Vasopressin elevation of Na+/H+ exchange is inhibited by genistein in human blood platelets.

The regulation of intracellular Na+ and pHi in human blood platelets is known to be controlled by the function of the Na+/H+ exchanger. The phosphorylation state of the Na+/H+ exchanger which determines the exchanger activity in human blood platelets is regulated by the activities of protein kinases and protein phosphatases. Observations in this study indicate that arginine vasopressin (AVP) that interacts with a V1 receptor, activates the Na+/H+ exchange in human blood platelets through a genistein-inhibited mechanism. The AVP-activated Na+/H+ exchange is probably not regulated by protein kinase C (PKC), since this activation is not inhibited by staurosporine. The multiple ways in which platelet Na+/H+ exchange can be modulated may indicate the critical role played by this exchanger in the homeostasis control of pHi in human blood platelets.     

Ligand-affected shift of Na+/H+ exchange pHi set point in human blood platelets, rapidly revealed by a novel approach.

The Na+/H+ exchange time-course of BCECF-loaded human platelets, suspended in isotonic media containing NaCl and sodium propionate and activated by intracellular acidification, was measured spectrofluorimetrically. Sequential alkalinization rates decline exponentially as a function of the changing intracellular pH (pHi) and its linear expression (log rate vs. pHi) extrapolates reproducibly to the pHi set point for the Na+/H+ exchange activation. The set point of control platelets (7.28 +/- 0.01) is shifted rapidly (discernibly less than or equal to 30 s) and markedly to alkaline pHi (7.62 +/- 0.03) by PMA, that activates protein kinase C and is shifted to acidic pHi (7.05 +/- 0.01) by staurosporine, which inhibits protein kinases. The addition of 5-N-(3-aminophenyl)amiloride reveals that the alkalinization measured is predominantly Na+/H+ exchange with only a minute contribution (delta pHi = 0.012 +/- 0.002 in 1 min) of an acid loading component, at pHi greater than 7.2. The results support recent studies concluding that the set point indeed reflects the phosphorylation state of the Na+/H+ exchanger.     

The Na+/H+ exchanger is phosphorylated in human platelets in response to activating agents.

alpha-Thrombin, phorbol esters (PMA) and 1,2-diacylglycerol (DAG), three activators of the amiloride-sensitive Na+/H+ exchange in human platelets, rapidly increase the intracellular pH and the level of phosphorylation of the Na+/H+ exchange protein (NHE1). This stimulatory effect is suppressed by staurosporine, a potent kinase inhibitor, and increased by okadaic acid, a potent inhibitor of phosphatase 1 and 2A. The modulations of NHE1 phosphorylation by these factors correlate well with their effects on platelet pH. Thus, we conclude that in platelets (i) Na+/H+ exchange is mediated by NHE1, and (ii) platelet activating agents stimulate NHE1 via the modulation of the kinase/phosphatase equilibrium.     

WS-1 human fibroblasts contain distinct calcium and protein kinase C-mediated pathways for activation of Na+/H+ exchange: contrasting effects of thrombin and PMA

PMA and thrombin were examined for their ability to activate Na+/H+ exchange in growth-arrested WS-1 human fibroblasts. PMA or thrombin caused a cytoplasmic alkalinization that required extracellular sodium and was sensitive to 1 mM amiloride, suggesting that the rise in pH was mediated by the Na+/H+ exchanger. However, PMA and thrombin activated Na+/H+ exchange by distinctly different mechanisms. The rate of cytoplasmic alkalinization caused by 30 nM PMA was slower than 10 nM thrombin. The PMA-induced pH change was sensitive to the protein kinase inhibitors staurosporine (50 nM) and H-7 (100 microM). No increase in intracellular calcium was observed after PMA treatment and the cytoplasmic alkalinization caused by PMA was not sensitive to the drug TMB8 (200 microM) or the intracellular calcium-chelator BAPTA. In contrast, the thrombin-induced rise in cytoplasmic pH was insensitive to 50 nM staurosporine and only partially reduced with 100 microM H-7. The thrombin-induced activation of Na+/H+ exchange was inhibited by 200 microM TMB8 or pretreatment with BAPTA. PMA caused translocation of PKC activity from a cytoplasmic to membrane fraction whereas thrombin did not. Pretreatment with 50 nM staurosporine significantly reduced measurable PKC activity with or without PMA treatment. PMA and thrombin were also examined for their ability to induce DNA synthesis in growth-arrested WS-1 human fibroblasts. Unlike thrombin, PMA did not stimulate [3H]-thymidine incorporation in cells serum-deprived for 48 hours. In addition, PMA inhibited thrombin-induced DNA synthesis when added at the same time or as late as 10 hours after thrombin addition. Therefore, thrombin and PMA activate Na+/H+ exchange by distinct pathways, but only the thrombin-induced pathway correlates with a mitogenic response.     

