TYROSINE KINASES AND CALCIUM-DEPENDENT ACTIVATION OF ENDOTHELIAL-CELLPHOSPHOLIPASE-D BY DIPEROXOVANADATE

Reactive oxygen species (ROS) mediated modulation of signal transducti on pathways represent an important mechanism of cell injury and barrie r dysfunction leading to the development of vascular disorders. Toward s understanding the role of ROS in vascular dysfunction, we investigat ed the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in b ovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [P-32] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several pr otein bands (Mr 20-200 kD), as determined by Western blot analysis wit h antiphosphotyrosine antibodies. The DPV-induced [P-32] PBt-accumulat ion was inhibited by putative tyrosine kinase inhibitors such as genis tein, herbimycin, tyrphostin and by chelation of Ca2+ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisi ndolylmaleimide showed minimal inhibition. Also down-regulation of PKC alpha and epsilon, the major isotypes of PKC in BPAECs, by TPA (100 n M, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as deter mined by comparing [P-32] PBt formation between DPV and TPA. In additi on to tyrosine kinase inhibitors, antioxidants such as N-acetylcystein e and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest th at oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation.