Abnormalities in the regulation of blood platelet free cytosolic calcium in malignant hyperthermia. I. Human platelets.

The effect of halothane on the regulation of blood platelet free cytosolic calcium was investigated in Quin-2-loaded cells from patients susceptible to Malignant Hyperthermia (MH) and healthy controls, respectively. The resting level of free cytosolic calcium was slightly, but statistically significantly, enhanced in platelets from patients (90 +/- 10 nM vs 110 +/- 35 nM). Halothane induced a dose-dependent, rapid Ca2+ release from intracellular stores both in normal and in MH derived cells, but the resulting increase in cytosolic calcium was significantly higher in the latter (2 mM halothane: [Ca2+]i = 117 +/- 12 nM vs 218 +/- 117 nM; 4 mM halothane: 225 +/- 35 nM vs. 417 +/- 201 nM). Whereas in platelets from healthy donors a complete reversibility of the halothane effect could be observed within 30-45 min, the cytosolic Ca2+ transients in platelets from patients were different from those in normals either in a higher initial peak or in a diminished decline velocity or in both. The basal Ca2+ permeability of the platelet plasma membrane was very low. Generally, halothane caused a dose-dependent increase in Ca2+ permeability. However, the influx of external calcium was significantly higher in platelets from patients than in controls (2 mM halothane: delta [Ca2+]i = 69 +/- 12 nM vs 135 +/- 63 nM; 4 mM halothane: 127 +/- 33 nM vs. 258 +/- 111 nM). Combining the results, the suggestion can be made that susceptibility to MH is characterized by a generalized membrane defect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular calcium and hormone secretion in clonal AtT-20/D16-16 anterior pituitary cells

Intracellular ionized Ca2+ concentration was measured in clonal mouse anterior pituitary tumor cells with the fluorescent Ca2+ indicator Quin-2. In control physiological solution, free cytoplasmic Ca2+ concentration was found to be 139 +/- 11 nM. Replacement of 50 mM NaCl by 50 mM KCl in the extracellular fluid caused a 29 mV depolarization and a 4.2-fold increase in the concentration of free cytoplasmic Ca2+. Under comparable depolarizing conditions, a specific influx of 2.66 nmole of 45Ca2+ per mg protein was detected 1 min after addition of high K+, accompanied by a marked increase in the initial rate of beta-endorphin secretion. In the absence of external Ca2+, depolarization by K+ produced little or no increase in either intracellular free Ca2+ or hormone release. Incubation of AtT-20/D16-16 cells in the secretagogue norepinephrine led to a depolarization accompanied by an increase in spontaneous action potential frequency and a marked elevation in cytosolic Ca2+ concentration. Exposure of cells to somatostatin, an inhibitor of hormone release, led to only transient decreases in burst frequency and no significant reduction in intracellular Ca2+ levels. These results indicate that in addition to intracellular Ca2+, other factors also control secretory activity in AtT-20/D16-16 anterior pituitary cells.     

Calcium mobilization and catecholamine secretion in adrenal chromaffin cells. A Quin-2 fluorescence study

To better understand the relation between cell calcium and exocytotic secretion, a quantitative dependence of adrenal catecholamine secretion on cytosolic free calcium has been determined for isolated, intact, bovine chromaffin cells, using the fluorescent probe Quin-2. The cells required a threshold of 250-300 nM cytosolic calcium to be reached before detectable secretion occurred and half-maximal secretion occurred near 2 microM cytosolic calcium. Nicotinic receptors mediated an increase of cytosolic calcium from resting levels near 100 nM to levels in the 1-10 microM range within seconds followed by a decay back to resting levels over several minutes. Muscarinic receptors mediated a smaller rise in cytosolic free calcium from 100 to about 200 nM, within seconds. The nicotinic response required extracellular calcium, while the muscarinic response was largely independent of extracellular calcium, suggesting the latter mobilizes intracellular calcium. The acetylcholine-evoked rise in cytosolic calcium decayed by at least two kinetically distinct processes with half-time constants: t1 = 0.6 min and t2 = 3.2 min. Extracellular Na+ deprivation caused a more prolonged elevation of the acetylcholine-evoked calcium transient, suggesting a possible role of Na+/Ca2+ exchange and/or other Na+ -dependent processes in lowering cytosolic calcium following stimulation. The possible perturbing effects of Quin-2 on resting and stimulated cytosolic calcium levels and on secretion were examined and a novel use of Quin-2 to measure membrane calcium flux was demonstrated.     