The effect of endothelin-1 on Na+/H+ metabolism in vascular smooth muscle cells]

The effects of endothelin on intracellular pH (pHi) were examined in cultured rat vascular smooth muscle cells (VSMC) using the fluorescent probe BCECF. Endothelin induced biphasic changes in pHi: initial decrease followed by a subsequent increase above the basal level due to activation of the Na+/H+ exchange. The elevation of pHi was slow and sustained, but depended on the dose of endothelin: IC50 was about 3 x 10(-8) M. Na+/H+ exchange inhibition by EIPA (10(-7) M) or by equimolar replacement of external Na+ by choline abolished the pHi increase by enhancing the first phase of cytoplasm acidification. Effects of endothelin were compared with the action of protein kinase C (PK-C) activator phorbol 12-13 myristate ester (PMA). PMA induced a monophasic slow and sustained increase in pHi. The treatments of VSMC with H-7 and staurosporine (PK-C) inhibitors prevented the pHi response to endothelin and PMA. These results suggest that protein kinase C may play an important role in mediating the effects of endothelin on Na+/H+ exchange in VSMC.     

Na+/H+ exchange is not necessary for protein kinase C-mediated effects in platelets

The role of Na+/H+ exchange in protein kinase C-mediated effects in platelets was investigated by studying the effect of removal of extracellular Na+ ([Na+]e) on the different responses induced by phorbol 12-myristate 13-acetate (PMA) and 1,2-dioctanoylglycerol (diC8). None of the responses studied, namely, protein phosphorylation, translocation of enzyme activity to the membrane fraction, potentiatory and inhibitory effect on platelet activation ([Ca2+]i, arachidonate and granule release) showed an absolute dependence on [Na+]e. With the exception of dense-granule release, which was clearly potentiated by the removal of [Na+]e and showed a negative correlation with exchanger activity, the other effects of PMA and diC8 were not affected by [Na+]e removal. It is concluded that Na+/H+ exchange is not essential for protein kinase C activation in platelets.     

Sodium fluoride prevents receptor- and protein kinase C-mediated activation of the human platelet Na+/H+ exchanger without inhibiting its basic pHi-regulating activity

When human platelets are stimulated with thrombin or activators of protein kinase C, cytosolic pH (pHi) increases due to activation of Na+/H+ exchange. In order to further elucidate the molecular mechanisms that regulate the exchanger, we used sodium fluoride, which is a known activator of guanine nucleotide-binding proteins (G proteins) in platelets. Although NaF induced the mobilization of Ca2+ from intracellular storage sites in fura2-loaded platelets, it failed to raise pHi as determined from the fluorescence of 2,7-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein-loaded platelets. Furthermore, when thrombin (0.1 unit/ml) or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) had raised pHi from 7.13 +/- 0.05 to 7.35 +/- 0.07 (n = 30), addition of NaF (2.5-10 mM) rapidly restored pHi to values found before stimulation. Conversely, preincubation of platelets with low concentrations of NaF (2.5 mM) completely prevented alkalinization in response to thrombin or TPA. Unlike ethylisopropylamiloride, which completely blocked Na+/H+ exchange, NaF did not prevent the recovery of pHi from an artificial acid load. Hence, the inhibitory action of NaF is restricted to receptor-mediated activation of the antiport. In order to investigate whether the NaF effect was attributable to a G protein, platelets were preincubated with N-ethylmaleimide (50 microM), which is known to inhibit the adenylyl cyclase-inhibitory G protein. N-Ethylmaleimide treatment not only prevented inhibition of adenylyl cyclase by epinephrine but also completely reversed the inhibitory effect of NaF on the Na+/H+ exchanger. Our data suggest the existence of a novel G protein which is activated by fluoride and functions as a negative regulator of the Na+/H+ exchanger in platelets.     

