EVIDENCE FOR THE ACTIVATION OF THE SIGNAL-RESPONSIVE PHOSPHOLIPASE A(2) BY EXOGENOUS HYDROGEN-PEROXIDE

The intracellular events that lead to arachidonic acid release from bovine endothelial cells in culture treated with hydrogen peroxide were characterized. The hydrogen peroxide-stimulated release of arachidonic acid was time- and dose-dependent, with maximal release achieved at 15 minutes after the addition of 100 mu M hydrogen peroxide. Hydrogen peroxide-stimulated release of arachidonic acid was blocked with the phospholipase A(2) inhibitor quinacrine. Treatment of the cells with hydrogen peroxide did not result in liberation of oleic acid, indicating that hydrogen peroxide exercised its effect on an arachidonate-specific phospholipase. Pretreatment of the cells with antioxidants, transition metal chelators, and hydroxyl radical scavengers did not affect the hydrogen peroxide-stimulated arachidonic acid release, indicating that the response to hydrogen peroxide is not oxygen radical-mediated. The response to hydrogen peroxide does not appear to be calcium-dependent, due to me following two observations: (a) No increase in intracellular calcium was seen upon exposure of the FURA2-loaded cells to hydrogen peroxide at concentrations sufficient to release arachidonic acid, and (b) no change in the release response was detected in cells loaded with the intracellular calcium chelator BAPTA. Significant inhibition of arachidonic acid release was seen when the cells were pretreated with inhibitors of protein kinase C, but not with inhibitors of tyrosine kinase. The results of these studies indicate that hydrogen peroxide-stimulated arachidonic acid release is mediated by a specific signal-responsive phospholipase A(2), and that this process is not mediated via the actions of either lipid peroxidation or calcium but, rather, that a stimulation of intracellular kinase activity is necessary for this response.