins of M , 120,000and 58,000. Second, the 120-kDa phosphorylated polypeptide is barely detectable in the Na+H+ antiport deficient mutant, PS120; and third, those changes pointed out by the arrows (Fig. 8B) were reproduced in three independent experiments. In addition, in a preliminary experiment on in vivo phosphorylation, the 120-kDa polypeptide was phosphorylated and stimulated by addition of thrombin or TPA in AR300 (M. Kohno, A. Franchi, and J. Pouyssegur, unpublished results). Although the altered levels of membrane phosphoproteins (120 kDa and 58 kDa) are specifically associated with mutants of Na+-H+ antiport system, that does not prove that these polypeptides are subunits of the transporter. Current studies combining affinity labeling of the antiporter and analysis by two-dimensional gel electrophoresis of mutants, “overproducer” or altered in the structural gene, will soon bring a definite identification of the transporter molecule(s). Finally, the isolation in two steps of mutants lacking the Na+-H+ antiport activity in the highly transfectable mouse L929 TK- cell line (Wigler el a/., 1978; Kuhn et al., 1983; A. Franchi and J . Pouyssegur, unpublished results) opens the way for the molecular cloning of this transport system by DNA-mediated gene transfer. VII.

CONCLUSIONS

The first advances in the genetics of the Na+-H+ antiport system have offered a new approach to analyzing the physiology of pH,-regulating system in fibroblasts, the role of pH, in growth control, and the identification of the Na+-H+ antiport at a molecular level. We have confirmed or established the following points. 1. The Na+-H+ antiporter is a major pH,-regulating system in fibroblasts. 2. In the presence of HCO;, the operation of a SITS-sensitive and Na+-dependent CIk-HCO; antiporter is a good substitute for Na+-H+ exchange in pH, regulation. 3. Growth factor-induced cytoplasmic alkalinization is strictly triggered by activation of the Na+-H+ antiporter. 4. Growth factor activation results from an increased affinity of the system for internal H + . 5 . pH, exerts a tight control on the rate of cell entry into S phase.

218


The last conclusion is drawn from the observation that PS120 mutant cells fail to reinitiate DNA synthesis over the range of external pHs 6.6-7.2. This restricted action of growth factors results from the incapacity of mutant cells to elevate their pHi above the critical threshold value (pHi = 7.2). Indeed, if pHi is increased, either by raising external pH or by adding bicarbonate, DNA synthesis and growth resume normal rates. However, raising pHi alone has no effect on the reinitiation of DNA synthesis. The extreme sensitivity of pHi dependence for DNA synthesis certainly reflects the cooperation of multiple pHi-dependent cellular functions. A classic example of highly pHi-sensitive process is the activation of glycolysis and of the regulatory enzyme phosphofructokinase (Fodge and Rubin, 1973; Wenner, 1975). Another pHi-dependent limiting step is the phosphorylation of ribosomal protein S6 (PouyssCgur et al., 1982; Chambard et al., 1983). This growth factor-induced phosphorylation, which appears to be necessary for the activation of protein synthesis (Thomas et al., 1982) does not occur at nonpermissive pHi values (Chambard and Pouysdgur, 1986). In contrast, growth factor-induced c-myc mRNA (Kelly et al., 1983), another early event stimulated by a-thrombin in CCL39 cells, occurs at nonpermissive pHi values (PouyssCgur et al., 1985, and unpublished results). Finally, an important finding is that a-thrombin, a potent activator of polyphosphoinositide breakdown in CCL39 cells (G. L’Allemain, S. Paris, and J.PouyssCgur, unpublished results), activates, as serum growth factors and phorbol esters, the Na+-H+ antiporter by increasing its affinity for internal H+ (Moolenaar et al., 1983; Paris and PouyssCgur, 1984; Grinstein et al., 1984). The molecular identification of the antiporter molecule(s) should soon demonstrate whether the change in pHi sensitivity reflects different states of kinase C-dependent phosphorylation. ACKNOWLEDGMENTS We would like to gratefully acknowledge Dr. W. Moolenaar for helpful advise and collaboration on the use of the pH-sensitive fluorescent probe, Dr. E. Van Obberghen-Schilling for critical reading of the manuscript, and Mrs. G. Cltnet for secretarial assistance. Dr. M. Kohno is on leave from the Institute for Virus Research, Kyoto University, and is supported by a fellowship from the Ministry of Education, Culture and Science of Japan. This work was supported by grants from the Centre National de la Recherche Scientifique (LP 7300, ATP 136, and ASP 394), the Institut National de la Santt et de la Recherche M6dicale (CRE 84-2015), the Fondation pour la Recherche Mtdicale, and the Association pour la Recherche contre le Cancer. REFERENCES Aronson, P. S., Nee, J., and Suhm, M. A. (1982). Nature (London) 299, 161-163. Boron, W. (1983). J . Membr. Biol. 72, 1-16.


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Boron, W., and De Weer, P. (1976). J. Gen. Physiol. 67, 91-1 12. Boron, W., and Russel, J . (1983). J. Gen. Phvsiol. 81, 373-399. Busa, W., and Nuccitelli, R. (1984). A m . J . Phy.viol. 246, R409-R438. Cassel, D., Rothenberg, P., Zhuang, Y., Deuel. T., and Glaser. L. (1983). Proc. Nurl. Acad. Sci. U . S . A . 80,6224-6228. Castagna, M., Takai, Y ., Kaibuchi, K . , Sano, K., Kikkawa, V., and Nishizuka, Y. (1982). J. B i d . Chem. 257, 7847-7851. Chambard, J . C., and Pouyssegur, J. (1986). Exp. Cell R e s . . in press. Chambard, J . C., Franchi, A., Le Cam, A., and Pouysstgur, J . (1983). J. Biol. Chem. 258, 1706- 17 13. Courtneidge, S., Levinson, A., and Bishop, M . (1980). Proc. Nut/. Acud. Sci. U . S . A . 77, 3783-3787. Fodge, D., and Rubin, H. (1973). Nature (London) Neuj Biol. 246, 181-183. Franchi, A., and PouyssCgur, J . (1984). Biol. Cell 52, 77a. Franchi. A ., and Pouysstgur, J. (1986). In preparation. Frelin, C., Vigne, P., and Lazdunski, M. (1983). J . B i d . Chem. 258, 6272-6276. Gillies, R. (1981). In "The Transformed Cell" (1. Cameron and T. Pool, eds.), pp. 347-395. Academic Press, New York. Grinstein, S., Cohen, S . , Goetz, J . , Rolhstein, A., and Gelfand, E. (1985). Proc. Nurl. Acad. Sci. U . S . A . 82, 1429-1433. Johnson, J., Epel, D., and Paul, M. (1976). Nature (London) 262, 661-664. Kelly, K . , Cochran, B.. Stiles, C., and Leder, P. (1983). Cell 35, 603-610. Koch, K . , and Leffert, H. (1979). Cell 18, 153-163. Kuhn, L., Barbosa, J., Kamarck, M.. and Ruddle, F. (1983). Mol. B i d . Med. 1, 335-352. L'Allemain, G., Franchi, A,, Cragoe. E., and Pouyssegur, J . (1984a). J. B i d . Chem. 259, 4313-4319. L'Allemain, G., Pans, S., and Pouyssegur. J . (1984b). J. B i d . Chem. 259,5809-5815. L'Allemain, G . , Paris, S.. and Pouysstgur, J . (1985). J. Biol. Chem. 260, 4877-4883. Leffert, H . , Koch, K., Fehlmann, M., Heiser, W., Lad, P. J . , and Skelly, H . (1982). Biochem. Biophys. Res. Commun. 108, 738-745. Lubin, M., and Cahn, F. (1981). J . Cell B i d . 91, 6a. Moolenaar, W., Yarden, Y., de Laat, S., and Schlessinger, J. (1982). J. B i d . Chem. 257, 8502-8506. Moolenaar, W., Tsien, R., Van der Saag, P.. and de Laat, S. (1983). Nature (London) 304, 645-648.

Moolenaar, W., Tertoolen, L., and de Laat, S . (1984). Nature (London) 312, 371-374. Nishisuka, Y. (1984). Nature (London) 308, 693-698. Nuccitelli, R., and Deamer, D. W. (1982). In "Intracellular pH,: Its measurement, regulation, and utilization in cellular functions." Liss. New York. Owen, N., and Villereal, M. (1983). Cell 32, 979-985. Pans, S., and Pouysstgur, J . (1983). J . Eiol. Chem. 258, 3503-3508. Paris, S., and Pouysstgur, J. (1984). J. B i d . Chem. 259, 10989-10994. Pouysstgur, J . , Franchi, A., and Silvestre, P. (1980a). Nature (London) 287, 445-447. Pouysstgur, J . , Franchi, A., Salomon, J . C., and Silvestre, P. (1980b). Proc. Null. Actrd. Sci. U . S . A . 77, 2698-2701. Pouysstgur, J . , Chambard, J. C., Franchi, A., Paris, S., and Van Obberghen-Schilling, E. (1982). Proc. Narl. Acud. Sci. U . S . A . 79, 3935-3939. Pouyssegur, J., Chambard, J. C., Franchi, A,, L'Allemain, G., Paris, S., and Van Obberghen-Schilling, E. (1983). I n "Hormonally Defined Media" (G. Fisher and R. Wieser, eds.), pp. 88-102. Springer-Verlag, Berlin.

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Pouysskgur, J., Sardet, C., Franchi, A., L’Allemain, G., and Paris, S. (1984). Proc. Nutl. Acad. S C ~ U.S.A. . 81, 4833-4837. Pouysstgur, J., Chambard, J. C., Franchi, A., L’Allemain, G., Paris, S., and Van Obberghen-Schilling, E. (1985). In “Conferences on Cell Proliferation and Cancer,” Vol. 12, pp. 409-415. Cold Spring Harbor Series. Roos, A., and Boron, W. F. (1981). Physiol. Rev. 61, 296-434. Rothenberg, P., Glaser, L., Schlesinger, P., and Cassel, D. (1983a). J . Biol. Chem. 258, 12644-12653. Rothenberg, P., Glaser, L., Schlesinger, P., and Cassel, D. (1983b). J . Biol. Chem. 258, 4883-4889. Rozengurt, E. (1981). Adu. Enzyme Regul. 19, 61-85. Schimke, R. (1984). Cell 37, 705-713. Schuldiner, S., and Rozengurt, E. (1982). Proc. Nut/. Acad. Sci. U . S . A . 79, 7778-7782. Stark, G., and Wahl, G. (1984). Annu. Rev. Biochem. 53, 447-491. Thomas, G., Martin-Perez, J., Siegmann, M., and Otto, A. (1982). Cell 30, 235-242. Thomas, R. C. (1977). J . Physiol. (London) 273, 317-338. Van Obberghen-Schilling, E., Perez-Rodriguez, R., Franchi, A., Chambard. J. C., and Pouyssegur, J. (1983). J . Cell. Physiol. 115, 123-130. Vigne, P., Frelin, C., Cragoe, E., Jr., and Lazdunski, M. (1984a). Mol. Phurmacol. 25, 131136. Vigne, P., Frelin, C., and Lazdunski, M. (1984b). E M B U J . 3, 1865-1870. Vigne, P., Frelin, C., Audinot, M., Borsotto, M., Cragoe, E., Jr., and Lazdunski, M. (1984~).EMBO J . 3, 2647-265 1. Wenner, C. (1975). In “Cancer: A Comprehensive Treatise” (F. F. Becker, ed.), Vol. 111, pp. 389-403. Plenum, New York. Wigler, M., Pellicer, A., Silverstein, S., and Axel, R. (1978). Cell 14, 725-731.

Part IV

Role of Na+-H+ Exchange in Hormonal and Adaptive Responses



Chapter 13 Hormonal Regulation of Renal Na+-H+ Exchange Activity BERTRAM SACKTOR AND JAMES L . KINSELLA Laboratory c?f Biological Chemistry Gerontology Research Center National Institute on Aging National Institutes of Heulth Baltimore, Marylnnd

I.

..................................... Proximal Tubule ....................

Introduction

enal Brush Border Membrane Vesicles ...................................................... IV. Extrinsic Effectors of Renal Na+-H+ Exchange Activity.. .................. A. Regulation of Na+-H+ Exchange Activity by Thyroid Hormones . . B. Regulation of Na+-H+ Exchange Activity by Glucocorticoids . . . . . . . . . . . C. Alteration in Na+-H+ Exchange Activity in Chronic Metabolic Acidosis.. V. Hierarchy of Hormonal Effects of Na+-H+ Exchange.. ..................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.

223 224

228 235 237 240 243

INTRODUCTION

The kidney proximal tubule, which carries out transepithelial transport of solutes and fluid, is characterized by cells with demonstrable polarity. This polarity is evident functionally by the observed differences in the mechanisms by which solutes enter and leave the cell and ultrastructurally by the differentiation of the plasma membrane into two distinct entities, the luminal (brush border) and basolateral membranes (Sacktor, 1977). The brush border membrane of the mammalian renql proximal tubule contains the Na+-H+ exchanger (Murer et al., 1976; Kinsella and Aronson, 1980). The carrier couples Na' flux down its concentration gradient (lumen to cell) to the secretion of H' against its concentration gradient (cell to lumen). In this communication, we will describe some 223

224

BERTRAM SACKTOR AND JAMES L. KINSELLA

properties of the Na+-H+ exchanger in brush border membrane vesicles isolated from the rat renal cortex and point out the large number of apparently disparate extrinsic factors which have been reported to alter the activity of the carrier. We will present some initial experiments describing how two hormones, thyroid hormones and glucocorticoids, regulate Na+-H+ exchange and how exchange activity responds in metabolic acidosis. Last, we will introduce a concept of hormonal hierarchy in the control of renal Na+-Ht exchange activity.

II.

