溶血性磷脂酰胆碱诱导血小板活化及蛋白质酪氨酸激酶和蛋白激酶C的作用

应用双道血小板聚集仪和荧光分光光度计分别测定溶血性磷脂酰胆碱(LysoPC)诱导的兔洗涤血小板聚集,细胞内钙离子浓度(［Ca²⁺］_i),细胞内pH值(pH_i)的变化及5-羟色胺(5-HT)的释放,并观察蛋白质酪氨酸激酶(PTK)抑制剂金雀异黄素(Gen)和蛋白激酶C(PKC)抑制剂星形孢菌素(Sta)对其作用的影响. 结果表明：LysoPC(30-300 μmol·L^-1)诱导血小板聚集,5- HT释放和［Ca²⁺］_i 升高,均呈现良好的浓度依赖性,100 μmol·L^-1以上时伴有pH_i的增加;Gen使LysoPC 诱导的聚集浓度效应曲线右移,半效聚集浓度值从(100±23) μmol·L^-1增加到(200±42) μmol·L^-1,明显抑制 ［Ca²⁺］_i 升高和胞浆碱化,对5-HT释放无明显影响;Sta对较低浓度LysoPC 诱导的血小板聚集,5-HT释放, ［Ca²⁺］_i 和pH_i的增加均有明显抑制作用,但对高浓度LysoPC 诱导的血小板活化无明显影响. 提示：LysoPC通过内Ca²⁺调节和Na^＋/H^＋交换介导血小板聚集和致密颗粒释放;PTK的激活参与了LysoPC诱导的血小板聚集,内Ca²⁺调节和Na^＋/H^＋交换,而在5-HT释放的机理中意义不大;PKC的激活在较低浓度LysoPC 诱导的血小板活化中起重要作用,高浓度LysoPC通过不依赖于PKC的途径激活血小板.     

Protein kinase C regulation of 12-lipoxygenase-mediated human platelet activation

Platelet activation is important in the regulation of hemostasis and thrombosis. Uncontrolled activation of platelets may lead to arterial thrombosis, which is a major cause of myocardial infarction and stroke. After activation, metabolism of arachidonic acid (AA) by 12-lipoxygenase (12-LOX) may play a significant role in regulating the degree and stability of platelet activation because inhibition of 12-LOX significantly attenuates platelet aggregation in response to various agonists. Protein kinase C (PKC) activation is also known to be an important regulator of platelet activity. Using a newly developed selective inhibitor for 12-LOX and a pan-PKC inhibitor, we investigated the role of PKC in 12-LOX-mediated regulation of agonist signaling in the platelet. To determine the role of PKC within the 12-LOX pathway, a number of biochemical endpoints were measured, including platelet aggregation, calcium mobilization, and integrin activation. Inhibition of 12-LOX or PKC resulted in inhibition of dense granule secretion and attenuation of both aggregation and αIIbβ(3) activation. However, activation of PKC downstream of 12-LOX inhibition rescued agonist-induced aggregation and integrin activation. Furthermore, inhibition of 12-LOX had no effect on PKC-mediated aggregation, indicating that 12-LOX is upstream of PKC. These studies support an essential role for PKC downstream of 12-LOX activation in human platelets and suggest 12-LOX as a possible target for antiplatelet therapy.     

Okadaic acid inhibits activation of phospholipase C in human platelets by mimicking the actions of protein kinases A and C.

1. The effect of okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A), on human platelets has been investigated. 2. Okadaic acid exerts a general increase in phosphorylation of platelet proteins but did not induce aggregation or secretion of 5-hydroxytryptamine (5-HT). Okadaic acid, however, did inhibit thrombin-induced functional responses. 3. Maximally effective concentrations of prostacyclin, to elevate adenosine 3'-5'-cyclic monophosphate (cyclic AMP), or phorbol dibutyrate, to activate protein kinase C, inhibited the formation of inositol phosphates by thrombin by approximately 60%. When used in combination, prostacyclin and phorbol dibutyrate reduced the levels of inositol phosphates induced by thrombin to 11%. 4. Okadaic acid (1 microM) decreased thrombin-induced formation of inositol phosphates by approximately 55% and increased the inhibitory action of prostacyclin or phorbol dibutyrate. Okadaic acid had no further effect when prostacyclin and phorbol dibutyrate were used in combination. 5. These results suggest that protein kinases A and C act to inhibit phospholipase C by distinct mechanisms and that their action is reversed by PP1 and/or PP2A.     

