Stimulation of mitogen-activated protein kinase and Na+/H+ exchanger in human platelets. Differential effect of phorbol ester and vasopressin.

Treatment of human platelets with phorbol 12-myristate 13-acetate (PMA) and arginine vasopressin (AVP) increase the phosphorylation and activation of mitogen-activated protein kinase (MAPK). Electrophoretic retardation of MAPK mobility on SDS-polyacrylamide gels was used for determination of MAPK phosphorylation. The activity of MAPK was tested in myelin basic protein (MBP)-containing polyacrylamide gels. In this study we compared the PMA and AVP signal transduction pathways leading to the activation of MAPKs and Na+/H+ exchanger (NHE). Both agonists stimulate MAPK and NHE activities in a similar time frame and concentration dependence. The MAPK and NHE activities induced by PMA were inhibited by staurosporine, a potent inhibitor for protein kinase C (PKC), and by MAPK kinase (MEK) inhibitor, PD98059, but were not affected by the tyrosine kinase inhibitor genistein. In contrast, both AVP-induced MAPK and NHE activities were inhibited by genistein and MEK inhibitor but were not affected by staurosporine. Immunoprecipitation studies demonstrate that PMA, but not AVP, enhances the basal phosphorylation of the NHE-1. In this study, MAPKs are suggested to be a part of converging signaling leading to NHE activation by PKC-dependent and AVP-tyrosine kinase-dependent pathways. We propose that the MAPK activation of the NHE-1 does not involve phosphorylation of this exchanger protein. On the other hand, PKC can lead to phosphorylation and to additional activation of the NHE-1 through a MAPK-independent pathway.

The Na ϩ /H ϩ exchange system is ubiquitous and is present in plasma membranes of essentially all mammalian cell types. The first Na ϩ /H ϩ exchanger isoform cloned, referred to as NHE-1 (10), is the predominant species in nonepithelial cells. This form of the antiporter was found in human platelets (11) and is thought to be primarily responsible for intracellular pH homeostasis. Immunoprecipitation studies of NHE-1 demonstrate that the antiporter is phosphorylated in unstimulated cells (11)(12)(13)(14). It was suggested that growth factor-activated NHE-1 is controlled by phosphorylation of the exchanger protein, since parallel to the induced pH i rise, increased phosphorylation of the NHE-1 was observed (12,15). Recently, the properties of the NHE-1 were further analyzed, using deletion variants expressed in the exchanger-deficient mutant cell line PS120 (16). It was found that deletion of all major phosphorylation sites including growth factor-sensitive ones reduced growth factor-induced cytoplasmic alkalinization by only 50%. Therefore, it was suggested that growth factor activation of the NHE-1 occurs at least in part by a mechanism that does not involve phosphorylation of the exchanger. In agreement with a NHE-1 phosphorylation independent mechanism, activation of the antiporter during volume regulation was not associated with increased phosphorylation (14). The understanding of the Na ϩ /H ϩ exchange regulation mechanism, by extracellular signals, is still far from complete. The MAPK pathway, which is essential for the propagation of growth factor signals, was suggested to be a good candidate for mediation of NHE activation (17). In agreement, it was found that the protooncogene product p39 c-Mos kinase activates Raf-1 kinase and p45 MAPK (18 -20) and up-regulates the Na ϩ /H ϩ exchange in Xenopus oocytes (21). In addition, long-term expression of c-Ha-Ras stimulates Na ϩ /H ϩ and Na ϩ -dependent Cl-HCO 3 exchange in NIH-3T3 fibroblasts (22).
In previous studies, we showed that PMA (23)(24)(25)(26) and arginine vasopressin (AVP) activate the NHE, in human platelets, by two different pathways (26). Recent observations demonstrate that AVP activates MAPKs in vascular smooth muscle cells (VSMC) (2) and induces tyrosine phosphorylation of protein(s) in human platelets (27). Thus, it was interesting to investigate whether AVP and PMA affect the Na ϩ /H ϩ ex-* This work was supported by the Gila and Arieh Keshet heart disease research fund and by the Ministry of Science and Arts fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
In this study, we report that in human platelets the phosphorylation and activation of the 42-and 44-kDa MAPK variants are stimulated by AVP and PMA through two different signaling pathways. Parallel to this activation of MAPKs, PMA and AVP stimulate Na ϩ /H ϩ exchange, while only PMA induces NHE-1 phosphorylation. We suggest a possible role for MAPKs in NHE activation by both agonists through a mechanism that does not involve phosphorylation of the exchanger. Our data also suggest that phosphorylation of the NHE-1, induced by protein kinase C (PKC) activation, may cause additional activation of the transporter.

