Chapter 7 Activation of the Na+-H+ Antiport by Changes in Cell Volume and by Pnorbol Esters; Possible Role of Protein Kinase

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 26

Chapter 7

Activation of the Na+-H+ Antiport by Chan es in Cell Volume and by P orbol Esters; Possible Role of Protein Kinase

3,

S . GRINSTEIN,* S . COHEN,* J . D . GOETZ,* A . ROTHSTEIN,* A . MELLORS,? AND E. W . GELFANDS Divisions of Cell Biology* and Immunology$ The Hospital for Sick Children Toronto, Ontario, Canada and ?Department of Chemistry and Biochemistry University qf Guelph Guelph, Ontario, Canada

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

11. Basic Properties of Na+-H+ Exchange in Lymphocytes 111. Na+-H+ Exchange in Volume Regulation. . . . . . . . . . . . .

IV. V. VI . VII. VIII.

A. The Nature of Volume Regulatory Increase.. ......... B. Mechanism of Osmotic Activation of the Na+-H+ Antiport.. . . . . . . . . . . Stimulation of Na+-H+ Exchange by Phorbol Esters .... A. Mechanism of Activation ........................ ........... B. Role of Protein Kinase C ........................ Similarities of the Phorbol Ester and Volume-Induced Activation.. ... Possible Involvement of a Protein Kinase in Volume Reg Osmotically Induced Changes in Phosphoinositide Turnover . . Concluding Remarks. .................................................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.

1 IS

120

122

133 134

INTRODUCTION

A system that catalyzes the exchange of Na+ for H + appears to be a ubiquitous feature of the plasma membrane of nucleated mammalian cells. The system was originally described by Murer et al. (1976) in the 115 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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S.GRINSTEIN ET AL.

brush border membrane of intestinal and renal epithelial cells, where it is thought to play a role in transepithelial transport (Aronson, 1983). In recent years similar exchangers or antiports have been described in a variety of cell types from many species. In nonepithelial tissues the exchangers are believed to be involved in cell volume regulation (Kregenow, 1981; Cala, 1983) and in the control of cytoplasmic pH (Roos and Boron, 1981; Paris and Pouysskgur, 1983; Thomas, 1984), and their activation is thought to be permissive for the early events leading to mitogenesis (Koch and Leffert, 1979; Villereal, 1981; L’Allemain et al., 1984a,b; Boron, 1984). Countertransport of Na+ for H+ has also been detected in the membranes of lymphocytes from human peripheral blood and rat thymus. In the latter the antiport can be activated by lowering the cytoplasmic pH (pHi), by osmotic shrinking, or by a family of tumor-promoting phorbol diesters (Grinstein et al., 1984a, 1985a,b). The relationships among these modes of stimulation and a tentative model of the activation process are the subjects of this review. II. BASIC PROPERTIES OF Na+-H+ EXCHANGE IN LYMPHOCYTES

The Na+-H+ exchange can be readily detected in rat thymocytes following acid loading, a procedure that greatly increases its activity. A convenient method for controlled acid loading involves the use of nigericin, an electroneutral K+-H+ exchanging ionophore. When added to cells suspended in Na+-and K+-freemedia, nigericin catalyzes the exchange of extracellular H+ (H:) for internal K+, with consequent cytoplasmic acidification. In suspensions of intact thymocytes, the course of the acidification can be followed by measuring the emission of the fluorescent pHi indicators dicarboxyfluorescein (DCF) or bis(carboxyethy1)carboxyfluorescein (BCECF) (Fig. 1). When nigericin is used, acid loading can be terminated when the desired pHi is reached by scavenging the ionophore with albumin. The addition of Na+ to acid-loaded cells results in a rapid cytoplasmic alkalinization (Fig. l), consistent with a Na+-induced efflux of cytoplasmic H+. This process is effectively blocked by amiloride (Fig. l), a K+sparing diuretic that blocks Na+-H+ exchange in other cell types (Benos, 1982). A transmembrane flux of H+ equivalents under these circumstances could be demonstrated by measuring the extracellular pH (pH,) in lightly buffered suspensions: cellular alkalinization was accompanied by an approximately equimolar acidification of the medium (Grinstein et al.,

7. ACTIVATION

OF THE Na+-H+ ANTIPORT

117

Nigericin 7.11

FIG. 1. Na+-induced alkalinization of acid-loaded rat thymic lymphocytes. Cytoplasmic pH (pHJ was measured from the fluorescence of dicarboxyfluorescein as described elsewhere (Grinstein ef ul., 1984a). The cells were suspended in N-methyl-o-glucamine' solution (Na+ free) and nigericin (0.3 pghnl) was added where indicated. Acid loading was terminated by addition of albumin (5 mg/ml, final). Finally, 50 mM NaCl was added to initiate Na+-H+ exchange. (Reproduced from the Annuls of rhc New York Academy of Sciences by copyright permission.)