Regulation of the phosphoinositide cycle by Na+/H+ exchange and intracellular pH in human platelets.

We have found that thrombin-induced activation of protein kinase C (PKC) in platelets, measured by phosphorylation of the 47 kDa protein, is synergistically enhanced by the amiloride analogue ethylisopropylamiloride (EIA), a specific inhibitor of Na+/H+ exchange. This EIA effect was further synergistically enhanced by lowering intracellular pH (pHi) with either nigericin or sodium propionate, and reversed by raising pHi with monensin or ammonium chloride. The synergistic enhancement of thrombin-activated PKC by EIA plus nigericin was not observed when PKC was directly activated by phorbol esters. EIA and EIA plus nigericin caused a 3- to 6-fold increase in thrombin-induced diacylglycerol (DAG), but not phosphatidic acid (PA), production. EIA and nigericin also caused a marked increase in thrombin-induced breakdown and inhibition of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2). In summary, we have presented evidence that inhibition of Na+/H+ exchange causes primarily a H(+)-mediated interruption of the phosphoinositide cycle in activated platelets, including the accumulation of DAG associated with the enhancement of PKC activation, the inhibition of conversion of DAG to PA, and increased PIP2 breakdown. These data suggest a model in which Na+/H+ and pHi play an important regulatory role in permitting the phosphoinositide cycle to proceed in thrombin-activated platelets.     

Mode-specific inhibition of sodium-calcium exchange during protein phosphatase blockade

The effects of the protein phosphatase inhibitors calyculin A and okadaic acid on Na(+)/Ca(2+) exchange activity were examined in transfected Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger. Incubating the cells for 5-10 min with 100 nM calyculin A reduced exchange-mediated (45)Ca(2+) uptake or Ba(2+) influx by 50-75%. Half-maximal inhibition of (45)Ca(2+) uptake was observed at 15 nM calyculin A. The nonselective protein kinase inhibitors K252a and staurosporine provided partial protection against the effects of calyculin A. Okadaic acid, another protein phosphatase inhibitor, nearly completely blocked exchange-mediated Ba(2+) influx. Chinese hamster ovary cells expressing a mutant exchanger in which 420 out of 520 amino acid residues were deleted from the central hydrophilic domain of the exchanger remained sensitive to the inhibitory effects of calyculin A and okadaic acid. Surprisingly, Na(o)(+)-dependent Ca(2+) efflux appeared to be only modestly inhibited, if at all, by calyculin A or okadaic acid. We conclude that protein hyperphosphorylation during protein phosphatase blockade selectively inhibits the Ca(2+) influx mode of Na(+)/Ca(2+) exchange, probably by an indirect mechanism that does not involve phosphorylation of the exchanger itself.     

Deficient protein kinase C-dependent Na+/H+ exchanger activity in T cells from bone marrow transplantation recipients

The early Na+/H+ exchanger-mediated alkalinization of intracellular pH (pHi) was analyzed in peripheral blood T cells from 23 bone marrow transplantation (BMT) recipients (17 allogeneic and 6 autologous) and a group of 13 healthy controls, in response to stimulation of protein kinase C (PKC) with a phorbol ester. In parallel we evaluated the proliferative response of peripheral blood T cells to an anti-CD3 mAb in the presence of either IL-2 or PMA. The pHi increase (delta pHi) observed in control samples ranged from 0.14 to 0.23 pH units (X +/- SD = 0.17 +/- 0.03). In 10 allogeneic and four autologous BMT recipients the delta pHi was under the lower limit of the control range (range: 0.01 to 0.09, X +/- SD = 0.05 +/- 0.02), whereas the remaining nine cases responded similarly to control samples (range: 0.14 to 0.24, X +/- SD = 0.17 +/- 0.04). The response of the Na+/H+ antiporter to a PKC-independent osmotic stimulation appeared to be normal, thus indicating that the intrinsic Na+/H+ exchanger activity was unaltered. The anti-CD3 induced proliferative response of the group of samples displaying a suboptimal delta pHi, was significantly lower (p less than 0.01) than that detected in control samples. T cell proliferation in samples from BMT recipients displaying a normal delta pHi was undistinguishable from the control group (p greater than 0.05). Our results provide the first evidence for a defective early metabolic event, closely related to PKC activity, in T cells from BMT recipients displaying a low proliferative response to T cell mitogens.     