FUNCTIONS OF Na+-H+ EXCHANGE IN THE PROXIMAL TUBULE

Na+-H+ exchange participates in multiple proximal tubular functions. A schematic model depicting the roles of the antiport reaction in these processes is shown in Fig. 1. The exchanger which removes Nat from the filtrate represents a mechanism for the reabsorption of a significant share of the filtered Na+ that is reclaimed by the proximal tubule. The Na+ that is transported into the cell is pumped out of the cell back into the blood by the Na+,K+-ATPase localized in the basolateral membrane. In the mammalian kidney proximal tubule, acidification of the glomerular filtrate leads to the reabsorption of about 90%of the filtered HCO; (Warnock and Rector, 1981). Protons, secreted by the exchanger, titrate HCO; in the lumen to form carbonic acid. Catalyzed by carbonic anhydrase, carbonic acid is dehydrated to C 0 2 , which then diffuses across the brush border membrane. Intracellular OH- generated by the secretion of H+ is buffered by the C 0 2 to reform HCO;. The HCO; exits the cell, crossing the basolateral membrane by a conductive pathway or by exchange for extracellular C1-. The Na+-H+ exchanger may also participate in a mechanism for generating new blood HCO; . As illustrated in Fig. 1, C02 may diffuse across the basolateral membrane into the cell, where it is converted into HCO; by titration with OH-. The H + , generated by the hydrolysis of H 2 0 , is removed to the lumen by the action of the brush border Na+-H+ exchange carrier. Here, H+ can acidify the urine, protonate the dibasic form of phosphate, and serve as a sink to convert NH3 to NH; . Thus, these latter reactions suggest that Na+-Ht exchange can play a role in regulating phosphate reabsorption and NH: excretion. 111.

MEASUREMENT OF Na+-H+ EXCHANGE ACTIVITY IN RENAL BRUSH BORDER MEMBRANE VESICLES

Nat-Ht exchange activity was determined by two independent methods, the uptake of 22Na+energized by a H + gradient and the flux of H t into

13. REGULATION OF RENAL Na+-H+ EXCHANGE ACTIVITY

FILTRATE

Pi -

CELL

225

BLOOD

i

NH3

the vesicle energized by an imposed Na+ gradient. These systems are shown in Figs. 2 and 3 , respectively. Rat renal cortical brush border membrane vesicles were prepared by a Mn2+aggregation method, as previously described (Freiberg et ul., 1982). The protocol for measurement of Na' uptake is illustrated schematically in Fig. 2A. Unless otherwise noted, the vesicles were preloaded with an intravesicular medium containing 150 mM KCI, 25 mM MES, 4 mM KOH, pH 5.5, by washing and suspending the brush border membranes with medium at least two times. The uptake of ??Na+was determined at 20°C by a rapid filtration technique (Aronson and Sacktor, 19751, with 0.65-pm Millipore filters. The uptake of Na' was initiated by the rapid mixing of 20 pl of membrane suspension (150 to 350 pg of protein) with 30 pI of uptake medium to give (final concentration) 145 m M KCI, 15 mM KOH, 10 mM MES, 9 mM HEPES; I mM NaCI, 0.2 pCi r2Na+,pH 7.5.

226


A

25 mM MES150 mM KCI pH 5.5

149 mM KCI 1 mM NaCl

H’

pH 7.5

10

20

30

1 hr

SECONDS

FIG. 2. Measurement of Na+-H+ exchange activity in renal brush border membrane vesicles. The uptake of zzNa+ energized by a H+ gradient, [HtIi > [H+],. (A) Protocol showing intravesicular and extravesicular media. (B) Time course of Na+ uptake in the absence and presence of amiloride. [Data taken from our earlier paper (Freiberg et ul., 1982) and redrawn.]

The uptakes were terminated by the addition of 3 ml of ice-cold stop solution containing 0.1 mM amiloride, 150 mM KCl, 15 mM HEPESKOH, pH 7.5. A typical experiment describing the uptake of Na+ in brush border membrane vesicles is illustrated in Fig. 2B. In the presence of an intravesicular > extravesicular H+ gradient, the approximate initial rate of Na+ uptake was rapid, 3.5 nmol/mg of protein 10 sec. Uptake was maximal in about 1 min. At the peak of the “overshoot,” the accumulation of Na+ reached a value several times the final equilibrium uptake (1 hr). Amiloride (1 mM), 30 times its Ki value (Kinsella and Aronson, 1981), inhibited the initial rate by 90%, suggesting that nearly all the Na+ uptake at incubation times of 10 sec or less was through Na+-H+ exchange. The difference between the uptakes in the absence and presence of amiloride represents the amiloride-sensitive Na+ uptake, i.e., Na+-H+ exchange activity .

227


A

150 mM Na gluconate

H+

10 mM TRlS HEPES 150mMTMAgluconate pH 7 5

B

1 0.04A

1 MIN 20mM Na

FIG.3. Measurement of Na+-H+ exchange activity in renal brush border membrane vesicles. The flux of H+ into the vesicles, measured by acridine orange absorbance, energized by an initially imposed Nat gradient [Na+]i > "a+],. (A) Protocol showing intravesicular and extravesicular media. (B) Trace of absorbance changes with time.

Figure 3A describes schematically the protocol used to measure the generation of a H+ gradient in membrane vesicles in which there was imposed initially a Na+ gradient. Here, H+ flux into the vesicles was measured by changes in absorbance of the weak base acridine orange, with 600-492 nm used as the reference and experimental wavelengths, respectively (Burnham et al., 1982). Membrane vesicles were loaded with 150 m M Na gluconate, 10 mM Tris-HEPES, pH 7.5, and diluted 100-fold into a solution containing 150 mM TMA gluconate, 10 mM Tris-HEPES, pH 7.5, and 20 pM acridine orange. The movement of Na+ from the intravesicular to the extravesicular medium via the exchanger generates the pH gradient [H+]i > [H+], . This effects the redistribution of acridine orange, the dye accumulating in the vesicle, resulting in a decrease in absorbance. An experimental trace describing these absorbance changes is illustrated in Fig. 3B. As shown, the mixing of the membrane suspension with the reaction mixture initiates the decrease in absorbance. Addition of Na+ to the medium perturbs the steady state of the system, causing the efflux of H+ and the increase in absorbance. These reversible absorbance changes could be blocked completely by amiloride (Kinsella et al., 1984a).

228


IV. EXTRINSIC EFFECTORS OF RENAL Na+-H+ EXCHANGE ACTIVITY

Table I lists the extrinsic factors currently reported to regulate renal Na+-H+ exchange activity. As shown, hormones may alter carrier function by increasing or decreasing activity. Thus, endocrines, such as thyroid hormones (Kinsella and Sacktor, 1984; Sacktor and Kinsella, 1985; Kinsella and Sacktor, 1985), glucocorticoids (Freiberg et al., 1982; Kinsella et d . , 1985a), prostaglandin E2 (Chaudhari et d . , 1984), and a ~ adrenergic agonists (Nord et al., 1984) were found to enhance renal Na+H+ exchange activity, whereas other hormones, such as parathyroid hormone (Cohn et al., 1983) and atrial natriuretic factor (Dousa et al., 1984), were reported to decrease activity. The mechanisms of action by which these hormones exert their effects on Na+-H+ exchange activity are not known. In general, it is believed that thyroid hormone and glucocorticoids act via genome mechanisms. Although studies on their effects on the Na+-H+ exchange system have yet to be carried out, their action on the sodium phosphate cotransport system in renal cells was blocked by inhibitors of transcription and protein synthesis (Sacktor et al., 1984).The inhibition of Na+-H+ exchange by parathyroid hormone is presumably mediated by cyclic AMP generation and protein kinase phosphorylation of specific membrane proteins. The effect of a2-agonists,a known attenuator of hormonally stimulated adenylate cyclase, in enhancing Na+-H+ TABLE I EXTRINSIC EFFECTORS OF RENAL BRUSH BORDERMEMBRANE Na+-H+ EXCHANGE ACTIVITY Hormone Thyroid hormone Glucocorticoids Prostaglandin E2 az-Adrenergic agonists Parathyroid hormone Atrial natriuretic factor Pathophysiological state Metabolic acidosis Chronic renal failure Streptozotocin-induced diabetes High-protein diet Low-K+ diet

Action Increase Increase Increase Increase Decrease Decrease Action Increase Increase Increase Increase Increase


229

exchange would be consistent with this presumption. On the other hand, atrial natriuretic factor has been reported to induce the synthesis of cyclic GMPin the kidney (Waldman et ul., 1984). Although an attractive hypothesis, it remains to be determined whether protein phosphorylation/dephosphorylation is involved in modulation of Na+-H+ exchange. In addition to hormones, a variety of pathophysiological states appear to alter the activity of the Na+-H+ exchanger in the brush border membrane (Table I). Thus, metabolic acidosis (Cohn el a/., 1983; Kinsella et al., 1984a), chronic renal failure (Cohn et ul., 1982), streptozotocin-induced diabetes (EI-Seifi et d., 1983), high-protein diets (Harris of a/., 1984), and'low-K diets (Seifter and Harris, 1984) all increase Na+-H+ exchange activity in the brush border membrane. A common denominator in these seemingly disparate conditions is that they all induced renal cortical hypertrophy and increases in glomerular filtrate rate. Indeed, in a series of rats, uninephrectomized and sham-operated, on diets containing different protein contents, positive correlations were found between Nat-H+ exchange activity and renal mass and glomerular filtration rate (Harris er al., 1984). Moreover, the transcription inhibitor actinomycin D prevented the adaptive increase in Na+ uptake and inhibited compensatory growth and increased filtration following unilateral nephrectomy. This correlation between increased Na+-H exchange activity and renal growth is of additional interest because in several different cell types growth factors and mitogenic stimuli were found to be associated with increases in the activity of the exchanger (Moolenaar et d.,1982; Pouyssegur et ul., 1982; Rosoff and Cantley, 1983; Benos and Sapirstein, 1983). The possibility that intracellular alkalinization and/or Na+ entry may be related to cell growth and differentiation constitutes an attractive and intriguing hypothesis. +

A. Regulation of Na+-H+ Exchange Activity by Thyroid Hormones

The previous reports that thyroid hormones increased glomerular filtration rate and Na+ load, net transport of Na' , isotonic fluid reabsorption in the proximal tubule, and Na+,K+-ATPaseactivity (Edelman, 1975; Katz and Lindheimer; De Santo et al., 1982) prompted us to examine whether thyroid hormones also enhanced the entry of Na' from the filtrate to the tubular cell across the luminal membrane by stimulating Na+-H+ exchange. To test this hypothesis, rats were made hypothyroid by adding thiouracil to their chow and drinking water (Kinsella and Sacktor, 1985). Thyroid powder was added to the thiouracil-containing diet to produce

230


hyperthyroid rats with different levels of serum T3 and T 4 . All animals were kept on the test diets for 3 weeks. It was observed that the size of the kidneys of the rats on the diets for only 3 weeks was markedly dependent of the thyroid status of the animal. As shown in Fig. 4, the kidney of the hypothyroid rat weighed 1.07 g. The kidney of the euthyroid animal had increased to 1.33 g, whereas the kidney of the hyperthyroid animal had become relatively immense, 1.58 g. This suggests that thyroid hormones, like the various pathophysiological conditions listed in Table I, cause renal cortical hypertrophy. Figure 5 shows that Na+-H+ exchange activity in renal brush border membrane vesicles was also affected by the thyroid hormone status of the rats from which the membranes were derived. The approximate initial rate (5 sec) of Na+ uptake in vesicles from euthyroid (T4 = 11 nglml) rats was 2.12 0.26 nmol/mg protein. Na+ uptake into vesicles from hypothyroid (0.4 ng/ml) animals was decreased by half, 0.90 & 0.13 nmol/mg protein. The rate of Naf uptake in vesicles from hyperthyroid (66 ng/ml) animals was 3.62 nmol/mg protein, almost twice that in membrane vesicles from the euthyroid rat. Accumulations of Na+ at equilibrium (1 hr) were essentially the same for the vesicles isolated from hypo-, eu- and hyperthyroid rats, suggesting that the differences in the initial rate of Na+ uptake were not due to an alteration in vesicle size. When 1 mM amiloride was present in the uptake medium, Na+ uptakes were decreased to the same level in the three experimental conditions, 0.13 nmollmg protein, indicating that thyroid status did not affect amiloride-insensitive Na+ uptake. These observations are consistent with the hypothesis that thyroid hormone altered Na+ uptake via Na+-H+ exchange in the luminal membrane of the renal proximal tubule. Because amiloride-sensitive, pH gradient-dependent Na+ uptake was increased in vesicles from hyperthyroid rats, membrane vesicles from the

*

FIG.4. The thyroid status of the rat and renal hypertrophy.

13. REGULATION OF RENAL Na*-H+ EXCHANGE ACTIVITY

5 sec 1 hr

231

TI

W

Y

a

I-

n 3

m '

z

-

0.4 nglml

11 nglml

66 ng/ml

T, CONCENTRATION

FIG.5. The effect of thyroid hormones on renal brush border Na+-H' exchange activity. [Data represent a replot of values reported elsewhere (Kinsella and Sacktor, 1985).]

hyperthyroid animals should also more rapidly generate a Na+ gradientdependent pH gradient. That this was indeed the case is shown in the experiment described in Fig. 6. By monitoring the change in absorbance of acridine orange, the rate of pH gradient change was shown to be faster in vesicles from hyperthyroid (T4 = 23.4 ng/ml) than in vesicles from hypothyroid (T4 = 1.4 ng/ml) animals. Amiloride ( 1 mM) completely blocked the changes in acridine orange absorbance. Figure 7 illustrates the correlation between Na+ uptake and log serum T4 concentration. Variation in the serum T4 level was effected by feeding rats diets that contained thyroid extract between 0.01 and 3 g/kg chow, in addition to thiouracil. As shown, there was a high correlation ( r = 0.827; p < 0.001) between Na+-H+ exchange activity and log serum T4 concentration. A similar correlation was found between Na+ uptake and serum levels of T3 (Kinsella and Sacktor, 1985). Kinetically, thyroid hormones may regulate Na+-H+ exchange activity by altering the affinities; changing the properties of the cytosolic H+ modifier site; changing the exchange stoichiometry ; increasing the incorporation of functional exchangers into the membrane; or increasing the turnover rate of existing carriers. Therefore, we next investigated some of these kinetic mechanisms (Kinsella et al., 1985b).