Noradrenaline release and the effect of endogenous activation of the phospholipase C/protein kinase C signalling pathway in rat atria

1. It has been proposed that protein kinase C (PKC) in sympathetic nerves is activated during action-potential evoked release of noradrenaline and helps maintain transmitter output. We studied this phenomenon further in rat atria radiolabelled with [³H]-noradrenaline.
2. Noradrenaline release was elevated by continuous electrical stimulation of the atria for 10 min at either 5 or 10 Hz. Two inhibitors of PKC, polymyxin B (21 μM) and Ro 318220 (3 μM), markedly inhibited the release of noradrenaline but only at the higher stimulation frequency.
3. Further experiments were conducted with 10 Hz stimulation but for shorter train durations. In this case polymyxin B inhibited noradrenaline release during a 10 or 15 s train of impulses but not during a 5 s train. This suggests that PKC effects are induced during the stimulation train by some process.
4. The diacylglycerol kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"R59949","term_id":"830644","term_text":"R59949"}}R59949 (10 μM), which prevents the breakdown of diacylglycerol, enhanced noradrenaline release elicited by stimulation at 10 Hz for 10 or 15 s. This effect was not seen if polymyxin B was present and suggests that diacylglycerol is the endogenous activator of PKC.
5. The source of the diacylglycerol may be through phospholipase C pathways, since the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (3 μM) inhibited noradrenaline release at 10 Hz for 10 s and the effect was not seen if polymyxin B was also present.
6. It is unlikely that phospholipase D is the source of diacylglycerol. Although the phospholipase D inhibitor wortmannin (1 μM) inhibited noradrenaline release, this effect was still observed in the presence of polymyxin B. Furthermore ethanol, which inhibits diacylglycerol formation by phospholipase D, had no effect on noradrenaline release.
7. We therefore suggest that during a train of high frequency pulses phospholipase C is activated and this results in the production of diacylglycerol which in turn activates PKC. This enables the neurones to maintain transmitter release at a high level.

     

Vasopressin-induced activation of human blood platelets: prominent role of Mg2+

Summary Arginine-vasopressin (AVP) caused a marked shape change reaction and rise in [Ca²⁺]_i in human blood platelets only when the extracellular buffer contained Mg²⁺ or Ca²⁺. At physiological concentrations of the cations the potency of AVP was higher in the presence of Mg²⁺ than of Ca²⁺. The amplitude of the shape change reaction was also greater with Mg²⁺ than with Ca²⁺, although the [Ca²⁺]_i-rise was slightly more marked with extracellular Ca²⁺. The concentration of Mg²⁺ at which AVP showed half of its maximal effects was below the physiological plasma level of the cation, whereas the corresponding value for Ca²⁺ was higher. Addition of Ca²⁺ to the Mg²⁺ containing medium did not further enhance the action of AVP on platelet shape. In platelet-rich plasma the potency and efficacy of AVP in causing a shape change were similar in the presence and absence of EGTA, whereas with EDTA in the medium AVP had no effect. In conclusion, Mg²⁺ has an essential physiological role in AVP-induced platelet activation, which is brought about partly by release of intracellular calcium and partly by some other intracellular mechanism.     

Protein kinase C epsilon-dependent pathway of extracellular signal-regulated protein kinase activation by P2Y1 and P2Y2 purinoceptors that activate cytosolic phospholipase A2 in endothelial cells.

