NO MEDIATED MODIFICATION OF NMDA RECEPTOR DURING HYPOXIA

这些研究将探讨缺氧诱导的n -甲基- d -天冬氨酸（NMDA）受体修饰导致细胞内钙离子浓度升高并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。体内脑缺氧程度将通过31p核磁共振波谱连续测量高能磷酸盐化合物并进行生化确认来监测。我们认为缺氧时一氧化氮介导的NMDA受体的硝化改变了NMDA受体的识别、共激活和离子通道位点的特征，而缺氧诱导的受体酪氨酸残基的去磷酸化为硝化提供了位点。实验方案将在新生仔猪上进行研究：(1)定量组织缺氧与NMDA受体NR1、NR2A和NR2B亚基酪氨酸残基硝化的关系；(2)缺氧对脑内NMDA受体亚基硝基酪氨酸残基免疫组化分布的影响；(3)缺氧对突触膜3-硝基酪氨酸水平的影响；(4) NMDA受体亚基的硝化水平与NMDA受体识别、共激活剂和离子通道位点特征改变的关系；(5)缺氧时NMDA受体的硝化作用增加与Ca++流入突触体的关系；(6)定量组织缺氧与NMDA受体NR1、NR2A、NR2B亚基去磷酸化的关系；(7)去磷酸化对随后过氧亚硝酸盐介导的NMDA受体亚基硝化的影响；(8)体内给药一氧化氮合酶抑制剂n -硝基- l -精氨酸（NNLA）对缺氧诱导的一氧化氮自由基增加和NMDA受体结构和功能变化的影响。所提议的实验将利用成熟的技术进行。这些研究将为NMDA受体功能的调控机制和缺氧诱导的NMDA受体修饰导致脑损伤的理解提供新的见解。阐明NMDA受体修饰对缺氧反应的分子机制将有助于开发新的预防新生儿缺氧引起的脑功能障碍的策略。

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.