232


TI-HYPERT

-

AMlLORlDE

I

HYPOTHYROID W

AMlLORlDE

1

f

0.01 A

_I I---2

s--l

FIG.6. Generation of a pH gradient in Na+-loadedbrush border membrane vesicles from hypothyroid and hyperthyroid rats. [Traces are redrawn from experiments reported elsewhere (Kinsella and Sacktor, 19851.1

0

r:

-

1

7

EUTHYROID EUTHYROID *THIOURACIL WITH

m

-

2

tn

In

w Y

1.0-

a

r = 0.827

t 3 m

Z I

O0

L

2

5

10

20

50

100

SERUM T4(ng I rnl)

FIG.7. Relationship between log serum T4 concentration and Na+-Ht exchange activity. [Date are from Kinsella and Sacktor (1983.1

233


The kinetic properties of Na+-H+ exchange, with respect to Nat, were examined to determine whether thyroid hormones altered the apparent KNat or the V , . The initial rates ( 2 sec) of Na+ uptake at various Na+ concentrations in the presence of a pH gradient (pH, = 5.5; pH, = 7.5) are presented in Fig. 8 as a Hanes-Woolf plot. Thyroid hormones had no effect on the apparent Na+ affinity (euthyroid, 6.5 0.9 mM; hyperthyroid, 7.3 ? 1.7 mM), but significantly enhanced the maximal velocity (euthyroid, 9.0 -+ 0.3 nmol/mg 2 sec; hyperthyroid, 18.9 5 1 .O nmol/mg 2 sec). Aronson et ul. (1982) previously reported that the Na+-H+ exchanger in rabbit renal microvillar vesicles contained a distinct intravesicular Hi modifier site and an internal H+ transporting site. We confirmed this finding in rat renal brush border membranes and showed additionally that the rate of Nat measured as a function of intravesicular pH represented a saturable process. Further, as shown in Fig. 9, when the data are presented in the form of an Eadie-Scatchard plot and fitted to the Hill equation, a positive cooperative mechanism for the influence of cytosolic H + on Na+-H+ exchange could be demonstrated and a [H+l0 of 0.41 p M and an interaction coefficient of 1.43 could be calculated. To determine how thyroid hormones altered the kinetic properties of Na+-H+ exchange, with respect to H', the experiments illustrated in Fig. 10 were carried out.

*

3.0 EUTHYROID

-

-

Vm=9.0t0.3 nmobmg-'.2s-' KNa=6.5t0.9mM

/ / I

-

2.0 2.5

>, +

2

/ $ , /$ $ A A ' A

1.5-

l.O0.5

0

HYPERTHYROID

;I /*

Vm=18.9?1.0nmol mg-'.Zs-' K ~ ~ = 7 . 3 ? 1mM .7 1

4

1

“a+] mM

FIG.8. Effect of thyroid hormone on brush border membrane Na+-H’ exchange kinetics, with respect to Na+. [Data are from Kinsella et c d . (1985b).]

234


PHI

FIG.9. The effect of intravesicular pH on Na+-H+ exchange activity indicating a positive cooperative mechanism for activation of H+ at the cytosolic H+ modifier site.

I

0.5

I

1.o

I

1.75

2.0

V (nmol Na+ MG PROTEIN-' 2 S-')

FIG.10. Effect of thyroid hormone on brush border membrane Na+-H+ exchange kinetics, with respect to H+. [Data are redrawn from values reported elsewhere (Kinsella ef al. 1985b).I


235

As shown, neither the [H+],.5 nor the n was affected by the hormones (euthyroid: [H+]o.5= 0.44 p M , n = 1.2; hyperthyroid; [H+],.5 = 0.39 p M , n = 1.2). Only the apparent V , was increased, from 0.96 in the control to 1.68 nmol/mg - 2 sec after thyroid hormone treatment. Other experiments (not illustrated) demonstrated that Na+-H' exchange activities in vesicles from rats in different thyroid states were not altered by membrane potential. These results indicated that the increased exchange activity found in hyperthyroidism was not due to a change in the rate-limiting step for translocation from a membrane potential insensitive to a sensitive step or to an alteration of the stoichiometry of the carrier. These results of the effects of thyroid hormones on the kinetics of Na+H+ exchange-namely, no change in the apparent affinity for Na+, the [H+]o,~, the Hill coefficient, and the stoichiometry or sensitivity to membrane potential, whereas the V , with respect to extravesicular Na+ and intravesicular H+ were both increased-suggest that thyroid hormones increase the number of carriers per unit membrane or cause a more rapid turnover of existing carriers. At this time, we cannot distinguish between the two possibilities. 6. Regulation of Na+-H

+

Exchange Activity by Glucocorticoids

Adrenal glucocorticoids are important regulators of acid-base balance in the kidney. For example, adrenalectomy decreases renal excretion of ammonium and net titratable acid (Sartorious et al., 1952; Dubrovsky et al., 1981); and glucocorticoids have been shown to increase endogenous acid production, to stimulate acid secretion, and to enhance ammonium production (Hulter et af.,1980; 1981). These findings raised the possibility that the physiological alterations in acid metabolism may reflect actions induced by the hormone on the Na+-H+ exchange carrier in the renal brush border membrane. To test this hypothesis, the following experimental protocol was formulated (Freiberg et al., 1982). In brief, rats were adrenalectornized, sham-operated, or sham-operated and given multiple pharmacological doses of dexamethasone (60 pg/IOO g body wt). In another series, animals were adrenalectomized and then given physiological dosages (5 pg/IOO g body wt) of the glucocorticoid, dexamethasone, or the mineralocorticoid aldosterone. The results of these experiments are illustrated in Fig. 1 1 . The most active uptake of Naf was found with vesicles from sham-operated rats injected with high doses of dexamethasone (DEX). The initial rate of uptake was 4.71 2 0.39 nmol/rng 10 sec, a value 70% greater than the uptake found with vesicles from control animals, 2.80 k 0.18. Na+ uptake

236


1

2

5 6 0

1

2

5

60

TIME IMlNUTESl

FIG. 11. The effect of glucocorticoids on Na+-H+ exchange activity in renal brush border membranes. [Data are from Freiberg et al. (1982J.l

in vesicles from adrenalectomized (ADX) rats was not significantly different from the uptake in vesicles from sham-operated animals. When adrenalectomized rats were given the low maintenance dose of dexamethasone, the rate of Na+ uptake was 3.49 ?Z 0.13, a 40% increase relative to the uptake with adrenalectomized animals. In contrast, when aldosterone (ALDO) was administered, Na+ uptake was not significantly different from that found with adrenalectomized rats. In the presence of amiloride, no differences in Nat uptake were found among the different groups of animals. Concomitant with the increase in Na+ uptake via the exchanger, Na+-dependent H+ flux was stimulated and dissipation of the H+ gradient in the absence of Na+ was not affected (Freiberg et al., 1982). These findings suggest that the dexamethasone effect found in the absence of amiloride was attributable to the action of the hormone on the amiloridesensitive Na+-H+ exchange and that the adrenal corticosteroid effect on Na+-H+ exchange was limited to glucocorticoids. To determine the time course of induction of Na+-H+ exchange activity by glucocorticoids, adrenalectomized rats were administered a single subcutaneous injection of dexamethasone (60 pg/lOO g body wt). As shown in Fig. 12, a rise in the amiloride-sensitive Na+ uptake was seen 2 hr after injection, and a further rise was found in 4 hr (Kinsella et af., 1985). The increase was highly significant by 12 hr and maximal uptakes were found at 24 hr. By 48 hr after the single injection the uptake of Nat had decreased slightly but still was enhanced relative to the uptake in vesicles from the adrenalectomized rat.

237


1 rnM AMlLORlDE c

-

o

.

o

-

o

-

I I I I I I I

0 4

12

o

~

I

I

24

48

HOURS

FIG. 12. The time course of induction of Na+-H' exchange activity by glucocorticoids. [Data are from Kinsella et ul. (1985a).]

C. Alteration in Na+-H+ Exchange Activity in Chronic Metabolic Acidosis

Because the kidney responds to metabolic acidosis by increasing secretion of acid, phosphate, and ammonium and by enhancing reabsorption of bicarbonate (Cogin et af., 19811, we examined the possibility that metabolic acidosis induces changes in renal brush border Na+-H+ exchange activity (Kinsella ec al., 1984a). Rats were made acidotic by adding I % NH4CI to their drinking water for 6 to 8 days. Blood pH and pCOz and urinary pH of the acidotic animals were 7.19, 25.4 mm of Hg, and 5.77, respectively, compared to 7.41, 38.5 mm, and 6.81, respectively, in control animals. The effect of metabolic acidosis on Na+-H+ exchange activity in the renal brush border membrane vesicles is shown in Fig. 13. Exchange activity was increased; with membrane vesicles from acidotic rats, the initial ( 5 sec) rate of uptake of Na' in the presence of a pH gradient was 3.27 0.17 nmol/mg, a value 43% greater than the uptake found with vesicles from control animals, 2.26 0.19. Accumulations of Na+ at equilibrium ( I hr) were the same. When amiloride was present in the incubations, Na+ uptake rates by vesicles from acidotic and control animals were very low and were essentially identical. These observations are consistent with the hypothesis that metabolic acidosis altered Na+ uptake mediated by an amiloride-sensitive process, i.e., Na+-H' exchange. The

*

*

238


30

60 1 hr TIME (sec)

FIG. 13. Effect of metabolic acidosis on brush border membrane Na+-H+ exchange activity. [Data are from Kinsella ef al. (1984a).]

view is supported additionally by other findings that the enhanced uptake of Na+ in metabolic acidosis was not due to a decreased rate of passive H+efflux and that Na+-loaded vesicles from acidotic animals had a increased rate of H+ flux (Kinsella et al., 1984a). The kinetic mechanism of the metabolic acidosis-induced increase in Na+-H+ exchange activity was examined next (Kinsella et a l . , 1984b), and the results compared with the kinetic findings obtained when exchange activity was enhanced by thyroid hormones and glucocorticoids (Table 11). As shown, the affinity for Na+, the [H+]o.s,and the Hill coefficient were unchanged as a result of metabolic acidosis. Only the V , of the exchanger was increased. Similar results were found for the kinetic actions of glucocorticoids and thyroid hormone. Therefore, we conclude that metabolic acidosis, glucocorticoids, as well as thyroid hormones lead to either an increase in the number of functioning carriers or an increase in the turnover rate of the limiting step in the exchange. Because it was known that metabolic acidosis is associated with the increased synthesis and circulating levels of adrenal corticosteroids (Sartorius et al., 1952; Dubrovsky et al., 1981) and that both metabolic acidosis (Kinsella et al., 1984a) and glucocorticoids (Freiberg et al., 1982) induce increases in Na+-H+ exchange activity, we tested the hypothesis

239

13. REGULATION OF RENAL Na+-Ht EXCHANGE ACTIVITY

TABLE 11 KINETIC EFFECTSOF HORMONES ON Na+-H+ EXCHANGE KN~+ (mM)

V

Condition

(nrnolhg . 2 sec)

Thyroid -

8.9 18.9

+

Glucocorticoid -

* 0.3

(ILICI)

n

0.9 1.7

0.44 0.39

1.2 1.2

1.1 1.6

0.32 0.33

1.3 1.3

10.2 ? 0.5 10.2 2 0.5

0.41 0.37

1.4 1.3

* 1.1

6.5 7.3

?

7.9 ? 0.5 10.9 0.6

8.3 7.9

?

*

+

[H t l O . ~

?

?

Metabolic acidosis 11.3 +- 0.9 15.3 0.7

-

*

+

that glucocorticoids have a role in mediating the renal adaption in metabolic acidosis. Initial (5 sec) rates of amiloride-sensitive Na+ uptake in brush border membrane vesicles from differently treated rats are illustrated in Fig. 14. In this series of experiments, the Na' uptakes in membrane vesicles from control and acidotic animals were 1.95 k 0.21 and 2.97 ? 0.17 nmol/mg, 4.0

-

T

i

u)

In

b

-E0

3.0

-E

W

Y

a ! i 3

+

2.0

m

z

a

J

0

!-

0

$0

u a

nu

x-I-

? !I

0

z

0

d

d a

FIG. 14. Role of the adrenals and glucocorticoids in the stimulation of Na+-H+ exchange activity in brush border membranes from acidotic rats. [Data are redrawn from values reported elsewhere (Kinsella er a / ., 1984a).]

240


respectively. The uptake in vesicles from adrenalectomized rats was essentially the same as in vesicles from the control group (not illustrated). However, if acidotic rats were also adrenalectomized, Na+ uptake was not enhanced, 2.15 ? 0.27 compared to 2.97 ? 0.17 nmol/mg in membrane vesicles from acidotic rats with intact adrenals. Na+ uptake in vesicles from adrenalectomized, acidotic animals given dexamethasone (30 pg/IOO g body wt, 24 and 16 hr prior to sacrifice) was increased to 3.56 0.26 nmol/mg. Amiloride-insensitive uptakes, amounts of Na+ accumulated at equilibrium, and rates of passive Ht gradient dissipation did not differ between experimental groups (Kinsella et al., 1984a). These findings suggest that an intact adrenal gland or glucocorticoid supplement is necessary for an increase in Na+-H+ exchange activity with metabolic acidosis. The other responses of the kidney to metabolic acidosis, e.g., increased excretions of titratable acids (mostly phosphate) and ammonium, were also shown to be mediated, at least in part, by glucocorticoids (Kinsella et al., 1984a).

*

V.

HIERARCHY OF HORMONAL EFFECTS ON Na+-H+ EXCHANGE

As pointed out in Table I, hormones of distinct class and presumed action regulate the activity of the renal proximal tubule Na+-H+ exchanger. Assuming that the endocrines act on the same proximal tubule cell, one may ask why there is this diversity in input. Do the hormones act independently, in a permissive or coordinate fashion, or synergistically? Are different hormones invoked because each induces a unique pattern of pleiotypic responses, only one of which is to modulate the exchange reaction? Is the response time for each effector different? The answers to these and other important questions are not known at this time. However, preliminary bits of information addressing these questions are emerging. For example, we know now that the glucocorticoid dexamethasone enhanced Na+-H+ activity in the euthyroid animal as well as in the hypothyroid rat in which plasma levels of thyroid hormones could not be detected (Fig. 15). This indicates that glucocorticoids may stimulate Na+-H+ exchange activity in the apparent absence of thyroid hormone. Moreover, the increment of increase induced by the glucocorticoid was the same in the hypo- and euthyroid rat. These findings suggest that glucocorticoids act independently of thyroid hormones and the responses to the two endocrines are additive. Whether the two hormones evoke additive responses when they are used at saturating concentrations has yet to be determined.

24 1

13. REGULATION OF RENAL Na+-H- EXCHANGE ACTIVITY

z r

I I ~ T H Y R O I D ~ EUTHYROD -

1

2

DEX

DEX

FIG. IS. Glucocorticoids act to enhance Na+-H+ exchange activity independently of thyroid hormone.

