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

Abstract

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 Ca 2+ 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, Ca 2+ movements, monitored by quin 2 fluorescence, were markedly impaired. The most pronounced effect was towards Ca 2+ influx, which presented a rapid inhibition with low doses. In the presence of external calcium, thrombin raised cytoplasmic free Ca 2+ concentration from 100 nM to 1277 nM. This was reduced to 466 nM and 175 nM with 10 −7 and 10 −4 M sin 1, respectively. Ca 2+ mobilization from internal stores was less inhibited, since cytoplasmic free Ca 2+ concentration was reduced from 148 nM with thrombin alone to 134 nM with 10 −4 M sin 1. In addition to Ca 2+ 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 Ca 2+ in the incubation medium. However, this effect appeared much more sensitive to sin 1 in the presence of external Ca 2+ (25% at 10 −7 M sin 1 with external Ca 2+ against 12% at the same sin 1 concentration with EGTA). This discrepancy might be explained by the difference ofcGMP level obtained when platelets were treated by sin 1 in the presence or in the presence or in the absence of Ca 2+ 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 Ca 2+ influx through plasma membrane.