Mechanism of Modification of the NR2A Subunit of the NMDA Receptor during Graded Hypoxia in the Cerebral Cortex of Newborn Piglets

Abstract 376

Previous studies have shown that phosphorylation of tyrosine and serine residues of the NR2A subunit of the NMDA receptor by protein kinases increases NMDA receptor activation and intracellular Ca⁺⁺ flux during normoxia in cultured rat neurons. During hypoxia, the NMDA receptor is modified resulting in increased receptor activation correlating with decreased cerebral energy metabolism. The present study tests the hypothesis that NMDA receptor modification during hypoxia is due to phosphorylation of the tyrosine, threonine and serine residues of the NR2A subunit of the NMDA receptor and that the degree of receptor phosphorylation is a function of the decrease in cerebral energy metabolism induced by hypoxia. Studies were performed in 15 anesthetized, ventilated newborn piglets. In the hypoxic group (n=9) varying degrees of cerebral energy metabolism were achieved by different concentrations of FiO₂ (5-9%) and documented biochemically by tissue phosphocreatine (PCr) levels. P₂ membrane proteins were immunoprecipitated with antiphosphoserine, antiphosphotyrosine or antiphosphothreonine antibodies and separated by 8% SDS-PAGE. Proteins were transblotted and probed with NR2A antibodies. The phosphorylated subunit proteins were visualized with horseradish peroxidase conjugate, analyzed by imaging densitometry (GS-700 Bio-Rad) and expressed as absorbance × mm². An internal standard was used as a reference. Results are expressed as percent of peak density for each gel. During hypoxia, there is an inverse linear relationship between tissue PCr levels and the % density of the NR2A subunit (r=0.7), phosphorylated tyrosine residues (r=0.7), and phosphorylated threonine residues (r=0.5) but not with phosphorylated serine residues. As tissue PCr values decrease from 3.5 to 0.5 µmol/g brain, the density of the NR2A subunit increases by 56%, however the densities for each of the phosphorylated residues do not increase to the same degree: phosphorylated tyrosine residues increase by 40%, phosphorylated threonine residues by 20% and phosphorylated serine residues by 27%. Due to the relative increase in density of the NR2A subunits during increasing hypoxia, the overall % of phosphorylated residues decreases - tyrosine by 29%, threonine by 64% and serine by 52%. The data demonstrate that as cerebral energy metabolism decreases, the ratio of phosphorylated / dephosphorylated tyrosine, threonine and serine residues of the NR2A subunit of the NMDA receptor decreases, indicating a shift to the left in the phosphorylation equilibrium of the NMDA receptor. We speculate that the decrease in phosphorylation of the NMDA receptor tyrosine, threonine and serine residues may be either due to a decrease in protein kinase activity or to a conformational change in the

(Funded by NIH HD-20337).