We also now know that the different hormones control the activity of more than one transport system in the renal brush border membrane. For instance, thyroid hormones, glucocorticoids, and parathyroid hormone regulate Na+-H' exchange activity and the sodium phosphate cotransport system (Sacktor and Kinsella, 1985). But the direction of modulation is not necessarily the same. Thus, thyroid hormones and glucocorticoids increase Na+-H+ exchange activity; however, the former stimulates phosphate uptake, whereas the latter inhibits phosphate uptake. On the other hand, glucocorticoids enhance exchange activity, whereas parathyroid hormone decreases the antiport; yet both hormones inhibit Na+-dependent phosphate transport. Thus, in a simplistic way, the specific hormone which becomes operative may be determined by the homeostatic systems that need to be regulated. For example, in the stress concomitant with metabolic acidosis, glucocorticoids are involved. This brings about the increased excretion of acid, phosphate, and ammonium, but also the increased reabsorption of bicarbonate, mediated by a stimulated Na+-H+ exchange. In acute metabolic alkalosis, on the other hand, both phosphate and bicarbonate reabsorption are depressed, perhaps involving, at least in part, parathyroid hormone action (Bank and Malnic, 1980). The response time, or the time elapsed between exposure of the tubular cell to the various effector hormones and a demonstrable change in the brush border membrane Na+-H+ exchange activity, differs with the different endocrines. These temporal related responses are illustrated schematically in Fig. 16. Na+-H+ activity can be up-regulated or down-regulated, depending on the thyroid status of the animal. However, at least a

242


-SC

MN

HR

DAY

FIG. 16. Time-dependent hierarchy of factors regulating renal Na+-H+ exchange activity.

day is required for the effects of an altered thyroid state to become manifest. This delayed response is consistent with the presumed genomic mechanism of action of the hormone. Moreover, the half-life of thyroid hormones in uiuo is relatively long. Hence, rapid fluctuations in hormone titer in plasma are not to be expected. Considering these observations, we would like to propose that thyroid hormones may serve to establish a basal level of Na+-H+ exchange activity, elevated in hyperthyroidism and depressed in the hypothyroid state, but whose activity is not subject to rapid modulation by thyroid hormone. We further hypothesize that the action of the other hormone effectors is superimposed on this thyroid hormone-imposed steady state. However, the response times for these other hormones also vary. As shown in Fig. 12, a significant increase in Na+-H+ exchange activity was not measurable for several hours after the injection of dexamethasone. Again, this is in accord with the presumed action of glucocorticoids in inducing de nouo protein synthesis. Moreover, as described in Fig. 14, glucocorticoid-dependentincreases in Na+H+ exchange were independent of, or superimposed on, the activity previously established by thyroid hormones. By analogy with the quick response times of other systems to a-adrenergic agonists, prostaglandin E2, parathyroid hormone, and atrial natriuretic factor, it is not unreasonable to propose that Na+-H+ exchange activity may also be responsive to minute-to-minute fluctuations in their titers. Increases in the plasma level of parathyroid hormone or the atrial natriuretic peptide may rapidly decrease exchange activity, whereas increases in the titer of q-adrenergic agonists or prostaglandin E2 may rapidly stimulate carrier function. Further, lowering of the hormonal titers or the presence of mechanisms for terminating the action of the endocrines, e.g., phosphoprotein phosphatases, may restore Na+-H+ exchange activity to basal levels. Last, Na+H+ exchange activity may be modulated by intrinsic effectors, such as by

13. REGULATION OF RENAL Na+-Hi EXCHANGE ACTIVITY

243

H+ at the cytosolic regulating site. Slight shifts in intracellular pH, within the physiological range, profoundly alter activity (Fig. 9). A decrease in intracellular pH enhances activity; thus, HS secretion, which in turn results in intercellular alkalization and decreases in activity. The response time of this intrinsic feedback mechanism is likely to be instantaneous. In summary, the present discussion of multiendocrine inputs and the temporal-dependent hierarchy of hormonal control of Na’-H+ exchange represents an initial attempt to develop a concept for the mechanism by which Na+-H+ exchange activity is regulated. It is patently clear that numerous and wide gaps exist in our current knowledge. However, despite this paucity of information, a formulation of a model at this time presents opportunities for insightful experimentation. Such studies will lead to modification and refinement of the idea and to the eventual development of a firmer picture of the mechanism and control of this crucial renal ion transport system. REFERENCES Aronson, P. S.. and Sacktor, B. (1975). J. B i d . Chem. 250, 6032-6039. Aronson, P. S., Nee, J.. and Suhm, M. A. (1982). Nature (London)299, 161-163. Bank, N., and Malnic, G. (1980). In “Renal Handling of Phosphate” (S. G. Massry and H . Fleisch, eds.), pp. 209-241. Plenum, New York. Burnham, C., Munzesheimer, C.. Rabon, E., and Sachs, G . (1982). Biochim. Biopbys. Acru 685, 260-272. Chaudhari, A., Badie-Dezfooly, B., Homadani. B. M., and Fine, L. G. (1984). Annu. Meet. A m . Soc. Nephrol. (Abstr.), p. 156A. Cogan, M. G.. Rector, F. C., Jr., and Seldin, L). W. (1981). In “The Kidney” (€3. M. Brenner, and F. C. Rector, Jr., eds.), pp. 841-907. Saunders, Philadelphia. Cohn, D. E., Hruska, K. A., Klahr. S., and Hammerman, M. R. (1982). A m . J . Pbysiol. 243, F292-F299. Cohn, D. E., Klahr, S., and Hammerman, M. R. (1983). A m . J. Pby.tio/. 245, F217-F222. DeSanto, N. G., Capasso, G., Kinne, R., Moewes, B., Carella, C., Anastasio, P . , and Giordano, C. (1982). PJliigers Arch. 394, 294-301. Dousa, T. P., Hammond, T. G., Yusufi, A. N. K.. and Knox, F. G. (1984). Annu. Meet. A m . Soc. Nephrol. (Absrr.), p. 208A. Dubrovsky, A. H. E., Nair, R. C., Byers, M. K . , and Levine, D. Z. (1981). Kidney lnt. 19, 5 16-528. Edelman, I. S . (1975). Med. Clin. North A m . 59, 605-614. El-Seifi, S., Freiberg, J. M., and Sacktor, B. (1983). Fed. Proc., Fed. A m . Soc. Exp. Biol.. 42, 1287 (Abstr.). Freiberg, J. M., Kinsella, J., and Sacktor, B. (1982). Proc. N U / / .Acrid. Sci. W.S.A. 79, 4932-4936. Hulter, H. N., Licht, J . H., Bonner, E. R.. Glynn, R. D., and Sebastian, A. (1980). A m . J. Physiol. 239, F30-F43. Hulter, H. N., Sigala, J. F., and Sebastian, A. (1981). Kidney In/. 20, 43-49. Katz, A. I., and Lindheimer, M. D. (1977). Annu. Reu. Pbysiol. 39, 97-134. Kinsella, J. L., and Aronson, P. S. (1980). A m . J. Physiol. 238, F461-469.

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Kinsella, J. L., and Aronson, P. S. (1981). A m . J. Physiol. 241, F374-F379. Kinsella, J., Cujdik, T., and Sacktor, B. (1984a). Proc. Nutl. Acad. Sci. U.S.A. 81, 630634. Kinsella, J., Cujdik, T., and Sacktor, B. (1984b). J. Biol. Chem. 259, 13224-13227. Kinsella, J., and Sacktor, B. (1984). Fed. Proc., Fed. Am. SOC.Exp. Biol. 43, 633 (Abstr.) Kinsella, J., and Sacktor, B. (1985). Proc. Null. Acad. Sci. U.S.A. 82, 3606-3610. Kinsella, J. L., Cujdik, T., and Sacktor, B. (1985b). Ann. N. Y . Acad. Sci. 456, 445-447. Kinsella, J. L., Freiberg, J . M., and Sacktor, B. (1985a). A m . J. Physiol. 248, F233-F239. Moolenaar, W. H., Yanden, Y., de Laat, S. W., and Schlessenger, J. (1982). J . Biol. Chem. 257, 8502-8506. Murer, H., Hopfer, U., and Kinne, R. (1976). Biochem. J. 154, 597-604. Nord, E. P., Meier, K. E., Goldfarb, D., Hafezi, A., Vaystub, S., Insel, P. A., and Fine, L. G. (1984). Annu. Meet. A m . SOC.Nephrol. (Abstr.),p. 219A. Pouysskgur, J., Chambard, J. C., Franchi, A., Paris, S., and Van Oblerghen-Schilling, E. (1982). Proc. Nutl. Acud. Sci. U . S . A . 79, 3935-3939. Rosoff, P. M., and Cantley, L. C. (1983). Proc. Nut/. Acud. Sci. U . S . A . 80, 7547-7550. Sacktor, B. (1977). In “Mammalian Cell Membranes” (G. A. Jamieson and D. M. Robinson, eds.), Vol. 4, pp. 221-254. Butterworths, London. Sacktor, B., Cheng, L., and Noronha-Blob, L. (1984). I n “Coupled Transport in Nephron, Mechanisms and Pathophysiology” (T. Hoshi, ed.), pp. 10-22. Miura Medical Research Foundation, Tokyo. Sacktor, B., and Kinsella, J . L. (1985). Ann. N . Y . Acud. Sci. 456, 438-444. Sartorius, D.W., Calhoon, D., and Pitts, R.F. (1952). Endocrinology 51, 444-450. Seifter, J. L., and Harris, R. C. (1984). Kidney Int. 24, 302 (Abstr.) Waldman, S. A., Rapoport, R. M., and Murad, F. (1984). J. Biol. Chem. 259, 14332-14334. Warnock, D. C., and Rector, F. C., Jr. (1981). In “The Kidney” (B. M. Brenner and F. C. Rector, Jr., eds.), pp. 440-494. Saunders, Philadelphia.

CURRENT TOPICS IN MEMBRANES A N D TRANSPORT, V O L U M E 26

Chapter 14

Adaptation of Na+-H+ Exchange in the Proximal Tubule: Studies in Microvillus Membrane Vesicles JULIAN L . SEIFTER AND RAYMOND C . HARRIS Laboratory of Kidney and Electrolyte Physiology and Department of Medicine Brigham and Women's Hospital Boston. Massachusetts and Haruard Medical School Boston. Massachusetts

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Experimental Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Effects of Uninephrectomy and Dietary Protein on Na+-H+ Exchange . . . . . . . IV. Effects of Potassium Depletion on Na+-H' Exchange ...................... .., .., ., .., . V. Other Models of Adaptation.. ........................... VI. Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . .

1.

245 247 249 256 257 258 259

INTRODUCTION

Na+-H+ exchange has been identified in a wide range of epithelial and nonepithelial cells (Ives and Rector, 1984). This transport mechanism participates in the regulation of cell pH (Roos and Boron, 1981) and volume (Cala, 1980) and is important for cell division in the fertilized egg (Johnson et al., 1976) and in cultured cells responding to growth factors and mitogenic stimuli (Moolenar et crl., 1983). In transporting epithelia such as the renal proximal tubule, Na+-H+ exchange present on the microvillus membrane is an important luminal mechanism for NaHC03 245 Copyright C 1986 hy Academic P r e s . Inc All nghts ot reproduction in any torm rererved

246

JULIAN

L. SEIFTER AND RAYMOND C. HARRIS

reabsorption (Warnock and Rector, 198 1). Although proximal HCO; reabsorption is determined by multiple factors including luminal fluid flow rates, peritubular Starling forces, and electrochemical ion gradients, it is possible that changes in intrinsic characteristics of the Na+-H+ antiporter may contribute to the regulation of HCOY transport. Proximal HCO; transport responds to changes in physiologic factors including the rate of glomerular filtration and the state of acid-base, electrolyte, and hormonal balance of the animal (Cogan and Alpern, 1984). Several studies have suggested that intrinsic changes in membrane transport occur during adaptation of the animal to an altered physiologic environment. For example, proximal tubules isolated from uremic animals have an increased rate of fluid absorption when studied in vitro (Fine et al., 1978; Trizna et al., 1981). The rate of volume absorption is also increased in tubules isolated from rabbits with an increased single-nephron glomerular filtration rate (SNGFR) induced by contralateral uninephrectomy or glucocorticoid administration (Tabei et al., 1983). Such changes are noted within 24 hr of the experimental manipulation. Several authors have utilized isolated renal cortical membrane vesicles to study the activity of individual transport systems in animals adapted to abnormal chronic conditions. For example, Na-dependent inorganic phosphate transport in rat renal brush border membranes increased in animals on a low-phosphate diet (Kempson and Dousa, 1979). This correlated with the finding in the intact animal that a low-phosphate diet resulted in increased phosphate reabsorption. Na-dependent phosphate transport was decreased in vesicles from animals with metabolic acidosis (Kempson, 1982) and in chronic renal failure (Hruska et al., 1982), conditions associated with decreased phosphate reabsorption. Acute respiratory acidosis and chronic metabolic acidosis are accompanied by increased ammoniagenesis in the proximal cell, an effect associated with increased vesicle glutamine transport across basolateral membranes of adapted animals (Windus et al., 1984a,b). Na+-H+ exchange has been studied similarly in models of adaptation in which NaHC03 reabsorption capacity is thought to be increased. For example, an enhanced rate of Na+-H+ exchange was found in renal brush border membranes isolated from the remnant canine kidney (Cohn et al., 1982). Several studies have demonstrated increased Na+-H+ exchange activity in vesicles from animals with metabolic acidosis (Cohn et ul., 1983; Tsai et al., 1984; Kinsella et al., 1984), a condition also associated with increased HCO; reabsorptive capacity (Alpern et al., 1983). Although the study of adaptation of the Na+-H+ exchanger in isolated renal microvillus membrane vesicles may yield important information

14. Na+-H+ EXCHANGE IN THE PROXIMAL TUBULE

247

about regulatory functions of proximal tubule transport, it should be noted that changes in vesicle transporter activity may have as its physiologic correlate functions other than HCO; transport. This may be particularly true for Na+-H+ exchange, given the broad range of functions attributed to this antiporter in nonreabsorptive cells. The following discussion will consider intrinsic changes in Na+-H+ antiporter activity that occur in microvillus membrane vesicles isolated from the rat renal cortex in models of chronic adaptation of the animals. First the adaptive response of Na+-H' exchange in vesicles isolated from remnant renal tissue following unilateral nephrectomy will be characterized. Ablation of renal mass is associated with compensatory hypertrophy and an increase in the filtration rate of remnant glomeruli with concordant increases in HCO; reabsorptive capacity per nephron (Bank and Aynedjian, 1978). Since restricting dietary protein intake is known to modify the increase in SNGFR after renal ablation (Hostetter et al., 1981), the effect of variations in dietary protein content will be assessed with respect to the activity of Na+-H+ exchange. Since severe dietary K+ depletion also results in renal cortical hypertrophy (Gustafson et al., 1973) and increases in HCO, reabsorptive capacity (Chan et al., 1981, 1982), the adaptation of vesicle Na+-H+ exchange will be considered in this model. II. EXPERIMENTAL APPROACH

Isolated microvillus membrane vesicles have proved to be of value in the study of membrane transport mechanisms. However, when they are employed to evaluate differences in transport among experimental groups of animals, certain criteria need be met to allow for such comparisons. We have considered several criteria in the evaluation of Na+-H+ exchange activities in renal microvillus membrane vesicles. Vesicles were prepared from homogenates of renal cortex by a method of Mg2+aggregation and differential centrifugation (Aronson, 1978). The specific activities of brush border marker enzymes including maltase, y-glutamyl transpeptidase, and alkaline phosphatase were comparable in the control and experimental vesicle membranes, as were activities of depurified enzyme markers including Na+,K+-ATPase and succinic dehydrogenase. Transport was determined for radioisotopically labeled solutes using rapid filtration techniques. Intravesicular volumes (expressed in microliters per milligram of vesicle protein) were estimated from the known concentrations of protein and solute and the amount of uptake of the labeled solute at equilibrium. In the case of isotopic D-glucose and ?"a+, equilibrium volumes were similar in control and experimental groups.