Involvement of inositol 1,4,5-trisphosphate in Ca2+ influx in thrombin stimulated human platelets

By incubating platelets at low temperature (10 degrees C), the relationship between Ca2+ mobilization and formation of inositol 1,4,5-trisphosphate (IP3) in thrombin stimulated platelets could be precisely investigated. In the presence of 1 mM EGTA, time dependent changes in the intracellular free calcium concentration [( Ca2+]i) were closely related to those in IP3 formation. Time course of the influx of external Ca2+, estimated by delta [Ca2+]i obtained by subtracting [Ca2+]i in the presence of 1 mM EGTA from that in the presence of 1 mM CaCl2 was also very similar to that of IP3 formed. Furthermore, the increase in delta [Ca2+]i was extremely well correlated with the amount of IP3 formed (Y = 49X - 34, r = 0.99). Thus, these data indicate that IP3 might be involved not only in intracellular Ca2+ mobilization but in Ca2+ influx of human platelets stimulated by thrombin.     

Changes in cytoplasmic free calcium caused by halothane. Role of the plasma membrane and intracellular Ca2+ stores

Malignant hyperthermia is a muscle disease characterized by an abnormal response to anaesthetics, stress, and exercise. It is typified by muscle contracture and a dramatic elevation in body temperature. A defect in the regulation of the concentration of cytoplasmic free calcium, [Ca2]i, is thought to underlie this disease, but the actual [Ca2+]i was not measurable until recently. We have shown that the anaesthetic halothane increases [Ca2+]i in isolated lymphocytes from malignant hyperthermia-susceptible humans and pigs but not in the normal counterparts. In this report we extend these observations to a larger number of cases and analyze the molecular mechanisms responsible for the increase. The halothane-mediated rise in [Ca2+]i required external Ca2+ and was prevented by nifedipine, an inhibitor of the voltage-sensitive Ca2+ channels of the cell membrane. In addition, the effect of halothane on the releasable Ca2+ from intracellular stores was determined by measuring the size of the releasable pool before and after addition of the anaesthetic. After addition of halothane, about 73% of this Ca2+ pool was still available for release by the Ca2+ ionophore ionomycin in cells from normal humans and pigs. In contrast, only about 45% of the free Ca2+ in intracellular stores was left after treatment with halothane in cells from malignant hyperthermia-susceptible humans and swine. These results indicate that halothane acts both at the cell membrane and at intracellular organelles, and that this action results in a net increase in [Ca2+]i in malignant hyperthermia, but not in normal cells. The action at the cell membrane appears to be on the voltage-sensitive Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pyrophosphate functions as phosphoryl donor with UDP-glucose-treated mammalian phosphofructokinase

CGP 28392, a novel compound structurally related to the dihydropyridine Ca2+-entry blockers, causes a dose-dependent increase in intracellular free Ca2+ in human platelets, as measured with the Quin-2 Ca2+ indicator, with a semimaximal effective concentration of 2.2 X 10(-7) M. This effect occurs in a concentration range in which CGP 28392 competes for specific [3H]nitrendipine binding in guinea pig heart membranes. It can be inhibited by nitrendipine. The data presented furnish direct evidence of the Ca2+-entry-stimulating properties of CGP 28392 and indicate the presence of dihydropyridine-susceptible structures in human platelets.     

Intracellular Ca2+ mobilization and not calcium influx promotes phorbol ester-stimulated thromboxane A2 synthesis in human platelets.