The aim of this study was to investigate the stimulating effects on arachidonic acid release of P2Y1 and P2Y2 receptor-selective agonists, 2-methylthio-ATP (2MeSATP) and UTP, respectively, in bovine pulmonary artery endothelial cells. Exposure of cells to 2MeSATP and UTP led to the release of arachidonic acid, a response which was abolished by the removal of extracellular Ca2+ and methyl arachidonyl fluorophosphonate. Phorbol 12-myristate 13-acetate (PMA) itself not only stimulated arachidonic acid release but also played a permissive role in the response to UTP. However, PMA failed to enhance the arachidonic acid response induced by 2MeSATP, probably due to greater attenuation of the [Ca2+]i increase caused by 2MeSATP than UTP. Inhibition of protein kinase C with Ro 31-8220 (1-[3-(amidinothio) propyl-1H-indoyl-3-yl]-3-(1-methyl-1H-indoyl-3-yl)-maleimide -methane sulphate) and staurosporine, but not with Go 6976 (12-(-2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-indolo(2, 3-a)pyrrolo(3,4-c)carbazole), reduced the arachidonic acid response of 2MeSATP, UTP and PMA. PMA-induced potentiation of the UTP response reached a maximum after a 1-h preincubation, then declined and eventually lost its effect when the preincubation lasted up to 8 h. Among the protein kinase C isoforms present in endothelial cells, betaI and epsilon could be down-regulated by treatment with PMA for 4-24 h. PD 098059 (2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) inhibited extracellular signal-regulated protein kinase activation, cytosolic phospholipase A2 phosphorylation and arachidonic acid release caused by 2MeSATP, UTP and PMA. Taken together, our results demonstrate that P2Y1 and P2Y2 purinoceptors mediate arachidonic acid release by activating cytosolic phospholipase A2 through an elevation of [Ca2+]i and protein kinase C epsilon-, extracellular signal-regulated protein kinase-dependent phosphorylation.     

Expression of matrix metalloproteinase-9 in human platelets: regulation of platelet activation in in vitro and in vivo studies

1. The aim of this study was to identify the presence of matrix metalloproteinase-9 (MMP-9) in human platelets and systematically examine its inhibitory mechanisms of platelet activation. 2. In this study, we report on an efficient method for the quantitative analysis of pro-MMP-9 in human platelets using capillary zone electrophoresis (CZE). To elucidate subcellular localization of MMP-9 in human platelets, we investigated intraplatelet MMP-9 by immunogold labeling and visualized it using electron microscopy. In an in vivo thrombotic study, platelet thrombus formation was induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium. 3. MMP-9-gold labeling was observed on the plasma membrane, alpha-granules, open canalicular system, and within the cytoplasma both in resting and activated platelets. Furthermore, activated MMP-9 concentration-dependently (15-90 ng ml(-1)) inhibited platelet aggregation stimulated by agonists. Activated MMP-9 (21 and 90 ng ml(-1)) inhibited phosphoinositide breakdown, intracellular Ca(2+) mobilization, and thromboxane A(2) formation in human platelets stimulated by collagen (1 microg ml(-1)). In addition, activated MMP-9 (21 and 90 ng ml(-1)) significantly increased the formation of nitric oxide/cyclic GMP. 4. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12, 13-dibutyrate (PDBu) (60 nm). This phosphorylation was markedly inhibited by activated MMP-9 (21 and 90 ng ml(-1)). Activated MMP-9 (1 microg g(-1)) significantly prolonged the latency period of inducing platelet plug formation in mesenteric venules. 5. These results indicate that the antiplatelet activity of activated MMP-9 may be involved in the following pathways. (1) Activated MMP-9 may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown, protein kinase C activation, and thromboxane A(2) formation, thereby leading to inhibition of intracellular Ca(2+) mobilization. (2) Activated MMP-9 also activated the formation of nitric oxide/cyclic GMP, resulting in inhibition of platelet aggregation. These results strongly indicate that MMP-9 is a potent inhibitor of aggregation. It may play an important role as a negative feedback regulator during platelet activation.     

Pyrimidinoceptor-mediated activation of phospholipase C and phospholipase A2 in RAW 264.7 macrophages.

