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 inhibitor pervanadate on activation of signal transduction in human umbilical vein endothelial cells. Endothelial cells responded to pervanadate treatment by increasing tyrosine phosphorylation of cellular proteins, including phospholipase C (PLC) gamma 1, generating inositol phosphates (IPs), releasing arachidonic acid, and producing prostacyclin (prostaglandin [PG] I2). The dose and time responses for these events were similar. Tyrosine phosphorylation and formation of IPs in response to pervanadate were reduced by both staurosporine and genistein. Short-term incubation with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, which inhibits thrombin-induced IP generation, did not affect the IP response to pervanadate. To investigate the possible involvement of tyrosine phosphorylation in thrombin or histamine-induced IP generation and PGI2 production, we examined the effects of costimulation with pervanadate and either thrombin or histamine. These responses proved to be different. While the tyrosine phosphorylation of PLC gamma 1 was enhanced after cotreatment with thrombin and pervanadate compared with pervanadate alone, costimulation with pervanadate and histamine resulted in no more tyrosine phosphorylation of PLC gamma 1 than after pervanadate alone. Similarly, while cotreatment with pervanadate and thrombin caused synergistic increase in IP generation, costimulation with pervanadate and histamine resulted in an additive response. However, PGI2 responses to costimulation of pervanadate with either thrombin or histamine were both synergistic. Furthermore, stimulation with histamine, thrombin, or pervanadate all caused tyrosine phosphorylation of a mitogen-activated protein kinase (ERK1/p44). The results suggest that a tyrosine phosphorylation-dependent mechanism has a role in the phosphoinositide signal transduction pathway of human endothelial cells. Moreover, thrombin- but not histamine-induced generation of IPs appears to be partly caused by tyrosine phosphorylation of PLC gamma 1.