The involvement of Na+/K(+)-ATPase in the development of platelet procoagulant response

In circulation, platelets may come into contact with both exogenous (cardiac glycoside treatment) and endogenously produced inhibitors of Na+/K(+)-ATPase. We examined whether blocking of platelet Na+/K(+)-ATPase by ouabain results in generation of procoagulant activity. It was shown that an in vitro treatment of platelets with ouabain (20-200 microM for 20 to 60 min) is associated with an intracellular accumulation of sodium ([Na+](i)), generation of a weak calcium signal, and expression of procoagulant activity. The ouabain-induced procoagulant response was dose- and time-related, less pronounced than that evoked by collagen and similar to that produced by gramicidin, not affected by EDTA or aspirin, and strongly reduced in the absence of extracellular Na+ or by hyperosmolality. Flow cytometry studies revealed that ouabain treatment results in a unimodal left shift in the forward and side scatter of the entire platelet population indicating morphological changes of the plasma membrane. The shift was dose related, weaker than that evoked by collagen and similar to that produced by gramicidin. Ouabain-treated platelets express phosphatidylserine (PS). The ouabain-evoked PS expression was dose- and time-dependent, weaker than that produced by collagen and similar to that evoked by gramicidin. Electronic cell sizing measurements showed a dose-dependent increase in mean platelet volume upon treatment with ouabain. Hypoosmotically-evoked platelet swelling resulted in the appearance of procoagulant activity. Thromboelastography measurements indicate that, in whole blood, nanomolar (50-1000 nM, 15 min) concentrations of ouabain significantly accelerate the rate of clot formation initiated by contact and high extracellular concentration of calcium. We conclude that inefficiently operating platelet Na+/K(+)-ATPase results in a rise in [Na+](i). An increase in [Na+](i) and the swelling associated with it may produce PS exposure and a rise in membrane curvature leading to the generation of a procoagulant activity.     

Involvement of the Na+/H+ exchanger in membrane phosphatidylserine exposure during human platelet activation

Platelet membrane phosphatidylserine (PS) exposure that regulates the production of thrombin represents an important link between platelet activation and the coagulation cascade. Here, we have evaluated the involvement of the Na+/H+ exchanger (NHE) in this process in human platelets. PS exposure induced in human platelets by thrombin, TRAP, collagen or TRAP+ collagen was abolished in a Na+ -free medium. Inhibition of the Na+/H+ exchanger (NHE) by 5-(N-Ethyl-N-Isopropyl) Amiloride (EIPA) reduced significantly PS exposure, whereas monensin or nigericin, which mimic or cause activation of NHE, respectively, reproduced the agonist effect. These data suggest a role for Na+ influx through NHE activation in the mechanism of PS exposure. This newly identified pathway does not discount a role for Ca2+, whose cytosolic concentration varies together with that of Na+ after agonist stimulation. Ca2+ deprivation from the incubation medium only attenuated PS exposure induced by thrombin, measured from the uptake of FM1-43 (a marker of phospholipid scrambling independent of external Ca2+). Surprisingly, removal of external Ca2+ partially reduced FM1-43 uptake induced by A23187, known as a Ca2+ ionophore. The residual effect can be attributed to an increase in [Na+]i mediated by the ionophore due to a lack of its specificity. Finally, phosphatidylinositol 4,5-bisphosphate (PIP2), previously reported as a target for Ca2+ in the induction of phospholipid scrambling, was involved in PS exposure through a regulation of NHE activity. All these results would indicate that the mechanism that results in PS exposure uses redundant pathways inextricably linked to the physio-pathological requirements of this process.     