1. As well as the presence of P2Z purinoceptors previously found in macrophages, we identified pyrimidinoceptors in RAW 264.7 cells, which activate phospholipase C (PLC) and phospholipase A2 (PLA2). 2. The relative potency of agonists to stimulate inositol phosphate (IP) formation and arachidonic acid (AA) release was UTP = UDP > > ATP, ATP gamma S, 2MeSATP. For both signalling pathways, the EC50 values for UTP and UDP (3 microM) were significantly lower than that for ATP and all other analogues tested (> 100 microM). 3. UTP and UDP displayed no additivity in terms of IP formation and AA release at maximally effective concentrations. 4. UTP-, but not ATP-, evoked AA release was 60% inhibited by pertussis toxin (PTX), while stimulation of IP formation by both agonists was unaffected. Short-term treatment with phorbol 12-myristate 13-acetate (PMA) led to a dose-dependent inhibition of IP responses to UTP and UDP, but failed to affect the AA responses. Removal of extracellular Ca2+ inhibited the PI response to UTP, but abolished its AA response. 5. ATP-induction of these two transmembrane signal pathways was decreased in high Mg(2+)-containing medium but potentiated by the removal of extracellular Mg2+. 6. Suramin and reactive blue displayed equal potency to inhibit the IP responses of UTP and ATP. 7. Both UTP and UDP (0.1-100 microM) induced a sustained increase in [Ca2+]i which lasted for more than 10 min. 8. Taken together, these results indicate that in mouse RAW 264.7 macrophages, pyrimidinoceptors with specificity for UTP and UDP mediate the activation of PLC and cytosolic (c) PLA2. The activation of PLC is via a PTX-insensitive G protein, whereas that of cPLA2 is via a PTX-sensitive G protein-dependent pathway. The sustained Ca2+ influx caused by UTP contributes to the activation of cPLA2. RAW 264.7 cells also possess P2z purinoceptors which mediate ATP(4-)-induced PLC and PLA2 activation.     

Carrageenan-induced activation of human platelets is independent of phospholipase A2 and of formation of thromboxanes

Aggregationof washed rabbit platelets by thrombin and by carrageenan is accompanied by the activation of phospholipase A₂ and by the synthesis of thromboxanes. Accordingly, aggregation, the accompanying release reaction and the activation of phospholipase are blocked by p-bromophenacyl bromide and by CB 874 (2,3-dibromo (4′-cyclohexyl-3′-chloro)-phenyl-4- oxo-butyric acid), two recognized inhibitors of the enzyme. Since these two reagents also inhibit aggregation and the release reaction induced by thrombin and by carrageenan on washed human platelets, it might have been anticipated that the mechanisms of aggregation of the platelets from the two species are similar. Nevertheless, no thromboxanes A₂ or B₂, nor activation of phospholipase A₂ could be demonstrated with the use of carrageenan on human platelets, under conditions where thrombin was effective. It is concluded that carrageenan activates the human platelets by phospholipase A₂- and thromboxane A₂-independent mechanisms, and that the inhibitors of phospholipase A₂ may block platelet functions by mechanisms other than inhibition of the expected enzyme.     

Carrageenan-induced activation of human platelets is independent of phospholipase A2 and of formation of thromboxanes

Aggregation of washed rabbit platelets by thrombin and by carrageenan is accompanied by the activation of phospholipase A2 and by the synthesis of thromboxanes. Accordingly, aggregation, the accompanying release reaction and the activation of phospholipase are blocked by p-bromophenacyl bromide and by CB 874 (2,3-dibromo (4'-cyclohexyl-3'-chloro)-phenyl-4-oxo-butyric acid), two recognized inhibitors of the enzyme. Since these two reagents also inhibit aggregation and the release reaction induced by thrombin and by carrageenan on washed human platelets, it might have been anticipated that the mechanisms of aggregation of the platelets from the two species are similar. Nevertheless, no thromboxanes A2 or B2, nor activation of phospholipase A2 could be demonstrated with the use of carrageenan on human platelets, under conditions where thrombin was effective. It is concluded that carrageenan activates the human platelets by phospholipase A2- and thromboxane A2-independent mechanisms, and that the inhibitors of phospholipase A2 may block platelet functions by mechanisms other than inhibition of the expected enzyme.     

Demethoxyviridin and wortmannin block phospholipase C and D activation in the human neutrophil.

