PROTEIN-TYROSINE KINASES REGULATE AGONIST-STIMULATED PROSTACYCLIN RELEASE BUT NOT VON-WILLEBRAND-FACTOR SECRETION FROM HUMAN UMBILICAL VEINENDOTHELIAL-CELLS

The rapid synthesis and release of prostacyclin (PGI(2)) and the exocy totic secretion of von Willebrand Factor (vWF) elicited by activation of G-protein-coupled receptors on endothelium occur via signalling mec hanisms which are incompletely defined. Activation of protein tyrosine kinases (PTKs) and modulation of the tyrosine-phosphorylation state o f endogenous proteins have been implicated in several cellular process es including arachidonate release and exocytosis. In the present study we have examined the regulatory role of PTKs in agonist-stimulated re lease of PGI(2) and vWF from human umbilical vein endothelial cells (H UVECs) using two chemically and mechanistically dissimilar PTK inhibit ors (genistein and ST271). Genistein, but not the less active analogue daidzein, dose-dependently attenuated PGI(2) release in response to t hrombin and histamine (IC50 approx. 20 mu M), and to the thrombinm-rec eptor-activating peptide. A more potent inhibition of thrombin- and hi stamine-induced PGI(2) synthesis was observed in cells exposed to ST27 1. In contrast, neither genistein nor ST271 modulated agonist-drive vW F secretion. At concentrations that abolished PGI(2) release, genistei n blocked thrombin- or histamine-evoked tyrosine phosphorylation of a 42 kDa protein. Ca2+ ionophore-induced PGI(2) generation, but not vWF secretion, was also inhibited by both genistein and ST271, suggesting that these agents modulate PGI(2) synthesis by acting at, or distal to , agonist-induced changes in intracellular Ca2+ ([Ca2+](i)). In fura-2 -loaded HUVECs genistein partially reduced the histamine-induced peak [Ca2+](i) but had no effect on the thrombin response. Ca2+-induced PGI (2) release from electrically permeabilized HUVECs was abolished in th e presence of ST271 or genistein, but not daidzein. The generation of PGI, in response to exogenous arachidonic acid was not modulated by ge nistein or ST271, suggesting that PTK inhibitors do not directly inhib it cyclo-oxygenase activity. Taken together, these results suggest tha t PTKs regulate PGI(2) synthesis and release in HUVECs by modulating, directly or indirectly, a Ca2+-sensitive step upstream of cyclo-oxygen ase.