Phorbol esters, potent activators of protein kinase C (PKC), greatly enhance the release of arachidonic acid and its metabolites (TXA2, HETES, HHT) by Ca2+ ionophores in human platelets. In this paper, we report the relationship between intracellular Ca2+ mobilization and external calcium influx into platelets and the ability of PMA plus A23187 to promote thromboxane A2 (TXA2) synthesis. The enhanced levels of TXA2 due to the synergistic stimulation of the platelets with A23187 and phorbol esters are not affected significantly by the presence of external Ca2+ or the calcium-chelator EGTA. PKC inhibitors, staurosporine and sphingosine, abolished phorbol myristate acetate (PMA) potentiation of TXA2 production which strongly supports the role of PKC in the synergism. Platelet aggregation is more sensitive to PMA and external calcium than TXA2 formation. PMA increased TXA2 production as much as 4-fold at low ionophore concentrations. The A23187-induced rise in [Ca2+]i was reduced by pretreatment of human platelets with phorbol esters, both in the presence and absence of EGTA, and staurosporine reversed this inhibitory effect. These results indicate that the synergistic stimulation of TXA2 production by A23187 and phorbol esters is promoted by intracellular Ca2+ mobilization and not by external calcium influx. Our data also suggest that PKC is involved in the regulation of Ca2+ mobilization from some specific intracellular stores and that PKC may also stimulate the Ca(2+)-dependent phospholipase A2 at suboptimal Ca2+i concentrations.     

Effects of sodium removal on calcium mobilization and dense granule secretion induced by thrombin in human platelets

Removal of extracellular sodium decreased calcium mobilization from intracellular stores induced by thrombin in aspirin-treated human platelets. ATP and serotonin secretion were also significantly reduced. Secretion was positively correlated with calcium mobilization, but the presence or absence of sodium did not modify the slope of the regression line. Half-maximal secretion was reached when [Ca2+]i was increased by about 0.1 microM. Calcium mobilization induced by the divalent cation ionophore ionomycin was not modified by sodium removal. Secretion induced by ionomycin was much smaller than the thrombin-induced one for the same increases of [Ca2+]i. These results suggest that the presence of external sodium is required for normal thrombin-induced calcium release from the intracellular stores and hence for dense granule secretion. However, secretion cannot be only attributed to the increase of cell [Ca2+]i but also to other process(es) which are not affected by external sodium.     

Regulation of free cytosolic Ca2+ in the isolated guinea pig gastric chief cells

Stimulation with COOH-terminal octapeptide of cholecystokinin (CCK8) or carbachol resulted in a rapid increase in Quin-2 fluorescence of isolated guinea pig gastric chief cells, whereas histamine, vasoactive intestinal peptide, secretin or forskolin had no effect. The minimum effective dose of CCK8 or carbachol to elicit the rise in Quin-2 fluorescence was almost similar to that for pepsinogen secretion. Removal of Ca2+ from extracellular medium or Ca2+ channel blockers did not affect CCK8- or carbachol-induced increase in Quin-2 fluorescence. Moreover, following addition of CCK8, carbachol was unable to stimulate a second increase in Quin-2 fluorescence. These results suggest that CCK8 and carbachol share common Ca2+ pools and an increase in free cytosolic Ca2+ concentration may mediate CCK8- or carbachol-induced pepsinogen secretion from gastric chief cells.     

Activation of human platelets by ADP causes a rapid rise in cytosolic free calcium without hydrolysis of phosphatidylinositol-4,5-bisphosphate

Phosphatidylinositol-4,5-bisphosphate decreased 40% within 10 seconds after the addition of thrombin to platelets. This thrombin-induced loss was accompanied by a corresponding increase of inositol phosphates. In contrast, within the first 60 seconds after exposure of platelets to ADP there was no detectable change in the amounts of phosphatidylinositol-4,5-bisphosphate or inositol phosphates. Both thrombin and ADP, however, caused a very rapid rise of cytosolic free calcium, as measured by Quin-2. The magnitude of this rise of calcium was similar for the two agonists. These results suggest that in platelets, agonist stimulation may lead to increased cytosolic free calcium independently of phosphatidylinositol-4,5-bisphosphate degradation.     