Methods
Preparation and Activation of Human Platelets-Venous blood was drawn from healthy volunteers, ages 22-40, who had not received any medication during the previous 14 days. The blood was anticoagulated with acid-citrate-dextrose solution comprising 65 mM citric acid, 11 mM glucose, and 85 mM trisodium citrate, at a volume ratio of blood:anticoagulant of 6:1. Platelet rich plasma (PRP) was obtained by centrifugation at 120 ϫ g for 10 min and had a pH of 6.5 Ϯ 0.1. PRP was supplemented with prostaglandin E 1 (2.8 M) and centrifuged at 1500 ϫ g for 10 min. The platelets were resuspended (about 8 -9 ϫ 10 8 cells/ml) in a standard Na ϩ medium, containing, in mM: 140 NaCl, 5 KCl, 10 glucose, 0.42 NaH 2 PO 4 , 20 HEPES, pH 6.8, and 1 mg/ml bovine serum albumin. For immunoprecipitation analysis, the platelets were resuspended in a phosphate-free standard Na ϩ medium and were incubated with 1 mCi/ml [ 32 P]phosphoric acid for 90 min at 37°C. After incubation, the platelets were gel-filtered through a Sepharose 2B column (10 ϫ 0.76 cm), in order to remove excess of [ 32 P]phosphoric acid. The solution used to equilibrate the column and to elute the platelets (about 5-6 ϫ 10 8 cells/ml) was the standard Na ϩ medium. After the addition of various modulators, at 37°C, for the times indicated in the figures, cells were mixed with an equal volume of 2 ϫ concentrated lysis buffer and frozen in liquid nitrogen (1 ϫ lysis buffer: 10% glycerol (v/v), 25 mM NaCl, 50 mM NaF, 10 mM sodium pyrophosphate, 2 mM EGTA, 2 mM dithiothreitol (DTT), 2 mM Na 2 VO 4 , 20 mM p-nitrophenyl phosphate, 0.1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 25 mM Tris-HCl, pH 7.4). For immunoblot analysis of cell extracts, containing phosphorylated and unphosphorylated MAPK, the following buffer was used: 1.5 mM EGTA, 1 mM EDTA, 1 mM DTT, 0.1 mM Na 2 VO 4 , 0.2 mM phenylmethylsulfonyl fluoride, 20 g/ml leupeptin, 50 mM ␤-glycerophosphate, pH 7.3. The samples were allowed to thaw on ice, and the cells were sonicated twice for 5 s at 0-4°C in a Branson B-30 cell disruptor, at 40% duty cycle. The resulting homogenate was centrifuged at 17,300 ϫ g for 20 min and the supernatant of the cell extracts was mixed with 0.25 volume of 5 ϫ concentrated sample buffer, followed by heating at 100°C for 5 min (1 ϫ sample buffer: 3% SDS (w/v), 0.0015% bromphenol blue (w/v), 5% 2-mercaptoethanol (v/v), 11% glycerol (v/v), 70 mM Tris-HCl, pH 6.8). For immunoprecipitation analysis, cells were lysed by the addition of an equal volume of 2 ϫ concentrated lysis buffer and 0.25 volume of 5 ϫ concentrated bromphenol blue free sample buffer, followed by heating at 100°C for 5 min. For spectrofluorimetric measurements of pH changes, PRP was loaded in the dark with BCECF acetoxymethyl ester (3 M, final) for 30 min and was then gel-filtered through a Sepharose 2B column as described above. The suspension of BCECF-loaded platelets was supplemented with 1 mM CaCl 2 , 1 mM MgCl 2 , and 0.2 mM probenecid.
