The rapid synthesis and release of prostacyclin (PGI2) and the exocytotic secretion of von Willebrand Factor (vWF) elicited by activation of G-protein-coupled receptors on endothelium occur via signalling mechanisms which are incompletely defined. Activation of protein tyrosine kinases (PTKs) and modulation of the tyrosine-phosphorylation state of endogenous proteins have been implicated in several cellular processes including arachidonate release and exocytosis. In the present study we have examined the regulatory role of PTKs in agonist-stimulated release of PGI2 and vWF from human umbilical vein endothelial cells (HUVECs) using two chemically and mechanistically dissimilar PTK inhibitors (genistein and ST271). Genistein, but not the less active analogue daidzein, dose-dependently attenuated PGI2 release in response to thrombin and histamine (IC50 approx. 20 μM), and to the thrombin-receptor-activating peptide. A more potent inhibition of thrombin- and histamine-induced PGI2 synthesis was observed in cells exposed to ST271. In contrast, neither genistein nor ST271 modulated agonist-drive vWF secretion. At concentrations that abolished PGI2 release, genistein blocked thrombin- or histamine-evoked tyrosine phosphorylation of a 42 kDa protein. Ca2+ ionophore-induced PGI2 generation, but not vWF secretion, was also inhibited by both genistein and ST271, suggesting that these agents modulate PGI2 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 PGI2 release from electrically permeabilized HUVECs was abolished in the presence of ST271 or genistein, but not daidzein. The generation of PGI2 in response to exogenous arachidonic acid was not modulated by genistein or ST271, suggesting that PTK inhibitors do not directly inhibit cyclo-oxygenase activity. Taken together, these results suggest that PTKs regulate PGI2 synthesis and release in HUVECs by modulating, directly or indirectly, a Ca2+-sensitive step upstream of cyclo-oxygenase.
Protein tyrosine kinases regulate agonist-stimulated prostacyclin release but not von Willebrand factor secretion from human umbilical vein endothelial cells
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Caroline P. D. WHEELER-JONES, Michael J. MAY, Anthony J. MORGAN, Jeremy D. PEARSON; Protein tyrosine kinases regulate agonist-stimulated prostacyclin release but not von Willebrand factor secretion from human umbilical vein endothelial cells. Biochem J 15 April 1996; 315 (2): 407–416. doi: https://doi.org/10.1042/bj3150407
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