Ca2+-independent activation of Bruton's tyrosine kinase is required for store-mediated Ca2+ entry in human platelets

Store-mediated Ca(2+) entry (SMCE), which is rapidly activated by depletion of the intracellular Ca(2+) stores, is a major mechanism for Ca(2+) influx. Several studies have involved tyrosine kinases in the activation of SMCE, such as pp60(src), although at present those involved in the early activation steps are unknown. Here we report the involvement of Bruton's tyrosine kinase (Btk) in the early stages of SMCE in human platelets. Cell treatment with thrombin or thapsigargin (TG) plus ionomycin (Iono) results in rapid activation of Btk, which was independent of rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) but dependent on H(2)O(2) generation. Platelet treatment with Btk inhibitors, LFM-A13 or terreic acid, significantly reduced TG+Iono- and thrombin-evoked SMCE. Btk was rapidly activated by addition of low concentrations of H(2)O(2), whose effect on Ca(2+) entry was prevented by Btk inhibitors. Our results indicate that pp60(src) and Btk co-immunoprecipitate after platelet stimulation with TG+Iono, thrombin or H(2)O(2). In addition, we have found that LFM-A13 impaired actin filament reorganization after store depletion and agonist-induced activation of pp60(src), while the inhibitor of pp60(src), a protein that requires actin reorganization for its activation, did not modify Btk activation, suggesting that Btk is upstream of pp60(src). We propose a role for Btk in the early steps of activation of SMCE in human platelets.     

Key role of PKC and Ca2+ in EGF protection of microtubules and intestinal barrier against oxidants

Using monolayers of human intestinal (Caco-2) cells, we showed that growth factors (GFs) protect microtubules and barrier integrity against oxidative injury. Studies in nongastrointestinal cell models suggest that protein kinase C (PKC) signaling is key in GF-induced effects and that cytosolic calcium concentration ([Ca2+](i)) is essential in cell integrity. We hypothesized that GF protection involves activating PKC and maintaining normal ([Ca2+](i)) Monolayers were pretreated with epidermal growth factor (EGF) or PKC or Ca2+ modulators before exposure to oxidants (H2O2 or HOCl). Oxidants disrupted microtubules and barrier integrity, and EGF protected from this damage. EGF caused rapid distribution of PKC-alpha, PKC-betaI, and PKC-zeta isoforms to cell membranes, enhancing PKC activity of membrane fractions while reducing PKC activity of cytosolic fractions. EGF enhanced (45)Ca2+ efflux and prevented oxidant-induced (sustained) rises in ([Ca2+](i)). PKC inhibitors abolished and PKC activators mimicked EGF protection. Oxidant damage was mimicked by and potentiated by a Ca2+ ionophore (A-23187), exacerbated by high-Ca2+ media, and prevented by calcium removal or chelation or by Ca2+ channel antagonists. PKC activators mimicked EGF on both (45)Ca2+ efflux and ([Ca2+](i)). Membrane Ca2+-ATPase pump inhibitors prevented protection by EGF or PKC activators. In conclusion, EGF protection of microtubules and the intestinal epithelial barrier requires activation of PKC signal transduction and normalization of ([Ca2+](i)).     

Mechanisms of thrombin-induced MAPK activation associated with cell proliferation in human cultured tracheal smooth muscle cells

The elevated level of thrombin has been detected in the airway fluids of asthmatic patients. However, the implication of thrombin in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study we investigated the effect of thrombin on cell proliferation and p42/p44 mitogen-activated protein kinase (MAPK) activation in human tracheal smooth muscle cells (TSMCs). Thrombin stimulated [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin (PTX) significantly inhibited [3H]thymidine incorporation and phosphorylation of MAPK induced by thrombin. These responses were attenuated by tyrosine kinase inhibitors genistein and herbimycin A, phosphatidyl inositide (PI)-phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor GF109203X, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and PI 3-kinase inhibitors wortmannin and LY294002. In addition, thrombin-induced [3H]-thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. Furthermore, overexpression of dominant negative mutants, RasN17 and Raf-301, significantly suppressed p42/p44 MAPK activation induced by thrombin and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results conclude that the mitogenic effect of thrombin was mediated through the activation of Ras/Raf/MEK/MAPK pathway. Thrombin-mediated MAPK activation was modulated by PI-PLC, Ca(2+), PKC, tyrosine kinase, and PI 3-kinase associated with cell proliferation in cultured human TSMCs.     