MAPK Activity Determined by Using MBP-containing Polyacrylamide Gels-This determination was performed according to the method of Kameshita and Fujisawa (29) with slight modifications. The proteins in the crude extracts were resolved by 12% SDS electrophoresis (30) through mini polyacrylamide gels (5.5 ϫ 8.5 cm) that contain the substrate (0.5 mg of MBP/ml) polymerized into the separation gel. After electrophoresis, the proteins in the gels were allowed to denature and renature, and then the gels were immersed for 30 min at 22°C in buffer containing 40 mM HEPES, pH 8.0, 2 mM DTT, and 0.1 mM EGTA. For determination of MBP kinase activity, the gels were incubated for 1 h at 22°C in 7.5 ml of the same buffer, supplemented with 5 mM MgCl 2 and 25 M [␥-32 P]ATP (25 Ci/ml). The location of any MBP kinases in the gels was then detected by autoradiography. The relative MBP kinase activity in the corresponding protein bands was determined by scanning the films and integrating the peaks using the ImageQuant and Personal Densitometer Molecular Dynamics system.
Western Blotting-After separation of extracts by 11% SDS-PAGE, proteins were transferred to nitrocellulose membrane filters (Schleicher & Schuell). Membranes were blocked using 3% non-fat dried milk in phosphate-buffered saline, pH 7.2, and incubated for 16 h with the same buffer containing immune serum at a serum:buffer ratio of 1:250. Immunoreactive protein bands were visualized using ECL (Amersham) Western blotting detection system. Agfa RP2 x-ray films were used in order to detect the enhanced chemiluminescence of the appropriate bands.
Determination of NHE Activity-Changes in cytoplasmic pH were determined as described (23)(24)(25)(26). Fluorescence was measured in a Jasco FP-770 spectrofluorimeter with wavelength settings at 495 and 525 nm for excitation and emission using 5 and 10 nm slits, respectively. For measuring the Na ϩ /H ϩ exchange, an aliquot of the gel-filtered platelets loaded with BCECF (1-2 ϫ 10 7 cells) was mixed with 1.8 ml of NaClsodium propionate solution, containing in mM: 80 NaCl, 60 sodium propionate, 5 KCl, 1 CaCl 2 , 1 MgCl 2 , 10 glucose, and 20 HEPES, pH 7.35. All tested compounds were added to the assay medium prior to the addition of the platelets. Fluorimetric tracings were recorded for 90 s, starting within Ͻ3 s of platelet addition. At termination, 9 l of 10% Triton X-100 was added and calibration of pH versus fluorescence was performed with increments of MOPS as titrant. The pH was monitored in the cuvette by GK2401C combined electrode, connected to Ion 83 Ion meter (Radiometer, Copenhagen), with a resolution of 0.001 pH unit. A predetermined factor was used to correct for the red shift of the intracellular dye. The Na ϩ /H ϩ exchange rate is the ⌬pH i per 9 s of the alkalinization process at pH i 7.0 as described (25).
Immunoprecipitation-Platelet extracts were diluted 15-fold in buffer A (100 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 10 mM Tris, pH 7.0), and activated Omnisorb (Calbiochem) was added. After 30 min at 4°C, nonspecific compounds, bound to the Omnisorb particles, were discarded by centrifugation at 17,300 ϫ g for 20 min. Nonimmune or immune serum was added to the supernatant at a serum:extract ratio of 1:30. After incubation for 16 h at 4°C, activated Pansorbin (Calbiochem) was added for another 30 min at 4°C. The bacteria immunocomplexes were washed three times with buffer A, once with high salt buffer (1 M NaCl, 10% Nonidet P-40, 10 mM Tris, pH 7.0), and once more with buffer A. The immunoprecipitated proteins were then dissolved in 1 ϫ sample buffer, boiled for 5 min, and separated by 7.5% SDS-PAGE. The radiolabeled proteins were visualized by autoradiography of the stained and dried gels. The relative phosphorylation of the NHE-1 was determined by scanning the films and integrating the peaks using the ImageQuant and Personal Densitometer Molecular Dynamics system. Activation of MAPK by PMA and AVP in Human Platelets-In previous studies, it was shown that MAPK can phosphorylate MBP, a commercially available substrate, at a significant rate (2,7,9). In the procedure presented in Fig. 3, MAPK activity was determined in MBP-containing polyacrylamide gels. The results illustrate that renatured 42-and 44-kDa variants of MAPK, obtained from PMA-and AVP-stimulated platelets (Fig. 3, A and B, respectively), expressed timedependent and transient stimulation of MBP kinase activity. The MAPK variants appeared as a doublet on the MBP-containing polyacrylamide gels, with higher activity of the 42 kDa protein. These results are in agreement with higher amounts of this variant in human platelets (Fig. 1). The effect of PMA is rapid, reaching almost maximal phosphorylation of MBP within 30 s (Fig. 3A). This maximal level of phosphorylation starts to decline only after 2 min. In AVP-stimulated platelets, the MBP kinase activity reaches a maximum at 1 min (Fig. 3B) and then returns to almost the basal level after 2 min of hormone addition.

