NMDA-INDUCED SUPEROXIDE PRODUCTION AND NEUROTOXICITY IN CULTURED RAT HIPPOCAMPAL-NEURONS - ROLE OF MITOCHONDRIA

Excitotoxic mechanisms are believed to be involved in the death of neu rons after trauma, epileptic seizures and cerebral ischaemia. We inves tigated 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 mu M, 10 min) induced significant neuronal death, which was sensitive to cy cloheximide (1 mu M) and the caspase-1 inhibitor, acetyl-Tyr-Val-Ala-A sp-chloromethylketone (10 mu M). Intracellular superoxide production w as monitored semiquantitatively on sister cultures from the same plati ngs using the oxidation-sensitive probe, hydroethidine. Brief exposure s to toxic NMDA concentrations induced significant increases in supero xide production which correlated with the degree of neuronal injury. H owever, subtoxic NMDA exposures also produced moderate, yet statistica lly 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), o r a combination of rotenone (2 mu M)and oligomycin (2 mu M). The mitoc hondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP, 1 mu M) mimicked the effect of NMDA on mitochondrial superoxid e production. Both NMDA-induced superoxide production and neurotoxicit y were potentiated by FCCP (1 mu M). Exposure to FCCP alone (1-10 mu M , 10 min), however, failed to produce any toxicity. Our data suggest t hat mitochondrial superoxide production per se is not sufficient to tr igger the degeneration of cultured hippocampal neurons, but that manip ulation of mitochondrial activity alters NMDA-induced superoxide produ ction and neurotoxicity.