Thrombin-stimulated cell proliferation mediated through activation of Ras/Raf/MEK/MAPK pathway in canine cultured tracheal smooth muscle cells.

The elevated level of thrombin has been detected in the airway fluids of asthmatic patients and shown to stimulate cell proliferation in tracheal smooth muscle cells (TSMCs). However, the implication of thrombin in the cell proliferation was not completely understood. In this study, thrombin stimulated [3H]thymidine incorporation and p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation in a time- and concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin (PTX) significantly inhibited [3H]thymidine incorporation and phosphorylation of MAPK induced by thrombin. These responses were attenuated by tyrosine kinase inhibitors genistein and herbimycin A, phosphatidyl inositide (PI)-phospholipase C (PLC) inhibitor U73122, protein kinase C inhibitor GF109203X, removal of Ca2+ by addition of BAPTA/AM plus EGTA, PI 3-kinase inhibitors wortmannin and LY294002, and inhibitor of MEK1/2 PD98059. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 MAPK activation induced by thrombin and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results conclude that the mitogenic effect of thrombin was mediated through the activation of Ras/Raf/MEK/MAPK pathway. Thrombin-mediated MAPK activation was modulated by PI-PLC, Ca2+, PKC, tyrosine kinase, and PI 3-kinase associated with cell proliferation in canine cultured TSMCs.     

The inositol trisphosphate receptor antagonist 2-aminoethoxydiphenylborate (2-APB) blocks Ca2+ entry channels in human platelets: cautions for its use in studying Ca2+ influx.

It has been reported that store-mediated Ca2+ entry (SMCE) in human platelets is likely to be mediated by a secretion-like coupling mechanism. Recently, 2-aminoethoxydiphenylborate (2-APB) has been used in the investigation of SMCE. Here, the mechanism of action of 2-APB is investigated in human platelets. In a Ca2+-free medium (EGTA added), addition of 0.1 U/ml thrombin caused an elevation in [Ca2+]i. Preincubation with 100 microM 2-APB for 170s abolished the release of internal Ca2+. In platelets whose internal Ca2+ stores had been depleted by treatment with 200 nM thapsigargin, addition of extracellular Ca2+ caused an elevation in [Ca2+]i indicative of SMCE. Preincubation with 2-APB decreased SMCE by 95.5+/-1.1%. After activation of SMCE, addition of 2-APB rapidly decreased [Ca2+]i to basal levels; in contrast, the coupling between Trp1 and IP3RII, which has been shown to play an important role in SMCE in platelets, remained intact at the same time points. The rate of decrease of [Ca2+]i and the absence of measurable latency in the effect of 2-APB were comparable to the effects of La3+ (a cation channel blocker). These data suggest that 2-APB may act as a blocker of Ca2+ permeable plasma membrane channels. These data provide further information regarding the mechanism and site of action of 2-APB and highlight the necessity of careful interpretation of work performed using this molecule.     

Regulation of sn-1,2-diacylglycerol second-messenger formation in thrombin-stimulated human platelets. Potentiation by protein kinase C inhibitors.

Stimulation of platelets with thrombin leads to rapid degradation of inositol phospholipids, generation of diacylglycerol (DAG) and subsequent activation of protein kinase C (PKC). Previous studies indicated that prior activation of PKC with phorbol myristate acetate (PMA) desensitizes platelets to thrombin stimulation, as indicated by a decreased production of inositol phosphates and decreased Ca2+ mobilization. This suggests that PKC activation generates negative-feedback signals, which limit the phosphoinositide response. To test this hypothesis further, we examined the effects of PKC activators and inhibitors on thrombin-stimulated DAG mass formation in platelets. Pretreatment with PMA abolishes thrombin-stimulated DAG formation (50% inhibition at 60 nM). Pretreatment of platelets with the PKC inhibitors K252a or staurosporine potentiates DAG production in response to thrombin (3-4-fold) when using concentrations required to inhibit platelet PKC (1-10 microM). K252a does not inhibit phosphorylation of endogenous DAG or phosphorylation of a cell-permeant DAG in unstimulated platelets, indicating that DAG over-production is not due to inhibition of DAG kinase. Sphingosine, a PKC inhibitor with a different mechanism of action, also potentiates DAG formation in response to thrombin. Several lines of evidence indicate that DAG formation under the conditions employed occurs predominantly by phosphoinositide (and not phosphatidylcholine) hydrolysis: (1) PMA alone does not elicit DAG formation, but inhibits agonist-stimulated DAG formation; (2) thrombin-stimulated DAG formation is inhibited by neomycin (1-10 mM) but not by the phosphatidate phosphohydrolase inhibitor propranolol; and (3) no metabolism of radiolabelled phosphatidylcholine was observed upon stimulation by thrombin or PMA. These data provide strong support for a role of PKC in limiting the extent of platelet phosphoinositide hydrolysis.     