Identification of MAPKs in Human
MAPK stimulation by PMA and AVP is concentrationdependent. Stimulatory saturation is apparent at 3 ϫ 10 Ϫ7 M PMA (Fig. 4A) and 10 Ϫ7 -10 Ϫ6 M AVP (Fig. 4B), while halfmaximal effect is attained at 10 Ϫ7 M PMA and 0.9 ϫ 10 Ϫ9 M AVP. The PMA effect is in agreement with considerable stimulation of 42-kDa MAPK in sheep platelets (7).
As shown in Fig. 5, the AVP effect is mediated through a V 1 receptor, since only the V 1 receptor-specific antagonist (32) (lane 4) decreased significantly the AVP-elevated MBP phosphorylation (lane 2). In addition, only the V 1 receptor-specific agonist (lane 6) mimics the AVP-elevated MBP phosphorylation (lane 2). These observations are consistent with the V 1 receptor existence (33) and with AVP-V 1 receptor-dependent tyrosine phosphorylation of protein(s) in human platelets (27).
Do MAPKs Mediate PMA and AVP Activation of the NHE?-We compared the effect of staurosporine, a potent and relatively specific inhibitor of PKC (34,35), genistein, a relatively specific tyrosine kinase inhibitor (36) and MEK inhibitor (4 -6) on MAPK (Fig. 6) and NHE (Table I) activities stimulated by PMA or AVP. The results show that both PMA and AVP (treatments 2 and 6, respectively) markedly increase the MBP kinase activity of the 42-and 44-kDa variants of MAPK and the Na ϩ /H ϩ exchange rate. Both PMA-induced MAPK and Na ϩ /H ϩ exchange activities are reduced by staurosporine (treatments 3) and MEK inhibitor (treatments 5), but are not affected by genistein (treatments 4). In contrast, both activities of the AVP-induced MAPK and NHE are not affected by staurosporine (treatments 7), but are reduced by genistein (treatments 8) and MEK inhibitor (treatments 9). Does MAPK Mediation Require Direct Phosphorylation of the NHE-1?-Antibody against the NHE-1 carboxyl-terminal peptide (residues 801-815), prepared in our laboratory, was used for Western immunoblots of human platelet extracts. As was previously reported (11), human platelets contain this antiporter with an apparent molecular mass of 100 -115 kDa (Fig.  7A). Addition of the carboxyl-terminal peptide of the NHE-1 (5 g/ml) abolished the observed chemiluminescence of the NHE-1 protein band (Fig. 7B). These results emphasize the specificity of the antibody toward this antiporter.
Immunoprecipitations of NHE-1-phosphorylated protein, using this antibody, are shown in Fig. 8. As reported for fibroblasts (12,13), MGH-U1 cells (14), and platelets (11) The proteins in the crude cell extracts were resolved by SDS electrophoresis through 12% mini polyacrylamide gels that contain MBP polymerized into the separation gel. After electrophoresis, the proteins in the gels were allowed to renature, and then the gels were immersed in a kinase assay buffer that contained [␥-32 P]ATP, as described under "Experimental Procedures." The location of any MBP kinases in the gels was then detected by autoradiography. preserved up to 10 min (data not shown). The phosphorylation levels of the NHE-1 in PMA-and AVP-treated platelets, for 1 min, were quantified by densitometric integration of autoradiograms. The ratio of AVP to control phosphorylation averaged 0.95 Ϯ 0.08 (mean Ϯ S.E., n ϭ 6), while the ratio of PMA to control phosphorylation averaged 1.40 Ϯ 0.06 (mean Ϯ S.E., n ϭ 9). Further characterization of the PMA-induced NHE-1 phosphorylation demonstrates that this phosphorylation is inhibited by staurosporine (lane d), but is not affected by genistein (lane e) and MEK inhibitor (lane f).