Activation of the neutrophil by calcium-mobilizing ligands. I. A chemotactic peptide and the lectin concanavalin A stimulate superoxide anion generation but elicit different calcium movements and phosphoinositide remodeling

The relevance of phosphoinositide remodeling to calcium movements and to the physiological response of superoxide anion (O2-) generation was probed in neutrophils stimulated by the chemotactic peptide fMet-Leu-Phe and the lectin concanavalin A. fMet-Leu-Phe and concanavalin A triggered O2- generation but elicited different patterns of calcium mobilization and phosphoinositide remodeling. fMet-Leu-Phe (10(-7) M) triggered a rise in cytosolic calcium by mobilization of intracellular calcium (fura-2) and increased calcium permeability (45Ca uptake), while concanavalin A (100 micrograms/ml) elicited a rise in cytosolic calcium, primarily by uptake of extracellular calcium (45Ca uptake). fMet-Leu-Phe triggered rapid breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate and phosphatidylinositol, and generation of inositol 1,4,5-trisphosphate (IP3). In contrast concanavalin A triggered breakdown of phosphatidylinositol, but not PIP2, nor was there a significant increase in IP3. However, both fMet-Leu-Phe and concanavalin A triggered a rapid biphasic increase in levels of labeled diacylglycerol (in [3H]arachidonate or [14C]glycerol prelabeled cells) and a 3-fold increase in [32P] phosphatidic acid. These results are concordant with a role for PIP2 breakdown and generation of IP3 specifically in intracellular calcium mobilization but not for other aspects of the signaling pathway for O2- generation. Calcium permeability changes were associated with elevated diacylglycerol and [32P]phosphatidic acid, although a cause and effect relationship is not apparent. Ligands such as concanavalin A enhance cytosolic calcium and trigger generation of O2- without significant PIP2 remodeling; elevated diacylglycerol and cytosolic calcium are the common events associated with ligand-induced O2- generation.     

Platelet aggregation induced by alpha 2-adrenoceptor and protein kinase C activation. A novel synergism.

Adrenaline or UK 14304 (a specific alpha 2-adrenoceptor agonist) and phorbol ester (phorbol 12,13-dibutyrate; PdBu) or bioactive diacylglycerols (sn-1,2-dioctanoylglycerol; DiC8) synergistically induced platelet aggregation and ATP secretion. The effect on aggregation was more pronounced than the effect on secretion, and it was observed in aspirinized, platelet-rich plasma or suspensions of washed aspirinized platelets containing ADP scavengers. No prior shape change was found. In the presence of adrenaline, DiC8 induced reversible aggregation and PdBu evoked irreversible aggregation that correlated with the different kinetics of DiC8- and PdBu-induced protein kinase C activation. Adrenaline and UK 14304 did not induce or enhance phosphorylation induced by DiC8 or PdBu of myosin light chain (20 kDa), the substrate of protein kinase C (47 kDa), or a 38 kDa protein. Immunoprecipitation studies using a Gcommon alpha antiserum or a Gi alpha antiserum showed that Gi alpha is not phosphorylated after exposure of platelets to PdBu or PdBu plus adrenaline. Adrenaline, PdBu or adrenaline plus PdBu did not cause stimulation of phospholipase C as reflected in production of [32P]phosphatidic acid. Adrenaline caused a small increase of Ca2+ in the platelet cytosol of platelets loaded with Indo-1; this effect was also observed in the absence of extracellular Ca2+. However, under conditions of maximal aggregation induced by adrenaline plus PdBu, no increase of cytosolic Ca2+ was observed. Platelet aggregation induced by PdBu plus adrenaline was not inhibited by a high intracellular concentration of the calcium chelator Quin-2. These experiments indicate that alpha 2-adrenoceptor agonists, known to interact with Gi, and protein kinase C activators synergistically induced platelet aggregation through a novel mechanism. The synergism occurs distally to Gi protein activation and protein kinase C-dependent protein phosphorylation and does not involve phospholipase C activation or Ca2+ mobilization.     

The diacylglycerol analogue, 1,2-sn-dioctanoylglycerol, induces an increase in cytosolic free Ca2+ and cytosolic acidification of T lymphocytes through a protein kinase C-independent process.