Factors affecting dense and alpha-granule secretion from electropermeabilized human platelets: Ca(2+)-independent actions of phorbol ester and GTP gamma S.

Electropermeabilized human platelets containing 5-hydroxy[14C]tryptamine ([14C]5-HT) were suspended in a glutamate medium containing ATP and incubated for 10 min with (in various combinations) Ca2+ buffers, phorbol 12-myristate 13-acetate (PMA), guanine nucleotides, and thrombin. Release of [14C]5-HT and beta-thromboglobulin (beta TG) were used to measure secretion from dense and alpha-granules, respectively. Ca2+ alone induced secretion from both granule types; half-maximal effects were seen at a -log [Ca2+ free] (pCa) of 5.5 and maximal secretion at a pCa of 4.5, when approximately 80% of 5-HT and approximately 50% of beta TG were released. Addition of PMA, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), GTP, or thrombin shifted the Ca2+ dose-response curves for secretion of both 5-HT and beta TG to the left and caused small increases in the maximum secretion observed. These results suggested that secretion from alpha-granules, like that from dense granules, is a Ca(2+)-dependent process stimulated by the sequential activation of a G-protein, phospholipase C, and protein kinase C (PKC). However, high concentrations of PMA and GTP gamma S had distinct effects in the absence of Ca2+ (pCa greater than 9); 100 nM PMA released approximately 20% of platelet 5-HT but little beta TG, whereas 100 microM GTP gamma S stimulated secretion of approximately 25% of each. Simultaneous addition of PMA greatly enhanced these effects of GTP gamma S. Phosphorylation of pleckstrin in permeabilized platelets incubated with [gamma-32P]ATP was used as an index of the activation of PKC during secretion. In the absence of Ca2+, 100 nM PMA caused maximal phosphorylation of pleckstrin and 100 microM GTP gamma S was approximately 50% as effective as PMA; neither GTP gamma S nor Ca2+ enhanced the phosphorylation of pleckstrin caused by 100 nM PMA. These results indicate that, although activation of PKC promoted secretion, GTP gamma S exerted additional stimulatory effects on secretion from both dense and alpha-granules that were not mediated by PKC. Measurement of [3H]inositol phosphate formation in permeabilized platelets containing [3H]phosphoinositides showed that GTP gamma S did not stimulate phosphoinositide-specific phospholipase C in the absence of Ca2+. It follows that in permeabilized platelets, GTP gamma S can both stimulate PKC and enhance secretion via G-protein-linked effectors other than this phospholipase.     

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.     

A role for hTRPC1 and lipid raft domains in store-mediated calcium entry in human platelets

We have previously suggested that store-mediated Ca2+ entry (SMCE) in human platelets may be activated by a secretion-like coupling model, involving de novo coupling of the type II inositol 1,4,5-trisphosphate receptor (IP(3)RII) to the putative Ca2+ entry channel, hTRPC1. In other cells, hTRPC1 has been reported to be associated with cholesterol-rich lipid raft domains (LRDs) in the plasma membrane. Here we have shown that hTRPC1 is largely associated with detergent-resistant platelet membranes, from which it is partially released when the cells are depleted of cholesterol by treatment with methyl-beta-cyclodextrin (MBCD). MBCD treatment inhibited thapsigargin (TG)-evoked SMCE in a concentration-dependent manner, reducing it to 38.1+/-4.1% at a concentration of 10mM. Similarly, the Ca2+ entry evoked by thrombin (1unit/ml) was reduced to 48.2+/-4.5% of control following MBCD (10mM) treatment. Thrombin- and TG-evoked coupling between IP(3)RII and hTRPC1 was also reduced following cholesterol depletion. These results suggest that hTRPC1 is associated with LRDs in human platelets and that these domains are important for its participation in SMCE.     

