An increased production of superoxide has been shown to mediate glutamate-induced neuron death. We monitored intracellular superoxide production of hippocampal neurons during and after exposure to the glutamate receptor agonist NMDA (300 microm). During a 30 min NMDA exposure, intracellular superoxide production increased significantly and remained elevated for several hours after wash-out of NMDA. After a 5 min exposure, superoxide production remained elevated for 10 min, but then rapidly returned to baseline. Mitochondrial membrane potential also recovered after wash-out of NMDA. However, recovery of mitochondria was transient and followed by delayed mitochondrial depolarization, loss of cytochrome c, and a secondary rise in superoxide production 4-8 hr after NMDA exposure. Treatment with a superoxide dismutase mimetic before the secondary rise conferred the same protection against cell death as a treatment before the first. The secondary rise could be inhibited by the complex I inhibitor rotenone (in combination with oligomycin) and mimicked by the complex III inhibitor antimycin A. To investigate the relationship between cytochrome c release and superoxide production, human D283 medulloblastoma cells deficient in mitochondrial respiration (rho(-) cells) were exposed to the apoptosis-inducing agent staurosporine. Treatment with staurosporine induced mitochondrial release of cytochrome c, caspase activation, and cell death in control and rho(-) cells. However, a delayed increase in superoxide production was only observed in control cells. Our data suggest that the delayed superoxide production in excitotoxicity and apoptosis occurs secondary to a defect in mitochondrial electron transport and that mitochondrial cytochrome c release occurs upstream of this defect.

Excitotoxic mechanisms are believed to be involved in the death of neurons after trauma, epileptic seizures and cerebral ischaemia. We investigated the role of mitochondrial superoxide production in excitotoxic cell death of cultured rat hippocampal neurons. Brief exposure to the selective glutamate agonist N-methyl-D-aspartate (NMDA; 100-300 microM, 10 min) induced significant neuronal death, which was sensitive to cycloheximide (1 microM) and the caspase-1 inhibitor, acetyl-Tyr-Val-Ala-Asp-chloromethylketone (10 microM). Intracellular superoxide production was monitored semiquantitatively on sister cultures from the same platings using the oxidation-sensitive probe, hydroethidine. Brief exposures to toxic NMDA concentrations induced significant increases in superoxide production which correlated with the degree of neuronal injury. However, subtoxic NMDA exposures also produced moderate, yet statistically significant increases in superoxide production. Both NMDA-induced superoxide production and neurotoxicity were reduced by inhibition of mitochondrial electron transport using either sodium cyanide (1 mM), or a combination of rotenone (2 microM) and oligomycin (2 microM). The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP, 1 microM) mimicked the effect of NMDA on mitochondrial superoxide production. Both NMDA-induced superoxide production and neurotoxicity were potentiated by FCCP (1 microM). Exposure to FCCP alone (1-10 microM, 10 min), however, failed to produce any toxicity. Our data suggest that mitochondrial superoxide production per se is not sufficient to trigger the degeneration of cultured hippocampal neurons, but that manipulation of mitochondrial activity alters NMDA-induced superoxide production and neurotoxicity.