The study of the intracellular mechanism of retinal neuronal death caused by ischemia

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This study was performed to elucidate the role of nitric oxide (NO) in N-methyl-D-aspartate (NMDA) -receptor mediated glutamate neurotoxicity in the retina. The experiments were done with primary retinal cultures obtained from 17-to 19-day-old rat fetuses. The nitric oxide synthase (NOS) activity measured by monitoring the conversion of [^3H] arginine to [^3H] citrulline was approximately 5 pmol/min/mg protein. A 10-min exposure of the cultured cells to glutamate (1 mM) or NMDA (1 mM) followed by a 1-h incubation in a normal medium consistently resulted in 60% cell death. Concomitant addition of an inhibitor of NOS,N^<omega>-nitro-L-arginine (300 muM) , with glutamate or NMDA reduced cell death by 70%. A briet exposure of the cells to sodium nitroprusside (SNP,500 muM) or S-nitrosocysteine (SNOC,500 muM) , NO generating agents, caused 60% cell death. Depletion of NO by reduced hemoglobin prevented the cell death induced by either glutamate, NMDA,or NO generating agents. Fiffy muM SNOC alone had no effect on the cell viability. However, pretreatment with 50 mM SNOC as well as simultaneous application of 50 muM SNOC with NMDA inhibited cell death induced by NMDA.Electrophysiological study using a patch-clamp technique demonstrated NO inhibited NMDA-receptor itself. These findings indicate that a low concentration of NO plays a protective role in glutamate neurotoxicity via closing the NMDA receptor gated ion channel. However, elevated concentrations of NO,interacting with oxygen radicals, become toxic and mediate glutamate-induced neurotoxicity in the cultured retinal neurons.