Inhibition of hydrogen peroxide-induced apoptosis but not arachidonic acidrelease in GH3 cell by EGF

Reactive oxygen species (ROS) and arachidonic acid (AA) can both function a s extra- and intra-cellular messengers to regulate various cell functions i ncluding cell death. The effect of ROS on phospholipase A(2) (PLA(2)) activ ity and/or AA release has not been extensively studied in neuronal cells. I n this study, we investigated the effects of H2O2 on AA release and apoptos is in GH3 cells, a clonal strain from rat anterior pituitary. Incubation wi th H2O2 for 1 h stimulated [H-3]AA release in a concentration-dependent man ner from prelabeled GH3 cells. [H-3]AA release was inhibited by arachidonyl trifluoromethyl ketone, a specific inhibitor of cytosolic PLA(2), and cyto solic PLA(2) protein with a molecular mass of 100 kDa was detected by immun oblotting. Culture with 0.2 mM H2O2 and 30 mu M AA for 24 h induced lactate dehydrogenase (LDH) leakage, DNA laddering and DNA fragmentation in GH3 ce lls. 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 [H-3 ]AA release was not modified. Mastoparan, a wasp venom peptide, induced [H- 3]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 PLA(2), (2) H2O2 and AA induce apop totic death of GH3 cells and (3) treatment with EGF protects H2O2- and AA-, but not mastoparan-, induced GH3 cell death. (C) 1999 Elsevier Science B.V . All rights reserved.