1. The fungal metabolite, wortmannin, has recently been shown to inhibit fMet-Leu-Phe-stimulated superoxide production and phospholipase D (PLD) activation in the human neutrophil. 2. We have found that a close structural analogue of wortmannin, demethoxyviridin, has a similar inhibitory profile but in addition blocks phosphatidylinositol 4,5-bisphosphate-specific phospholipase C and hence inositol 1,4,5-trisphosphate (IP3) formation. 3. Inhibition of fMet-Leu-Phe-stimulated PLD by demethoxyviridin was characteristically non-competitive (IC50 = 31 +/- 10 nM). 4. Inhibition of fMet-Leu-Phe-stimulation IP3 formation required concentrations almost 10 times higher (IC50 = 250 +/- 130 nM). 5. Surprisingly, demethoxyviridin only inhibited fMet-Leu-Phe-induced intracellular calcium mobilization at concentrations 100 times greater than those needed to block IP3 formation. 6. Demethoxyviridin also inhibited PLD activation induced by sodium fluoride or phorbol myristate acetate (PMA) but the concentrations required were 100 times those needed to block fMet-Leu-Phe-stimulated PLD. 7. These observations support the contention that PLD plays an important role in signal transduction in the human neutrophil and indicate that wortmannin and demethoxyviridin inhibit PLD activation at a common step in the signalling pathway. 8. Furthermore, these results suggest that demethoxyviridin may block the interaction between the chemotactic peptide receptor and a GTP-binding protein that is intimately involved in PLD activation.     

Synergistic interaction of adrenaline and histamine in human platelet aggregation is mediated through activation of phospholipase, map kinase and cyclo-oxygenase pathways.

This study was conducted to examine the mechanism(s) of synergistic interaction of histamine- and adrenaline-mediated human platelet aggregation. We found that platelet aggregation mediated by subthreshold concentrations of histamine (1-4 microm) plus adrenaline (0.5-2 microm) is inhibited by both an alpha(2)-adrenoceptor blocker (yohimbine) and a histamine (H1) receptor antagonist (diphenhydramine). In examining the role of the downstream signalling pathway, we found that such an interaction is inhibited by the calcium channel blockers verapamil and diltiazem. However, platelet aggregation by adrenaline plus histamine was inhibited by very low concentrations of the phospholipase C (PLC) inhibitor, U73122 (IC(50)= 1.2 microm), the MEK inhibitor, PD98059 (IC(50)= 1.1 microm) and the cyclo-oxygenase (COX) inhibitor, indomethacin (IC(50)= 7 microm). However the inhibition of receptor tyrosine kinase, protein kinase C and phosphatidylinositol 3-kinase by genistien, chelerythrine and wortmannin, respectively, had no significant effect on aggregation. Similarly the nitric oxide donor (SNAP) had no effect on this synergism. These data suggest that the synergistic effect of histamine and adrenaline during human platelet aggregation is receptor mediated and involves activation of PLC, COX and MAP kinase signalling pathways.     

Characterization of the L-arginine:nitric oxide pathway in human platelets.

1. The activation of the L-arginine: nitric oxide (NO) pathway during aggregation of human platelets by adenosine 5'-diphosphate (ADP), arachidonic acid, thrombin and the calcium ionophore A23187 and its inhibition by NG-monomethyl-L-arginine (L-NMMA), NG-nitro-L-arginine methyl ester (L-NAME) and N-iminoethyl-L-ornithine (L-NIO) were studied. The inhibition of the cytosolic platelet NO synthase by these compounds was also examined. 2. Platelet aggregation induced by ADP (1-10 microM) and arachidonic acid (0.1-10 microM), but not that induced by thrombin (1-30 mu ml-1) or A23187 (1-10 nM), was inhibited by L-, but not D-arginine (1-30 microM). However, in the presence of a subthreshold concentration of prostacyclin (0.1 nM) or of M & B 22948 (1 microM), a selective inhibitor of guanosine 3':5'-cyclic monophosphate (cyclic GMP) phosphodiesterase, L-arginine caused concentration-dependent inhibition of aggregation induced by all of these aggregating agents. 3. L-NMMA, L-NAME and L-NIO (all at 1-30 microM), but not their D-enantiomers, enhanced to the same extent platelet aggregation induced by ADP, arachidonic acid and thrombin without affecting that induced by A23187. 4. In the presence of 300 microM L-arginine, the NO synthase in platelet cytosol was inhibited by L-NMMA, L-NAME and L-NIO with IC50s of 74 +/- 9, 79 +/- 8 and 8.5 +/- 1.5 microM (n = 3), respectively. 5. These results indicate that the L-arginine: NO pathway in human platelets plays a role in the modulation of platelet aggregation.     