1984a). Furthermore, measurements of cellular cation contents and of isotopic fluxes indicated that Na+ is transported into the cells by an amiloride-sensitive process concomitantly with the alkalinization (Grinstein et al., 1984a,b). Taken together these observations support the existence of a coupled Na+-H+ exchange mechanism in the plasma membrane of thymic lymphocytes. Some of the basic properties of the antiport were defined by using the experimental model already described. Na+ was found to interact with the external transport site with an apparent affinity of 59 mM and could be competitively displaced by %+,Nai was also found to compete with amiloride, which inhibits transport with an apparent K, of 2.5 pM. A comparison of the amiloride-sensitive uptake of Na+ with the efflux of H+ (calculated as the product of ApH, and the buffering power) indicated an approximate one-to-one stoichiometry . Independent evidence for an equimolar stoichiometry was obtained from measurements of transmembrane potential, which showed that the exchange was electroneutral, indicating an obligatory coupling of the movement of equal numbers of Na+ and H+. In lymphocytes, the principal determinant of the rate of Na+-H+ exchange is the pH,. As illustrated in Fig. 2, the rate of Na+-dependent H+ extrusion is negligible when pH, is 27.1, but it increases markedly as the

118

S. GRINSTEIN ET AL.

-I

*'

7.5

0 0

V

a 6.2

+

I

I

A

d o 0

6.0

6.2

6.4

6.6

A v

6.8

7.0

7.2

PHi

FIG.2. Effect of pHi on Na+-induced H+ efflux from thymocytes. Inset: typical fluorescence traces of Na+-induced alkalinization from experiments like that in Fig. 1 , where pHi was preset at varying levels. Main panel: relationship between pHi and H+ extrusion rates. The latter were calculated from the initial rate of ApHi and the buffering power (approximately 25 mMlpH over the pHi 6.2-7.2 range). (Reproduced from The Journal of General Physiology, 1984, Vol. 83, pp. 341-369, by copyright permission of the Rockefeller University Press.)

cytoplasm is acidified. At pHi = 6.3, "a+], = 140 mM, and 22"C, the rate of exchange exceeds 10 mmol liter-' min-'. This is a rapid flux: in severely acid-loaded cells, in which the Na+ pump is presumably inhibited by the low pHi (Boron and Russell, 1983), the cellular Na+ content can double within 2 min. The relative inactivity of the antiport at near-physiological pHi (about 7. I ) cannot be attributed to the attainment of electrochemical equilibrium, which for an electroneutral 1 : 1 exchanger driven by the combined Na+H+ chemical gradient is expected to occur at pHi > 8.0 (see Grinstein et al., 1984b, for detailed discussion). Moreover, although net Na+ fluxes would not be expected at thermodynamic equilibrium, unidirectional amiloride-sensitive ZZNa+uptake should be detectable if the transporter were operating. As shown by the triangles in Fig. 3, the observed rate of 22Na+ influx at pHi > 7.1 is marginal but increases very markedly at lower pHi . It must be concluded, therefore, that the antiport is not thermodynamically limited but rather kinetically controlled. In other words, transport is

119

7. ACTIVATION OF THE Na+-H+ ANTIPORT

0.41

-1 u)

al

0

E

0.1

I

6.0

6.3

1

6.6

I

I

6.9

7.2

PHi

FIG.3. Effect of pH, on 22Na+influx in thymocytes in the presence (solid symbols) or

absence (open symbols) of amiloride. Triangles: untreated cells. Circles: pH,-clamped cells. Clamping was accomplished by pretreatment with nigericin in K+-rich media of the appropriate pH (Grinstein et a / . , 1984b). Data are means t SE of three experiments, each with duplicate determinations. (Reproduced from The Jortrnul ofceneral Physiology,1984, Vol. 84, pp. 585-600, by copyright permission of the Rockefeller University Press.)

“turned off” at pHi > 7.1 in spite of the existing chemical gradients which could, in principle, still support the forward (Nd-H;) operation of the exchanger. The existence of such a pHi-sensitive controlling mechanism was originally proposed by Aronson et al. (1982), who suggested that an allosteric modifier site on the cytoplasmic face of the antiport activates transport upon H+ binding. This hypothesis would also explain (1) the non-Michaelian [H+],dependence of transport, which departs from the expectations for a 1 : 1 exchanger that obeys Michaelis-Menten kinetics with respect to N d and (2) the finding that increasing [H+],does not inhibit 22Na+efflux (Grinstein et d.,1984b) as predicted for a simple competitive interaction. What is the physiological role of the modifier site? The observation that the “set point,” i.e., the threshold pHi for activation of the exchanger, is practically identical to the resting pH, of the cells suggests a role in pH, homeostasis. The modifier site appears to operate as the pH-sensing de-

120

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vice of a cellular pH-stat, maintaining pHi by controlling the rate of H+ efflux through the transport site. The latter is thermodynamically poised to extrude H+, utilizing the potential energy inherent in the pre-existing Na+ gradient generated by the Na+-K+ pump. 111.