DISCUSSION
Agonist stimulation of platelets results in activation of many protein kinases. In this study, we have examined the stimula-tion of MAPKs during PMA-and AVP-induced platelet activation. Two forms of MAPK, 42 and 44 kDa, were identified in human platelets. The change in the electrophoretic mobility of MAPKs (Fig. 2) indicates the conversion of these proteins to their phosphorylated and activated forms by PMA and AVP. Accordingly, the in-gel MBP kinase activity of both MAPK variants was increased (Fig. 3). Recently, several studies also identified two isoforms of MAPK in sheep platelets (7) and in human platelets (8,9). Thrombin (8,9), PMA, and PAF (7,9) were shown to activate the 42-and 44-kDa MAPKs. Our findings are in agreement with these and other studies in which both 42-and 44-kDa MAPK variants are coactivated in response to various stimuli (2,37,38). PMA and AVP stimulate the same class of MAPKs since each of the agonist results in the phosphorylation of both variants. Moreover, at maximal stimulating conditions, these agonists cause a mobility shift of all visualized MAPK molecules (Fig. 2). Tyrosine phosphorylation and stimulation of immunoprecipitated MAPKs from    human platelets, induced by 10 Ϫ7 M PMA, were below the detection range in the work of Nakashima et al. (9). PMA concentration-dependent studies indicate that a concentration of 10 Ϫ7 M is sufficient to induce 50% of maximal MAPK stimulation (Fig. 4A). These results are in agreement with a significant stimulation of cytosolic MBP and S6 kinase activities, in sheep platelets, using 2 ϫ 10 Ϫ7 M PMA (7).
The activation of MAPKs in response to PMA and AVP is transient (Fig. 3). Activation of MAPKs requires both threonine and tyrosine phosphorylation, which is catalyzed by the single dual-specific enzyme-MAPK kinase (3). Therefore, it is likely that MAPK dephosphorylation is regulated by the activity of a dual specificity protein phosphatase, capable of dephosphorylating threonine and tyrosine residues (31, 39 -42). Recently 3CH134, a dual specificity protein phosphatase, was established as a dynamically regulated, immediate early gene product. This protein phosphatase was suggested to have a role in attenuating signaling pathways initiated by angiotensin II in VSMC (42) and by serum in fibroblasts (31). Platelets are nonproliferative, terminally differentiated cells that do not contain a nucleus or active machinery for protein synthesis (43,44). Thus, deactivation of MAPKs following agonist-dependent activation, does not involve induction of protein phosphatase expression.
Several lines of evidence indicate a possible regulatory role for MAPKs in NHE activation in human platelets. 1) Both PMA and AVP rapidly increase, in a similar time frame, the phosphorylation and activation of the MAPKs and the activity of the NHE. When platelets are suspended in NaCl-sodium propionate solution, a rapid intracellular acidification takes place followed by alkalinization process that represents the activity of the NHE. If the NaCl-sodium propionate solution contains PMA or AVP, the platelets respond by an increased rate of Na ϩ /H ϩ exchange that can be measured within 10 s (26). The results in Fig. 3 indicate that both PMA and AVP induce an increase in the in-gel MBP kinase activity within this period of time.
2) The concentration dependence is very similar for activation of both MAPKs (Fig. 4) and NHE (24,26). Maximal activation of the PMA and AVP effects are observed at 3 ϫ 10 Ϫ7 M and 10 Ϫ6 -10 Ϫ7 M, respectively. The concentrations for halfmaximal effect for MAPK and NHE activation are: (a) 10 Ϫ7 M and 1.2 ϫ 10 Ϫ7 M PMA, respectively, and (b) 0.9 ϫ 10 Ϫ9 M and 0.3 ϫ 10 Ϫ9 M AVP, respectively. 3) Both PMA-induced MAPK activity and Na ϩ /H ϩ exchange are inhibited by staurosporine and MEK inhibitor but are not affected by genistein. In contrast, both AVP-induced MAPK activity and Na ϩ /H ϩ exchange are inhibited by genistein and MEK inhibitor but are not affected by staurosporine.
These observations, summarized in Fig. 9, indicate the existence of: 1) distinct pathways for PMA and AVP stimulation of the NHE; 2) a possible regulatory role for MAPKs in NHE activation by both PMA and AVP; 3) MEK and MAPK as a part of a converging signaling pathway leading to the activation of the NHE; 4) PKC but not tyrosine kinase involvement, upstream to MAPKs, in the signaling pathway initiated by PMA; 5) tyrosine kinase but not PKC involvement, upstream to MAPKs, in the signaling pathway initiated by AVP.
