Effects of various nitric oxide synthase inhibitors on NMDA-induced neuronal injury in rat cortical neurons

Using an in vitro primary cell culture model in which cortical neurons undergo a gradual and delayed neuronal death after a brief (5min) challenge with glutamate receptor agonist N-methyl-Daspartate (NMDA, 300μM), the neuroprotective effects of various nitric oxide synthases (NOS) inhibitors were compared with that of the NMDA receptor antagonist dizocilpine maleate (MK-801). Our rat cortical cultures consisted of approximately 80-96% neurons and 5-20% astroglia as determined by immunocytochemical staining with antibodies against glial fibrillary acidic protein (GFAP) or neuron specific enolase (NSE). The delayed type of NMDA-induced neurotoxicity was examined by the morphological estimate of cell injury and was further confirmed by the activity of lactate dehydrogenase (LDH) in the extracellular fluid measured 24 hrs after the 5-min NMDA exposure. The accumulation of nitrite, the stable metabolite of nitric oxide (NO), was also measured 24 hrs after the 5-min NMDA exposure. The brief NMDA exposure caused about 60% neuronal death, as compared with persist (24 hr) NMDA exposure at 24 hr after NMDA exposure. Effects of drugs were studied by pretreating the cultures for 10mins prior to the induction of NMDA neurotoxicity. Both the nonselective NOS inhibitor N(superscript ω)-nitro-L-arginine methyl ester (L-NAME, 100μM) and the selective neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindozale (7-NI, 100μM) suppressed nitrite accumulation and attenuated neuronal damage induced by NMDA. However, the selective inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine (AG, 100μM) exhibited no neuroprotective effects and no reduction in the nitrite production. The NMDA-induced neurotoxocity and nitrite production was abolished by pretreatment with the NMDA receptor antagonist MK-801 (100μM). Thus the results indicate that a brief NMDA exposure leads to delayed neuronal damage with concomitant increase in NO production in cortical neuronal cultures. We suggest that the NO may originate primarily from nNOS. The neuroprotective effects of NOS inhibitors are weaker than that of MK-801.