The role of Na+/H+ exchanger in serotonin secretion from porcine blood platelets

This study was undertaken to evaluate whether a link exists between the activation of protein kinase C (PKC), operation of Na(+)/H(+) exchanger (NHE), cell swelling and serotonin (5-HT) secretion in porcine platelets. Activation of platelets by thrombin or phorbol 12-myristate 13-acetate (PMA), a PKC activator, initiated a rapid rise in the activity of Na(+)/H(+) exchanger and secretion of 5-HT. Both thrombin- and PMA-evoked activation of Na(+)/H(+) exchanger was less pronounced in the presence of ethyl-isopropyl-amiloride (EIPA), an NHE inhibitor, and by GF 109203X, a PKC inhibitor. Monensin (simulating the action of NHE) caused a dose-dependent release of 5-HT that was not abolished by GF 109203X or EGTA. Lack of Na(+) in the suspending medium reduced thrombin-, PMA-, and monensin-evoked 5-HT secretion. GF 109203X nearly completely inhibited 5-HT release induced by PMA-, partly that induced by thrombin, and had no effect on 5-HT release induced by monensin. EIPA partly inhibited 5-HT release induced by thrombin and nearly totally that evoked by PMA. Electronic cell sizing measurements showed an increase in mean platelet volume upon treatment of cells with monensin, PMA or thrombin. The PMA- and thrombin-evoked rise in mean platelet volume was strongly reduced in the presence of EIPA. As judged by optical swelling assay monensin and PMA produced a rapid rise in platelet volume. The swelling elicited by PMA was inhibited by EIPA and its kinetics was similar to that observed in the presence of monensin. Hypoosmotically evoked platelet swelling did not affect platelet aggregation but significantly potentiated thrombin-evoked release of 5-HT and ATP. Taken together, these results show that in porcine platelets PKC may promote 5-HT secretion through the activation of NHE. It is hypothesized that enhanced Na(+)/H(+) antiport may result in a rise in cell membrane tension (due to cell swelling) which in turn facilitates fusion of secretory granules with the plasma membrane leading to 5-HT secretion.     

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.     

Stimulation of human platelets by collagen occurs by a Na+/H+ exchanger independent mechanism

In stimulated human platelets dense-granule secretion in response to the 'weak agonists' ADP, adrenaline, platelet activating factor and low concentrations of thrombin as well as Ca2+ mobilisation in response to thrombin are enhanced by a Na+/H+ exchanger. In the present study the role of this antiport in collagen stimulated human platelets was examined. While stimulation of platelets loaded with the fluorescent intracellular pH-sensitive dye, bis-carboxyethyl-5-(6)-carboxyfluorescein (BCECF) with thrombin resulted in the activation of the Na+/H+ exchanger, activation of this antiport did not occur in collagen-stimulated platelets. The lack of antiport activity in response to collagen using BCECF-loaded platelets correlated with the lack of any functional role of the antiport in collagen stimulated platelets. In the presence of a Na+/H+ exchange inhibitor, ethylisopropylamiloride, neither collagen-induced platelet aggregation or dense-granule secretion was affected. Furthermore, while the removal of extracellular Na+ (Na+ext), a condition that also prevents activation of the antiport, inhibited dense-granule secretion in response to a low concentration of thrombin, collagen-induced secretion was potentiated. This potentiatory effect could not be attributed to changes in either the membrane potential or in collagen-induced phospholipase C or protein kinase C activity. The present results indicate that in contrast to the 'weak agonists' (1) collagen-induced platelet activation does not require activation of the Na+/H+ exchanger and (2) Na+ext per se is an inhibitor of collagen-induced secretion.     

Activation of sodium-proton exchange is not a prerequisite for Ca2+ mobilization and aggregation in human platelets

Recently it has been suggested [(1987) Nature 325, 456-458; (1987) FEBS Lett. 212, 123-126] that the activation of Na+/H+ exchange is a prerequisite for platelet aggregation and the development of the Ca2+ signal. As direct evidence for the role of the Na+/H+-exchange pathway the inhibition of the Ca2+ signal by EIPA, a specific inhibitor of Na+/H+ exchange, was offered. Here we demonstrate that low concentrations of EIPA (below 1 microM) completely block Na+/H+ exchange while EIPA inhibits aggregation or Ca2+ mobilization only in concentrations 100-times greater than 1 microM. Moreover, another amiloride analogue, CBDMB, developed to act predominantly on Na+/Ca2+ exchange, does not affect Na+/H+ exchange in platelets but blocks aggregation and Ca2+ mobilization. We conclude that while Na+/H+ exchange has a fundamental role in platelet functions it is not prerequisite for the development of Ca2+ signal and aggregation.     

