EVIDENCE FOR REQUIREMENT OF TYROSINE PHOSPHORYLATION IN ENDOTHELIAL P-2Y-PURINOCEPTOR AND P-2U-PURINOCEPTOR STIMULATION OF PROSTACYCLIN RELEASE

1 The release of prostacylin (PGI(2)) from vascular endothelial cells is stimulated by ATP acting at G protein-coupled P-2-purinoceptors. Here we investigate the hypothesis that tyrosine protein phosphorylations are involved in this response. 2 The use of Western blots with anti-phosphotyrosine antibodies showed that 30 mu M 2MeSATP (selective for P-2Y-purinoceptors), 300 mu M UTP (selective for P-2U-purinoceptors) and 300 mu M ATP (effective at both these purinoceptors), each stimulate the tyrosine phosphorylation of proteins in bovine cultured aortic endothelial cells. Each of these agonists also stimulates 6-keto PGF(1 alpha) accumulation in the medium (an index of PGI(2) release) in these cells in the same period. 3 The tyrosine kinase inhibitor, genistein, inhibits the 6-keto PGF(1 alpha) response with the same concentration-dependency (1-100 mu M) as the tyrosine phosphorylation response. 4 Tyrphostin, a structurally and functionally distinct tyrosine kinase inhibitor, is also a potent inhibitor (0.1-10 mu M) of the 6-keto PGF(1 alpha) response. 5 Neither tyrphostin nor genistein inhibit the phospholipase C response to P-2-purinoceptor stimulation. Furthermore, these inhibitors do not affect the 6-keto PGF(1 alpha) response to ionomycin. 6 These results show that the regulation of vascular endothelial cells by ATP acting at both P-2Y- and P-2U-purinoceptors involves the stimulation of tyrosine phosphorylation, and suggest that this is a necessary event for the purinoceptor-mediated stimulation of PGI(2) production.