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.
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