Na+/H+ exchange modulates Ca2+ mobilization in human platelets stimulated by ADP and the thromboxane mimetic U 46619

According to recent observations ADP stimulates platelets via activation of Na+/H+ exchange which increases cytosolic pH (pHi). This event initiates formation of thromboxane A2 (via phospholipase A2) and, thereafter, inositol 1,4,5-trisphosphate (via phospholipase C) which is known to mobilize Ca2+ from intracellular storage sites. We investigated changes in pHi and cytosolic free Ca2+, [Ca2+]i, activating platelets with ADP and the thromboxane mimetic U 46619. We found that ADP (5 microM) increased pHi from 7.15 +/- 0.08 to 7.35 +/- 0.04 (n = 8) in 2'-7'-bis-(carboxyethyl)-5,6-carboxyfluorescein-loaded platelets, whereas thromboxane A2 formation was inhibited by indomethacin. ADP also induced a dose-dependent Ca2+ mobilization in fura2-loaded platelets which again was not affected by indomethacin. [Ca2+]i increased by 54 +/- 10 nM (n = 8) at 1 microM and by 170 +/- 40 nM (n = 7) at 10 microM ADP above the resting value of 76 +/- 12 nM (n = 47). Inhibition of Na+/H+ exchange by ethylisopropylamiloride (EIPA) reduced ADP-induced Ca2+ mobilization by more than 65% in indomethacin-treated platelets. This inhibition could be completely overcome by artificially raising pHi using either NH4Cl or the Na+/H+ ionophore monensin. We found that U 46619 increased pHi by 0.18 +/- 0.05 at 0.1 microM and by 0.29 +/- 0.07 (n = 7) at 1.0 microM above the resting value via an EIPA-sensitive mechanism. In conflict with the proposed role of the Na+/H+ exchange we found that U 46619 raised [Ca2+]i via a mechanism that for more than 50% depended on intact Na+/H+ exchange. Again, artificially elevating pHi restored U 46619-induced Ca2+ mobilization despite the presence of EIPA. Thus, our data show that Na+/H+ exchange is a common step in platelet activation by prostaglandin endoperoxides/thromboxane A2 and ADP and enhances Ca2+ mobilization independently of phospholipase A2 activity.     

Influence of sodium on the alpha 2-adrenergic receptor system of human platelets. Role for intraplatelet sodium in receptor binding

The affinity of many types of membrane receptors for agonists is decreased by Na+ in radioligand binding experiments. We studied the alpha 2-adrenergic receptor of human platelets to determine whether Na+ acts at an intracellular or extracellular location. The Na+ content of intact platelets in an isotonic saline buffer was 38 nmol/10(8) platelets. This increased to 138 nmol/10(8) platelets with the Na+-selective ionophore monensin and decreased to 13 nmol/10(8) platelets with incubation in a Na+-free buffer. Epinephrine-induced platelet aggregation was increased by the addition of monensin and was decreased in the Na+-free buffer, while thrombin-induced aggregation was unaltered by either condition. Monensin, gramicidin, and ouabain (which all increased intraplatelet Na+) caused a 2-3-fold increase in the Kd of epinephrine (in competition with [3H]yohimbine) for alpha 2-adrenergic receptors on intact platelets. Conversely, incubation in a Na+-free buffer (which decreased intraplatelet Na+) decreased the Kd of the receptors for epinephrine 2-3-fold. These experiments suggest that changes in intracellular Na+ alter epinephrine binding. Control studies eliminated several alternative explanations for the effect of monensin on epinephrine binding: 1) monensin altered epinephrine binding only with intact platelets and not with platelet membranes; 2) although monensin depolarized platelets (assessed by [3H]methyltriphenylphosphonium uptake), other depolarizing conditions did not change epinephrine binding; 3) although monensin may increase intracellular pH (by exchanging Na+ for H+) such an increase in pH decreased the Kd of alpha 2-receptors on platelet membranes for epinephrine, an effect opposite to that produced by monensin in intact platelets. We conclude that alterations in the intracellular concentration of Na+ may change the affinity of platelet alpha 2-receptors for epinephrine. These results suggest a key role for intracellular Na+ in modulating binding at cell surface receptors in vivo.     

Role of Na+/H+ exchange in thrombin- and arachidonic acid-induced Ca2+ influx in platelets

Platelet activation is accompanied by an increase of cytosolic free Ca2+ concentration, [Ca2+]i, (due to both extracellular Ca2+ influx and Ca2+ movements from the dense tubular system) and an Na+ influx associated with H+ extrusion. The latter event is attributable to the activation of Na+/H+ exchange, which requires Na+ in the extracellular medium and is inhibited by amiloride and its analogs. The present study was carried out to determine whether a link exists between Ca2+ transients (measured by the quin2 method and the 45CaCl2 technique) and Na+/H+ exchange activation (studied with the pH-sensitive intracellular probe, 6-carboxyfluorescein) during platelet stimulation. Washed human platelets, stimulated with thrombin and arachidonic acid, showed: (1) a large and rapid [Ca2+]i rise, mostly due to a Ca2+ influx through the plasma membrane; (2) a marked intracellular alkalinization. Both phenomena were markedly inhibited in the absence of extracellular Na+ or in the presence of an amiloride analog (EIPA). Monensin, a cation exchanger which elicits Na+ influx and alkalinization, and NH4Cl, which induces alkalinization only, were able to evoke an increase in [Ca2+]i, mostly as an influx from the extracellular medium. Our results suggest that Ca2+ influx induced by thrombin and arachidonic acid in human platelets is strictly dependent on Na+/H+-exchange activation.     

