GTP gamma S and phorbol ester act synergistically to stimulate both Ca(2+)-independent secretion and phospholipase D activity in permeabilized human platelets. Inhibition by BAPTA and analogues.
We have tested the hypothesis that phospholipase D (PLD) is the effector of the unidentified G protein (GE) mediating Ca(2+)-independent exocytosis in platelets. Although GTP gamma S, and to a lesser extent phorbol 12-myristate 13-acetate (PMA), caused some secretion of 5-HT from electropermeabilized human platelets in the effective absence of Ca2+ (pCa > 9), these stimuli had much more potent synergistic effects when added together. In all cases, secretion of 5-HT was closely correlated to the stimulus-induced formation of [3H]phosphatidic acid ([3H]PA) from [3H]arachidonate-labelled phospholipids. Addition of ethanol inhibited both secretion and [3H]PA formation and led to the accumulation of [3H]phosphatidylethanol ([3H]PEt), indicating that [3H]PA was formed largely by activation of PLD. BAPTA and analogues caused dose-dependent inhibitions of both GTP gamma S-induced secretion and PLD activity in the permeabilized platelets. This action of BAPTA did not appear to be mediated by chelation of Ca2+ or by direct inhibition of protein kinase C (PKC). The results suggest that PLD is the target of GE in platelets and that BAPTA can block PLD activation.
Coorssen
J R
JR
Department of Pathology, McMaster University, Hamilton, Ont., Canada.
Haslam
R J
RJ
D000345:Affinity Labels; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D001798:Blood Proteins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004357:Drug Synergism; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004789:Enzyme Activation; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D006801:Humans; D010712:Phosphatidic Acids / Q000096:biosynthesis; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010750:Phosphoproteins; D010766:Phosphorylation; D012701:Serotonin / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000345:Affinity Labels; D001798:Blood Proteins; D010712:Phosphatidic Acids; D010750:Phosphoproteins; C039077:platelet protein P47; D012701:Serotonin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004533:Egtazic Acid; D010739:Phospholipase D; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium