The blocker of receptor-mediated calcium entry SK&F 96365 was used to evaluate the contribution of calcium influx to the formation of biologically active endothelial prostanoids and endothelium-derived relaxing factor (EDRF). SK&F 96365 inhibited histamine-stimulated calcium entry into human umbilical vein endothelial cells but not its discharge from intracellular stores as determined spectrofluorometrically by changes of intracellular calcium concentration in fura-2-loaded cells. Concordantly, SK&F 96365 inhibited histamine-induced endothelial synthesis of 6-keto-prostaglandin F la and thromboxane B, in a dose-dependent manner. To assess the functional significance of endothelial formation of prostacyclin and EDRF to platelets, the cAMP-and cGMPdependent phosphorylation of two platelet proteins, raplB and a 50-kD vasodilator-stimulated phosphoprotein (VASP), was analyzed in coincubation experiments of endothelial cells with platelets. Autacoids released by histamine-stimulated E ndothelium-derived autacoids are crucial for the maintenance of local vascular homeostasis and endothelium-platelet interactions. Endothelial cells (ECs) respond to hormonal, chemical, and physical stimuli with the generation and release of prostacyclin and endothelium-derived relaxing factor (EDRF; nitric oxide or nitric oxide-releasing compounds), both of which are known to dilate vessels and inhibit platelet function in a paracrine manner. 15 Concomitantly, ECs also release the potent vasoconstrictor and stimulator of platelet aggregation thromboxane A 2 (TXA 2 ).6 ' 7The increase of cytosolic calcium concentration ([Ca 2+ ],), a key step in the stimulus-response coupling of ECs, leads to the activation of calcium-dependent enzymes such as phospholipase A 2 and nitric oxide synthase. 89 A variety of blood-borne mediators interact with endothelial surface receptors and raise [Ca 2+ ], by both inducing second messenger-mediated discharge of calcium from internal stores and promoting influx from the extracellular milieu.1 Calcium entry into nonexcitable cells occurs voltage independently via receptorcoupled channels of the plasma membrane that are not well characterized.
10Formation and release of EDRF and prostanoids by the vascular endothelium are coupled to an increase in Received May 31, 1994; accepted Jury 25, 1994. From the Institut fur Prophylaxe und Epidemiologie der Kreislaufkrankheiten, University of Munich, FRG.Correspondence to H.-J. Kruse, Institut fur Prophylaxe und Epidemiologie der Kreislaufkrankheiten, UniversitSt Munchen, Pettenkoferstr 9, 80336 Munchen, Germany.O 1994 American Heart Association, Inc.endothelial cells caused the phosphorylation of rap IB and VASP in platelets, which was only partly inhibited by either indomethacin or A r°-monomethyl-L-arginine but was almost completely suppressed by SK&F 96365. The concomitant endothelial release of thromboxane A 2 had no effect on protein kinase C-and calcium-dependent phosphorylation of platelet proteins. The results demonstrate that blockade of recepto...