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.     

Mechanism of collagen activation in human platelets

The mechanism of collagen-induced human platelet activation was examined using Ca2+, Na+, and the pH-sensitive fluorescent dyes calcium green/fura red, sodium-binding benzofuran isophthalate, and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Administration of a moderate dose of collagen (10 microg/ml) to human platelets resulted in an increase in [Ca2+](i) and platelet aggregation. The majority of this increase in [Ca2+](i) resulted from the influx of calcium from the extracellular milieu via the Na+/Ca2+ exchanger (NCX) functioning in the reverse mode and was reduced in a dose-dependent manner by the NCX inhibitors 5-(4-chlorobenzyl)-2',4'-dimethylbenzamil (KD(50) = 4.7 +/- 1.1 microm) and KB-R7943 (KD(50) = 35.1 +/- 4.8 microm). Collagen-induced platelet aggregation was dependent on an increase in [Ca2+](i) and could be inhibited by chelation of intra- and extracellular calcium through the administration of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) and EGTA, respectively, or via the administration of BAPTA-AM to platelets suspended in no-Na+/HEPES buffer. Collagen induced an increase in [Ca2+](i) (23.2 +/- 7.6 mm) via the actions of thromboxane A(2) and, to a lesser extent, of the Na+/H+ exchanger. This study demonstrates that the collagen-induced increase in [Ca2+](i) is dependent on the concentration of Na+ in the extracellular milieu, indicating that the collagen-induced increase in [Ca2+](i) causes the reversal of the NCX, ultimately resulting in an increase in [Ca2+](i) and platelet aggregation.     

Role of Na+/H+ exchange in the granulocyte-macrophage colony-stimulating factor-dependent growth of a leukemic cell line

The growth of the human leukemia cell line AML-193 in a serum-free medium is strictly dependent on the presence of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), which is one of the major regulators of the myelomonocytic lineage. At present, little is known about the mechanisms by which this growth factor transduces the signal intracellularly. The results of this study demonstrate that GM-CSF needs the operation of a Na+/H+ exchanger, which is located in the plasma membrane of almost every vertebrate cell. In fact, the GM-CSF-dependent proliferation of AML-193 cells is strongly reduced in the presence of the amiloride analog EIPA, a specific inhibitor of the Na+/H+ exchanger. When acidified, AML-193 cells are able to recover the original pHi in a Na(+)-dependent and EIPA-inhibitable way; this demonstrates for the first time the presence of the Na+/H+ exchanger in these cells. Finally, GM-CSF, at doses superimposable to those needed for triggering proliferation, induces in AML-193 cells a sustained alkalinization, which is dependent on a operating Na+/H+ exchange, as it is inhibited by EIPA. These results suggest that GM-CSF, like other growth factors in other cell systems, exerts its mitogenic activity in AML-193 cells by inducing a Na+/H+ exchanger-mediated rise in pHi.     

Low density lipoproteins inhibit the Na+/H+ antiport in human platelets via activation of p38MAP kinase

Low density lipoproteins (LDL) inhibit the Na+/H+ antiport and thereby sensitize platelet towards agonist. However, mechanisms underlying the suppressing effect of LDL on Na+/H+ exchange are unclear. We here show that the lowering of intracellular pH and the suppression of the sodium propionate-induced Na+/H+ exchange in the presence of LDL are abolished by SKF86002, a selective inhibitor of p38MAP kinase (p38MAPK). The inhibitory effect of LDL on Na+/H+ exchange was mimicked by H2O2, which directly activates p38MAPK. Exposure of platelets to LDL or H2O2 led to phosphorylation of p38MAPK, its upstream regulator MAP kinase kinase 3/6 (MKK 3/6), and its downstream target heat shock protein 27 (HSP27), and this effect was abrogated in SKF86002-pretreated platelets. In addition, both LDL and H2O2 produced the SKF86002-sensitive phosphorylation of an oligopeptide encompassing p38MAPK phosphorylation sequence derived from NHE-1, a major Na+/H+ exchanger in platelets. We further show that the sensitizing effects of LDL on the thrombin-induced platelet activation, as reflected by aggregation and granule secretion, are abolished in cells pretreated with SKF86002. We conclude that activation of p38MAPK is required for the inhibitory effect of LDL on Na+/H+ antiport and thereby for LDL-dependent sensitization in human platelets.     

