Direct evidence for the role of nitric oxide on the glutamate-induced neuronal death in cultured cortical neurons.

It has been reported that glutamate-induced neurotoxicity is related to an increase in nitric oxide (NO) concentration. An NO-sensitive electrode has been developed to measure NO concentration directly. Using this electrode, we examined NO concentration and neuronal survival after glutamate application in rat cultured cortical neurons. We also examined the effects of NMDA receptor antagonists, MK-801 and ketamine, and the NO synthetase inhibitor, L-NMMA on NO production and neuronal death. After 7 days in culture, application of glutamate (1 mM) or L-arginine (0.3 mM) to the cultured medium increased NO concentration, and decreased the number of anti-microtubule-associated protein 2 positive neurons. Both pretreatment with MK-801 (300 microns) and ketamine (300 microns) prevented glutamate-, but not L-arginine-induced increase in NO concentration and neuronal death. L-NMMA prevented both glutamate- and L-arginine-induced NO production and neuronal death. The nitric oxide donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP) also caused neuronal death, and MK-801, ketamine and L-NMMA did not prevent SNAP-induced toxicity. We have demonstrated excitatory amino acid-induced changes of NO concentration and the parallel relationship between changes of NO concentration and neuronal death. In conclusion, an increase in NO concentration does induce neuronal death, and the inhibition of the production of NO prevents glutamate-induced neuronal death.