Expression and Phosphorylation of N-Methyl-D-Aspartate Receptor Subunits during Graded Hypoxia in the Cerebral Cortex of Newborn Piglets

The present study tests the hypothesis that during graded hypoxia, N-methyl-D-aspartate (NMDA) receptor expression and phosphorylation are altered in the cerebral cortex of newborn piglets. Studies were performed in anesthetized, ventilated piglets, 6 normoxic and 9 exposed to different lengths of decreased fractions of inspired oxygen to achieve varying biochemical levels of phosphocreatine (PCr). P 2 membrane proteins were immunoprecipitated with antiphosphoserine, antiphosphotyrosine, or antiphosphothreonine antibodies and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were transblotted and probed with NMDA receptor subunit 1 (NR1), NR2A or NR2B antibodies. As tissue PCr levels decreased from 3.5 to 0.5 µmol/g brain during hypoxia, NR1, NR2A and NR2B protein increased by 84, 56 and 38%, respectively. Phosphorylated serine, tyrosine and threonine residues also increased during hypoxia on the three subunits. However, the increase in subunit protein exceeded the increase in phosphorylated residues for all three subunits. Therefore, the ratio of phosphorylated/dephosphorylated serine, tyrosine and threonine residues decreased with worsening hypoxia. We speculate that an alteration in the ratio of phosphorylated/dephosphorylated residues of the NMDA receptor may regulate receptor activation during hypoxia.