Bisdemethoxycurcumin promotes apoptosis in human platelets via activation of ERK signaling pathway

Curcumin, a major bioactive component of turmeric (Curcuma longa), is known for its multiple health benefits. Curcumin as such is a mixture of its analogs: bisdemethoxycurcumin (BDMC)-3%, and demethoxycurcumin (DMC)-17%. Although the effect of curcumin on platelets is documented, the effect of BDMC and DMC on platelets is less studied. Considering the indispensable role played by platelets in hemostasis, thrombosis, inflammation, and immunity, the present study evaluates the effect of curcumin, DMC and BDMC on platelet apoptosis. The components of curcumin were purified by silica-gel column chromatography. The purity and mass analysis of the purified curcuminoids was determined by RP-HPLC and LC-MS respectively. When analyzed for platelet apoptotic markers, only BDMC demonstrated increased incidence of platelet apoptotic markers including increase in intracellular Ca²⁺, decrease in ∆ψm, alteration in BCl-2 family proteins, the release of cytochrome c, caspase activation, and PS externalization via activation of ERK activation. ERK inhibitor PD98059 significantly alleviated BDMC induced decrease in ∆ψm, alteration in BCl-2, caspase-8 activation and PS externalization. Our results demonstrate that curcumin, DMC and BDMC differentially act on platelet in inducing apoptosis and the study highlights that the toxicity associated with curcumin therapy might be attributed to BDMC in the mammalian system.     

KF4939, a new anti-platelet agent, inhibits activation of phospholipase C and A2 in rabbit platelets

Effect of KF4939 on activation of phospholipase C and A2 was examined by using rabbit platelets prelabelled with [14C]-arachidonic acid. The results showed that KF4939 inhibited the activation of the two phospholipases, separately. The inhibition of phospholipase C activation is thought to be a possible mechanism of KF4939 to inhibit aggregation and secretion which are independent of the synthesis of cyclooxygenase products such as thromboxane A2.     

Vanadate-induced activation of cytosolic phospholipase A2alpha in L929 cells: Roles of tyrosine kinase, protein kinase C, and extracellular signal-regulated kinase

Orthovanadate (Na3VO4), which acts as an inhibitor of protein tyrosine phosphatases, has a various pharmacological effects including the release of arachidonic acid (AA) from cells. We investigated roles of alpha-type cytosolic phospholipase A2 (cPLA2alpha), Src family kinases (Src) and protein kinase C (PKC) in the release of AA induced by Na3VO4 from a murine fibroblast cell line, L929. C12 cells, a variant of L929 that lacks expression of cPLA2alpha, were used along with a clone of C12 cells that are stably expressing cPLA2alpha (C12-cPLA2alpha cells). In the presence of a Ca2+ ionophore (10 microM A23187), 5 and 10mM Na3VO4 synergistically stimulated AA release from L929 and C12-cPLA2alpha cells, and to a much lesser extent from control C12 cells. The release of AA by Na3VO4/A23187 was inhibited by a selective cPLA2alpha inhibitor (3 microM pyrrophenone). The release of AA by Na3VO4/A23187 was significantly inhibited by a PKC inhibitor (10 microM GF109203X), in PKC-depleted cells, by a Src inhibitor (2 microM PP2) and by an inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2) kinase (10 microM U0126). The phosphorylation of ERK1/2 was stimulated by Na3VO4, and the response was significantly decreased by inhibitors of Src, PKC and ERK1/2 kinase. Our data show that Na3VO4 stimulates AA release largely via cPLA2alpha activation in Ca2+-dependent manner, and the cross-talk between Src and PKC and the ERK-dependent pathways are involved in Na3VO4-induced AA release from L929 cells.     

