NO MEDIATED MODIFICATION OF NMDA RECEPTOR DURING HYPOXIA

拟开展的研究将探讨缺氧诱导N-甲基-D-天冬氨酸(NMDA)受体修饰导致细胞内钙浓度升高并导致新生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将与缺氧的严重程度相关。体内脑缺氧的程度将通过31P-核磁共振波谱连续测量高能磷酸化合物并经生化证实来监测。我们认为，缺氧时NO介导的NMDA受体硝化改变了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。本实验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NMDA受体这些亚基硝基酪氨酸残基在脑内免疫组织化学分布的影响；(3)缺氧对突触膜3-硝基酪氨酸水平的影响；(4)NMDA受体亚基硝化程度与NMDA受体识别、辅助激活和离子通道位置特征改变的关系；(5)缺氧时N-甲基-D-天冬氨酸受体硝化增加与钙内流到突触神经体的关系；(6)定量组织缺氧与N-甲基-D-天冬氨酸受体NR1、NR2 A和NR2 B亚单位去磷酸化的关系；(7)去磷酸化对过氧亚硝酸盐介导的N-甲基-D-甲基-D-天冬氨酸受体亚单位随后硝化的影响；(8)体内应用一氧化氮合酶抑制剂N-硝基-L-精氨酸(N-NLA)对低氧诱导的一氧化氮自由基增加和N-甲基-D-天冬氨酸受体结构和功能变化的影响。拟议的实验将利用成熟的技术进行。这些研究将对NMDA受体功能的调节机制以及对缺氧诱导的NMDA受体修饰导致脑损伤的理解提供新的见解。阐明NMDA受体修饰对低氧反应的分子机制将有助于开发新的低氧所致新生儿脑功能障碍的预防策略。

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.