Phorbol myristate acetate inhibits thrombin-stimulated Ca 2 l mobilization and phosphatidylinositol 4 , 5-bisphosphate hydrolysis in human platelets ( phorbol esters / phosphatidic acid / quin-2 / secretion / protein kinase C )

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Author	Zavoico, George B. Halenda, Stephen P. Feinstein, Maurice B.
Abstract	The tumor-promoting phorbol diester 4,8phorbol 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 ofPMA, 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 byPMA 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 implicated as a second messenger that causes the release of Ca2+ from intracellular storage sites in various cells (8, 9), including platelets (10), thereby providing a mechanism by which receptor-mediated activation of the hydrolysis of Ptdlns-4,5P2 can potentially lead to elevation of [Ca2+]i. The stimulation ofprotein kinase C by exogenous acyl2Gro or phorbol diesters is accompanied by slow and partial secretion from platelets (7) without a rise of [Ca2+]i that is detectable by quin-2 (2). However, secretion is strikingly enhanced when [Ca2+]J is slightly elevated by ionophore A23187 or by ionomycin concurrently with the activation of protein kinase C by exogenous acyl2Gro or 4,-phorbol 12-myristate 13-acetate (PMA) (11). Currently, the only known pathway that antagonizes receptor-linked increase of [Ca2+]i (12-15), the hydrolysis of Ptdlns-4,5-P2 (16), and the formation of Ins-1,4,5-P3 (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. METHODS AND MATERIALS Fresh platelet concentrates obtained from the Connecticut Red Cross Blood Center were centrifuged at 120 x g for 10 min to remove residual erythrocytes and the platelets were then sedimented at 1000 x g for 10 min. The platelet pellet was resuspended in about 25 ml of buffer A (10 mM sodium Pipes, pH 6.5/0.2 mM EGTA/135 mM NaCl/5 mM KCl/5.5 mM glucose) and then recentrifuged at 1000 x g for 10 min. The platelet pellet was resuspended with 15-20 ml of buffer B (10 mM sodium Hepes, pH 7.4/135 mM NaCl/5 mM KC1/5.5 mM glucose) and incubated for 30 min with 0.2 mCi (1 Ci = 37 GBq) of [32P]orthophosphate and 10-15 ,uM quin-2 acetoxymethyl ester in dimethyl sulfoxide [final dimethyl sulfoxide concentration, 0.15% (vol/vol)] with gentle agitation in a water bath at 37°C. The platelets were then washed with buffer B containing 0.2 mg of bovine serum albumin per ml and resuspended in the same solution or in buffer C (buffer B with 0.2 mg of bovine serum albumin per ml/1 mM CaCl2/MgCl2) to obtain a final concentration of 1.5-2.6 x 109 platelets per ml. [Ca2+]i was measured, as described previously (12, 15), in quin-2-loaded platelets diluted to 108 platelets per ml in either (i) buffer B with bovine serum albumin, (ii) buffer C with or without CaCl2, or (iii) buffer C with 2 mM EGTA and no added Ca2+. Responses to PMA and thrombin (with or without PMA) were measured under the same conditions. In Abbreviations: [Ca2i]i, cytoplasmic free Ca21 concentration; PMA, 4f3-phorbol 12-myristate 13-acetate; PDec2, 4a-phorbol 12,13didecanoate; acyl2Gro, 1,2-diacylglycerol; PtdOH, phosphatidic acid; PtdIns-4-P, phosphatidylinositol 4-phosphate; Ptdlns-4,5-P2, phosphatidylinositol 4,5-bisphosphate; Ins-1,4,5-P3, myo-inositol 1,4,5-trisphosphate; PAF-acether, 1-0-alkyl-2-acetyl-sn-glyceryl-3phosphocholine. 3859 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3860 Medical Sciences: Zavoico et al. several experiments trypsin and PAF-acether were also used as agonists. [Ca2"]i was calculated from the quin-2 fluorescence measurements by the calibration procedure described by Tsien et al. (18) and Hallam et al. (2) in which the cells were lysed with 50 uM digitonin and fluorescence was determined in the presence of 1 mM Ca2l (Fmax) and at "zero" Ca2+ (Fmin) by adding 4 mM EGTA at pH 8.3. [Ca2+]j was calculated from the equation (18): [Ca2+]i = Kd(F Fmin)/(Fmax F), using a Kd of 115 nM (18). Corrections for autofluorescence and quin-2 in the medium were negligible. Secretion of dense granule constituents was monitored simultaneously with quin-2 fluorescence by measuring the release of Ca2l into the medium (when extracellular Ca2+ = 35-50 ,uM) using a Radiometer Ca2+-selective electrode placed in the fluorometer cuvette (12, 15). PtdIns-4,5-[32P]P2 and [32P]PtdOH were measured in platelets that were preloaded with [32P]orthophosphate (19) and quin-2. The platelets were washed (12, 15) and resuspended in buffer B (without Ca2+) or buffer C (with Ca2+) for stimulation by thrombin. Under the conditions of our experiments the intracellular quin-2 concentration was determined (18) to be 0.6-1.0 mM, which had no substantial effect on lipid metabolism (unpublished). Lipid extracts were prepared according to Billah and Lapetina (20) and the phospholipids were separated by thin-layer chromatography (4) with a solvent system consisting of chloroform/methanol/20% aqueous methylamine, 60:36:10 (vol/vol). Radioactive lipid bands were located by autoradiography and quantitated by liquid scintillation spectroscopy. Most experiments were carried out at 23°C, but all effects described in this paper were verified at 37°C as well. Human thrombin (2000 units/mg) was obtained from United States Biochemical (Cleveland, OH), PAF-acether, trypsin, PMA, 4a-phorbol 12,13-didecanoate (PDec2), and bovine serum albumin were from Sigma, A23187 and quin2/acetoxymethyl ester were from Calbiochem, and digitonin was from Pfaltz & Bauer (Stamford, CT). All other chemicals were of analytical grade from various sources.
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