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.     

Regulation of intracellular pH in human platelets. Effects of thrombin, A23187, and ionomycin and evidence for activation of Na+/H+ exchange and its inhibition by amiloride analogs

Intracellular pH (pHi) of human platelets was measured with the fluorescent dye 2',7'-bis(carboxyethyl)5,6-carboxyfluorescein under various conditions. Stimulation by thrombin at 23 degrees C caused a biphasic change in pHi (initial pHi 7.09); a rapid fall of 0.01-0.04 units (correlated with the rise of [Ca2+]i measured with quin2) followed after 10-15 s by a sustained rise of 0.1-0.15 units pHi. The fall of pHi and [Ca2+]i mobilization was reduced by early (5 s) addition of hirudin, but the later elevated pHi was not reversed by hirudin added after 30 s, although this strips thrombin from receptors and rapidly returns [Ca2+]i to basal levels. In Na+-free medium, or in presence of the Na+/H+ antiport inhibitors, 5-(N,N-dimethyl)amiloride (DMA) or 5-(N-ethyl-N-isopropyl)amiloride (EIPA), thrombin caused a greater fall of pHi (0.22-0.26 units) that was sustained. DMA or EIPA could also reverse the alkalinization response to thrombin. Ca2+ ionophores (ionomycin, A23187) decreased platelet pHi by 0.02-0.15 units, but without an increase of pHi comparable to that following thrombin; DMA and EIPA enhanced the fall of pHi (0.14-0.33 units). Cytoplasmic acidification produced by nigericin (K+/H+ ionophore) was followed by return towards normal that was abolished by Na+/H+ antiport inhibitors. The phorbol diester phorbol 12-myristate 13-acetate had little effect on resting pHi but increased the rate of recovery 2-3-fold after cytoplasmic acidification by nigericin, ionomycin, or sodium propionate. These results indicate that elevation of [Ca2+]i by thrombin enhances H+ production, but the subsequent alkalinization is independent of receptor occupancy or elevated [Ca2+]i and stimulation of the Na+/H+ antiporter by thrombin probably involves some mechanism apart from regulation by H+ and protein kinase C.     

Na+/H+ exchange and aggregation of human platelets activated by ADP: the exchange is not required for aggregation

Isolated human blood platelets, loaded with the pH-sensitive fluorescence dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein show cytoplasmic alkalinization upon stimulation with thrombin but acidification with ADP stimulation. In both cases a Na+/H+ exchange is activated. This can be revealed by the sensitivity of the induced pH changes to amiloride and to 5-N-(3-aminophenyl)amiloride (APA), known inhibitors of the Na+/H+ exchanger, and by a dependence on sodium in the external medium. ADP-induced platelet aggregation is not affected by omission of sodium from the external medium. Furthermore, aggregation is barely inhibited (less than 10%) by amiloride or APA at concentrations up to 50 microM while the Ki values in affecting the Na+/H+ exchange are 5.9 and 1.6 microM for amiloride and APA, respectively. Platelet aggregation is inhibited by amiloride or APA at concentrations higher than 50 microM, but this inhibition is apparently due to a secondary effect of the agents. It is concluded that platelet aggregation induced by ADP is not dependent on activation of Na+/H+ exchange.     

Inhibition of Na+/H+ exchange reduces Ca2+ mobilization without affecting the initial cleavage of phosphatidylinositol 4,5-bisphosphate in thrombin-stimulated platelets

Stimulation of human platelets increases cytoplasmic pH (pHi) via activation of Na+/H+ exchange. We have determined the effect of inhibiting Na+/H+ exchange on (i) thrombin-induced Ca2+ mobilization and (ii) turnover of 32P-labelled phospholipids. Blocking Na+/H+ exchange by removal of extracellular Na+ or by ethylisopropylamiloride (EIPA) inhibited Ca2+ mobilization induced by 0.2 U/ml thrombin, whereas increasing pHi by NH4Cl enhanced the thrombin-induced increase in cytosolic free Ca2+. The effect of EIPA was bypassed after increasing pHi by moneasin. The thrombin-induced cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) was unaffected by treatments that blocked Na+/H+ exchange or increased pHi. It is concluded that activation of Na+/H+ exchange is a prerequisite for Ca2+ mobilization in human platelets but not for the stimulus-induced hydrolysis of PIP2.     