Na+-H+ EXCHANGE IN VOLUME REGULATION

A. The Nature of Volume Regulatory Increase

Under certain conditions, osmotically shrunken cells will regain nearnormal volumes by a process known as regulatory volume increase (RVI) (Kregenow, 1981; Cala, 1983). In the case of lymphocytes, reswelling in hypertonic solutions is strictly dependent on the availability of external Na+, is associated with an increased rate of 22Na+uptake, and in the presence of ouabain, is associated with an increased cellular Na+ content (Grinstein et al., 1983). Both RVI and the associated translocation of Na+ are inhibited by amiloride, suggesting a possible role of the Na+-H+ antiport. As shown in Fig. 4A, this hypothesis was substantiated by measurements of the intracellular pH. In nominally HC0;-free medium, hypertonic shrinking of thymocytes results in a substantial cytoplasmic alkalinization (in eight experiments ApHi averaged 0.26 -+ 0.04 units, mean 2 SE). Two lines of evidence indicate that the alkalinization is a consequence of countertransport of extracellular Na+ ( N d ) for internal H+ (HT): (1) The ApHi is dependent on the presence of N d ; (2) the alkalinization is completely abolished by amiloride. A direct demonstration that the internal alkalinization is due to the transmembrane displacement of H+ equivalents was achieved by extracellular pH determinations (not illustrated) (Grinstein et al., 1985~).The disappearance of H+ from the cytoplasm was found to be accompanied by a commensurate acidification of the extracellular solution. As expected, the latter was similarly Na: dependent and amiloride sensitive. Taken together, the available data indicate that osmotic shrinking produces an activation of the Na+-H+ antiport in blood and thymic lymphocytes. B. Mechanism of Osmotic Activation of the Na+-H+ Antiport

The mechanism of activation of Na+-H+ exchange by osmotic shrinking was evaluated by comparing the kinetic parameters of transport in resting (isotonic) and hyperosmotically stressed cells. Measurable rates of transport in isotonic conditions were obtained by manipulation of pHi, as in Fig. 2. In these experiments, the “a+], dependence of the relative rate

121

7. ACTIVATION OF THE Na+-H+ ANTIPORT A

Hypertonic

140 mM Na' +amiloride

7.0

7.3 -

Nigericin Albumin

1

pHi 6.9

-

6.5

-

140 mM Na'

0 rnM Na'

Hypertonic

1

70 mM Na'

FIG. 4. Effect of osmotic shrinking on pH, in rat thymocytes. ( A ) Effect of hypertonic stress on pH,. Cells were suspended in isotonic medium (285 mOsm). Where indicated, the cells were sedimented and resuspended in hypertonic (550 mOsm) medium containing 140 mM Na+ with or without amiloride, or 5 mM Na+. (B) Cells were suspended in N-methyl-D-glucamine+ medium and acid loading was performed as described for Fig. I . The cells were then sedimented (dotted lines) and resuspended in either isotonic or hypertonic medium. Both media contained 70 mM Na' and were osmotically balanced with N-methyl-D-glucamine+. [Reproduced from Federation Proceedings ( 1985).]

of H+ extrusion was found to be essentially identical in iso- and hypertonic media. Thus, a change in the K , for Na; cannot account for the observed osmotic activation of the antiport. Indeed, because the K , for Nd in isotonic cells is in the range of 50 to 60 mM (Grinstein et a l . , 1984a,b), it is theoretically unlikely that osmotic activation in media containing physiological concentrations of N d (-140 mM, i.e., more than twice the K,,,) is due to an increased affinity for this cation. As discussed earlier, in lymphocytes, as well as in other cells (Aronson et al., 1983; Paris and Pouyssegur, 1983), Na:-H: exchange is inhibited by extracellular H+(H;), at least partly by competition with Na; for the externally facing transport site. Therefore, a change in the inhibitory potency of H; could conceivably result in activation of forward (NalH:) exchange. However, a comparison of the relative inhibitory potency of H : in normal (acid-loaded) and shrunken cells (with or without acid loading) revealed no significant differences. It was concluded that changes in the extracellular cation affinity of the antiport are not responsible for the osmotically induced activation.