胞浆Ca~(2+)和某些激酶对NADPH氧化酶激活和肌动蛋白聚合的作用

 为澄清中性粒细胞胞浆 Ca2 +和某些 O-·2 产生相关激酶对 NADPH氧化酶激活和肌动蛋白聚合的作用 ,利用分化为中性粒细胞样的 HL- 60细胞研究了胞浆 Ca2 +螯合剂 BAPTA- AM和激酶抑制剂对这些激酶激活、NADPH氧化酶激活和肌动蛋白聚合的影响 .使用 1 0 μmol/L的 Ca2 +螯合剂 BAPTA- AM去除胞浆 Ca2 +后 ,趋化肽 f MLP诱导的 O-·2 产生明显减少 ,但不影响 f MLP诱导的肌动蛋白聚合 ;8μmol/L的 PKC激酶抑制物 GF1 0 92 0 3x几乎完全抑制 O-·2 产生 ;50 μmol/L的p38激酶抑制物 SB2 0 3580、50 μmol/L的 ERK激酶抑制物 PD0 980 59和 0 .1 μmol/L的 PI3激酶抑制物渥曼青霉素 (Wortmannin)使 f MLP诱导的 O-·2 产生大约减少一半 ;其中 Wortmannin还抑制 f MLP诱导的肌动蛋白聚合 ;f MLP刺激细胞后 ,PI3- K、p38和 ERK激酶迅速激活 ,但这些激酶的激活对 Ca2 +是非必需的 .这些结果说明 Ca2 +依赖途径 (PKC)和 Ca2 +非依赖途径 (PI3- K、p38和ERK)对 NADPH氧化酶激活都起着重要作用 ,而 Ca2 +非依赖途径中的 PI3- K激酶还参与中性粒细胞样 HL- 60细胞的肌动蛋白聚合 .     

Translocation-independent activation of protein kinase C by platelet-activating factor, thrombin and prostacyclin. Lack of correlation with polyphosphoinositide hydrolysis in rabbit platelets.

The relationship between polyphosphoinositide hydrolysis and protein kinase C (PKC) activation was explored in rabbit platelets treated with the agonists platelet-activating factor (PAF), thrombin and 12-O-tetradecanoylphorbol 13-acetate (TPA), and with the anti-aggregant prostacyclin (PGI2). Measurement of the hydrolysis of radiolabelled inositol-containing phospholipids relied upon the separation of the products [3H]inositol mono-, bis- and tris-phosphates by Dowex-1 chromatography. PKC activity, measured in platelet cytosolic and Nonidet-P40-solubilized particulate extracts that were fractionated by MonoQ chromatography, was based upon the ability of the enzyme to phosphorylate either histone H1 in the presence of the activators Ca2+, diacylglycerol and phosphatidylserine, or protamine in the absence of Ca2+ and lipid. Treatment of platelets for 1 min with PAF (2 nM) or thrombin (2 units/ml) led to the rapid hydrolysis of inositol-containing phospholipids, a 2-3-fold stimulation of both cytosolic and particulate-derived PKC activity, and platelet aggregation. Exposure to TPA (200 nM) for 5 min did not stimulate formation of phosphoinositides, but translocated more than 95% of cytosolic PKC into the particulate fraction, and induced a slower rate of aggregation. PGI2 (1 microgram/ml) did not enhance phosphoinositide production, and at higher concentrations (50 micrograms/ml) it antagonized the ability of PAF, but not that of thrombin, to induce inositol phospholipid turnover, even though platelet aggregation in response to both agonists was blocked by PGI2. On the other hand, PGI2 alone also appeared to activate (by 3-5-fold) cytosolic and particulate PKC by a translocation-independent mechanism. The activation of PKC by PGI2 was probably mediated via cyclic AMP (cAMP), as this effect was mimicked by the cAMP analogue 8-chlorophenylthio-cAMP. It is concluded that this novel mechanism of PKC regulation by platelet agonists may operate independently of polyphosphoinositide turnover, and that activation of cAMP-dependent protein kinase represents another route leading to PKC activation.     

Tyrosine phosphorylation of an SH2-containing protein tyrosine phosphatase is coupled to platelet thrombin receptor via a pertussis toxin-sensitive heterotrimeric G-protein.

