Evidence for requirement of tyrosine phosphorylation in endothelial P2y‐ and P2u‐ purinoceptor stimulation of prostacyclin release

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Abstract
1 . The release of prostacylin (PGI2) from vascular endothelial cells is stimulated by ATP acting at G protein-coupled P2-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 μm 2MeSATP (selective for P2Y-purinoceptors), 300 μm UTP (selective for P2U-purinoceptors) and 300 μm 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 PGF1α accumulation in the medium (an index of PGI2 release) in these cells in the same period. 3 . The tyrosine kinase inhibitor, genistein, inhibits the 6-keto PGF1α response with the same concentration-dependency (1–100 μm) as the tyrosine phosphorylation response. 4 . Tyrphostin, a structurally and functionally distinct tyrosine kinase inhibitor, is also a potent inhibitor (0.1–10 μm) of the 6-keto PGF1α response. 5 . Neither tyrphostin nor genistein inhibit the phospholipase C response to P2-purinoceptor stimulation. Furthermore, these inhibitors do not affect the 6-keto PGF1α response to ionomycin. 6 . These results show that the regulation of vascular endothelial cells by ATP acting at both P2Y- and P2U-purinoceptors involves the stimulation of tyrosine phosphorylation, and suggest that this is a necessary event for the purinoceptor-mediated stimulation of PGI2 production.

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