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 bo vine 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 1 5 minutes after the addition of 100 mu M hydrogen peroxide. Hydrogen p eroxide-stimulated release of arachidonic acid was blocked with the ph ospholipase A(2) inhibitor quinacrine. Treatment of the cells with hyd rogen peroxide did not result in liberation of oleic acid, indicating that hydrogen peroxide exercised its effect on an arachidonate-specifi c phospholipase. Pretreatment of the cells with antioxidants, transiti on metal chelators, and hydroxyl radical scavengers did not affect the hydrogen peroxide-stimulated arachidonic acid release, indicating tha t the response to hydrogen peroxide is not oxygen radical-mediated. Th e response to hydrogen peroxide does not appear to be calcium-dependen t, due to me following two observations: (a) No increase in intracellu lar calcium was seen upon exposure of the FURA2-loaded cells to hydrog en 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 w ith inhibitors of protein kinase C, but not with inhibitors of tyrosin e 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 v ia the actions of either lipid peroxidation or calcium but, rather, th at a stimulation of intracellular kinase activity is necessary for thi s response.