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Because [H+]i is one of the most important determinants of the rate of Na+-H+ exchange in isotonic cells, the pHi sensitivity of the antiport is a likely target for the activating effects of osmotic shrinking. For this reason, the pHi dependence of the rate of H+ extrusion in normal and osmotically shrunken cells was compared. Cells were acid-loaded to varying degrees by using the nigericin-albumin technique described earlier and then resuspended in Na+-containing medium of either normal or elevated tonicity (Fig. 4B). The maximal rate of H+ extrusion was then calculated from the rate of ApHi. When the results obtained at different pHi values were compiled in graphs like that in Fig. 2, it was found that the approximately linear form of the curve was similar for normal and shrunken cells (not illustrated). However, osmotic shrinking caused a 0.2-0.3-unit alkaline shift of the pHi-dependence curve of the antiport, which intercepts the abscissa (i.e., becomes inactive) at pHi 7.35. This shift, which renders the quiescent transporter functional at physiological pHi (-7. l ) , can account for the osmotic activation of Na+-H+ exchange during RVI. The shift in the pHi dependence of the antiport presumably reflects an altered behavior of the modifier site inasmuch as this site largely determines the pHi sensitivity of the exchanger. According to this model, the set point of the modifier, which normally prevents transport at pHi 2 7.1, is adjusted upward. As a result, the nearly quiescent exchanger is activated, but the activation persists only until pHi attains a value of -7.35, the new set point. It is noteworthy that even at this elevated pHi a 1 : 1 electroneutral exchange is not at electrochemical equilibrium (see preceding discussion and Grinstein et al., 1985b), indicating that the allosteric control of transport has not been lost but only shifted to a more alkaline setting.

-

IV.

STIMULATION OF Na+-H+ EXCHANGE BY PHORBOL ESTERS

A. Mechanlsm of Activatlon

In several cell types, including lymphocytes, Na+ uptake is stimulated by the addition of phorbol esters, which are structural analogs of diacylglycerol (Burns and Rozengurt, 1983; Rosoff et al., 1984; Moolenaar et al., 1984). As shown in Fig. 5 , this increased uptake occurs concomitantly with a transmembrane efflux of H+ equivalents and is therefore attributable to an activation of the Na+-H+ antiport. In these experiments intracellular alkalinization was detected in thymocytes treated with 12-0tetradecanoylphorbol-13-acetate(TPA), consistent with H+ extrusion. As expected for a process mediated by the antiport, the alkalinization oc-

123

7. ACTIVATION OF THE Na+-H+ ANTIPORT

A

[

v

L

e

M

.-%o.= .c = E &0.50-

7.3 PHI

5

7.1

5fjs ;

TPA

1.

25 sG025-

3 min

li'. \. -.,,/ B

8

'

Control

'.-,

L.4 o-

O-O-O-~

+ Amiloride

0-0

v

0.00 Time (rnin)

FIG.5 . (A) Effect ofTPA on pH,. Cells were suspended in either K+-richsolution or Na solution with or without 100 pM amiloride. Where indicated, 10 * M TPA was added. (B) TPA-induced extracellular acidification. The pH of a lightly buffered suspension of thymocytes was measured with a combination electrode. Where indicated, lo-’ M TPA was added. The rate of acidification of the medium (ordinate) was calculated by determining the buffering power of the suspension by addition of known aliquots of KOH of HCI. [Reproduced from Proceedings of the National Academy of Sciences (1985) by copyright permission.]

curred only in the presence of external Na+ and was blocked by micromolar concentrations of amiloride (Fig. 5A). As in the case of hypertonic shrinking, the TPA-induced intracellular alkalinization was accompanied by an acidification of the external medium, measurable with a combination electrode in a poorly buffered medium (Fig. 5B).The external acidification is blocked by amiloride (Fig. SB),requires extracellular Na+, and is roughly commensurate with the internal alkalinization, indicating transmembrane displacement of H'+equivalents. This evidence, together with the stimulation of amiloride-sensitive Na+ fluxes, strongly supports the activation of coupled Na+-H+ exchange by phorbol esters. The mechanism of activation by TPA was studied essentially as described for osmotically shrunken cells. It was found that the apparent K , for N d was not significantly affected by the phorbol ester. Instead, as shown in Fig. 6, TPA seems to alter the pHi sensitivity of the antiport. This shift in the set point is remarkably similar to the one produced by hypertonic solutions and is also likely due to an altered behavior of the modifier site. 9. Role of Protein Kinase C

Specific high-affinity receptors for biologically active phorbol esters have been detected in the cytosol and membrane fractions of several cell

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PHI

FIG. 6. pHi dependence of the rate of H+ efflux in control and TPA-treated thymic lymphocytes. Determined as in Fig. 2, except at 37"C,using either untreated or TPA-treated cells. [Reproduced from Proceedings of the National Academy of Sciences (1985) by copyright permission.]

