Evidence for requirement of tyrosine phosphorylation in endothelial P2Y- and P2U- purinoceptor stimulation of prostacyclin release.
1. The release of prostacyclin (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 microM 2MeSATP (selective for P2Y-purinoceptors), 300 microM UTP (selective for P2U-purinoceptors) and 300 microM 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 PGF1 alpha 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 PGF1 alpha response with the same concentration-dependency (1-100 microM) as the tyrosine phosphorylation response. 4. Tyrphostin, a structurally and functionally distinct tyrosine kinase inhibitor, is also a potent inhibitor (0.1-10 microM) of the 6-keto PGF1 alpha response. 5. Neither tyrphostin nor genistein inhibit the phospholipase C response to P2-purinoceptor stimulation. Furthermore, these inhibitors do not affect the 6-keto PGF1 alpha 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.
Bowden
A
A
Department of Cell Physiology and Pharmacology, University of Leicester.
Patel
V
V
Brown
C
C
Boarder
M R
MR
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000648:ultrastructure*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D011464:Epoprostenol / Q000096:biosynthesis / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D058906:Purinergic P1 Receptor Agonists; D058910:Purinergic P2 Receptor Agonists; D018047:Receptors, Purinergic P1 / Q000502:physiology*; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D013268:Stimulation, Chemical; D014443:Tyrosine / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000494:pharmacology
D004791:Enzyme Inhibitors; D058906:Purinergic P1 Receptor Agonists; D058910:Purinergic P2 Receptor Agonists; D018047:Receptors, Purinergic P1; D018048:Receptors, Purinergic P2; D014443:Tyrosine; D015759:Ionomycin; D000255:Adenosine Triphosphate; D011464:Epoprostenol; D011505:Protein-Tyrosine Kinases; D014544:Uridine Triphosphate