FORMATION OF BIOLOGICALLY-ACTIVE AUTACOIDS IS REGULATED BY CALCIUM INFLUX IN ENDOTHELIAL-CELLS

The blocker of receptor-mediated calcium entry SK&F 96365 was used to evaluate the contribution of calcium influx to the formation of biolog ically active endothelial prostanoids and endothelium-derived relaxing factor (EDRF). SK&F 96365 inhibited histamine-stimulated calcium entr y into human umbilical vein endothelial cells but not its discharge fr om intracellular stores as determined spectrofluorometrically by chang es of intracellular calcium concentration in fura-2-loaded cells. Conc ordantly, SK&F 96365 inhibited histamine-induced endothelial synthesis of 6-keto-prostaglandin F-1 alpha and thromboxane B-2 in a dose-depen dent manner. To assess the functional significance of endothelial form ation of prostacyclin and EDRF to platelets, the cAMP- and cGMP-depend ent phosphorylation of two platelet proteins, rap1B and a 50-kD vasodi lator-stimulated phosphoprotein (VASP), was analyzed in coincubation e xperiments of endothelial cells with platelets. Autacoids released by histamine-stimulated endothelial cells caused the phosphorylation of r ap1B and VASP in platelets, which was only partly inhibited by either indomethacin or N-G-monomethyl-L-arginine but was almost completely su ppressed by SK&F 96365. The concomitant endothelial release of thrombo xane A(2) had no effect on protein kinase C- and calcium-dependent pho sphorylation of platelet proteins. The results demonstrate that blocka de of receptor-mediated calcium entry by SK&F 96365 markedly reduced t he release of biologically active prostacyclin and EDRF from endotheli al cells. Thus, calcium influx but not calcium release from intracellu lar stores plays a critical role in the receptor-stimulated formation and liberation of prostacyclin and EDRF in endothelial cells.