In this paper, we demonstrate that low concentrations (0.5-2.5 microM) of 1,2-sn-dioctanoylglycerol (DiC8), a potent diacylglycerol used in many previous studies to probe the role of protein kinase C (PKC) in cell activation, cause cytosolic alkalinization of human, mouse and pig T lymphocytes through PKC-mediated activation of the Na+/H+ antiport. However, at higher concentrations (greater than or equal to 12.5 microM), the effect on cytosolic pH (pHi) is reversed, resulting in a marked cytosolic acidification, followed by a gradual return of pHi to baseline values. DiC8 also induces marked changes in cytosolic free calcium concentrations ([Ca2+]i), initially by releasing calcium from intracellular stores, followed by a net transmembrane influx of calcium. The DiC8-induced cytosolic acidification, the resultant return to baseline pH and the increase in [Ca2+]i are independent of activation of PKC. Unlike many other agents which increase [Ca2+]i, DiC8 does not induce phosphatidylinositol hydrolysis with the resultant production of inositol phosphates. Other compounds known to activate PKC, including the closely related diacylglycerol analogues, 1,2-sn-dihexanoylglycerol and 1,2-sn-didecanoylglycerol, phorbol esters and mezerein, did not induce changes in [Ca2+]i or cytosolic acidification in T lymphocytes. Thus the action of DiC8 on intact lymphocytes is different from that of phorbol esters and other diacylglycerols, and is specific to the length of the acyl chains. Because changes in [Ca2+]i are often associated with cell proliferation and cell differentiation, some effects of DiC8 on intact cells may be a consequence of changes in [Ca2+]i.     

Effects of prostaglandins, cAMP, and changes in cytosolic calcium on platelet aggregation induced by a thromboxane A2 mimic (U46619).

The effects of U46619, a thromboxane mimic, on cytosolic Ca2+ concentration and platelet aggregation were determined in human platelets. Cytosolic Ca2+ concentration was determined by Quin-2 fluorescence and platelet aggregation quantitated with an aggregometer. Addition of U46619 (1 x 10(-7) M) to the platelet suspension produced a rapid increase in cytosolic Ca2+ and platelet aggregation. Pretreatment of platelets with EGTA (3 x 10(-3) M), verapamil (5 x 10(-4) M), a calcium entry blocker, or 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (1 x 10(-3) M), an inhibitor of intracellular Ca2+ release, either blunted or markedly delayed the rate, but not the magnitude, of increase in cytosolic Ca2+ and prevented platelet aggregation by U46619. Pretreatment of platelets with prostaglandin I2 (PGI2) (5 x 10(-8) M), PGD2 (5 x 10(-8) M), PGE1 (5 x 10(-8) M), PGF2 alpha (1 x 10(-5) M), dibutyryl cAMP (5 x 10(-3) M), or forskolin (1 x 10(-6) M) prevented both the increase in cytosolic Ca2+ and the associated platelet aggregation induced by U46619. These data suggest that U46619 may induce platelet aggregation through an increase in cytosolic Ca2+, and that both Ca2+ entry and its release from intracellular storage sites probably contribute to the increase in cytosolic Ca2+. Furthermore, the rate of the increase in cytosolic Ca2+ concentration, as well as the magnitude of the increase, appear to be critical for platelet aggregation induced by U46619. Our data are consistent with the hypothesis that PGs inhibit U46619-induced platelet aggregation by preventing the increase in cytosolic Ca2+, and that these effects may be mediated via an increase in cAMP, since they were induced by PGs and cAMP.     

AG555对猪血小板胞浆钙离子浓度的影响

将荧光标记物Ｆｕｒａ２－ＡＭ参入到血小板中,利用荧光分光光度计检测胞浆钙离子浓度的变化来研究ＡＧ５５５（一种合成的酪氨酸蛋白激酶抑制剂）对猪血小板胞浆钙离子浓度的影响．结果发现ＡＧ５５５可降低血小板胞浆钙离子浓度,并对凝血酶诱导的胞浆钙离子浓度的升高有明显的抑制作用．ＡＧ５５５可能对血小板功能有一定的影响,这对于进一步阐明ＡＧ５５５的作用机理将有重要意义．     

Cholera toxin and its B subunit do not change cytosolic free calcium concentration