SH-PTP1 is a protein tyrosine phosphatase (PTP) predominantly expressed in haematopoietic cells and containing two src homology-2 (SH2) domains. Here we report that SH-PTP1 is phosphorylated on both serine and tyrosine residues in response to thrombin or phorbol myristate acetate (PMA), which increased by 60 and 40%, respectively, SH-PTP1 activity. Thrombin-induced phosphorylation of SH-PTP1 is an early signalling event (maximal within 10 s) involving neither integrin signalling, nor calcium, nor release of ADP or thromboxane A2. Moreover, in contrast with PMA, the effect of thrombin on the tyrosine phosphorylation of SH-PTP1 was hardly affected by GF109203X, a specific protein kinase C (PKC) inhibitor. Finally, phosphorylation of SH-PTP1 could be provoked in permeabilized platelets by thrombin or GTP gamma S. This was abolished by pertussis toxin, the specificity of this effect being verified with the megakaryocytic cell line Dami cell. Our data thus identify SH-PTP1 as an in vivo substrate of a putative protein tyrosine kinase linked to the thrombin receptor by a Gi protein. This might offer some clue to unravel the mechanism of thrombin not only in platelets but also in nucleated cells, where its mitogenic effect is known to involve pertussis toxin-sensitive G-proteins, tyrosine phosphorylation and the ras pathway.     

Thapsigargin-stimulated MAP kinase phosphorylation via CRAC channels and PLD activation: inhibitory action of docosahexaenoic acid

This study was conducted on human Jurkat T-cells to investigate the role of depletion of intracellular Ca(2+) stores in the phosphorylation of two mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated kinase (ERK) 1 and ERK2, and their modulation by a polyunsaturated fatty acid, docosahexaenoic acid (DHA). We observed that thapsigargin (TG) stimulated MAPK activation by store-operated calcium (SOC) influx via opening of calcium release-activated calcium (CRAC) channels as tyrphostin-A9, a CRAC channel blocker, and two SOC influx inhibitors, econazole and SKF-96365, diminished the action of the former. TG-stimulated ERK1/ERK2 phosphorylation was also diminished in buffer containing EGTA, a calcium chelator, further suggesting the implication of calcium influx in MAPK activation in these cells. Moreover, TG stimulated the production of diacylglycerol (DAG) by activating phospholipase D (PLD) as propranolol (PROP) (a PLD inhibitor), but not U73122 (a phospholipase C inhibitor), inhibited TG-evoked DAG production in these cells. DAG production and protein kinase C (PKC) activation were involved upstream of MAPK activation as PROP and GF109203X, a PKC inhibitor, abolished the action of TG on ERK1/ERK2 phosphorylation. Furthermore, DHA seems to act by inhibiting PKC activation as this fatty acid diminished TG- and phorbol 12-myristate 13-acetate-induced ERK1/ERK2 phosphorylation in these cells. Together these results suggest that Ca(2+) influx via CRAC channels is implicated in PLD/PKC/MAPK activation which may be a target of physiological agents such as DHA.     

Comparison of the role of protein kinase C in platelet functional responses induced by three different mechanisms, PAF, ionomycin and arachidonic acid.

The role of protein kinase C (PKC) in modulating platelet activation has been examined in platelets pre-incubated with either the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) or the non-specific protein kinase inhibitor, staurosporine. In order to determine where in the signal transduction pathway PKC is exerting its effect platelets were activated either with a receptor-operated stimulus platelet activating factor (PAF) or by direct elevation of [Ca2+]i (ionomycin) or with arachidonic acid which is converted into thromboxane B2 (TxB2). In PAF-stimulated platelets activation of PKC inhibited both [Ca2+]i elevation and TxB2 generation but had no effect on 5-hydroxytryptamine (5-HT) release whilst staurosporine increased the duration of [Ca2+]i elevation and potentiated TxB2 generation but inhibited 5-HT release. In ionomycin-stimulated platelets modulation of PKC had no effect on [Ca2+]i elevation but in contrast to PAF-stimulated platelets PKC activation caused potentiation of TxB2 generation and 5-HT release whilst inhibition of PKC caused inhibition of TxB2 generation and 5-HT release. Modulation of PKC did not affect arachidonic acid-induced TxB2 generation. These findings suggest that in receptor activated platelets endogenously activated PKC is exerting a negative feedback role, however, when [Ca2+]i elevation is not modified by PKC activation or inhibition (such as in ionomycin stimulated platelets) the relationship between the state of PKC activation and subsequent platelet functional responses corresponds more closely. The findings from this study suggest a different relationship between PKC and TxB2 generation than between PKC and dense granule release in PAF-stimulated platelets.     