Alpha-thrombin, epidermal growth factor, and okadaic acid activate the Na+/H+ exchanger, NHE-1, by phosphorylating a set of common sites.

The ubiquitous and amiloride-sensitive Na+/H+ exchanger (NHE-1), a plasma membrane phosphoglycoprotein that regulates intracellular pH, is rapidly activated by growth factors. We showed previously that epidermal growth factor (EGF), alpha-thrombin, or serum stimulates Na+/H+ exchange activity in growth-arrested Chinese hamster lung fibroblasts (ER22 cells) in a time-dependent manner which correlates with increased phosphorylation of NHE-1 at serine residues (Sardet, C., Counillon, L., Franchi, A., and Pouysségur, J. (1990) Science 247, 723-726). Here we show that the tumor promoter, okadaic acid, a potent in vivo inhibitor of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A), stimulates Na+/H+ exchange in G0-arrested ER22 cells and in exchanger-deficient fibroblasts transfected with the human NHE-1 cDNA. Okadaic acid effects are maximal at 1 microM (EC50 = 500 nM), detected in 2 min, complete within 15-20 min, and are additives when combined with EGF or alpha-thrombin. Parallel to the pHi-induced rise, okadaic acid alone or together with growth factors stimulated the phosphorylation of NHE-1. More importantly tryptic phosphopeptide maps of NHE-1, immunoprecipitated from cells treated with EGF, alpha-thrombin, or okadaic acid, show a common pattern of phosphorylation. This pattern consists of five major 32P-labeled peptides (P1-P5) present in lower amounts in resting cells. One of them, P5, barely detectable in resting cells is increased up to 15-fold in mitogen-stimulated cells. Taken together these results reinforce the notion that phosphorylation of NHE-1 controls the set point value of the exchanger and suggest that: (i) the proximate step in Na+/H+ exchange activation is mediated by as yet unidentified growth factor-activatable serine "NHE-1 kinase(s)" and (ii) this NHE-1 kinase(s), partly active in resting cells, integrate signals from receptor tyrosine kinases and G protein-coupled receptors.     

Epidermal growth factor and cyclic AMP stimulate Na+/H+ exchange in isolated rat hepatocytes

Na+/H+ exchange in acid-loaded isolated hepatocytes was measured using the intracellular pH indicator biscarboxyethyl-carboxyfluorescein (BCECF) to follow intracellular pH (pHi). The rate of amiloride-sensitive Na(+)-dependent recovery from cytoplasmic-acid-loading was found to be increased in cells treated with epidermal growth factor (EGF), 8-(4-chlorophenylthio)adenosine 3',5'-monophosphate (ClPhScAMP) or phorbol 12-myristate 13-acetate (PMA). These three agents increased the rate of Na+/H+ exchange to similar extents and their effects were not additive. The stimulation was shown in all three cases to be due an alkaline shift of 0.1 in the set point pH of the Na+/H+ exchanger. Experiments measuring the uptake of 22Na+ into acid-loaded primary hepatocyte monolayer cultures confirmed these results. EGF, ClPhScAMP and PMA significantly increased the amiloride-inhibitable accumulation of 22Na+, thus providing further evidence that Na+/H+ exchange is stimulated by these effectors.     

The Na+/H+ antiport is activated by serum and phorbol esters in proliferating myoblasts but not in differentiated myotubes. Properties of the activation process

The properties of the Na+/H+ exchange system have been studied with 22Na+ uptake techniques at two stages of muscle development: proliferating myoblasts and differentiated myotubes. The characteristics of the interactions of the exchanger with external H+, with external Na+, and with amiloride or its more potent analogs are the same at both stages of development. Differences between the two stages of development concern: (i) the internal pH (pHi) dependence of the Na+/H+ exchanger, and (ii) the activation of the Na+/H+ exchanger by serum and phorbol ester which is observed in myoblasts but not in myotubes. Properties of the Na+/H+ exchanger in myoblasts after serum activation seem to be identical to those observed in myotubes with or without serum as if myotube formation stabilized a fully activated state of the exchanger. The activation of the myoblast Na+/H+ exchange system by serum is due to a shift of the pHi dependence towards alkaline pHi values and to an increase in the maximal activity of the Na+/H+ exchange system at acidic pH. Phorbol esters which are well-known activators of protein kinase C can only partially mimic the effects of serum on the Na+/H+ exchanger: they produce a shift of the pH dependence, but they do not increase the maximal activity at acidic pH.     