types, including lymphoid cells (Shoyab and Todaro, 1980). It is now generally accepted that the main and perhaps the sole target of the phorbol esters is the Ca2+ and phospholipid-dependent protein kinase C (Nishizuka, 1984a,b). This enzyme has widespread occurrence in various tissues of most animals (Kuo et al., 1980) and has been detected in lymphocytes from thymus and other sources (Kuo et al., 1980; Ogawa et al., 1981). Three lines of evidence suggest that activation of Na+-H+ exchange by phorbol esters is mediated by stimulation of C kinase: First, the concentrations of TPA required for activation of countertransport are similar to those reported to activate the kinase. Second, only those phorbol derivatives that accelerate kinase activity had an effect on Na+-H+ exchange, detected as an amiloride-sensitive cytoplasmic alkalinization (Table I). Third, the activation of Na+-H+ exchange by TPA could be blocked by trifluoperazine and by TMB-8, which can inhibit C kinase activity (Couturier et al., 1984; Simpson et al., 1984). Phorbol diesters are structural analogs of diacylglycerols, which are thought to be the physiological activators of protein kinase C. Further

TABLE I EFFECTOF PHOREOL DERIVATIVES ON pH, AND CORRELATION WITH TUMOR-PROMOTING AND PROTEIN KINASEC-STIMULATING ACTIVITY=

Analog

Concentration (M)

Maximal rate of alkalinization (ApH min-I)

Tumor-promoting activity (relative units)b

+++

100

++

81

++

88

+

NA

TPA

I x 10-8

4p-Phorbol-12,13-didecanoate

5 x 10-8

0.003 (7) 100%1 0.051 2 0.005 (3)

4p-Phorbol-12,13dibutyrate

I x 10-7

0.052

I x 10-8

4p-Phorbol-12,13-dibenzoate 4p-Phorbol 4a-Phorbol 4a-Phorbol-12,13-didecanoate

1 x 10-6

2.5 x 2.5 x 1 x 10-6

0.058

_t

raw

5 0.003 (3) [900/01 0.015 _t 0.009 (3) [26%] 0.052 2 0.004 (3) r9o"roo]

0 0 0

Nonpromoter Nonpromoter Nonpromoter

Protein kinase C activation

(%Y

0 0

0

pH, was measured in BCECF-loaded cells suspended in Na' solution at 37°C. The maximal rate of alkalinization was recorded after addition of the indicated concentration of the phorbol analogs. Data are means 5 SE of the number of determinations in parenthesis. The activity, calculated as percentage of that of TPA, is indicated in brackets. NA means not available. Taken from Shoyab and Todaro (1980). Taken from Nishizuka (1984a), where the kinase C-stimulating activity was compared by using 5 pM of the phorbol derivatives.

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S. GRINSTEIN ET AL.

OAG

Controt

-tAmiloride

r

7.4

7.1

' 3 min'

FIG.7. Effect of 1-oleoyl-2-acetylglycerol(OAG) on cytoplasmic pH (pHJ in rat thymocytes. Cells were suspended in Na+ medium with or without 100 p M amiloride and pHi was measured by using BCECF. Where indicated by the arrow, OAG (25 pg ml-', final concentration) was added to the medium. Temperature was 37°C. The figure is a composite representative of three similar experiments.

evidence that this enzyme is involved in the activation of the Na+-H+ antiport was obtained by testing the effects of an exogenously added a diacylglycerol. We determined the effect of 1-oleoy1-2-acetylglycero1, relatively permeant diacylglyceride, on pHi, measured fluorimetrically with BCECF. As illustrated in Fig. 7, the diacylglycerol produced a cytoplasmic alkalinization, resembling the effect of TPA. The alkalinization was found to be Na; dependent and inhibitable by amiloride, indicative of activation of the antiport. Together with the results obtained with phorbol diesters, the effects of diacylglycerol strongly suggest that protein kinase C is responsible for the activation of the Na+-H+ exchanger. V. SIMILARITIES OF THE PHORBOL ESTER AND VOLUME-INDUCED ACTIVATION

When measured as the cytoplasmic alkalinization, the activations of the Na+-H+ exchanger induced by shrinking and by phorbol esters are remarkably similar (compare Figs. 4 and 5). The time course and extent of the responses are alike, as are the Na; dependence and amiloride sensitivity. In addition, both responses were found to be reversible: Return to isotonicity (in the case of osmotically shrunken cells) or removal of the phorbol ester resulted in a gradual recovery of the original pHi. The time course of the recovery was also similar (half-time 3 to 4 min), but only if 4/3-phorbol-12,13-dibutyratewas used as a stimulus. The activation by TPA, a more potent but more hydrophobic phorbol ester, reverted much more slowly, presumably owing to a reduced rate of dissociation from C kinase.