Calcium mobilization in human platelets by receptor agonists and calcium-ATPase inhibitors.

Inhibitors of the endoplasmic reticulum Ca(2+)-ATPase like thapsigargin (TG) and 2,5-di (tert-butyl)-1,4-benzohydroquinone (tBuBHQ) cause increases in cytosolic calcium in intact human platelets resulting from prevention of reuptake. A maximal concentration of TG (0.2 microM) mobilized 100% of sequestered Ca2+ compared to the action of a receptor agonist like thrombin (0.1 U/ml). A maximal dose of tBuBHQ (50 microM) stimulated release of about 40% of intracellular calcium compared to thrombin and TG. The reduced ability of tBuBHQ to release calcium can be explained with an inhibitory effect on the cyclooxygenase pathway (Ki approximately 7 microM). Therefore tBuBHQ is not able to cause platelet aggregation compared to TG. In the presence of a cyclooxygenase inhibitor or a thromboxane A2 receptor antagonist the action of TG is identical to that observed with tBuBHQ. Generally, inhibition of calcium sequestration does not automatically result in platelet activation. In contrast to a receptor mediated activation Ca(2+)-ATPase inhibitors require the self-amplification mechanism of endogenously formed thromboxane A2 to cause a similar response. We conclude that the calcium store sensitive to Ca(2+)-ATPase inhibitors is a subset of the receptor sensitive calcium pool.     

Thrombin-induced translocation of protein kinase C in human megakaryoblastic leukemia cells (MEG-01)

Our previous immunocytochemical study showed that Ca2+ ionophore-induced translocation of protein kinase C (PKC) in human megakaryoblastic leukemia cells (MEG-01) was potentiated by a synthetic diacylglycerol (T. Ito, T. Tanaka, T. Yoshida, K. Onoda, H. Ohta, M. Hagiwara, Y. Itoh, M. Ogura, H. Saito, and H. Hidaka, 1988, J. Cell Biol. 107, 929). In the present study, we analyzed the roles of the intracellular Ca2+ levels ([Ca2+]i) and diacylglycerol (DG) levels in thrombin-induced translocation of PKC using MEG-01 cells. When the cells were treated with thrombin (0.5 U/ml), PKC was translocated from the cytosol to the plasma membrane after 15 s, and the maximal membrane association was observed after 90 s. The [Ca2+]i of the cells rapidly increased (15 s) and reached a maximum level at 60 s which was sustained for a total of 600 s after thrombin addition. The increase in DG was biphasic with the first phase occurring in the first 15 s and the increase during the second phase lasting less than 600 s. The experiments without extracellular Ca2+ indicated that Ca2+ efflux accompanied by DG in the first phase was sufficient to initiate the membrane association of the PKC and that the large Ca2+ influx enhanced the binding. PKC returned to the cytosol within 600 s despite high levels of both [Ca2+]i and DG. We found that a relatively selective PKC inhibitor, H-7, enhanced thrombin-induced translocation of PKC without modulating [Ca2+]i or DG levels. These results indicate that certain protein phosphorylation events, potentially those mediated by PKC, may be responsible for, at least in part, inhibiting membrane association and further activation of the enzyme.     

Oxidative stress-induced intestinal epithelial cell apoptosis is mediated by p38 MAPK

Free oxygen radicals are involved in the pathogenesis of necrotizing enterocolitis (NEC) in premature infants. The stress-activated p38 mitogen-activated protein kinase (MAPK) has been implicated in gut injury. Here, we found that phosphorylated p38 was detected primarily in the villus tips of normal intestine, whereas it was expressed in the entire mucosa in NEC. H(2)O(2) treatment resulted in a rapid phosphorylation of p38 MAPK and subsequent apoptosis of rat intestinal epithelial (RIE)-1 cells; this induction was attenuated by treatment with SB203580, a selective p38 MAPK inhibitor, or transfection with p38alpha siRNA. Moreover, SB203580 also blocked H(2)O(2)-induced PKC activation. In contrast, the PKC inhibitor (GF109203x) did not affect p38 activation, indicating that p38 MAPK activation occurs upstream of PKC activation in H(2)O(2)-induced apoptosis. H(2)O(2) treatment also decreased mitochondrial membrane potential; pretreatment with SB203580 attenuated this response. Our study demonstrates that the p38 MAPK/PKC pathway plays an important role as a pro-apoptotic cellular signaling during oxidative stress-induced intestinal epithelial cell injury.     