Thrombin activation of the Na+/H+ exchanger in vascular smooth muscle cells. Evidence for a kinase C-independent pathway which is Ca2+-dependent and pertussis toxin-sensitive

The mechanism by which human alpha-thrombin activates the Na+/H+ exchanger was studied in cultured neonatal rat aortic smooth muscle cells. Thrombin (0.4 unit/ml) caused a rapid cell acidification followed by a slow, amiloride-inhibitable alkalinization (0.10-0.14 delta pHi above base line). In protein kinase C down-regulated cells (exposed to phorbol 12-myristate 13-acetate for 24 or 72 h), the delta pHi induced by thrombin was only partially attenuated. This protein kinase C-independent activation of the Na+/H+ exchanger was blocked by pertussis toxin (islet activating protein (IAP)), reducing delta pHi by 50%. IAP did not directly inhibit Na+/H+ exchange activity as assessed by the response to intracellular acid loading. Thrombin also stimulated arachidonic acid release by 2.5 fold and inositol trisphosphate release by 6.2 fold. IAP inhibited both of these activities by 50-60%. Intracellular Ca2+ chelation with 120 microM quin2 prevented the thrombin-induced Ca2+ spike, inhibited thrombin-induced arachidonic acid release by 75%, and inhibited thrombin-induced activation of the Na+/H+ exchanger in protein kinase C-deficient cells by 65%. Increased intracellular [Ca2+] alone was not sufficient to activate the Na+/H+ exchanger, since ionomycin (0.3-1.5 microM) failed to elevate cell pH significantly. 10 microM indomethacin inhibited thrombin-induced delta pHi in both control and protein kinase C down-regulated cells by 30-50%. Thus, thrombin can activate the Na+/H+ exchanger in vascular smooth muscle cells by a Ca2+-dependent, pertussis toxin-sensitive pathway which does not involve protein kinase C.     

Extracellular Na+, but not Na+/H+ exchange, is necessary for receptor-mediated arachidonate release in platelets.

The effect of extracellular Na+ removal and replacement with other cations on receptor-mediated arachidonate release in platelets was studied to investigate the role of Na+/H+ exchange in this process. Replacement with choline+, K+, N-methylglucamine+ (which abolished the thrombin-induced pHi rise) or Li+ (which allowed a normal thrombin-induced pHi rise) significantly decreased arachidonate release in response to all concentrations (threshold to supra-maximal) of thrombin and collagen. This inhibition was not reversed by NH4Cl (10 mM) addition, which raised the pHi in the absence of Na+, but, on the contrary, NH4Cl addition further decreased the extent of thrombin- and collagen-induced arachidonate release, as well as decreasing 'weak'-agonist (ADP, adrenaline)-induced release and granule secretion in platelet-rich plasma. No detectable pHi rises were seen with collagen (1-20 micrograms/ml) and ADP (10 microM) in bis-(carboxyethyl)carboxyfluorescein-loaded platelets. Inhibition of thrombin-induced pHi rises was seen with 0.5-5 microM-5-NN-ethylisopropylamiloride (EIPA), but at these concentrations EIPA had little effect on thrombin-induced arachidonate release. At higher concentrations such as those used in previous studies (20-50 microM), EIPA inhibited aggregation/release induced by collagen and ADP in Na+ buffer as well as in choline+ buffer (where there was no detectable exchanger activity), suggesting that these concentrations of EIPA exert 'non-specific' effects at the membrane level. The results suggest that (i) Na+/H+ exchange and pHi elevations are not only necessary, but are probably inhibitory, to receptor-mediated arachidonate release in platelets, (ii) inhibition of receptor-mediated release in the absence of Na+ is most likely due to the absent Na+ ion itself, and (iii) caution should be exercised in the use of compounds such as EIPA, which, apart from inhibiting the Na+/H+ exchanger, have other undesirable and misleading effects in platelets.     