Using the fluorescent Ca2+ probe Quin-2 it has been reported that cholera toxin (CT) and its B subunit (B-CT) increase cytosolic free Ca2+ concentration ([Ca2+]i) in entherocytes, thymocytes and fibroblasts. In this work we show, however, that the fluorescence increases of Quin-2-loaded cells (rat thymocytes, mouse splenocytes, P-388 macrophages and 3T3 fibroblasts) observed upon addition of CT or B-CT are not caused by an increase in [Ca2+]i. The observed effect appears to be accounted for by EDTA-2Na admixtures (present as conservation agent in all CT and B-CT preparations) which 'unquenches' the fluorescence of Quin-2 acid leaked out from the cells into the extracellular medium and produces influorescent complexes with contaminating heavy metal ions. Thus the mitogenic effect of B-CT is not obviously connected with the cytosolic free Ca2+ increase but is probably due to ganglioside-mediated protein phosphorylation.     

Cytosolic and stored calcium antagonistically control tyrosine phosphorylation of specific platelet proteins.

Depletion of intracellular calcium stores appears to increase plasma membrane permeability for calcium by an as yet obscure mechanism. We found that the Ca2+ ionophore, A23187, and thrombin elevate cytosolic calcium ([Ca2+]i) equally and cause tyrosine phosphorylation of a 130-kDa protein and to a lesser extent 80- and 60-kDa proteins. Chelation of [Ca2+]i by 1,2-bis(2-aminophenoxyethane)-N,N,N',N'-tetraacetic acid/acetomethoxy ester decreased thrombin-induced tyrosine phosphorylation responses. These results suggested that [Ca2+]i elevation promotes tyrosine phosphorylation. Tyrosine phosphorylation persisted in the presence or absence of extracellular calcium after thrombin stimulation but subsided rapidly after A23187 addition if extracellular calcium was present. When Ca2+/ATPase activity, which is apparently required to maintain calcium stores, is inhibited by low temperature, tyrosine phosphorylation of the 130-kDa protein occurs. Rewarming platelets reverses tyrosine phosphorylation only if extracellular calcium is present. Thapsigargin, a calcium ATPase inhibitor, also induces tyrosine phosphorylation of the 130-kDa protein and prevents dephosphorylation of this protein when added prior to rewarming. These observations suggest that homeostatic levels of calcium in storage compartments favor tyrosine dephosphorylation of specific proteins. Thus the levels of [Ca2+]i and stored calcium appear to control tyrosine phosphorylation antagonistically. Tyrosine phosphorylation may play a role in regulating calcium channel function.     

Free calcium concentrations in the nerve endings in the brain of rats with spontaneous hypertension

Fluorescent indicator Quin-2 was used for the determination of free calcium (Ca2+in) in synaptosomes incubated in the normal medium and media where sodium is replaced by potassium or choline. At external calcium concentration of 1 mM, Ca2+in in all three media was 20-30% higher in synaptosomes of spontaneously hypertensive rats (SHR) than in control animals. At external calcium concentration of 5 mM, the increase in Ca2+in values induced by K+-depolarization in sodium- or choline-containing media was 50-80% higher in synaptosomes of SHR. These differences are suggested to be the basis for the mechanism of increased peripheral chain activity in the sympathetic nervous system in primary hypertension.     

Thrombin and C-kinase activators potentiate calcium-stimulated arachidonic acid release in human platelets.

Human platelets were depleted of intracellular Ca2+ and then made selectively permeable to external Ca2+ by addition of the ionophore ionomycin. In this cell system a rapid release of arachidonic acid was seen in direct response to added Ca2+ at concentrations corresponding to cytosolic Ca2+ levels measured in thrombin-stimulated platelets. Thrombin and other activators of Ca2+/phospholipid-dependent protein kinase (C-kinase) potentiated the Ca2+-stimulated arachidonic acid release while exerting little or no effect in the absence of added Ca2+. Agents which increase (R59022) or decrease (isoquinolinesulphonylmethylpiperazine) the activation of C-kinase correspondingly enhanced or inhibited, respectively, the potentiation of arachidonic acid release caused by thrombin. These results support the hypothesis that arachidonic acid release in human platelets is regulated by a co-operative action between intracellular Ca2+ and C-kinase.     

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.