Cytosolic alkalinization alone is not sufficient for Ca2+ mobilization, phosphatidic acid formation, and protein phosphorylation in human platelets

One of the earliest events following stimulation of human platelets with thrombin is a rise in the cytosolic pH, pHi, mediated by Na+/H+ exchange, and an increase in the cytosolic free Ca2+ concentration, [Ca2+]i. In the present study we investigated whether an increase in pHi alone, induced by the Na+/H+ ionophore monensin, is sufficient for platelet activation. Although monensin (20 microM) raised pHi from 7.10 +/- 0.05 (n = 21) to 7.72 +/- 0.17 (n = 13), neither Ca2+ influx nor mobilization were detectable upon this treatment in fura2-loaded platelets. In contrast, thrombin (0.05 U/ml) raised pHi to 7.31 +/- 0.10 (n = 10) and increased [Ca2+]i by more than 250 nM both in the presence and absence of extracellular Ca2+. Thrombin also caused the formation of phosphatidic acid and phosphorylation of the 20 kDa and 47 kDa proteins in platelets labeled with 32P. Monensin, however, induced none of these responses. It is concluded that an increase in pHi alone is not a sufficient trigger for platelet activation but enhances intracellular signal transduction in platelets stimulated by natural agonists.     

Relative contribution of V-H+ATPase and NA+/H+ exchanger to bicarbonate reabsorption in proximal convoluted tubules of old rats

With aging, the kidney develops a progressive deterioration of several structures and functions. Proximal tubular acidification is impaired in old rats with a decrease in the activity of brush border Na+/H+ exchange and a fall of H-ion flux measured with micropuncture experiments. In the present work we evaluate the contribution of 5-N-ethyl-n-isopropyl amiloride- (EIPA) and bafilomycin-sensitive bicarbonate flux (JHCO3-) in proximal convoluted tubules of young and aged rats. We performed micropuncture experiments inhibiting the Na+/H+ exchanger with EIPA (10(-4) M) and the V-H+ATPase with bafilomycin (10(-6) M). We used antibodies against the NHE3 isoform of the Na+/H+ exchanger and the subunit E of the V-H+ATPase for detecting by Western blot the abundance of these proteins in brush border membrane vesicles from proximal convoluted tubules of young and old rats. The abundance of NHE3 and the V-H+ATPase was similar in 18-month-old and 3-month-old rats. The bicarbonate flux in old rats was 30% lower than in young rats. EIPA reduced by 60% and bafilomycin by 30% in young rats; in contrast, EIPA reduced by approximately 40% and bafilomycin by approximately 50% in old rats. The inhibited by bafilomycin was the same in young and old rats: 0.62 nmol.cm-2.s-1 and 0.71 nmol.cm-2.s-1, respectively. However, the EIPA-sensitive fraction was larger in young than in old rats: 1.26 nmol.cm-2.s-1 vs. 0.85 nmol.cm-2.s-1, respectively. These results suggest that the component more affected in bicarbonate reabsorption of proximal convoluted tubules from aged rats is the Na+-H+ exchanger, probably a NHE isoform different from NHE3.     

Inhibition and labeling of the rat renal Na+/H+-exchanger by an antagonist of muscarinic acetylcholine receptors

A covalently binding label for muscarinic acetylcholine receptors, propylbenzilylcholine mustard (PrBCM), irreversibly inhibits the Na+/H+ exchanger in rat renal brush-border membrane vesicles. Substrates of the antiporter, Na+ and Li+, as well as inhibitors, amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and propranolol, protect the antiporter from inactivation by PrBCM. With [3H]PrBCM a band with an app. Mr of 65 kDa is predominantly labeled. Amiloride protects this band from labeling with [3H]PrBCM and [14C]-N,N'-dicyclohexylcarbodiimide (DCCD) proving its identity with the renal Na+/H+ exchanger. Our data reveal a specific interaction of PrBCM with the Na+/H+ exchanger and suggest structural relations between antiporter and receptors.