Oxidative stress inhibits ionomycin-mediated cell death in cortical neurons

Thiol‐proteases play important roles in many cellular processes, including maintenance of protein homeostasis and execution of cell death. Therefore, determining how this family of enzymes is regulated is critical for our understanding of both physiological and pathological conditions. Because these proteases require a reduced cysteine residue for activity, the cellular redox state plays a crucial role in regulating the activity of thiol proteases. Importantly, increased oxidative stress can result in the direct modification of the active site cysteine, leading to enzyme inactivation. This would suggest that oxidative stress that occurs during pathological insults could prolong cell survival by preventing the execution of thiol‐protease‐dependent cell death pathways. To test this hypothesis, cultured rat cortical neurons were treated with the oxidizing agent diamide or doxorubicin in the presence or absence of the calcium ionophore ionomycin. Under normoxic conditions, ionomycin treatment resulted in ∼70% cell death, which was prevented by addition of the calpain‐selective inhibitor benyzloxycarbonyl‐Leu‐Leu‐Tyr fluoromethylketone. Similarly, pretreatment of neurons with either oxidant was also protective. Protection resulting from oxidative stress was not due to new protein synthesis, insofar as cycloheximide did not affect oxidant‐mediated protection. Interestingly, treatment with the antioxidant Trolox to reverse or prevent oxidative stress blocked the protective effects of both oxidants against ionomycin‐induced cell death. We interpret these findings to suggest that, in diseases or conditions in which oxidative stress is increased, the ability of thiol‐proteases to execute cell death pathways fully is decreased and may prolong cell survival. © 2004 Wiley‐Liss, Inc.