7. ACTIVATION OF THE Na+-H+ ANTIPORT

127

The many similarities between the two modes of activation are suggestive of a common underlying mechanism. If there is a common mechanism, then maximal stimulation by either procedure should preclude further stimulation by the other; i.e., the responses would not be additive. Conversely, additivity would be expected if independent processes are involved. To distinguish between these alternatives, cells were osmotically shrunk under conditions known to produce maximal activation of the antiport, while pH, was monitored fluorimetrically with BCECF. TPA was then added and no further alkalinization was observed, even at concentrations of the phorbol ester known to induce a maximal response. Similar experiments in which TPA was added initially, followed by hypertonic shrinking, confirmed that the responses were not additive. In contrast, subsequent addition of monensin (an exogenous Na+-H+ exchange ionophore) or of NH: (which is in equilibrium with NH3, a permeable weak base) produced the expected alkalinization. This indicates that the Na+ gradient can still drive protons outward and that the lack of additivity of the volume and TPA responses was not due to failure of the fluorescent dye to detect more alkaline levels of pH,. Further similarities between the phorbol ester and hypertonically induced responses were revealed by pharmacological studies. As mentioned earlier, the phorbol ester-mediated activation of the antiport was found to be blocked by trifluoperazine, an inhibitor of protein kinases, including C kinase. In parallel experiments, the response to hypertonicity was also blocked by this drug. In fact, a detailed study of the concentration dependence of the inhibitory effect showed that half-maximal inhibition was attained in both cases at the same concentration: -20 p M . It is worthwhile noticing that this is similar to the reported K , for inhibition of protein kinase C activity by trifluoperazine (Couturier et al., 1984). Both the osmotically induced and the TPA responses were also found to be inhibited by N-ethylmaleimide. Prolonged incubations in comparatively high concentrations of this alkylating reagent (e.g., 30 min at I mM) also inhibited the response of cytoplasmic acidification, suggesting that the transport function of the exchanger is impaired. However, milder treatment conditions, which fail to significantly affect activation of the antiport by acid (0.25 mM for 5 min at 22"C), completely abolish the activation by shrinking or by phorbol esters. These data again support the notion that both modes of stimulation share a common mechanism. Moreover, the results allow a distinction between the basal (acid-activated) and stimulated forms of transport. It is likely that the process of readjustment of the set point of the modifier, rather than the operation of the modifier itself, is impaired by alkylation with N-ethylmaleimide. Because stimulation of a protein kinase appears to be involved, it was reasoned that depletion of ATP, the putative substrate of the kinase,

128

S. GRINSTEIN ET AL.

might prevent activation of the antiport by phorbol esters and perhaps also by osmotic shrinking. As in the case of N-ethylmaleimide, the basal (acid-induced) Na+-H+ exchange was found to be affected by extensive ATP depletion. However, after short depletion periods (20 min at room temperature) the basal response was only slightly inhibited (-20%). In contrast, the responses to both phorbol esters and to shrinking were more drastically reduced (>70%). Thus, the basal and activated forms of the transporter can also be distinguished on the basis of their ATP dependence. Moreover, the ATP requirement for activation by either shrinking or TPA appears to be similar, reinforcing the analogy between the two stimuli. VI. POSSIBLE INVOLVEMENT OF A PROTEIN KINASE IN VOLUME REGULATION

An involvement of protein kinase(s) in the osmotic activation of Naf-H+ exchange is suggested by (1) the striking physiological and pharmacological similarities with the phorbol ester-induced response; (2) the fact that phorbol esters act by stimulating protein kinase C; (3) the reported ATP dependence of the activation; (4) the finite lagtime observed following shrinking (e.g., Fig. 4) suggestive of a chemical process such as phosphorylation; and ( 5 ) the slow recovery after returning the cells to isotonicity, which could be indicative of a slow dephosphorylating process. A hypothetical sequence of events would involve activation of a kinase upon osmotic shrinking, followed by phosphorylation of the Na+-H+ exchanger itself or of an ancillary protein. Phosphorylation would then result in an alkaline shift in the set point of the modifier site, rendering the antiport active at physiological pHi . Cellular volume increase would ensue owing to intracellular buffering of the ejected H+, to the accumulation of intracellular HCO; (a consequence of the cytoplasmic alkalinization), and to the exchange of intracellular HCO; for C1- (see Cala, 1980, for detailed discussion). As a partial test of this hypothesis, we undertook measurements of protein phosphorylation in normal and hypertonically treated cells. For comparison, cells were also treated with TPA, which is known to stimulate phosphorylation in a variety of cells (Nishizuka, 1984a,b). Thymocyte suspensions were preloaded with 32P-phosphateto allow incorporation of the isotope into the nucleotide pool. This was followed either by addition of TPA or by osmotic shrinking of the cells. After 5 min, a period that suffices for the full development of the transport response (Figs. 4A