Sphingosine differentially inhibits activation of the Na+/H+ exchanger by phorbol esters and growth factors

The role of protein kinase C in activation of the plasma membrane Na+/H+ exchanger was studied in cultured vascular smooth muscle cells. The basic lipid, sphingosine, was used to block enzymatic activity of protein kinase C. Na+/H+ exchange was activated by phorbol 12-myristate 13-acetate (PMA), diacylglycerols, platelet-derived growth factor (PDGF), thrombin, or by osmotically-induced cell shrinkage. Intracellular pH and Na+/H+ exchange activity were measured using the intracellular pH indicator, 2',7'-bis(carboxyethyl)-5(6) carboxyfluorescein. Acting alone, both crude sphingosine and pure, synthetic C18 D-(+)-erythro-sphingosine raised pHi in a dose-dependent manner (from 6.95 +/- 0.02 to 7.19 +/- 0.09 over 10 min for 10 microM sphingosine). This alkalinization was not due to Na+/H+ exchange as it was not altered by t-butylamiloride (50 microM) nor by replacement of the assay medium with a Na(+)-free solution. Sphingosine-induced alkalinization did not require protein kinase C activity, since it was fully intact in protein kinase C-depleted cells. It was also not due to a detergent action of sphingosine on the cell membrane, since both ionic and non-ionic detergents caused cell acidification. Rather, alkalinization induced by sphingosine appeared to be due to cellular uptake of NH3 groups since N-acetylsphingosine showed no alkalinization. After the initial cell alkalinization, cellular uptake of [3H]sphingosine continued slowly for up to 24 h. The ability of PMA or dioctanoylglycerol to activate Na+/H+ exchange fell to 20% of control after 24 h of sphingosine exposure. At all times, C11 and N-acetylsphingosine failed to block PMA-induced activation of the exchanger. Activation of the Na+/H+ exchanger by sucrose, which does not depend on protein kinase C activity, was unaffected by sphingosine. Activation of Na+/H+ exchange by thrombin and PDGF was partially inhibited by 30 and 20%, respectively. These data indicate that both thrombin and PDGF activate Na+/H+ exchange by pathway(s) that are primarily independent of protein kinase C.     

Okadaic acid, a phosphatase inhibitor, induces activation and phosphorylation of the Na+/H+ antiport.

We determined the effect of okadaic acid (OA), a potent phosphoprotein phosphatase inhibitor, on the intracellular pH (pHi) of rat thymic lymphocytes and human bladder carcinoma cells. OA induced a rapid and sustained cytosolic alkalinization. This pHi increase was Na(+)-dependent and was inhibited by 5,N-disubstituted analogs of amiloride, indicating mediation by the Na+/H+ antiport. As described for other stimulants, such as mitogens and hypertonic challenge, activation of the antiport by OA is attributable to an upward shift in its pHi dependence. Accordingly, the alkalinization produced by the phosphatase inhibitor was not additive with that induced osmotically. Activation of the antiport by OA was accompanied by a marked increase in phosphoprotein accumulation, revealing the presence of active protein kinases in otherwise unstimulated cells. We considered the possibility that phosphorylation of the antiport itself or of an ancillary protein is responsible for activation of Na+/H+ exchange. Consistent with this notion, the alkalinization induced by OA was absent in ATP depleted cells. More importantly, immunoprecipitation experiments demonstrated increased phosphorylation of the antiport following treatment with OA. We conclude that, upon inhibition of phosphoprotein phosphatase activity, constitutively active kinases induce the activation of Na+/H+ exchange, possibly by direct phosphorylation of the antiport.     

The Na+/H+ exchange system in glial cell lines. Properties and activation by an hyperosmotic shock

The properties of the Na+/H+ exchange system in the glial cell lines C6 and NN were studied from 22Na+ uptake experiments and measurements of the internal pH (pHi) using intracellularly trapped biscarboxyethyl-carboxyfluorescein. In both cell types, the Na+/H+ exchanger is the major mechanism by which cells recover their pHi after an intracellular acidification. The exchanger is inhibited by amiloride and its derivatives. The pharmacological profile (ethylisopropylamiloride greater than amiloride greater than benzamil) is identical for the two cell lines. Both Na+ and Li+ can be exchanged for H+. Increasing the external pH increases the activity of the exchanger in the two cell lines. In NN cells the external pH dependence of the exchanger is independent of the pHi. In contrast, in C6 cells, changing the pHi value from 7.0 to 6.5 produces a pH shift of 0.6 pH units in the external pH dependence of the exchanger in the acidic range. Decreasing pHi activates the Na+/H+ exchanger in both cell lines. Increasing the osmolarity of the external medium with mannitol produces an activation of the exchanger in C6 cells, which leads to a cell alkalinization. Mannitol action on 22Na+ uptake and the pHi were not observed in the presence of amiloride derivatives. Mannitol produces a modification of the properties of interaction of the antiport with both internal and external H+. It shifts the pHi dependence of the system to the alkaline range and the external pH (pHo) dependence to the acidic range. It also suppresses the interdependence of pHi and pHo controls of the exchanger's activity. NN cells that possess an Na+/H+ exchange system with different properties do not respond to mannitol by an increased activity of the Na+/H+ exchanger. The action of mannitol on C6 cells is unlikely to be mediated by an activation of protein kinase C.     