248

JULIAN L. SEIFTER AND RAYMOND C. HARRIS

Na+-H+ exchange was assayed as rates of 22Na+uptake into vesicles under conditions of an inside acid pH gradient (stimulating Na+ uptake) and in the absence of a pH gradient. It has been shown that the major route of 1 mM 22Natransport under both conditions is via Na+-H+ exchange (Kinsella and Aronson, 1980). In addition Na+-H+ exchange rates were determined as the component of 22Nat flux inhibitable by I mM amiloride, a known inhibitor of vesicle Na+-H+ exchange activity (Kinsella and Aronson, 1981). In all experimental groups electrodiffusive flux of I mM 22Na+was shown to be small and without difference among groups. Similarly, rates of non-Na-dependent pH gradient dissipation, studied as fluorescent quenching of the weak base acridine orange, a probe of pH gradients (Reenstra et al., 1981), were found to be the same in control and experimental models. Since Na D-glucose and L-alanine cotransport processes were found to be unchanged in the models studied, changes in Na+-H+ exchange activity were specifically altered during adaptation. Transport rates were expressed per milligram of vesicle membrane protein. Therefore any changes in activity suggest a disproportionate change in Na+-H+ antiport relative to other membrane protein. If during adaptation the number of functional transporters increased in proportion to membrane protein, an effect would not be observed on vesicle transport. Because solute concentrations can be defined on either side of the vesicle membrane, estimates of true unidirectional flux can be made, and applying such data to Michaelis-Menten kinetics, the parameters V,,, and K , for a solute can be determined for a given transport system. Although these kinetic properties may or may not conform to findings in the intact animal, they are useful for comparing transport in membranes from control and experimental animals, provided that the quality of the preparation is comparable and the conditions under which transport is studied are identical. The V,,, for a solute may be a reflection of the total number of carrier proteins available for transport. However, an observed increase in V,,, need not imply new carrier synthesis. For example, changes in membrane fluidity may activate carriers already in the membrane (Rasmussen et al., 1979). Alternatively, presynthesized transporters may be in an intracellular location available for incorporation into the membrane under the appropriate stimulus. The V,,, may also be increased for a given solute if the maximal turnover rate of a constant number of transport sites were to be increased. Other modifications of transport activity include changes in the Na+ requirement, K , , for transport and intrinsic changes of known modifier sites (Aronson et al., 1983).


249

111. EFFECTS OF UNINEPHRECTOMY AND DIETARY PROTEIN ON Na+-H+ EXCHANGE

In the first model of chronic adaptation we evaluated the effects of contralateral nephrectomy on vesicle Na+-H+ exchange activity in remnant renal tissue. Several well-known changes in renal structure and function occur in this model. Within hours of loss of contralateral function there is an increase in GFR and proximal reabsorption in the remaining kidney (Tabei et al., 1983). Both structural and biochemical evidence indicate that renal growth begins soon after decreases in functioning renal mass. On the first day following loss of contralateral excretory function, there occur increases in the numbers of free ribosomes and microtubular structures. There is dilation of endoplasmic reticulum and of the Golgi apparatus as the proximal cells increase in size. Multinucleated cells and cell division indicating hyperplasia appear by 72 hr following uninephrectomy (Anderson, 1967). Although there are increases in diameter and length of the proximal tubule associated with the hypertrophic response, increases in GFR and proximal fluid absorption precede these changes (Tabei et al., 1983). The increase in wet renal weight of the remnant kidney is due to an increase in tissue solids, largely protein (Halliburton, 1969). There are increased biosynthetic rates of RNA (Malt, 1969) and protein (Coe and Korty, 1967) within the first hours of uninephrectomy. Following these changes, after I to 2 days, an increase in DNA synthesis occurs in the remnant tissue (Northrup and Malvin, 1976). There is also evidence for an enhanced rate of new membrane phospholipid synthesis occurring within the first hours of contralateral nephrectomy (Toback et al., 1974). It has been shown that the hypertrophy of proximal cells in primary culture is blocked by amiloride, an inhibitor of Na+-H+ exchange in these cells (Lowe et al., 1984). Functionally, the increases in SNGFR are accompanied by concordant changes in the absolute proximal reabsorption of fluid, Na+, and HCO? , so that glomerulotubular balance is preserved. Preliminary experiments suggest that increases in luminal fluid flow rates in the isolated nephron may induce an amiloride-sensitive increase in fluid absorption within I hr of perfusion (Trizna et al., 1984). Dietary protein excess has many of the same effects on renal structure and function as does renal ablation. Thus GFR is increased as is renal mass due to hypertrophy and hyperplasia (Halliburton, 1969). Also, increases in synthesis of NH3 and glucose occur in both models (Schoolwerth et al., 1974, 1975). In addition, dietary protein restriction is

250


A

B

-.C

a

c

2

a

2'1

6

f

P

7 2.0f

\

u)

a 0

E

c

Y

sU

U c

I .o

c

a

41

3

U

2 N

z N N

?I

Time (sec)

N

2 hrs

05

0

10

30

60 2 hrs

Ti me (sec 1

FIG.1. Time course of **Na+uptake into microvillus membrane vesicles in the presence (A) or absence (B) of an inside-acid pH gradient. Vesicles were equilibrated at pH 6.1 with HEPES-MES buffer in (A) and at pH 7.5 with K-HEPES (B). Influx of 1 m M ?*NaCIwas assayed at 20°C. External pH was 7.2 in (A) and 7.5 in (B). Values represent the mean SEM for six or seven experiments in each group. UNx means uninephrectomy. Animals were fed 6% or 40% protein diets for 2 weeks before study. [Reproduced with permission from H a m s et al. (1984).]

*

known to modify increases in GFR and renal mass that result from renal ablation (Hostetter et al., 1981 ;Meyer et al., 1983). Therefore the independent effects of dietary protein on vesicle Na+-H+ exchange and the effects of altering protein intake in combination with uninephrectomy were evaluated. The uninephrectomy model of renal ablation is not complicated by uremia or metabolic acidosis, although net acid excretion and nitrogen level per nephron are increased (Schoolwerth et al., 1974). Figure 1 shows the time course of I mM 22Na+uptake into vesicles isolated from four groups of experimental Sprague-Dawley rats. Animals were studied after 2 weeks on a diet of either high-protein (40%) or lowprotein (6%) content. Rats on each of these diets had undergone prior unilateral nephrectomy or a sham operation. As shown in Fig. lA, the presence of an inside-acid pH gradient gave greater stimulation of Na+ uptake into vesicles isolated from animals fed the 40% protein diet when compared to the animals on the 6% diet. At each level of dietary protein intake, 22Na+uptake was greater in uninephrectomized animals compared to the sham-operated groups. As demonstrated in Fig. IB, Na+ uptake in the absence of a pH gradient was again greater in rats being fed high levels of dietary protein or having undergone prior uninephrectomy. Figure 2

251


shows that the increased NaS uptake rates were due to increases in amiloride-sensitive *"a+ flux and, in addition, demonstrates that the standard 24% protein diet gave intermediate values of Na+-H' exchange activity. As demonstrated in Figs. I and 2, low-protein diets modify the increased **Na+transport that otherwise follows uninephrectomy . Since increments in renal mass and GFR are known to occur within hours of renal ablation, ?*Na+ transport was studied I day after uninephrectomy. Stimulation of ??Na+uptake was apparent at this earlier time interval. Furthermore, when uninephrectomized animals were treated with a single intraperitoneal injection of actinomycin D (0.12 mg/kg) at the time of surgery, the increased Na+-H+ exchange activity did not occur. This inhibitor of protein synthesis is known to prevent the increases in GFR and renal mass that follow uninephrectomy (Northrup and Malvin, 1976). Whether actinomycin D is acting to inhibit synthesis of new NafH+ exchange proteins or other regulators of antiporter activity is not known. The kinetics of 22Na+transport in vesicles from uninephrectomized animals on 6% or 40% protein intake is shown in Fig. 3 . The effect of dietary protein was to increase the V,,,, , represented as the y intercept of the Eadie-Hofstee plot. The K , for Na+, indicated by the negative slope of the line, was unchanged. A similar effect on V,, was observed for uninephrectomized rats compared to sham-operated animals. The adaptive increase in vesicle Na+-H+ exchange in metabolic acidosis is also characterized by an increased V,,, (Tsai ef al., 1984; Kinsella ef al., 1984).

lli

Dietary Protein = 6%

24%

SHAM

L

+Amilor#da

+ Amiloride

6%

4 0%

UNINEPHRECTOMY

FIG. 2. Amiloride-sensitive ]?Na+ transport in memhrane vesicles from uninephrectomized and sham-operated animals fed 6% (low), 24% (standard), or 40% (high) protein diets for 2 weeks. 10-sec uptakes of 1 mM 22NaCIwere assayed in the presence of an insideacid pH gradient (pH,, = 6.1, pH,,, = 7.2). Values represent the mean r SEM for three to six experiments in each group. [Reproduced with permission from Harris er a / . (1984).]

252


nmoles Na uptake /mim mg protein/ "a+](
0.5) the effect of insulin upon pHi in frog skeletal muscle (Moore, 1979), confirming this prediction. This prediction is also supported by the finding that depleting frog oocytes of Na+ by soaking in Na+-free Ringer (which by decreasing the Na+ driving force should bring (AG)Na-Htoward zero) blocks the effect of insulin upon pHi (Morrill et al., 1983). Third, lowering "a+], still further reverses the sign of (AG)Na-Hand thus reverses the direction of Na-H exchange. Therefore, removing extracellular Na+ should convert the action of insulin from an increase to a decrease in pHi. Confirmation: When either Mg2+or choline is used to replace the Na+ in the Ringer, actually about 0.12 mM Na+ (Moore, 1981a), the effect of insulin upon pHi is converted to a statistically significant decrease ( P < 0.005) (Moore, 1981a). Of equal importance, the magnitude of A G N is ~ sufficient to move H+ against its energy gradient as is indicated by the fact that (AG)N~-H always has the proper sign for the observed flux of Hf (Moore, 1981b).

15. ROLE OF INTRACELLULAR pH

IN INSULIN ACTION

273

It is possible that all of the preceding results could be due to the operation of a Na+-CO:- cotransport system (Funder et al., 1978). However, the elevation of pH, by insulin in HC0;-free Ringer rules out this possibility (Moore et al., 1979a; Moore, 1981a) and also argues against the effect being due to HCO; exchange. Fourth, because the diuretic drug amiloride (3,5-diamino-6-chloropyrazinoylguanidine) blocks Na-H exchange (Benos, 1982), this drug should block all the preceding effects of insulin; i.e., it should block the effect of insulin upon both H+ efflux and the associated Na' influx and upon the decrease in pH, produced by insulin in Na-free Ringer. Conjrrnation: Insulin increases pH, in frog sartorius muscle whether or not 5% CO2/3OmM HCO; is present (Moore, 1977). At a concentration of 0.5 mM, amiloride blocks this elevation by insulin of pH, in the presence (Moore et al., 1979a) or the absence (Moore, 1981b; Putnam and Roos, 1983) of C02/HCO;. Of considerable importance is the finding that 1 mM amiloride inhibits the stimulation by insulin of pH, recovery which occurs in acid-loaded frog semitendinous muscles in the presence of 15 mM K; (Putnam and Roos, 1983). Finally, in the presence of 1 mM ouabain, 0.5 mM amiloride also blocks the increase in Na; due to exposure of muscles to insulin for 90 min (Moore, 1981). Finally, in muscles placed in Ringer in which Na' is replaced by osmot0.5 mM amiloride blocks the decrease ically equivalent amounts of Mg' +, in pH, produced by insulin (Moore, 1981). These effects of amiloride are not limited to amphibian skeletal muscle; the elevation of pH, in frog oocytes by insulin is also blocked by this drug (Morrill et al., 1983). Moreover, the elevation of pH, reported in rat hemidiaphragms by in uitro addition of insulin is also blocked by (5 x M ) amiloride (Clancy et a l . , 1983). There is evidence that the change in pH, is not secondary to metabolic changes. The results of Putnam and Roos (1983) are especially important because they clearly demonstrate that the effect of amiloride is to block the stimulation by insulin of acid trarzsport (as opposed to metabolic- or buffer-induced pH, changes). could Moreover, it is difficult to see how amiloride or nulling (AG)N~-" block metabolic- or buffer-induced pH, changes. In that context, insulin stimulates both glycolysis and, by increasing the activity of the Na+-K+ pump, MgATP hydrolysis. Although glycolysis does not produce intracelMar H' under anaerobic conditions (Busa and Nuccitelli, 1984). the accompanying hydrolysis of MgATP does according to the reaction (at pH > 8) MgATP2

+ H20

-

MgADP

+ Pi + H'

274

RICHARD D. MOORE

Even at lower pH values, the combined effect of glycolysis and hydrolysis of the ATP produced is still to produce protons according to the reaction (Busa and Nuccitelli, 1984) glucose

-

2 lactate-

+ 2 H’

Accordingly, under anaerobic conditions, if insulin does not stimulate acid extrusion, the hormone would be expected to produce a decrease in pHi due to stimulation of ATP hydrolysis by the Na+-Kf pump. C. Conclusions

It seems reasonably well established that in frog skeletal muscle and oocytes, insulin can stimulate the Na-H exchange system in the plasma membrane. The evidence that insulin exerts this effect upon pHi in mammalian cells is not yet as clear, but a growing body of evidence suggests that under physiological conditions, insulin regulates pHi in mammals as well. What little evidence exists suggests this is also due to activation of Na-H exchange. 111.