7. ACTIVATION OF THE Na+-H+ ANTIPORT

129

and 5 ) , the cells were sedimented, lysed, and separated into soluble and particulate fractions. The latter was further purified to obtain a plasma membrane-rich fraction. These fractions were then analyzed by polyacrylamide gel electrophoresis in sodium dodecyi sulfate, followed by autoradiography of the dried gels. One of six similar experiments analyzing the soluble fraction is illustrated in Fig. 8. Neither TPA nor osmotic shrinking significantly affected the overall polypeptide composition of the samples, as determined by silver staining (Fig. 8A). However, significant differences were observed in the phosphorylation pattern. TPA produced increased phosphorylation of a number of polypeptides (lane T in Fig, 8B), detectable also as an increased specific activity in the trichloroacetic acidprecipitable material. Some of the polypeptides phosphorylated by TPA were also labeled in osmotically stressed cells (solid arrowheads in Fig. 8B). In addition, some bands were only phosphorylated either by TPA or by shrinking (open arrowheads), indicating that although the procedures show similarities, they are not entirely identical. The fact that label incorporation into the majority of the bands was not affected by shrinking indicates that a specific process is involved, and not a general increase in phosphorylation due to elevated concentrations of nucleoside triphosphates. The correlation between increased transport and phosphorylation was investigated using trifluoperazine. The phenothiazine, which is a potent blocker of both volume- and TPA-induced Na+-H+ exchange, prevented the increased phosphorylation (not illustrated), consistent with its reported effect on both C kinase and calmodulin-dependent kinases. Perhaps more relevant to the activation of Na+-H+ exchange is the analysis of phosphorylation in the plasma membrane-rich fraction. In eight similar experiments TPA was found to increase markedly the 32P incorporation into polypeptides of approximately 60 and 65 kDa (not illustrated). In the same experiments, membranes isolated from hypertonically stressed cells also showed a marked increase in the labeling of the 60-kDa polypeptide. Small but consistent increases were noted in minor bands in the 50-55 kDa region of membranes treated with TPA or in hypertonic medium. As in the case of the supernatant, the many other polypeptides displayed normal intensity, ruling out the argument that increased phosphorylation is simply due to the elevation of 32P-ATPconcentration upon shrinking. These results provide direct evidence that cellular shrinking triggers a sequence of events that culminate in increased phosphorylation of cytosolic and membrane proteins. The similarities in the phosphorylation patterns of TPA and osmotically activated cells and their common susceptibility to inhibition by trifluoperazine suggest that an enzyme activated by cellular shrinking is protein kinase C.

FIG.8. Effects of TPA and of hypertonic shrinking on protein phosphorylation. Thymocytes were preloaded with 32P-phosphate and then incubated in either isotonic (285 mOsm) Na+ medium with (T) or without (C) lo-’ M TPA, or in hypertonic (550 mOsm) Na+ medium (H)for 5 min at 37°C. The cells were then lysed in cold hypotonic medium and sedimented at 30,000g for 20 min at 4°C. Samples of the supernatant were precipitated with trichloroacetic acid and analyzed by electrophoresis in 10% polyacrylamide gels with sodium dodecyl sulfate by the method of Laemmli. (A) Polypeptide patterns as revealed by silver staining. (B)Autoradiogram. The scale on the left indicates molecular weight in kilodaltons, determined using Sigma Mark VI standards. Solid arrowheads indicate polypeptides labeled in both TPA and hypertonic samples. Open arrowheads indicate bands labeled by only one of the treatments. Representative of six similar gels.

7. ACTIVATION OF THE Na+-H+ ANTIPORT

131

VII. OSMOTICALLY INDUCED CHANGES IN PHOSPHOlNOSlTlDE TURNOVER

It is generally thought that diacylglycerides are the natural ac ivators of protein kinase C (Nishizuka, 1984a,b; Berridge, 1984). Physiologically, as in the case of receptor-mediated activation, agonist binding is believed to induce an activation of a phosphodiesterase (phospholipase C) which releases diacylglycerol primarily from phosphoinositides. The main if not the sole substrate affected appears to be phosphatidylinositoI4,5-bisphosphate (Nishizuka, 1984a,b; Berridge, 1984). Because C kinase appears to be activated in hypertonic media, we investigated whether phosphoinositide metabolism was affected in shrunken cells. We hypothesized, by analogy with receptor-mediated activation, that diacylglycerol was released in hypertonically treated cells. In very responsive cell types, such as platelets and neutrophils, activation of the phosphodiesterase can be detected as a decrease in the phosphatidylinositol4,5-biphosphatepool. However, because of the rapid rate of interconversion of phosphoinositides and because of the resynthesis of phosphatidylinositol, this net change is not always observed. Instead, the system is capable of buffering the concentration of the various phosphoinositides by balancing the increased degradation with faster synthesis. In the latter case, activation of the cycle can be detected as an increased rate of turnover of intermediates. Because it is comparatively simpler, we used the latter experimental approach to determine whether phosphoinositide metabolism is affected in hypertonically stressed cells. For comparison, cells were also treated with TPA, which affects C kinase directly and is not expected a priuri to have any direct effect on phosphoinositide metabolism. Far these experiments, the cells were loaded with 32P-phosphate as described earlier for phosphoprotein determinations and then treated hypertonically or with TPA. The reaction was stopped after 5 or 60 min and the samples were extracted in acid chloroform methanol. The lipid extracts were then analyzed by thin-layer chromatography, individual lipids were identified by comparison with standards, and the respective spots were scraped off the plates and counted. The results of three experiments, each performed in triplicate, are summarized in Fig. 9. To allow comparison among experiments, the data are normalized with respect to the individual control. As expected, treatment with the phorbol ester had no significant effect in the rate of 32Pincorporation into either phosphatidylcholine (PC in Fig. 9), phosphatidylinositol (PI), or phosphatidylinositol 4-phosphate (DPI). In contrast, hypertonic shrinking had marked effects, particularly on phosphoinositides: At 5 min the extent of phosphorylation of phosphatidylinositol had increased by

