Oxidative stress induced by glutathione depletion in the mouse HT22 neuroblastoma cell line and embryonic rat immature cortical neurons causes a delayed, sustained activation of extracellular signal-regulated kinase (ERK) 1/2, which is required for cell death. This sustained activation of ERK1/2 is mediated primarily by a selective inhibition of distinct ERK1/2-directed phosphatases either by enhanced degradation (i.e., for mitogenactivated protein kinase phosphatase-1) or as shown here by reductions in enzymatic activity (i.e., for protein phosphatase type 2A). The inhibition of ERK1/2 phosphatases in HT22 cells and immature neurons subjected to glutathione depletion results from oxidative stress because phosphatase activity is restored in cells treated with the antioxidant butylated hydroxyanisole. This leads to reduced ERK1/2 activation and neuroprotection. Furthermore, an increase in free intracellular zinc that accompanies glutathioneinduced oxidative stress in HT22 cells and immature neurons contributes to selective inhibition of ERK1/2 phosphatase activity and cell death. Finally, ERK1/2 also functions to maintain elevated levels of zinc. Thus, the elevation of intracellular zinc within neurons subjected to oxidative stress can trigger a robust positive feedback loop operating through activated ERK1/2 that rapidly sets into motion a zinc-dependent pathway of cell death.

Oxidative stress after cerebral ischemia and reperfusion activates extracellular signal-regulated kinases (ERK) in brain. However, the mechanism of this activation has not been elucidated. We have previously reported that in an in vitro model of oxidative stress in immature cortical neuronal cultures, the inhibition of ERK phosphatase activity contributes to ERK1/2 activation and subsequent neuronal toxicity. This study examined whether ERK activation was associated with altered activity of ERK phosphatases in a rat cardiac arrest model. Rats in experimental groups were subjected to asphyxial cardiac arrest for 8 min and then resuscitated for 30 min. Significant ERK activation was detected in both cortex and hippocampus following ischemia/reperfusion by immunoblotting. ERK phosphatase activity was reversibly inhibited in cerebral cortex but not affected in hippocampus following ischemia/reperfusion. MEK1/2 was activated in both cerebral cortex and hippocampus following ischemia/reperfusion. Using a specific inhibitor of protein phosphatase 2A (PP2A), okadaic acid (OA), we have identified PP2A to be the major ERK phosphatase that is responsible for regulating ERK activation in ischemic brain tissues. Orthovanadate inhibited ERK phosphatase activity in brain tissues, suggesting that tyrosine phosphatases and dual specificity phosphatases may also contribute to the ERK phosphatase activity in brain tissues. Together, these data implicate ERK phosphatase in the regulation of ERK activation in distinct brain regions following global ischemia.