Modification of the Cerebral NMDA Receptor NR2B Subunit during Graded Hypoxia in Newborn Piglets

Abstract 1158 Poster Session II, Sunday, 5/2 (poster 66)

Previous studies have shown that phosphorylation of serine residues of the cerebral N-methyl-D-aspartate (NMDA) receptor by protein kinase C increases NMDA receptor activation in normoxic neuronal cultures. During hypoxia, the NMDA receptor is modified resulting in increased receptor activation that correlates with decreased cerebral energy metabolism. The present study tests the hypothesis that NMDA receptor modification during hypoxia is due to phosphorylation of the serine, tyrosine and threonine residues of the NR2B subunit of the NMDA receptor and that the degree of receptor phosphorylation correlates with decreased cerebral energy metabolism. Studies were performed in 15 anesthetized, ventilated newborn piglets. In the hypoxic group (n=9) varying degrees of cerebral energy metabolism were achieved by administration of different concentrations of FiO₂ (5-9%) and documented biochemically by tissue phosphocreatine (PCr) levels. P₂ membrane proteins were immunoprecipitated with antiphospho -serine, -tyrosine and -threonine antibodies and separated by 8% SDS-PAGE. Proteins were transblotted and probed with NR2B antibodies. 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 peak density for each gel. During hypoxia, there is an inverse linear relationship between tissue PCr levels and the % density of the NR2B subunit (r=0.6) and phosphorylated tyrosine residues (r=1.0), but not with phosphorylated threonine or serine residues. As tissue PCr decreases from 3.5 to 0.5 µmol/g brain, the density of the NR2B subunit increases by 35%, however the densities for each of the phosphorylated residues do not increase to the same degree: phosphorylated serine increases by 37%, threonine by 15% and tyrosine by 3%. Due to the relative increase in density of the NR2B subunits with increasing hypoxia, the overall density of phosphorylated serine residues increases by 6%, the density of phosphorylated threonine residues decreases by 57% and tyrosine decreases by 91%. The data demonstrate that there is preferential phosphorylation of the NR2B subunit of the NMDA receptor during graded hypoxia with the ratio of phosphorylated / dephosphorylated serine residues increasing but the ratio of phosphorylated / dephosphorylated threonine and tyrosine residues decreasing with increasing hypoxia. We speculate that the increase in phosphorylation of the serine residues of the NR2B subunit of the NMDA receptor may be due to an increase in protein kinase C activity and may result in receptor activation during hypoxia in the cerebral cortex of newborn piglets.

(Funded by NIH HD-20337).