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

缺血引起视网膜神经元死亡的细胞内机制研究

基本信息

批准号：
06404062
负责人：
HONDA Yoshihito
金额：
$ 17.86万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

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.

本研究旨在探讨一氧化氮(NO)在N-甲基-D-天冬氨酸(NMDA)受体介导的视网膜谷氨酸神经毒性中的作用。这些实验是用从17到19天大鼠胚胎获得的原代视网膜培养进行的。通过监测精氨酸向瓜氨酸的转化，测得一氧化氮合酶(NOS)活性约为5pmol/min/mg蛋白。培养的细胞在谷氨酸(1 MM)或NMDA(1 MM)中暴露10分钟，然后在正常培养液中孵育1小时，始终导致60%的细胞死亡。同时加入一氧化氮合酶抑制剂N^&lt；omega&gt；-硝基-L-精氨酸(300um)，与谷氨酸或N-甲基-D-天冬氨酸一起使细胞死亡减少70%。将细胞短暂暴露于硝普钠(SNP，500um)或S-亚硝半胱氨酸(Snoc，500um)，可导致60%的细胞死亡。还原的血红蛋白耗尽NO可防止谷氨酸、NMDA或NO生成剂诱导的细胞死亡。FIFFY MUM SNOC单独作用对细胞活力无影响。然而，50 mM SNOC预处理以及50 mM SNOC与NMDA同时作用可抑制NMDA诱导的细胞死亡。膜片钳电生理研究表明，NMDA受体本身不受抑制。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到保护作用。然而，NO浓度升高，与氧自由基相互作用，成为有毒的，并介导谷氨酸诱导的神经毒性在培养的视网膜神经元。