ROLE OF PROTEIN-TYROSINE PHOSPHORYLATION IN H2O2-INDUCED ACTIVATION OF ENDOTHELIAL-CELL PHOSPHOLIPASE-D

Oxidant-induced activation of phospholipase D (PLD) in bovine pulmonar y 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 (H2O2)-induced PLD activation and protein tyrosine phosphorylation wer e examined in BPAEC. Pretreatment of BPAEC with putative tyrosine kina se inhibitors genistein, tyrphostin, and herbimycin attenuated H2O2 (1 mM)-induced PLD activation. The inhibitory effect of the tyrosine kin ase inhibitors was highly specific for H2O2-induced modulation and sho wed no effect on PLD activation mediated by 12-O-tetradecanoylphorbol 13-acetate of bradykinin. Furthermore, addition of H2O2 increased in a time-dependent manner tyrosine phosphorylation of several proteins (1 7-200 kDa), as determined by immunoblot analysis with antiphosphotyros ine antibodies. H2O2-mediated protein tyrosine phosphorylation precede d PLD activation, and a good correlation was observed on the effect of genistein in H2O2-induced PLD activation and protein tyrosine phospho rylation. Addition of vanadate, a phosphotyrosine phosphatase inhibito r, synergistically increased both PLD activation and protein tyrosine phosphorylation mediated by H2O2. Moreover, vanadate by itself had min imal effect on basal PLD activity in BPAEC; however, at 10 mu M vanada te, an increase in protein tyrosine phosphorylation was observed. In a ddition to vanadate, phenylarsine oxide and diamide potentiated H2O2-i nduced PLD activation. These results suggest that tyrosine kinase acti vation may be involved in H2O2-induced PLD activation in vascular endo thelial cells.