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

Abstract

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 A 2 (PLA 2) activity and/or AA release has not been extensively studied in neuronal cells. In this study, we investigated the effects of H 2O 2 on AA release and apoptosis in GH3 cells, a clonal strain from rat anterior pituitary. Incubation with H 2O 2 for 1 h stimulated [ 3 H ]AA release in a concentration-dependent manner from prelabeled GH3 cells. [ 3 H ]AA release was inhibited by arachidonyl trifluoromethyl ketone, a specific inhibitor of cytosolic PLA 2, and cytosolic PLA 2 protein with a molecular mass of 100 kDa was detected by immunoblotting. Culture with 0.2 mM H 2O 2 and 30 μM 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 H 2O 2 and AA were inhibited, although H 2O 2-induced [ 3 H ]AA release was not modified. Mastoparan, a wasp venom peptide, induced [ 3 H ]AA release and cell death in GH3 cells. Neither effect of mastoparan was inhibited by EGF treatment. These findings suggest that (1) H 2O 2 stimulates AA release via activation of cytosolic PLA 2, (2) H 2O 2 and AA induce apoptotic death of GH3 cells and (3) treatment with EGF protects H 2O 2- and AA-, but not mastoparan-, induced GH3 cell death.