EFFECTS OF INSULIN-MEDIATED CHANGES IN pH1 UPON GLYCOLYSIS

Although the elevation of pHi may play a role in insulin action upon several cell functions including protein synthesis, nucleic acid synthesis, and cell division, the best-established example, based upon experimental results to date, is the stimulation of glycolysis by insulin. The hypothesis that the stimulation of glycolysis by insulin is due to activation of PFK by an increase in pHi consequent to stimulation by insulin of Na-H exchange at the plasma membrane leads to the following predictions. First, blocking the effect of insulin upon pHi should block the stimulation of glycolysis by this hormone. This prediction was verified by the finding that in a glucose-free Ringer (30 mM HC07/5% COz), either amiloride or a 15-fold reduction in the concentration of extracellular Na+, [Na+Io,also blocks the action of insulin upon glycolysis as well as upon pHi (Moore et a)., 1979a). Second, it follows from Eq. (2) that ( A G ) N a is - ~a function of log[Na+].. Therefore, if the effect of insulin upon glycolysis is due to Na-H exchange, this effect should be a function of log[Na+],, with a reversal of (AG)N,-~by decreasing “a+],, resulting in an inhibition of glycolysis by insulin.

275

15. ROLE OF INTRACELLULAR pH IN INSULIN ACTION

The effect of insulin upon glycol ytic flux varied approximately linearly with log[Na+], (Fig. 2), with the intercept at "a+], = 5.8 mM representing the value at which (AG)Na-Hshould equal zero in the preceding hypothesis. Reducing "a+], from 104 to 0.12 mM changes the insulin effect upon glycolytic flux from an increase of 42.9 ? 3.5% ( P < 0.001) to a decrease of 51.5 -+ 8.4% (P < 0.001). This may be the first example of a model of hormone action that successfully predicts a means to reuerse the direction of the hormone effect. Such a demonstration is more convincing than just blocking an effect. The latter can always be due to nonspecific actions, including damage to the system, whereas reversal of an effect requires a functioning transduction system. Third, if the central hypothesis that pHi represents the connection between insulin activation of Na-H exchange at the plasma membrane and the effect of insulin upon glycolysis is correct, changes in pHi produced by any other means (e.g., by varying COZ)should produce the same effect upon glycolysis as the pHi produced by insulin.

0.I

I 10 Extracellular Na* Concentration (mM)

100

on the action of insulin upon glycolytic flux, as FIG.2. The effect of lowering "a'], reflected by anaerobic lactate production, in frog sartorius. Percentage change in glycolytic flux due to insulin is plotted versus log[Na+],. Solid circles: mean t SE for 7 to 14 determinations. Open circles: mean t SE for 3 or 4 determinations. The mean glycolytic flux for all controls was 23.1 t 1.2 nmol lactate rnin-'/g wet wt. For the Na-free Ringer, a value of = 0.12 mM was used (see the text). Varying "a'], does have some effect upon "a'], control values of "a+], and pH,. However, since control values of "a'], and Z H , , varied by at most a factor of 2, their effect on (AG)N~-H was extremely small [see Eq. (l)] compared to that of "a'],, which varied by almost three orders of magnitude. A least-squares linear regression was performed on the 60 actual data points ( r = 0.823, P < 0.001; slope > 0, P < 0.001). Semilogarithmic scale. [Figure reproduced from Fidelman P I NI. (1982).]

276

RICHARD D. MOORE

Figure 3 demonstrates that the relationship between pHi and percentage change in glycolytic flux was independent of whether the effects are produced by insulin or by varying COZ since the means of the experimental points all lie on essentially the same line. The results demonstrate that the change in pHi produced by insulin was sufficient to account for the entire effect of insulin upon glycolysis in the time period (130 min) of these experiments. Fourth, stimulating glycolysis either by insulin or by increasing pH, secondary to a decrease in C 0 2 should be associated with activation of PKF. Also, inhibiting glycolysis either by insulin in a Na-free Ringer or by increasing COz to decrease pHi should be associated with a decrease in the activity of this rate-limiting enzyme. For a nonequilibrium process such as that catalyzed by PFK (Rolleston and Newsholme, 1967; Williamson, 1970), an inverse change in the amount of substrate (F-6-P in these experiments) and the flux through the

Change in lntracellular pH

FIG.3. The relationship between the change in intracellular pH and the percentage change in glycolytic flux, as reflected by the percentage change in anaerobic lactate production, produced in frog sartorius under identical experimental conditions. Solid circles: changes due to insulin a 5% C 0 2and (from left to right) “a+], = 6.8 mM, 28.7 mM, or 104 mM. Open circles: changes produced (in absence of insulin) by C 0 2 levels of (from left to right) lo%, 2.7%, or 2%, compared to paired controls at 5% C 0 2 . “a+], = 104 mM at 2% and 2.7% COz and in order to mimic the effect of insulin when the Na+ gradient is reversed. ”a+], = 0.12 mM at 10% C 0 2 .Each point represents the means ? SEs. The plotted line is a least-squares fit to the points. [Figure reproduced from Fidelman et al. (1982).]

277


enzyme (represented by anaerobic lactate production in these experiments) will unambiguously indicate a change in the activity of the enzyme (see the discussion in Fidelman et a / . , 1982). Stimulation or depression of glycolysis by insulin was associated in each individual experiment with, respectively, an activation or a deactivation of PFK. Using COZto produce an elevation or depression of pH, of the same magnitude produced by insulin was associated in 21 of 23 individual experiments with, respectively, an activation or a deactivation of PFK (see Fig. 4).

rn 0

c-20

c

u

+

-40

0)

2

6-60

-80

t

-loot-

, -80

,

, -40

, 0

40

80

Percent Change in F-6-P

FIG. 4. The change in activity of phosphofructokinase (PKF) associated with changes in glycolytic flux due to either insulin or to changes in CO? levels. The percentage change in anaerobic lactate production is plotted versus the percentage change in the level of fructose6-phosphate (F-6-P) in the same muscle. If PKF is activated. the data points should lie in the upper left quadrant, whereas if PKF is inhibited. data points should lie in the lower right quadrant. If the model is correct. data points should lie only in these two quadrants Glycolysis was stimulated either by adding insulin to Ringer containing the usual 104 mM Na' or by decreasing the COz level from 5% to 2.7% in the same Ringer. Glycolysis was inhibited either by adding insulin to Na-free Ringer or by increasing the CO? level from S%j to 10% in Na-free Ringer. Three data points due to insulin and one due to CO? are omitted from the lower right quadrant because their F-6-P levels were off scale ( 1 IS-240%). I n eight experiments, insulin was added to Ringer containing a level of Na'. 6.8 mM. that should bring (AG)Na-Hnear zero. In these experiments. as predicted by the model, there was no effect of insulin on glycolytic flux. and the change in F-6-P was not significant ( P> 0.05) as indicated by the square and SE bars. [Figure reproduced from Fidelman er ril. (1982).]

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RICHARD D. MOORE

A. Summary

These results provide very strong evidence that, at least in frog skeletal muscle, the acute effect of insulin upon glycolysis is mediated by an increase in pHi secondary to activation of Na-H exchange. The thesis that the immediate signal which mediates this insulin effect is the change in pHi is confirmed by the correlation between changes in pHi , whether induced by insulin or by changes in CO;! levels. That the insulin-induced change in pHi is due to Na-H exchange is supported by the fact that blocking this exchange system either with amiloride or by nulling its driving force, ( A G ) ~ J ~blocks - ~ , not only the change in pHi , but also the effect of insulin upon glycolysis. The fact that the Na+ concentration, or activity, component of (AG)N~-H can determine both the magnitude and the direction of the acute action of insulin upon glycolysis is especially convincing. That merely lowering “a+], reuerses the action of insulin on glycolytic flux indicates that ionic phenomena play the predominant role in mediating the acute action of insulin upon glycolysis. The confirmation of this particular prediction provides especially powerful support for the model and strongly implies a direct, functional relationship between the Na+ activity, or effective concentration, gradient and the intracellular signal that mediates the insulin effect upon glycolysis. The results, obtained in live muscle, of the plot of substrate versus rate indicate that the effect of changes in pHi is to affect the activity of phosphofructokinase (PFK), the pacemaker or rate-limiting enzyme (Karpatkin et al., 1964) of glycolysis. This is consistent with the extreme

z 140

Y

20

t

o 6.6

6.8

7.0

7.2

7.4

7.6

7.8

PH FIG.5 . The effect of pH on phosphofructokinase activity at various concentrations of fructose-6-phosphate in the presence or absence of 0. I m M 5‘-AMP. [The data are taken from Figs. I and 2 in Trivedi and Danforth (1966), with permission of W. H . Danforth.]


279

sensitivity (Trivedi and Danforth, 1966) to small changes in pH of PFK: phosphofructokinase isolated from frog skeletal muscle can be maximally activated by pH elevations as small as 0.1 to 0.2 units (Trivedi and Danforth, 1966), i.e., a 20-37% decrease in H + activity (see Fig. 5). IV.

ROLE OF INTRACELLULAR pH IN INSULIN ACTION

It is possible that the rise in pHi which results from insulin stimulation of Na-H exchange is part of the intracellular signal for perhaps several effects of this hormone. At least in frog skeletal muscle, the increase in pH, most likely mediates the acute effect of insulin upon glycolysis. Other systems which are stimulated by insulin and which can be stimulated by an increase in pHi include type A amino acid transport (see the review by Moore, 1983), hexose transport (Sonne et al., 1981), DNA synthesis (Winkler and Steinhardt, 1981), and protein synthesis (Winkler and Steinhardt, 1981; Winkler, 1982) (see the review by Busa and Nuccitelli , 1984). A. How Can a Change in pHI Affect Cell Function?

The proton is unique as a metabolic regulator. Whereas molecular signals (second messengers) represent discrete signals analogous to letters, the proton is a less discrete factor producing a more pervasive and therefore a more general effect. Whereas second messengers such as cyclic AMP may be analogous to letters entering a city, changes in pHi are more analogous to the fog rolling in. A letter goes only to a discrete address, but everyone is affected by the fog.

1. PROTONEFFECTSUPON ENZYME ACTIVITY A N D RESPONSE TO ALLOSTERIC MODIFIERS The obvious way pHi can regulate cell function is to produce allosteric effects upon enzyme activity. So far the best example of an effect of pH upon enzyme activity is provided by phosphofructokinase. The effect of pH on this enzyme is highly cooperative, resulting in a very steep pH profile (see Fig. 5 ) . Phosphorylation and dephosphorylation of key cell proteins are involved in amplification of many effects of insulin. Either amiloride or dissipation of membrane H+ gradients blocks the phosphorylation of ribosomal S6 proteins (PouyssCgur et al., 1982). In isolated hepatocytes, the

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RICHARD D. MOORE

effect of insulin to change the phosphorylated state of six protein fractions is inhibited by amiloride (Le Cam et al., 1982). But pH profiles are not sufficient to identify all possible enzyme control points for ApHi . Changes in pH can also affect the binding of effector molecules, thus modifying the response of an enzyme to other allosteric effectors (Busa and Nuccitelli, 1984; Moore et al., 1982). However, there are four other ways in which pHi can affect cell function, and each of these is a process.

2. INTERACTIONS BETWEEN pHi

AND

OTHERINTRACELLULAR SIGNALS

Busa and Nuccitelli (1984) have pointed out that the level of pHi interacts with other intracellular signals. These include interaction between pHi and Ca;' and possible interactions between pHi and cyclic AMP.

