Phosphorylation of Serine Residues of the NR1 Subunit of the NMDA Receptor During Graded Hypoxia in the Cerebral Cortex of Newborn Piglets † 195

Previous studies have shown that N-methyl-D-aspartate (NMDA) receptor - mediated intracellular Ca⁺⁺ flux increases with receptor phosphorylation by protein kinase C (PKC) during normoxia and increases with the hypoxia-induced decrease in cerebral energy metabolism. The present study tests the hypothesis that the serine residues of the NMDA receptor NR1 subunit are phosphorylated during hypoxia, and that the degree of phosphorylation correlates with the decrease in cerebral energy metabolism induced by hypoxia. Studies were performed in 3 normoxic (Nx) and 3 hypoxic (Hx) ventilated newborn piglets. In the Hx group varying degrees of cerebral energy metabolism were achieved by administration of different concentrations of O₂(5-9%) and documented biochemically by tissue ATP and phosphocreatine (PCr) levels. P₂ membrane proteins were immunoprecipitated with antiphosphoserine antibodies and separated by 8% SDS-PAGE. Proteins were transblotted and probed with NR1 antibody. The phosphorylated NR1 subunit protein was visualized with horseradish peroxidase conjugate. Protein bands were analyzed by imaging densitometry and expressed as absorbance/mm². During hypoxia, absorbance/mm² of the phosphorylated serine NR1 residues was 1.7±0.7, significantly greater than in the Nx group 0.6±0.1,(p< 0.05). The ATP, PCr and absorbance/mm² of phosphorylated serine residues were as follows: (5.9, 0.7, 1.4), (5.2, 1.2, 0.5), (4.9, 2.0, 0.7),(4.6, 0.6, 0.8), (4.4, 0.7, 2.4) and (0.0, 0.0, 2.2), respectively, demonstrating an inverse linear correlation between both ATP (r=0.5) and PCr(r=0.7) levels and phosphorylation of NR1 serine residues during graded hypoxia. The data suggest that as cerebral energy metabolism decreases, phosphorylation of serine residues of the NR1 subunit of the NMDA receptor increases, with phosphorylation and potential activation of the NMDA receptor increasing despite a decrease in tissue high energy phosphates. We speculate that increased phosphorylation of the NMDA receptor serine residues may be due to either an increase in PKC phosphorylation or to conformational change in the receptor exposing new serine residues to PKC. Phosphorylation of the NMDA receptor may be a potential mechanism of receptor activation and intracellular Ca⁺⁺ flux during hypoxia.