Ca2+ mobilization can occur independent of acceleration of Na+/H+ exchange in thrombin-stimulated human platelets

Intracellular free Ca2+ [( Ca2+]i) and pH (pHi) were measured simultaneously by dual wavelength excitation in thrombin-stimulated human platelets double-labeled with the fluorescent probes fura2 and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein to determine the relationship between changes in [Ca2+]i and pHi, respectively. At 37 degrees C, thrombin (0.5 or 0.1 units/ml) increased [Ca2+]i with no detectable lag period to maximum levels within 13 s followed by a slow return to resting levels. There was a transient decrease in pHi within 9 s that was immediately followed by an alkalinization response, attributable to activation of Na+/H+ exchange, that raised pHi above resting levels within 22 s. At 10-15 degrees C, thrombin-induced changes in [Ca2+]i and pHi were delayed and therefore better resolved, although no differences in the magnitude of changes in [Ca2+]i and pHi were observed. However, the increase in [Ca2+]i had peaked or was declining before the alkalinization response was detected, suggesting that Ca2+ mobilization occurs before activation of Na+/H+ exchange. In platelets preincubated with 5-(N-ethyl-N-isopropyl)amiloride or gel-filtered in Na+-free buffer (Na+ replaced with N-methyl-D-glutamine) to inhibit Na+/H+ exchange, thrombin stimulation caused a rapid, sustained decrease in pHi. Under these conditions there was complete inhibition of the alkalinization response, whereas Ca2+ mobilization was only partially inhibited. Nigericin (a K+/H+ ionophore) caused a rapid acidification of more than 0.3 pH unit that was sustained in the presence of 5-(N-ethyl-N-isopropyl)amiloride. Subsequent stimulation with thrombin resulted in slight inhibition of Ca2+ mobilization. These data show that, in human platelets stimulated with high or low concentrations of thrombin, Ca2+ mobilization can occur without a functional Na+/H+ exchanger and in an acidified cytoplasm. We conclude that Ca2+ mobilization does not require activation of Na+/H+ exchange or preliminary cytoplasmic alkalinization.     

Protein kinase C enhances Ca2+ mobilization in human platelets by activating Na+/H+ exchange

Control of cytoplasmic pH (pHi) by a Na+/H+ antiport appears a general property of most eukaryotic cells. In human platelets activation of the Na+/H+ exchanger enhances Ca2+ mobilization and aggregation induced by low concentrations of thrombin (Siffert, W., and Akkerman, J. W. N. (1987) Nature 325, 456-458). Several observations indicate that the exchanger is regulated by protein kinase C. (i) Inhibitors of protein kinase C (trifluoperazine, sphingosine) inhibit the increase in pHi seen during thrombin stimulation as well as Ca2+ mobilization; artificially increasing pHi by monensin or NH4Cl then restores Ca2+ mobilization. (ii) Direct activation of protein kinase C by 1-oleoyl-2-acetylglycerol initiates an increase in pHi that depends on the presence of extracellular Na+ and is sensitive to inhibition by ethylisopropylamiloride. The pHi sensitivity of thrombin-induced Ca2+ mobilization is particularly evident in the range between pH 6.8 and 7.4 and at low thrombin concentrations, whereas thrombin concentrations of more than 0.2 unit/ml bypass the pH sensitivity. In the absence of thrombin an increase in pHi, either induced artificially (by addition of the ionophores nigericin or monensin) or via activation of protein kinase C (by addition of 1-oleoyl-2-acetylglycerol), does not induce Ca2+ mobilization. We conclude that activation of protein kinase C is essential for Ca2+ mobilization in platelets stimulated by low concentrations of thrombin and that protein kinase C exerts this effect via activation of the Na+/H+ exchanger.