Inhibition of hydrogen peroxide-induced apoptosis but not arachidonic acid release in GH3 cell by EGF.

Reactive oxygen species (ROS) and arachidonic acid (AA) can both function as extra- and intra-cellular messengers to regulate various cell functions including cell death. The effect of ROS on phospholipase A2 (PLA2) activity and/or AA release has not been extensively studied in neuronal cells. In this study, we investigated the effects of H2O2 on AA release and apoptosis in GH3 cells, a clonal strain from rat anterior pituitary. Incubation with H2O2 for 1 h stimulated [3H]AA release in a concentration-dependent manner from prelabeled GH3 cells. [3H]AA release was inhibited by arachidonyl trifluoromethyl ketone, a specific inhibitor of cytosolic PLA2, and cytosolic PLA2 protein with a molecular mass of 100 kDa was detected by immunoblotting. Culture with 0.2 mM H2O2 and 30 microM AA for 24 h induced lactate dehydrogenase (LDH) leakage, DNA laddering and DNA fragmentation in GH3 cells. In GH3 cells pretreated with EGF (50 ng/ml) for 24 h, LDH leakage and DNA fragmentation by H2O2 and AA were inhibited, although H2O2-induced [3H]AA release was not modified. Mastoparan, a wasp venom peptide, induced [3H]AA release and cell death in GH3 cells. Neither effect of mastoparan was inhibited by EGF treatment. These findings suggest that (1) H2O2 stimulates AA release via activation of cytosolic PLA2, (2) H2O2 and AA induce apoptotic death of GH3 cells and (3) treatment with EGF protects H2O2- and AA-, but not mastoparan-, induced GH3 cell death.