Epidermal growth factor, ␣-thrombin, serum, and okadaic acid stimulate Na ϩ /H ϩ exchange activity in fibroblasts in a time-dependent manner that correlates with increased phosphorylation of the NHE-1 (12,13). Activation and phosphorylation of the Na ϩ /H ϩ antiport by okadaic acid were also demonstrated in lymphocytes (15). These results suggest that the proximate step in Na ϩ /H ϩ exchange activation is mediated by growth factor-activable NHE-1 kinase(s). Unlike the effects of growth promoters, activation of the antiport during volume regulation was not associated with increased phosphorylation of the NHE-1 (14). Furthermore, deletion of all major phosphorylation sites, mapped to the cytoplasmic tail between amino acids 636 and 815, still preserves 50% of the growth factorinduced cytoplasmic alkalinization (16). These results support the existence of a mechanism that does not require direct phosphorylation of the NHE-1. In this study, we are demonstrating that MAPK may be a part of a converging signaling pathway leading to the activation of Na ϩ /H ϩ exchange by PMA and AVP. We anticipated that if phosphorylation of the NHE-1 is essential for its activation, by MAPK downstream kinase(s), then both PMA and AVP will increase its phosphorylation. Our results demonstrate that treatment of platelets with PMA resulted in increased phosphorylation of the NHE-1. In contrast, exposure of platelets to AVP did not change the phosphorylation of the antiporter (Fig. 8). Therefore, we propose that the control of the Na ϩ /H ϩ exchange by the MAPK pathway, in human platelets, probably does not require direct phosphorylation of the NHE-1. Both PMA and AVP activate Na ϩ /H ϩ exchange in human platelets, with higher activation induced by PMA (Table I and Ref. 26). On the other hand, both PMA and AVP phosphorylate and activate MAPKs to the same degree (Figs. 2 and 3). Nevertheless, Na ϩ /H ϩ exchange activation induced by AVP is completely abolished by the MEK inhibitor while Na ϩ /H ϩ exchange activation induced by PMA is inhibited only by 50%. Interestingly, only PMA induces NHE-1 phosphorylation that is inhibited by staurosporine but is not affected by MEK inhibitor (Fig. 8). Taken together, these data support the existence of an additional distinct mechanism for PMA activation of the antiporter. In an earlier study, we investigated the phosphorylation of pleckstrin, a major substrate for PKC in human platelets (26), and a similar pattern of phosphorylation was observed. We propose, as illustrated in Fig. 9, that PKC activated by PMA can lead to Na ϩ /H ϩ exchange activation through two separate mechanisms. One mechanism involves the phosphorylation of the NHE-1 and the other one involves MEK and MAPK activation but does not require the phosphorylation of the NHE-1.
The binding of AVP to the V 1 receptor is known to induce the rapid hydrolysis of inositol phospholipids to generate the intracellular second messengers diacylglycerol, which activates PKC, and inositol 1,4,5-trisphosphate that mobilizes Ca 2ϩ (45). Interestingly, tyrosine phosphorylation induced by neuropeptide hormones, in glomerular mesangial cells, was mediated through PKC-dependent and -independent pathways (46). In addition, PKC was not responsible for the rapid stimulation of p125 FAK tyrosine phosphorylation by bombesin, in Swiss 3T3 cells (47). Furthermore, although PAF-induced activation of 42-kDa MAPK variant in sheep platelets was relatively low, it was achieved through PKC-dependent and independent mechanisms (7).
The AVP-induced stimulation of MAPKs, although mediated through a V 1 receptor (Fig. 5), does not involve activation of PKC since the AVP-stimulated MAPK activity was not inhibited by staurosporine. These results are consistent with our previous study (26) in which NHE activation by AVP through a V 1 receptor was not mediated by PKC. The present study provides evidence that, in human platelets, AVP stimulates the phosphorylation and activation of MAPKs and the activity of the NHE through a signal transduction pathway that is independent of PKC. MAPK-induced activation of the NHE probably does not involve the phosphorylation of the antiporter. Further studies are needed for the discovery of additional elements in the signaling pathways leading to the activation of the NHE by extracellular signals.