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

本研究旨在探讨一氧化氮（NO）在N-甲基-D-天冬氨酸（NMDA）受体介导的谷氨酸神经毒性中的作用。这些实验是用从17- 19日龄的大鼠胎儿中获得的原代视网膜培养物进行的。通过监测[^3H]精氨酸转化为[^3H]瓜氨酸，测得一氧化氮合酶（NOS）活性约为5 pmol/min/mg蛋白。将培养的细胞暴露于谷氨酸（1 mM）或NMDA（1 mM）10分钟，然后在正常培养基中孵育1小时，始终导致60%的细胞死亡。同时加入NOS抑制剂N^<omega>-硝基-L-精氨酸（300 μ M）和谷氨酸盐或NMDA可使细胞死亡减少70%。将细胞暴露于硝普钠（SNP，500 μ M）或S-亚硝基半胱氨酸（SNOC，500 μ M），NO产生剂，导致60%的细胞死亡。通过减少血红蛋白消耗NO防止由谷氨酸、NMDA或NO生成剂诱导的细胞死亡。单独的5 μ M SNOC对细胞活力没有影响。然而，50 mM SNOC预处理以及同时应用50 μ M SNOC与NMDA抑制NMDA诱导的细胞死亡。使用膜片钳技术的电生理研究表明NO抑制NMDA受体本身。这些结果表明，低浓度的NO通过关闭NMDA受体门控离子通道对谷氨酸神经毒性起保护作用。然而，浓度升高的NO，与氧自由基相互作用，成为有毒的和介导谷氨酸诱导的神经毒性在培养的视网膜神经元。

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.