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FIG.9. Effects of TPA and hypertonicity on the incorporation of 32Pinto phospholipids extracted from thymic lymphocytes. Cells were preloaded with 32P-phosphateand then incubated with or without TPA in isotonic solution or in hypertonic solution as in Fig. 8. The cells were then sedimented in an Eppendorf Microfuge and the cellular pellet extracted with chloroform :methanol :HCI. Nonradioactive carrier lipids were added to the extracts, which were then analyzed by one-dimensional thin-layer chromatography. Lipids were located by iodine staining and 32Pradioactivity was measured by liquid scintillation counting. The amounts of phosphatidylcholine (PC), phosphatidylinositol (PI), and phosphatidylinositol4phosphate (DPI) are expressed as percent cpm in the control sample. The data are means k SE of three experiments, each performed in triplicate.

- 140% and that of phosphatidylinositol 4-phosphate by over 500%.' Though quantitatively somewhat different, these changes persisted after 60 min (not illustrated). A small (60%) and barely significant effect was also measurable for phosphatidylcholine at 5 min (Fig. 9), but it was no longer detectable after 60 min (not shown). These data are indicative of a specific increase in phosphoinositide turnover and are consistent with a stimulation by hyperosmotic shrinking of phosphodiesterase-mediated hydrolysis of phosphoinositides . 'In more recent experiments, when lipids were analyzed by two-dimensional thin-layer chromatography, the osmotically induced increase in phosphatidylinositol4-phosphatewas significantly smaller (142%). This suggests that an impurity comigrates with phosphatidylinositol 4-phosphate in the one-dimensional system.

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VIII.

133

CONCLUDING REMARKS

Little is presently known of the molecular events underlying cellular volume regulation, particularly RVI. At the present time, the majority of the observations summarized in this review can be explained by a working hypothesis that involves the following sequence of events: ( I ) Shrinking triggers activation of a phosphodiesterase (phospholipase C) which breaks down phosphoinositides, presumably in the plasma membrane. (2) The resultant release of diacylglycerol stimulates protein kinase C. (3) The kinase phosphorylates the Na+-H+ exchanger or a neighboring protein which affects the operation of the antiport. (4) Phosphorylation produces a shift in the pH, dependence of the exchanger, resulting from a readjustment of the set point of the modifier site to a more alkaline pH,. (5) Na+-H+ exchange is activated, with simultaneous uptake of osmotically active Na+ and cytoplasmic alkalinization. (6) In the presence of external HCO; (which is in equilibrium with the permeant (COz), the internal HCO; concentration increases. (7) Extracellular C1- exchanges for internal HCO;. (8) The intracellular accumulation of Na+, HCO;, and CI- drives osmotically obliged water into the cells, which swell toward their normal (isotonic) volume. Though attractive, this model is only tentative and fails to explain a few observations. First, the protein phosphorylation pattern is not identical for TPA and shrunken cells (Fig. 8). Second, migration of soluble C kinase to the membranous fraction, which has been reported to occur in phorbol ester-treated cells (Kraft and Anderson, 1983), is also observed in TPA-treated thymocytes but not in shrunken cells (E. Mack, unpublished observations). These observations can be rationalized if it is assumed that, whereas phorbol esters diffuse throughout the cell, the more labile diacylglycerol generated upon shrinking acts only locally at or near the plasma membrane. Alternatively, it is conceivable that shrinking activates a different type of protein kinase, which in turn stimulates phosphoinositide turnover. Activation of the phosphorylation of mono- and diphosphatidylinositol by CAMP- or calmodulin-dependent kinases and even by tyrosine kinases has been reported (Berridge and Irvine, 1984). However, even if the original hypothesis has to be modified to accommodate these findings, the explanation for the primary event underlying RVI (i.e., the alteration of the set point of the modifier site that results from phosphorylation at or near the antiport) would still prevail. ACKNOWLEDGMENTS The experimental work summarized in this review was supported by the National Cancer Institute (Canada) and by the Medical Research Council of Canada. S. Grinstein is the recipient of a Medical Research Council Scientist Award.

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