Hydrogen peroxide-mediated phosphorylation of ERK1/2, Akt/PKB and JNK in cortical neurones:: dependence on Ca2+ and PI3-kinase

Primary cortical neurones exposed to an oxidative insult in the form of hydrogen peroxide (H2O2) for 30 min showed a concentration-dependent increase in oxidative stress followed by a delayed NMDA receptor-dependent cell death measured 24 h later. Extracellular signal-regulated protein kinase (ERK1/2), c-jun N-terminal kinase (JNK) and the kinase Akt/PKB may regulate neuronal viability in response to oxidative insults. Using phospho-specific antibodies, a 15-min stimulation of neurones with H2O2 (100 muM-1 mm) produced a concentration-dependent phosphorylation of ERK1/2 and Akt/PKB that was partly dependent on extracellular Ca2+ and phosphatidylinositol 3-kinase (PI3-K). Higher concentrations of H2O2 (1 mm) also stimulated a phosphorylation of JNK which was totally dependent on extracellular Ca2+ but not PI3-K. H2O2-induced phosphorylation of ERK1/2, Akt/PKB or JNK were unaffected by the NMIDA channel blocker MK801. Blocking ERK1/2 activation with the upstream inhibitor U0126 (10 muM) enhanced H2O2-induced (100-300 muM range) neurotoxicity and inhibited H2O2-mediated phosphorylation of the cyclic AMP regulatory binding protein (CREB), suggesting that ERK1/2 signals to survival under these conditions. At higher concentrations (mm), H2O2-Stimulated a phosphorylation of c-jun. It is likely, therefore, that subjecting neurones to moderate oxidative-stress recruits pro-survival signals to CREB but during severe oxidative stress pro-death signals through JNK and c-jun are dominant.