Role of Nitric Oxide in neuronal death and its modulation by neuroprotective factors

本研究旨在阐明脑血管疾病、阿尔茨海默病等中枢神经系统退行性疾病中神经元死亡的机制。我们利用培养的大鼠皮质神经元，研究了一氧化氮(NO)介导的谷氨酸神经毒性的机制以及尼古丁对谷氨酸神经毒性的保护作用。1.检测了其对尼古丁的保护作用。当谷氨酸或N-甲基-D-天冬氨酸(NMDA)短暂作用于培养物时，细胞存活率明显降低。在谷氨酸暴露前用尼古丁(10µM)孵育培养物可降低其细胞毒性。尼古丁对谷氨酸神经毒性的保护作用可被烟碱型乙酰胆碱受体特异性拮抗剂六甲溴铵所拮抗。短暂接触钙离子载体离子霉素和一氧化氮供体S亚硝基半胱氨酸均可诱导迟发性神经元死亡。尼古丁预防离子霉素诱导的Cy…在不影响SNOC诱导的细胞毒性的情况下，TO毒性更大。这些结果表明，刺激烟碱受体可通过减少NO的生成而对谷氨酸细胞毒性产生神经保护作用。2.我们先前已经证明抗磷脂酶C-Delta(PLC-Delta)抗体在阿尔茨海默病患者的脑内产生神经纤维缠结、围绕老年斑核心的轴突和神经纤维线的强烈染色。用抗PLC-Delta抗体检测谷氨酸对PLC-Delta免疫反应的影响。暴露于谷氨酸后，抗PLC-Delta抗体的免疫染色增强。N-甲基-D-天冬氨酸受体拮抗剂MK-801和一氧化氮合酶抑制剂N-硝基-L-精氨酸均可阻止PLC-Delta免疫反应性的增加。这些结果表明，谷氨酸激活NMDA受体后继发的NO形成导致PLC-Delta的类似于阿尔茨海默病的修饰。本研究结果将为推动神经保护药物的开发研究提供基础资料。较少

The purpose of this study was to elucidate the mechanism underlying neuronal death in neurodegeneration diseases in central nervous system (CNS), such as cerebrovascular disease and Alzheimer's disease. We studied the mechanisms underlying glutamate neurotoxicity mediated by nitric oxide (NO) and protective effect of nicotine against glutamate neurotoxicity using cultured rat cortical neurons. 1.We examined the protective effect on nicotine. The cell viability was markedly reduced when cultures were briefly exposed to glutamate or N-methyl-D-aspartate (NMDA). Incubating the cultures with nicotine (10muM) prior to glutamate exposure reduced its cytotoxicity. The neuroprotective effect of nicotine against glutamate neurotoxicity was antagonized by hexamethonium, an antagonist specific to nicotinic acetylcholine receptor. Both brief exposures to ionomycin, a calcium ionophore, and S-nitrosocysteine (SNOC), an NO donor, induced delayd neuronal death. Nicotine prevented ionomycin-induced cy … More totoxicity without affecting SNOC-induced cytotoxicity. These results suggest that the nicotinic receptor stimulation induces neuroprotection against glutamate cytotoxicity by reducing NO-formation. 2.We have previously shown that anti-phospholipase C-delta (PLC-delta) antibody produced intense staining of neurofibrillary tangles, the neurite surrounding senile plaque cores and neuropil threads in the brain of patients with Alzheimer's disease. We examined the influence of glutamate on PLC-delta immunoreactivity in the cultures using anti-PLC-delta antibody. Exposure to glutamate exhibited increased immunostaining with the anti-PLC-delta antibody. The increase in PLC-delta immunoreactivity was prevented by both application of MK-801, an NMDA receptor antagonist, and N^<omega>-nitro-L-arginine, an NO synthase inhibitor. These results suggest that NO formation secondary to NMDA receptor activation by glutamate leads to similar modifications of PLC-delta to those seen in Alzheimer's disease. The results in this study will offer basic materials to drive forward developmental research for neuroprotective drugs. Less