3. MEMBRANE TRANSPORT SYSTEMS Changes in pHi can affect membrane transport by two different mechanisms: (1) direct modification of the transport system; for example, an increase in pHi increases Na-Ca exchange in the plasma membrane (see the review by Moore, 1983);(2) a change in pHi could modify the number of transport systems in the plasma membrane. 4. METABOLIC PROCESSES PER SE By the law of mass action, metabolic reactions, such as the hydrolysis of MgATP, in which protons are either a substrate or a product are affected by changes in pH. 5 . REGULATION OF ENERGY STATEOF THE CELL

While the action of the insulin upon energy flow in the cell is widely recognized, it may not be so well appreciated that much of the increase in energy stored in the cell due to insulin is not in molecular form, but in biophysical parameters such as electric fields and activity (or concentration) gradients (see Moore, 1983). Insulin can also produce an actual increase in the average energy AGMgATP available from the hydrolysis of each ATP molecule according to the equation (Moore, 1983)

Since insulin not only increases pHi but can decrease aP; (Bailey et al., 1982) as well as restore the depressed levels of aMgADP found in diabetes

281


to normal (Moore et al., 1983), this hormone clearly has the ability to increase AGMgATP. The ability of insulin to increase AGMgATl’ and the electrochemical potential difference across the plasma membrane for both Na’ and H + suggests the concept that insulin increases not only the biochemical, but also the biophysical energy state of the whole cell (Moore. 1983). Once in the higher energy state, the “energized cell” is poised for energy-demanding chores such as synthesis of macromolecules, cell division, and other forms of work. Thus, by affecting the cell as a whole biophysical system and taking it into a higher biophysical energy state, insulin is preparing anabolic processes in the cell to respond to more specialized iztracellular signals, other than pH,, such as Ca” or messenger peptides. 6. Feedback Relations

The interactions among the level of pH, and other ions affected by insulin suggest the existence of novel ionic feedback mechanisms, both positive and negative, of probable biological significance. Although insulin stimulates both the Na+-K+ pump and Na-H exchange independently, the consequences produced by stimulation of either transport system feeds back on the other. The increase in pH, due to insulin activation would be expected to produce some increase in the activity of the Na+-Kt pump (Skou, 1982). Conversely, the “direct” intramembrane action of insulin to increase activity of the Na -Kt pump would lead to a decrease in [Nal-],,increasing the driving force for Na-H exchange and thus providing a “boost” to Na-H exchange after its activation by insulin. +

V. MODEL OF IONIC PART OF MECHANISM OF INSULIN ACTION

In view of the factors discussed in the preceding section, it appears that the proton, more specifically changes in pH,, represents the intracellular signal for insulin activation of glycolysis and probably part of the signal for the hormone’s effect upon several cell processes. On the other hand, there is reason to believe that Ca’+ and other signals including messenger peptides also play a role in mediating insulin action. The model proposed here, and illustrated in Fig. 6, is a holistic as opposed to a purely molecular system; it is driven by the interaction of thermodynamic gradients. In this model, the entire cell is the transducing system for insulin with the main energy, and information, transduction

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RICHARD D. MOORE

FIG.6. The model for the insulin transduction system to mediate the acute action of insulin upon glycolysis and probably participate in mediating this hormone’s effect upon other cell functions such as protein synthesis. In this model, part of the intracellular signal for the action of insulin is a change in the ionic atmosphere, especially pH, of the cell interior. When insulin binds to its receptor on the plasma membrane, it sends a signal, presumably within the membrane, which simultaneously stimulates (presumably by decreasing the activation energy) the Na-H exchange system and increases the activity of the sodium pump. The simultaneous stimulation of the sodium pump not only prevents Na+ from increasing inside the cell, but by increasing the Na+ energy gradient across the membrane, would tend to increase the rate of other transport systems such as Na-Ca exchange and Na-coupled amino acid transport. [Figure reproduced from Moore (1981).]

processes occurring at the plasma membrane. The primary energizer of this system is the Na+-K+ pump. Stimulation of this primary active transport system by insulin results in maximization of both the electrical and “chemical” (or activity) component of A ~ Nwhile ~ , activation of the NaH exchange system serves primarily as an information transduction process. The pHi-regulating aspects of the “insulin transduction system,” e.g., activation of Na-H exchange, are almost certainly involved not only in insulin action, but also in that of other hormones including mitogens (see other chapters, this volume). An analogy to this model in which the intact cell is required for transduction of a primary (extracellular) message into an intracellular signal would be the operation of a pipe organ. To play a tune, the system must be energized by turning on the bellows of the pipe organ, or by stimulating the Na+-K+ pump. The identity of each tune then is determined by which stops are pushed, or by which Na-coupled transport systems are activated. In the cell model presented here, Na-H exchange is also involved in the energization of intracellular processes including levels of ATP and


283

energy available from the hydrolysis of ATP (Winkler and Steinhardt, 1981).

Although these processes probably operate in the action of any hormone or mitogen which requires the cell to enter an energized state, activation of other specific transport systems and/or generation of molecular “second messengers” provides the opportunity for individualization of the message. In the case of insulin, the other transport systems very likely include the high-affinity Ca2+-ATPase,Na-Ca exchange, and possibly Na-Mg exchange. The inhibition of the high-affinity Ca?+-ATPaseby physiological concentrations of insulin in itself strongly suggests an important role for Ca2+.Moreover, both the increase in ApNa and the increase in pH, would be expected to increase Na-Ca exchange (see the preceding discussion). Finally, as mentioned earlier, H + and Ca2+can be expected to compete for binding to macromolecules and for transport sites on intracellular organelles. The increase in ApN, must play at least a secondary role in stimulation of amino acid uptake by this hormone since uptake of type A amino acids is directly coupled to ApNa and uptake of other amino acids is coupled to the distribution of type A amino acids. This model does not imply that other mechanisms such as a peptide mediator or direct intracellular action (Goldfine, 1981) do not play a role in insulin action. However, the model of the ionic aspects of insulin action has led to testable predictions and accounts for previously unexplainable observations. From a clinical standpoint, the ionic aspects of insulin action have the unique potential importance of being detectable in humans by noninvasive means, therefore being operationally testable in the clinical setting (see the next section). VI.

CLINICAL IMPLICATIONS

A. Rethinking the Role of Insulin

As suggested in Section I, there is reason to consider the hypothesis that the preoccupation with blood glucose, as opposed to electrochemical processes, is a historical accident. Given the situation at the time when insulin was discovered, it was inevitable that both clinicians and researchers would focus upon regulation of blood sugar, especially since the early results were so promising. Indeed, they had no other choice. In addition to the fact that we now know that insulin regulates biophysical parameters such as membrane potential, ion gradients, pHi, and A G M ~ ~ Tresults P, from comparative physiology and studies of evolution suggest that we rethink the fundamental role of insulin in physiology.

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1 . EVOLUTION Insulin is found not only in vertebrates, but also in invertebrates (Falkmer 1972), and a molecule that appears to be insulin has been found even in protozoa, Tetrahymena pyriformis, unicellular fungi, and Escherichia coli (Roth et al., 1982). If during evolution, insulin did appear while life was at a single-cell stage, the question arises as to what the primitive function of this “hormone” is. Certainly at that point it was not regulating blood glucose, but it might have been regulating fundamental events within these cells and, as Roth has suggested, could have been an ancient form of cell-to-cell communication. 2. COMPARATIVE PHYSIOLOGY

All birds and reptiles have normal blood glucose levels which are, by human standards, in the diabetic range, with blood glucose levels around 220-150 g/dl in the lizard Anolis (Rhoten, 1973, 1974, 1978), 210 g/dl in ducks (Laurent and Mialhe, 1978), and 250 g/dl in chickens (Langslow and Freeman, 1972). As in human prediabetics and diabetics (Cerasi and Luft, 1967; Cerasi, et al., 1972), the /3 cells of birds and reptiles are less sensitive to glucose than are normal /3 cells. The /3 cells of chickens are insensitive to glucose until the concentration reaches 500 mg/dl (Naber and Hazelwood, 1977) and a similar insensitivity exists in islets from the lizard Anolis carolinensis (Rhoten, 1974). Moreover, birds such as the chicken are extremely insensitive to injected insulin. Doses as high as 50 IU/kg body weight produce nothing more than a mild hypoglycemia and an increase (as opposed to the decrease seen in mammals) in plasma free fatty acids (Lepkovsky et al., 1967; Langslow and Hales, 1970). These studies suggest that, at least in birds and reptiles, perhaps the role of insulin may be to regulate something other than glucose. B. Implications of the Model for Diabetes Mellitus: An Alternative Hypothesis to Explain the Pathophysiology of Diabetes Mellitus

The model for mediation of the ionic effects of insulin, the “insulin transduction system,” predicts that in hypoinsulinemic states such as diabetes not only will Na: be elevated, but pHi will be decreased with a consequent decrease in production of ATP. Since the Na+-K+ pump can contribute directly and indirectly to the plasma membrane potential Vm,

285


the model also implies that the increase in Na: would be associated with a decrease in the magnitude of V , . The predicted increase in Na: has been confirmed by the finding that in rats made hypoinsulinemic by streptozotocin (SZ) diabetes (Moore et a / ., 1979b, 1983) or by fasting (Moore e t d., 1983) the level of Na: in the soleus muscles of these animals increases by about 30%. In the diabetic rats, the elevation of Na: was at least as well correlated with the diabetic state as was the elevation of plasma glucose (Moore et a / . , 1979b). In the hypoinsulinemic rats, either starved or diabetic, ATP, decreases after Na, increases (Moore et al., 1983). The predicted decrease in the magnitude of V , has been confirmed by Grossie (1982) in skeletal muscle of alloxan diabetic rats. Preliminary evidence (Table I) supports the predicted decrease in pH, in nonacidotic SZ diabetic rats. As expected, intraperitoneal injection of SZ significantly lowered plasma insulin levels and raised blood glucose. However, the degree of diabetes was sufficiently mild that there was no evidence of metabolic acidosis as reflected by the lack of decrease in blood pH. However, the pH, of the soleus muscle was significantly decreased by 0.076 units 7 days after SZ injection and by 0.152 units 10 days after SZ injection. I . INTRACELLULAR PARAMETERS A N D PATHOPHYSIOLOGY In light of today's knowledge, including that in this volume, it would be hard to believe that a cell which had a depolarized membrane potential, depressed pH,, elevated Na,' , probably elevated levels of free Caf ', diminished energy per ATP molecule, and depressed levels of ATP, could TABLE I STREPTOZOTOCIN DIABETES IN SPRAGUE-DAWLEY RATS: PLASMA GLUCOSE, INSUI.IN, pCOz. BLOODpH (pH,,), A N D INTRACEI.I.UI.AR pH (pH,)OF SOI.EUSMUSCLE"

T H E EFFEC'T OF ON

Days postinjection of 75 mg streptozotocin/kg Control (sham-injected) Glucose (mg/dl) Insulin (p,U/ml) PCO, (mm Hg) PHO PHI

165 + 8.6 58.7 + 7.6 35.2 + 1.0 7.485 + 0.009 7.026 + 0.017

7 437.5 + 13.5 + 35.6 + 7.472 + 6.950 +

I0

30.3* 5.1* 0.6 0.012 0.030*

448.7 = 6.0 + 37.2 + 7.474 + 6.874 +

i9.6*

2.1* 3.6 0.031 0.043*

Values marked with an asterisk are significantly different from controls at

P of less than 0.05. From Brunder c't crl. (1983).

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RICHARD

D. MOORE

function normally even if these changes were modest, especially over a 10- to 20-year period. One consequence which these abnormal cell parameters would suggest would be a higher anticipated incidence of hypertension. Elevated intracellular Na+ of arteriolar smooth muscle cells has been implicated in essential hypertension since, as just outlined, a decrease in Na-Ca exchange would be expected to elevate intracellular Ca2+and thus increase the contractile tone of these muscle cells (see Fregly and Kare, 1982). Na-Ca exchange has been reported to exist in arteriolar smooth muscle cells (Daniel et al., 1982). In addition, activation of voltage-sensitive pathways by a decrease in membrane potential would allow an increase in intracellular free Ca2+.Thus, the increase in Ni$ and the decrease in V, observed in hypoinsulinemia could provide an explanation for the observation that human diabetics have a higher incidence of hypertension than do nondiabetics. C. Rethlnklng Diabetes Therapy

Any approach which has been as unsuccessful as the current approach to the long-term treatment of diabetics should be suspect. One should be wary of relying on the assumption that “fine tuning” a previously unsuccessful approach will result in a significant change in results. On the other hand, there is now reason to suspect that one of the fundamental defects evident in diabetes mellitus is an intracellular acidosis. The accepted view is that most pathology in diabetes is due to hyperglycemia per se. Suggested mechanisms involve increased glycosylation of proteins and sorbitol metabolism. Recent developments have led to the view that finally one diabetic complication, diabetic polyneuropathy, can be explained by deranged sorbitol metabolism (Winegrad et al., 1983). This view involves postulating a decreased activity of the Na+-K+ pump in nerves consequent to the change in carbohydrate metabolism. But, in addition, it should be kept in mind that physiological levels of insulin do regulate the activity of the Na+-K+ pump in muscle and several other tissues independent of glucose transport (see the review by Moore, 1983). This effect occurs at physiological concentrations and may play a role in the pathophysiology of diabetes. Supporting this hypothesis is the finding (Moore et al., 1983) that intracellular Na’ in rat soleus muscle is elevated by hypoinsulinemia produced either by diabetes or by fasting. Moreover, studies of comparative physiology raise some serious questions as to the extent to which hyperglycemia per se is a likely cause of the pathophysiology observed in human diabetics. Since glycosylation of the


287

N-terminal group of proteins and peptides is nonenzymatic, it is a simple mass-action effect which should be the same in all species. Therefore, in view of the “diabetic” level of blood glucose in birds and reptiles, these animals should have levels of glycosylated proteins comparable to those found in human diabetics with very poor glucose control. If so, why are not birds and reptiles developing the bathology of the chronic diabetic? Why don’t they appear to go blind, to develop kidney disease, etc.? None of these questions has been addressed. For example, the hemoglobin of these animals has not been tested for glycosylation. On the other hand, the fact that in addition to elevated plasma glucose, diabetic animals have a decreased biophysical energy state of the cell (decreased membrane potential, elevated Na: , depressed pH,, and depressed levels of both ATP and the free energy available from ATP) offers additional possibilities to explain the pathophysiology of this disease. It has long been known that untreated juvenile diabetics become acidotic. This acidosis, reflected by a decrease in blood pH, is attributed to increased production of inorganic acids, i.e., ketoacids. However, while this undoubtedly is true, the finding that insulin increases intracellular pH by activation of a Na-H exchange system in the plasma membrane leads to the prediction that in hypoinsulinemic states, such as diabetes, there should be an initial acidosis, due to decreased activity of this biophysical transport mechanism, which may occur before metabolic acidosis becomes established. As discussed in preceding sections, even in the state of mild diabetes in which metabolic acidosis is not in evidence, pH, in rat tissues may be decreased by at least 0.15 units and intracellular ATP decreased by about 24% and Na: increased by up to 50%. This suggests a fundamental reconsideration of the pathophysiology of diabetes. Moreover, the fact that this increase in intracellular Na+ could be correlated with the diabetic state just as plasma glucose could be suggests that parameters within the cell may reflect the pathophysiological state of a diabetic at least as well as do extracellular levels of glucose. It is not an unreasonable hypothesis that if intracellular parameters, such as levels of Na: , ATP, , and pH,, can be normalized in the diabetic, the pathophysiology of this disease might be decreased. Until recently, this concept would have been of academic interest. However, as mentioned earlier, 3’P-NMRhas been used to follow noninvasively the effect of insulin upon pH, in frog skeletal muscle (Moore and Gupta, 1980). It is now possible (Havrankova er al., 1978) to follow noninvasively pH, and ATP, in the intact human by using wide-bore FT-NMR. This line of reasoning suggests the urgency of using FT-NMR to measure pH, and other intracellular parameters to determine whether they provide

