Role of protein tyrosine phosphorylation in H2O2-induced activation of endothelial cell phospholipase D
Natarajan, V.; Vepa, S.; Verma, R.S.; Scribner, W.M.
American Journal of Physiology 271(3 Pt 1): L400-L408
1996
ISSN/ISBN: 0002-9513 PMID: 8843788 Document Number: 457247
Oxidant-induced activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAEC) is independent of protein kinase C and calcium. In the present study, the effects of tyrosine kinase and protein tyrosine phosphatase (PTPase) inhibitors on hydrogen peroxide (H-2O-2)-induced PLD activation and protein tyrosine phosphorylation were examined in BPAEC. Pretreatment of BPAEC with putative tyrosine kinase inhibitors genistein, tyrphostin, and herbimycin attenuated H-2O-2 (1 mM)-induced PLD activation. The inhibitory effect of the tyrosine kinase inhibitors was highly specific for H-2O-2-induced modulation and showed no effect on PLD activation mediated by 12-O-tetradecanoylphorbol 13-acetate or bradykinin. Furthermore, addition of H-2O-2 increased in a time-dependent manner tyrosine phosphorylation of several proteins (17-200 kDa), as determined by immunoblot analysis with antiphosphotyrosine antibodies. H-2O-2-mediated protein tyrosine phosphorylation preceded PLD activation, and a good correlation was observed on the effect of genistein in H-2O-2-induced PLD activation and protein tyrosine phosphorylation. Addition of vanadate, a phosphotyrosine phosphatase inhibitor, synergistically increased both PLD activation and protein tyrosine phosphorylation mediated by H-2O-2. Moreover, vanadate by itself had minimal effect on basal PLD activity in BPAEC; however, at 10 mu-M vanadate, an increase in protein tyrosine phosphorylation was observed. In addition to vanadate, phenylarsine oxide and diamide potentiated H-2O-2-induced PLD activation. These results suggest that tyrosine kinase activation may be involved in H-2O-2-induced PLD activation in vascular endothelial cells.