ANTAGONISM BY NG-NITRO-L-ARGININE OF L-GLUTAMATE-INDUCED NEUROTOXICITY IN CULTURED NEONATAL RAT CORTICAL-NEURONS - PROLONGED APPLICATION ENHANCES NEUROPROTECTIVE EFFICACY

The effects of N(G)-nitro-L-arginine on L-glutamate-induced neurotoxicity have been evaluated on primary cultures of neonatal rat cortical neurons. Treatment of cultures with increasing concentrations of L-glutamate during 5 min produced a delayed neuronal death, as measured by lactate dehydrogenase release in the medium 24 h later. Maximal toxicity was obtained with 500 mu M Of L-glutamate. Substantial nitric oxide synthase activity was detected in these cortical cultures. Nitric oxide synthase activity and cellular L-glutamate-induced cyclic guanosine 3',5'-monophosphate accumulation were totally inhibited by 100 mu M N(G)-nitro-L-arginine. Addition of N(G)-nitro-L-arginine (100 mu M) to the medium either 5 min prior to and during L-glutamate exposure (500 mu M, 5 min) or for 24 h after L-glutamate exposure decreased the amino acid-induced neurotoxicity by 23% (not significant) and 43%, respectively. When added 5 min before L-glutamate and just after L-glutamate removal and kept in contact with neurons for the following 24 h, N(G)-nitro-L-arginine (100 mu M) antagonized by 74% the L-glutamate-induced neurotoxicity. This effect was not reversed by a co-application Of L-arginine (1 mM). The neuroprotective effect of N(G)-nitro-L-arginine was concentration-dependent, a half-maximal inhibition of L-glutamate-induced neurotoxicity being observed with the addition (before and after L-glutamate) of 4 mu M of the drug. These results suggest that the neuroprotective effect of N(G)-nitro-L-arginine previously observed in vivo is exerted at the neuronal level. The increased neuroprotective efficacy of N(G)-nitro-L-arginine, with prolonged application indicates that a nitric oxide-dependent delayed neuronal death is taking place during the maturation phase that folloWS L-glutamate exposure.