A ROLE FOR TYROSINE PHOSPHORYLATION IN GENERATION OF INOSITOL PHOSPHATES AND PROSTACYCLIN PRODUCTION IN ENDOTHELIAL-CELLS

We have examined the effects of the protein tyrosine phosphatase inhib itor pervanadate on activation of signal transduction in human umbilic al vein endothelial cells. Endothelial cells responded to pervanadate treatment by increasing tyrosine phosphorylation of cellular proteins, including phospholipase C (PLC)(gamma 1), generating inositol phospha tes (IPs), releasing arachidonic acid, and producing prostacyclin (pro staglandin [PG] I-2). The dose and time responses for these events wer e similar. Tyrosine phosphorylation and formation of IPs in response t o pervanadate were reduced by both staurosporine and genistein. Short- term incubation with the phorbol ester 12-O-tetradecanoylphorbol 13-ac etate, which inhibits thrombin-induced IP generation, did not affect t he IP response to pervanadate. To investigate the possible involvement of tyrosine phosphorylation in thrombin or histamine-induced IP gener ation and PGI(2) production, we examined the effects of costimulation with pervanadate and either thrombin or histamine. These responses pro ved to be different. While the tyrosine phosphorylation of PLC(gamma 1 ) was enhanced after cotreatment with thrombin and pervanadate compare d with pervanadate alone, costimulation with pervanadate and histamine resulted in no more tyrosine phosphorylation of PLC(gamma 1) than aft er pervanadate alone. Similarly, while cotreatment with pervanadate an d thrombin caused synergistic increase in IP generation, costimulation with pervanadate and histamine resulted in an additive response. Howe ver, PGI(2) responses to costimulation of pervanadate with either thro mbin or histamine were both synergistic. Furthermore, stimulation with histamine, thrombin, or pervanadate all caused tyrosine phosphorylati on of a mitogen-activated protein kinase (ERK1/p44). The results sugge st that a tyrosine phosphorylation-dependent mechanism has a role in t he phosphoinositide signal transduction pathway of human endothelial c ells. Moreover, thrombin- but not histamine-induced generation of IPs appears to be partly caused by tyrosine phosphorylation of PLC(gamma 1 ).