NO MEDIATED MODIFICATION OF NMDA RECEPTOR DURING HYPOXIA

缺氧期间 NMDA 受体没有介导的修饰

• 批准号: 6182595
• 负责人: OM P MISHRA
• 金额: $ 24.04万
• 依托单位: MCP HAHNEMANN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2002-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6182595
• 关键词: NMDA receptors; arginine; calcium flux; calcium ion; cerebral cortex; cerebral ischemia /hypoxia; glutamates; immunocytochemistry; immunoprecipitation; neurons; neurotoxicology; newborn animals; nitric oxide; nitric oxide synthase; nuclear magnetic resonance spectroscopy; oxidative phosphorylation; peroxynitrites; phosphorylation; protein structure function; radiotracer; receptor binding; swine; synapses; western blottings

The proposed studies will investigate molecular mechanisms of hypoxia-induced modification of the N-methyl-D-aspartate (NMDA) receptor that lead to increased intracellular Ca++ concentration and result in neuronal injury in the newborn. We propose that alteration of the NMDA receptor ion-channel structure and function will correlate with the severity of hypoxia. The degree of brain hypoxia in vivo will be monitored by continuous measurement of high energy phosphate compounds with 31P-nuclear magnetic resonance spectroscopy and confirmed biochemically. We propose that NO-mediated nitration of the NMDA receptor during hypoxia alters the characteristics of the recognition, co-activator and ion-channel sites of the NMDA receptor and that hypoxia-induced dephosphorylation of the tyrosine residues of the receptor provides sites for nitration. Experimental protocols will be carried out on newborn piglets investigating: (1) the relationship of quantitative tissue hypoxia to nitration of tyrosine residues of the NR1, NR2A and NR2B subunits of the NMDA receptor; (2) the effect of hypoxia on the immunohistochemical distribution of nitrotyrosine residues of these subunits of the NMDA receptor in the brain; (3) the effect of hypoxia on levels of 3-nitrotyrosine in synaptic membrane; (4) the relationship between the level of nitration of NMDA receptor subunits and alteration of characteristics of the recognition, co-activator and ion-channel site of the NMDA receptor; (5) the relationship of increased nitration of the NMDA receptor during hypoxia to Ca++-influx into synaptoneurosomes; (6) the relationship of quantitative tissue hypoxia to the dephosphorylation of the NR1, NR2A and NR2B subunits of the NMDA receptor; (7) the effect of dephosphorylation on subsequent peroxynitrite-mediated nitration of the NMDA receptor subunits; and (8) the effect of the in vivo administration of nitric oxide synthase inhibitor, N-nitro-L- arginine (NNLA) on hypoxia-induced increase in nitric oxide free radicals and changes in NMDA receptor structure and function. The proposed experiments will be performed by utilizing well established techniques. These studies will provide new insights into the mechanisms of regulation of NMDA receptor function and to the understanding of hypoxia-induced modification of the NMDA receptor that lead to brain injury. The elucidation of molecular mechanisms of NMDA receptor modification in response to hypoxia will aid in the development of novel preventive strategies for hypoxia-induced brain dysfunction in the newborn.

拟议的研究将探讨缺氧诱导的N-甲基-D-天冬氨酸（NMDA）受体的修饰，导致细胞内Ca++浓度增加，导致新生儿神经元损伤的分子机制。 我们认为NMDA受体离子通道结构和功能的改变与缺氧的严重程度有关。 将通过使用31 P-核磁共振光谱连续测量高能磷酸盐化合物来监测体内脑缺氧的程度，并进行生化确认。 我们建议，NO介导的硝化的NMDA受体在缺氧过程中改变的识别，共激活剂和离子通道的NMDA受体的网站和缺氧诱导的去磷酸化的受体的酪氨酸残基的特点提供硝化的网站。 本实验以新生仔猪为研究对象，研究了（1）组织缺氧与NMDA受体NR 1、NR 2A和NR 2B亚基酪氨酸残基硝化的关系，（2）缺氧对NMDA受体NR 1、NR 2A和NR 2B亚基硝基酪氨酸残基在脑组织中分布的影响，（3）缺氧对NMDA受体NR 1、NR 2A和NR 2B亚基硝基酪氨酸残基在脑组织中分布的影响，（4）缺氧对NMDA受体NR 1、NR 2A和NR 2B亚基硝基酪氨酸残基的影响。（3）缺氧对突触膜3-硝基酪氨酸水平的影响：（4）NMDA受体亚单位的硝化水平与NMDA受体的识别、辅激活和离子通道位点特性改变的关系;（5）缺氧时NMDA受体硝化增加与突触体Ca ~（++）内流的关系，（6）定量组织缺氧与NMDA受体NR 1、NR 2A和NR 2B亚基去磷酸化的关系;（7）去磷酸化对随后的过氧亚硝酸盐介导的NMDA受体亚单位硝化的影响;和（8）体内施用一氧化氮合酶抑制剂的效果，N-硝基-L-精氨酸（NNLA）对缺氧诱导的一氧化氮自由基增加及NMDA受体结构和功能的改变。将利用完善的技术进行拟定实验。 这些研究将为了解NMDA受体功能的调节机制和理解缺氧诱导的NMDA受体修饰导致脑损伤提供新的见解。 阐明NMDA受体对缺氧反应的分子机制将有助于开发新的预防策略，以预防新生儿缺氧诱导的脑功能障碍。