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a more rational, and more successful, basis for determining the dosage of insulin in diabetes therapy than does the present exclusive reliance upon plasma glucose. In this approach, cellular parameters would be used for the determination of insulin dosages and the diet then adjusted in order to normalize plasma glucose. This is not the first suggestion that glucose regulation may not be the most appropriate primary objective in diabetic therapy. Turner and Holman (1976) have suggested that glucose regulation is secondary in importance to maintenance of basal insulin secretion. These authors postulated that the basal plasma-insulin concentration needs to be kept above a certain minimum value for normal cell function and that the insulin requirement for cell growth is more vital than strict nutrient regulation. Their suggestion is in keeping with the views emerging from our studies of the effect of insulin upon pHi and from the several studies, summarized in this volume, of the role of pHi in the regulation of protein synthesis and in cell growth. The hypothesis presented here goes one step further than Turner and Holman’s: not only is insulin homeostatsis probably more important than glucose homeostasis, but homeostasis of intracellular parameters, such as Na: , ATPi , and pHi , is probably still more important. Moreover, with the advent of whole-body NMR, homeostasis of ATPi , pHi , and AGMMgATP can be followed noninvasively. The alternative to testing this new hypothesis is to continue to hope that the aproach which is now failing, regulation of blood glucose, will begin to work once the regulation is better. In view of the electrochemical, or biophysical, effects of insulin, that would seem rather unlikely. REFERENCES Bailey, 1. A., Radda, G . R. R., Seymour, A. L., and Williams, S. R. (1982). Biochim. Biophys. Acra 720, 17-27. Benos, D. J. (1982). A m . J . Physiol. 242, C131-CI45. Brunder, D. G., Oleynek, J . J . , and Moore, R. D. (1983). J . Gen. Physiol. 82, 15a. Busa, W. B., and Nuccitelli, R. (1984). A m . J . Physiol. 246, R409-R438. Cerasi, E . , and Luft, R. (1967). Diabetes 16, 615. Cerasi, E., Luft, R . , and Efendic, S. (1972). Diabetes 21, 224-234. Clancy, R . L., Gonzalez, M. C., Shaban, M . , and Cassmayer, V . (1983). Cclt. Physiol. 11, A-107. Clausen, T., and Kohn, P. J . (1977). J . Physiol. (London) 265, 19-42. Daniel, E. E., Groves, A. K.,and Kwan, C. Y.(1982). Fed. Pror.. Fed. Am. Soc. E x p . B i d . 41, 2898-2904. Falkmer, S. (1972). Gen. Comp. Endocrinol. Suppl. 3, 184-191. Fehlmann, M., and Freychet, P. (1981). J . B i d . Chem. 256, 7449-7453. Fidelman, M. L., Seeholzer, S . H . , Walsh, K. B., and Moore, R. D. (1982). Am. J . Physiol. 242, ~ 8 7 - ~ 9 3 .


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Fregley. M. J., and Kare, M. K . (1982). "The Role of Salt in Cardiovascular Hypertension." Academic Press, New York. Funder, J . . Tosteson, D. C., and Weith, J. 0. (1978). J . Gen. Physiol. 71, 721-746. Gavryck. W. A.. Moore. R. D.. and Thompson. K . C . (1975). J . Pltysiol. (London) 252, 43-58, Goldfine. 1. D. (19x1). Biodtim. Bioplrys. Acitr 650, 53-67. Grossie, J. (1982). Diubert,s 31, 194-202. Harrop. G. W.. and Benedict, E. M . (1924). J . B i d . C'lrrm. 59. 683-697. Havrankova. J., Koth, J.. and Brownstein. M . ( 1978). Ntrrrirc (London) 272, 827-829. Holler. J. W. ( 1946). J . A m . Med. A s s w . 131. I 1x6- I 189. Karpatkin, S . , Helrnreich, E . . and Cori. C. F. (1964). J . Biol. Chem. 239, 3139-314s. Langslow, D. K . , and Freeman. B. M. (1972). Diuhivfhgiu 8, 206-210. Langslow, D. R., and Hales, C. N. (1970). Lunc,e/ 1, 1151-1 152. Laurent, F . , and Mialhe, P. (1978). Diaherologiu 15, 313-321. Le Cam, A,, Auberger, P., and Sampson, M. (1982). Biochem. Biophys. R P S .Commun. 106, 1062- 1070. Lepkovsky, S . . Dimick. M. K., Furtrta, F.. Snapir. N.. Park, R., Narita, N.. and Komatus, K. (1967). Endocrinology 81, 1001-1006. Manchester, K. L . (1970). Hormones I, 342-351. Meyer, K . A , . Kushmerick. M. J.. Dillon. P. F.. and Brown. T. K.(19x3). Fed. P r o c . , Fed. A m . S o c . Exp. B i d . 42, a1248. Moolenaar, W. H., Tsien, R. V., Van der Saag, P. T.. and de Laat. S . W. (1983). Nutrirc, (London) 304, 645-648. Moore, R. D. (1973). J . Physiol. (London) 232, 23-45. Moore, R. D. (1977). Biophys. J . 17, 259a. Moore, R. D. (1979). Biochem. Biophys. R P S . Commrrn. 91, 900-904. Moore, R. D. (1981a). Biophys. J . 33, 203-210. Moore, R. D. (1981b). I n t . J . Qiran/tim CIiem. Qritrntrrm B i d . Symp. 8, 365-371, Moore, R. D. (1983). Biochim. BifJpllq's.Actu 737, 1-49. ii Qrrunrrim Biol. S y m p . 7,83Moore, R. D., and Gupta, R. K. (1980). Int. J . Q i ~ ~ i r t i i iCham. 92. Moore, R. D.. Fidelman. M. L.. and Seeholzer. S. H. (197%). Biochem. B i o p h y . ~ .Rc..s. Commun. 91, 905-910. Moore, R. D., Munford. J . W.. and Popolizio. M . (1979b). FEBS L e f t . 106, 375-378. Moore, R. D.. Fidelman, M. L.. Hansen, J . C.. and Otis. J . N. (1982).In "lntracellular pH: Its Measurement. Regulation, and Utilization in Cellular Functions'' ( K . Nuccitelli and D. W. Deamer, eds.), pp. 385-416. Liss. New York. Moore, R. D..Munford. J . W., and Pilloworth, Jr.. T. J. (1983). J . Phvsiol. (London) 338, 177-294. Morrill. G.. Kostellow. A . Weinstein. S. P.. and Gupta. R. J . (1983). F'cd. Proc.. Frtl. A m . Soc. E.4-p. Biol. 42, 1791. Naher. S.P., and Hazelwood. R. L. (1977). Gcn. Comp. Endoc.rinol. 32, 495-504. Podo. R..Carpinelli, G.. and D'Agnolo, G. (1982). I n / . Coqf. M a g n . Rc,son. Biol. Syst. 10th. Stanford 14 (1982). Pouyssegur, J., Chambard, J. C . . and Paris, S. (1982). I n "Symposium on Ions, Cell Proliferation and Cancer" (A. L. Boynton. W. L. McKeehan, and J . F. Whitfield. eds.), pp. 205-218. Academic Press, New York. Putnam, R . W.. and Roos. A. (1983). A m . Zool. 23, 996. Putnam, K . W. (1985). A m . J . Phvsiol. 248. (Crll Pliysiol.) C330LC336. Kesh, M . D., Nemenoff, R. A.. and (hidotti. G . (1980). J . Biol. C'hcm. 255, 10938-10945.

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Rhoten, W. B. (1973). Comp. Biochem. Physiol. 45A, 1001-1007. Rhoten, W. B. (1974). Am. J . Physiol. 227, 993-997. Rhoten, W. B. (1978). Proc. Soc. Exp. Biol. Med. 157, 180-183. Rolleston, F. S., and Newsholme, E. A. (1967). Biochem. J . 104, 524-533. Roos, A., and Boron, W. F. (1981). Physiol. Rev. 61, 296-433. Roth, J . , Le Roith, D., Shiloach, J., Rosenzweig, J. L., Lesniak, M. A., and Havrankova, J. (1982). N . Engl. J . Med. 306, 521-527. Skou, J. C. (1982). Ann. N . Y . Acad. Sci. 11, 169-184. Sonne, O., Gliemann, J., and Linde, S. (1981). J. B i d . Chem. 256, 6250-6254. Trivedi, B . , and Danforth, W. H. (1966). J . B i d . Chem. 241, 41 10-41 14. Turner, R. C., and Holman, R. R. (1976). Lancet June 12, 1272-1274. Williamson, J. R. (1970). Adu. Exp. Med. B i d . 6, 117-136. Winegrad, A. I., Simmons, D. A,, and Martin, D. B. (1983). N. Engl. J . Med. 308, 152-154. Winkler, M. M. (1982). I n “Intracellular pH: Its Measurement, Regulation, and Utilization in Cellular Functions” (R. Nuccitelli and D. W. Deamer, eds.), pp. 325-340. Liss, New York. Winkler, M. M., and Steinhardt, R. A. (1981). Deu. Biol. 84, 432-439. Zierler, K . L. (1957). Science 126, 1067-1068. Zierler, K . L. (1959). A m . J . Physiol. 197, 524-526.

CURRENT TOPICS IN MEMBKANES A N D TRANSPORT. VOLUME 26

Chapter 16 The Proton as an Integrating Effector in Metabolic Activation WILLIAM B . BUSA Department of Zoology University of California Davis, California

I. Introduction ............................................ 11. Four Systems Involving Regulatory pH, Changes.. ......................... A. Sea Urchin Egg Fertilization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Insulin Stimulation of Frog Skeletal Muscle . . . . ............ C. Growth Factor Stimulation of Serum-Starved Fib ............... D. Dormancy Termination in Artemiu Embryos E. Common Features of Metabolic Activation in ems.. . . . . . . . . . . 111. Comparison of in Vitro pH/Activity Profiles with in A. Phosphofructokinase and Glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Protein Synthesis C. Ca?+-RegulatedProcesses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusion: Why Use pH, as a Metabolic Regulator? ....................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.

291 292 292 293 293 294 294 295 295 297 300 302 304

INTRODUCTION

The reader of this volume may well wonder what are the metabolic consequences of the intracellular pH (pH,) changes documented here by several authors. Unfortunately, while the efforts of many workers have demonstrated that numerous cell types undergo substantial intracellular pH changes during defined metabolic transitions and have shown in several cases that these pH, changes appear to play a role as metabolic effectors (see Busa and Nuccitelli, 1984, for a review), nevertheless we are today little closer to an understanding of the molecular targets of pH,mediated metabolic regulation than we were I 1 years ago, when Daniel Mazia and his colleagues at Berkeley first demonstrated the importance of 291 Copyright 0 19x6 by Academic Press, In' All nghts of reproduction in any form reserved

292

WILLIAM B. BUSA

pHi’s role in regulating the metabolic activation of the fertilized sea urchin egg. If metabolic regulation via pH, involves a limited number of specialized receptors (as, for instance, regulation via Ca2+often involves calmodulin, while CAMP’Seffects are mediated via CAMP-dependent protein kinase), then we must, at present, admit to a nearly complete ignorance of such mechanisms. The purpose of the present essay, however, is to point out that we already possess a substantial fund of knowledge concerning a much more general means by which pHi changes might affect metabolism-that is, through the well-established pH dependencies of enzyme and structural protein activities. With but few exceptions (e.g., Londesborough, 1977; Hochachka and Mustafa, 1972; Trivedi and Danforth, 1966), biochemists have generally failed to consider the regulatory implications of the pH/activity profiles they collect in the process of characterizing enzyme activities; indeed, it is still all too common to find published characterizations which entirely neglect to report an enzyme’s pH dependence. But the fact that so many cells experience significant pHi changes, in combination with the fact that so many enzymes display pronounced pH dependence within the physiological range of pHi values, demands an effort t o make sense of in vitro pHlactivity projiles in light of the in vivo behavior of these enzymes in cells which experience p H i changes. II. FOUR SYSTEMS INVOLVING REGULATORY pH1 CHANGES

In what follows, I will focus on four cell systems (Table I) for which some evidence is available that pHi changes in fact play a regulatory role in the cellular responses they accompany. A fifth such system, the activating sea urchin spermatozoan, will not be considered because of its rather specialized lifestyle. For detailed discussion of the data concerning pHi changes and their roles in these cells, see Busa and Nuccitelli (1984); in this section I shall briefly summarize these observations. A. Sea Urchin Egg Fertilization

Eggs of the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus alkalinize by -0.4 pH unit within 5 min following fertilization (Shen and Steinhardt, 1978; Johnson and Epel, 1981; Winkler et a l . , 1982). Numerous studies involving either inhibition of this natural pHi change or artificial manipulation of pHi have demonstrated its regulatory role in a number of processes accompanying egg activation (see Busa and

293

16. THE PROTON AS AN INTEGRATING EFFECTOR

TABLE I COMMON CHARACTtRISTICS OF METAROI I( ACTlVAllON I N FOUR CFI I PHYSlOLOGlCAl LY SIGNIFICANT pH, CHANGFS“

Increased glycolysis Sea urchin egg (fertilization) Frog skeletal muscle (insulin stimulation) Arternirr embryo (dormancy /development transition) Fibroblast (serum stimulation)

Increased Calmodulinprotein mediated Increased synthesis re5ponses respiration

+

+ +

t‘

+

’?

+

-t

t

+

SYSTEM5 UNDEKGOINCI

Cytoskeletal changes

Membrane potential changes

+

+

t

+

t~

+

t”

+

+ $?

)

Current Topics in Membranes and Transport, Volume 26 Na+ - H+ Exchange, Intracellular Ph, and Cell Function

CURR TOPICS IN MEMBRANES & TRANSPORT V15, Volume 15 (Current Topics in Membranes and Transport)

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Current Topics in Membranes and Transport, Volume 26 Na+ - H+ Exchange, Intracellular Ph, and Cell Function

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Current Topics in Membranes and Transport, Volume 07

Current Topics in Membranes and Transport, Volume 10

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Current Topics in Membranes and Transport, Volume 08

Current Topics in Membranes and Transport, Volume 02

Current Topics in Membranes and Transport, Volume 06

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Current Topics in Membranes and Transport, Volume 19: Structure, Mechanism, and Function of the Na K Pump

Current Topics in Membranes and Transport, Volume 19: Structure, Mechanism, and Function of the Na K Pump

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Current Topics in Membranes & Transport Vol. 25: Regulation of Calcium Transport Across Muscle Membranes (Current Topics in Membranes and Transport) (v. 25)

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