Evidence that cytosolic phospholipase A2 is down-regulated by protein kinase C in intact human platelets stimulated with fluoroaluminate.

We reported that protein kinase C (PKC) inhibitors increase the release of arachidonic acid induced by fluoroaluminate (AlF4-), an unspecific G-protein activator, in intact human platelets. Now we demonstrate that this effect is independent of the extracellular Ca2+ concentration and that AlF4(-)-induced release of AA is abolished by BAPTA, an intracellular Ca2+ chelator, even in the presence of GF 109203X, a specific and potent PKC inhibitor. This compound also blocks the liberation of the secretory phospholipase A2 in the extracellular medium, indicating that this enzyme is not involved in the potentiation of arachidonic acid by PKC inhibitors. On the other hand, the latter effect is completely abolished by treatment of platelets with AACOCF3, a specific inhibitor of cytosolic phospholipase A2 (cPLA2). These observations indicate that cPLA2 is responsible for the AlF4(-)-induced release of arachidonic acid by a mechanism that is down-regulated by PKC.     

Glucose upregulates plasminogen activator inhibitor-1 gene expression in vascular smooth muscle cells

We investigated the effects of high concentrations of glucose on plasminogen activator inhibitor-1 (PAI-1) gene expression in cultured rat vascular smooth muscle cells (VSMC). In response to a high glucose concentration (27.5 mM), PAI-1 mRNA increased within 2 h, peaked at 4 h, remained elevated for another 4 h, then decreased to basal levels at 24 h. On the other hand, mannose at the same concentration (22.5 mM mannose plus 5.5 mM glucose) as an osmotic control had little effect on PAI-1 mRNA expression. The expression of PAI-1 mRNA that was also increased by H(2)O(2), angiotensin II, or phorbol myristate acetate, was reversed by the MAPK kinase (MEK) inhibitor PD98059 or the specific protein kinase C (PKC) inhibitor GF109203X. High glucose appeared to activate MAPK and PKC in VSMC judging from Elk-1 and AP-1 activation, respectively. PD98059 inhibited and GF109203X prevented subsequent PAI-1 induction by glucose. These results suggest that glucose at high concentrations induces PAI-1 gene expression in VSMC at least partially via MAPK and PKC activation. This direct effect of glucose might have important implications for the increased plasma concentrations of PAI-1 and possibly atherosclerosis that are associated with diabetes.     

Dual role of platelet protein kinase C in thrombus formation: stimulation of pro-aggregatory and suppression of procoagulant activity in platelets

Protein kinase C (PKC) isoforms regulate many platelet responses in a still incompletely understood manner. Here we investigated the roles of PKC in the platelet reactions implicated in thrombus formation as follows: secretion aggregate formation and coagulation-stimulating activity, using inhibitors with proven activity in plasma. In human and mouse platelets, PKC regulated aggregation by mediating secretion and contributing to alphaIIbbeta3 activation. Strikingly, PKC suppressed Ca(2+) signal generation and Ca(2+)-dependent exposure of procoagulant phosphatidylserine. Furthermore, under coagulant conditions, PKC suppressed the thrombin-generating capacity of platelets. In flowing human and mouse blood, PKC contributed to platelet adhesion and controlled secretion-dependent thrombus formation, whereas it down-regulated Ca(2+) signaling and procoagulant activity. In murine platelets lacking G(q)alpha, where secretion reactions were reduced in comparison with wild type mice, PKC still positively regulated platelet aggregation and down-regulated procoagulant activity. We conclude that platelet PKC isoforms have a dual controlling role in thrombus formation as follows: (i) by mediating secretion and integrin activation required for platelet aggregation under flow, and (ii) by suppressing Ca(2+)-dependent phosphatidylserine exposure, and consequently thrombin generation and coagulation. This platelet signaling protein is the first one identified to balance the pro-aggregatory and procoagulant functions of thrombi.