The effect of beta2-adrenoceptor agonists on phospholipase C (beta1) signalling in human airway smooth muscle cells

Prolonged use of beta2-adrenoceptor agonists has been associated with asthma exacerbation. Recently, phospholipase C-beta1 (PLC-beta1) induction in airway smooth muscle was proposed to underlie this phenomenon. We aimed to evaluate this mechanism in primary human airway smooth muscle cells. Stimulation of cells (1 microM isoprenaline or salbutamol for 2-48 h or 10(-9)-10(-4)M for 24 h) had no effect on PLC-beta1 gene expression. 1 microM beta2-adrenoceptor agonist treatment for 24 h did not alter sodium fluoride Inositol Phosphate (IP) responses, however augmented histamine IP responses (P<0.05). Therefore, beta2-adrenoceptor agonists can augment spasmogen responses in airway smooth muscle but not via a G-protein/PLC-beta1 mediated mechanism.     

Effect of a stimulant of guanylate cyclase, sin 1, on calcium movements and phospholipase C activation in thrombin-stimulated human platelets

The effects of sin 1, a metabolite of an antianginal agent, molsidomine, were investigated on human platelet activation induced by thrombin. This drug promoted a slight inhibition of serotonin release in a medium containing 1 mM Ca2+ or 1 mM EGTA (from 63% to 46% and from 57% to 41% of total serotonin secretion, respectively, with the highest dose used). Under these conditions, Ca2+ movements, monitored by quin 2 fluorescence, were markedly impaired. The most pronounced effect was towards Ca2+ influx, which presented a rapid inhibition with low doses. In the presence of external calcium, thrombin raised cytoplasmic free Ca2+ concentration from 100 nM to 1277 nM. This was reduced to 466 nM and 175 nM with 10(-7) M and 10(-4) M sin 1, respectively. Ca2+ mobilization from internal stores was less inhibited, since cytoplasmic free Ca2+ movements, sin 1 was tested on [32P] phosphatidic acid synthesis resulting from phospholipase C activation induced by thrombin. Phosphatidic acid labelling displayed a maximal inhibition of 43-50% with the highest doses of sin 1 (10(-4) M-10(-3) M) with or without Ca2+ in the incubation medium. However, this effect appeared much more sensitive to sin 1 in the presence of external Ca2+ (25% at 10(-7) M sin 1 with external Ca2+ against 12% at the same sin 1 concentration with EGTA). This discrepancy might be explained by the difference of cGMP level obtained when platelets were treated by sin 1 in the presence or in the absence of Ca2+ in the medium. This study shows that the major target of sin 1 via cGMP is not platelet phospholipase C as previously described, but inhibition of Ca2+ influx through plasma membrane.     

Involvement of cyclooxygenase, phospholipase C and MAP kinase pathways in human platelet aggregation mediated by the synergistic interaction of adrenaline and PAF

This study was conducted to examine the mechanism(s) of synergistic interaction of adrenaline and platelet-activating factor (PAF) mediated human platelet aggregation. We found that platelet aggregation mediated by subthreshold concentrations of PAF (5-8 nM) plus adrenaline (0.5-2 M) was inhibited by both ₂-adrenoceptor blocker (yohimbine) and PAF receptor antagonist (WEB2086). While examining the role of the downstream signalling pathways, we found that this synergism was inhibited by calcium channel blockers, verapamil, and diltiazem. In addition, platelet aggregation by co-addition of adrenaline and PAF was also inhibited by very low concentrations of phospholipase C (PLC) inhibitor (U73122; IC₅₀ = 0.2 _M), the MAP kinase inhibitor, PD98059 (IC₅₀ = 3 _M) and cyclooxygenase (COX-1) inhibitors including indomethacin (IC₅₀= 0.25 _M), flurbiprofen (IC₅₀ = 0.7 _M) and piroxicam (IC₅₀ = 7 _M). However, the COX-2 inhibitor, nimesulide, was also effective in inhibiting the aggregation. The inhibitors of tyrosine kinase (genistien) and phosphatidylinositol 3-kinase inhibitor (wortmannin) had no significant effects on platelet aggregation. These data suggest that the synergistic effect of adrenaline and PAF on human platelet aggregation is receptor-mediated and involves the activation of PLC/calcium, COX and MAP kinase signalling pathways.