Phorbol myristate acetate inhibits thrombin-stimulated Ca2+ mobilization and phosphatidylinositol 4,5-bisphosphate hydrolysis in human platelets.

Abstract

The tumor-promoting phorbol diester 4,8-phorbol 12-myristate 13-acetate (PMA) inhibited mobilization of intracellular Ca2' in platelets by thrombin (also trypsin and 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine). PMA was effective over the same concentration range that activates protein kinase C in intact platelets; ICse vs. thrombin = 2 ng/ml, 3.4 nM: >90% inhibition at 10-20 ng/ml. Suppression of thrombin-induced Ca2' mobilization was evident within 30 sec of pretreatment with PMA and was essentially complete by 6-10 min at 10-20 ng of PMA per ml. Thrombin-induced secretion was initially accelerated in the presence of PMA, but after 1 min it was progressively inhibited when Ca2+ mobilization was depressed by >60%. PMA did not inhibit Ca2+ mobilization or secretion caused by A23187. Thrombininduced phosphatidylinositol 4,5-[32P]bisphosphate breakdown and [32Plphosphatidic acid production were also initially increased by PMA and then progressively depressed. Inhibition of thrombin-induced lipid metabolism required higher concentrations of PMA (IC5s = 10 ng/ml), and it was not overcome by A23187. 4a-Phorbol 12,13-didecanoate, which lacks the ability to activate protein kinase C, did not inhibit any responses to thrombin. These results suggest that activation of protein kinase C, which initially fosters secretion and aggregation, may subsequently exert negative feedback on the receptor-mediated mobilization of intracellular Ca2+ and the hydrolysis of phosphatidylinositol 4,5-bisphosphate.The stimulation of platelets to secrete and aggregate by agonists, such as thrombin and 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine (PAF-acether), is associated with a rapid increase of the cytoplasmic free Ca2l concentration ([Ca2+]i) (1,2) and the hydrolysis of the phosphodiester bond of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) (3, 4). The latter reaction is closely correlated with stimulusresponse coupling in many cell types, including platelets (5). The products of this reaction, 1,2-diacylglycerol (acyl2Gro) and myo-inositol 1,4,5-trisphosphate (Ins-1,4,5-P3), appear to play important roles in various cellular activation processes.Acyl2Gro acts as a second messenger to stimulate Ca2+/ phosphatidylserine-dependent protein kinase C by increasing the affinity of the enzyme-lipid complex for Ca2+, so that the enzyme can be activated even at resting levels of [Ca2+]i (6).Acyl2Gro is subsequently metabolized by further hydrolysis to release arachidonic acid and by phosphorylation to form phosphatidic acid (PtdOH). The action ofacyl2Gro on protein kinase C can be duplicated by tumor-promoting phorbol esters (7). Ins-1,4,5-P3, on the other hand, has been impli- (17) is mediated by cyclic AMP. In this paper we report that PMA inhibits Ca2+ mobilization, breakdown of Ptdlns-4,5-P2, formation of PA, and secretion caused by thrombin. This suggests that the protein kinase C pathway can exert feedback inhibition on receptor-mediated platelet activation subsequent to its initial role in promoting secretion. METH...

Lack of inhibition of thrombin‐induced rise in intracellular Ca2+ levels and 5‐hydroxytryptamine secretion by 1‐oleoyl‐2‐acetylglycerol in human platelets

“…It is significant that, apart from the potentiatory effects described above, OAG had no inhibitory effect on thrombin-induced rise in [Ca2+Ji at both low and high thrombin concentrations and this represents a major divergence in the effects of GAG and phorbol esters, which have been shown to inhibit agonist-induced [Ca'+]i mobilisation in platelets [1,2]. Furthermore OAG had no significant effect on high-dose thrombin-induced…”

Distinct functions of down‐regulation‐sensitive and ‐resistant types of protein kinase C in rabbit aortic smooth muscle cells

“…Subsequently, we have clarified that protein kinase C has additionally an antiproliferative action in this cell type, and have proposed that this enzyme may serve as not only a positive but also a negative regulator in the proliferation of rabbit aortic SMCs [10]. During the course of these studies, we have found that protein kinase C inhibits the WBS-induced increase in [Ca2+]i in rabbit aortic SMCs as described in several cell types [11][12][13][14][15]. Moreover, we have found that protein kinase C is partially downregulated by prolonged treatment of rabbit aortic SMC with PDBu, and that the partial downregulation of protein kinase C abolishes completely both the proliferative and antiproliferative actions of this enzyme but does not abolish the inhibitory action of this enzyme in the regulation of [Ca2*]i.…”

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

“…5]. However these kinases have a large variety of different molecular targets in platelets that could mediate the effect of this kinase on platelet Ca 2+ signalling, with a number of publications suggesting a variety of mechanisms including inhibition of phospholipase C [6,7], stimulation of inositol-1,4,5-trisphosphate (IP 3 ) breakdown [8,9], stimulation of the plasma membrane Ca 2+ -ATPase (PMCA) [10,11], stimulation of sarco/endoplasmic reticulum Ca 2+ -ATPases (SERCAs) [12] and in the case of ADP, desensitization of the receptor [13]. Since inhibition of PMCAs does not abolish the potentiatory effect of PKC inhibitors on thrombin-evoked Ca 2+ rises, PMCAs do not appear to be involved in the response [14].…”