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.
Simon
M F
MF
INSERM Unité 101, Biochimie des Lipides, Hôpital Purpan, Toulouse, France.
Chap
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H
Douste-Blazy
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D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000032:analysis*
D010712:Phosphatidic Acids; D012701:Serotonin; C002385:linsidomine; D008981:Molsidomine; D010738:Type C Phospholipases; D013917:Thrombin; D006152:Cyclic GMP; D002118:Calcium