Previous studies have shown that during hypoxia the N-methyl-D-aspartate(NMDA) receptor ion-channel complex is modified, resulting in activation of the receptor and cellular injury through intracellular Ca++ flux. The degree of NMDA receptor modification correlates with the decrease in cerebral energy metabolism induced by hypoxia. The present study tests the hypothesis that expression of the NR1, NR2A and NR2B subunits of the NMDA receptor increases as cerebral energy metabolism decreases. Studies were performed in 3 normoxic (Nx) and 7 hypoxic (Hx) ventilated piglets. In the Hx group varying degrees of cerebral energy metabolism were achieved by administration of different concentrations of O2 (5-9%) for 20 min to 1 hr, and were documented biochemically by tissue ATP and phosphocreatine (PCr) levels. Immunoprecipation of NR1, NR2A and NR2B NMDA receptor subunits was performed with primary rat, mouse and mouse antibodies respectively, and separated using 8% SDS-PAGE. Subunit expression was quantified by imaging densitometry and expressed as absorbance × mm2. During hypoxia, expression(absorbance × mm2) of the NR1 (4.6±1.5 Hx vs 0.4±0.0 Nx, p <0.01), NR2A (5.0±1.2 Hx vs 1.7±0.0 Nx, p<0.05) and NR2B (3.1±0.6 Hx vs 1.3±0.0 Nx, p<0.05) subunits were increased compared to normoxic controls. The increased expression of the NMDA receptor NR1 subunit correlated inversely with tissue ATP and PCr levels (r=0.62, r=0.66) as did the increase in NR2A (r=0.99, r=0.99) and NR2B (r=0.99, r=0.97) subunit expression, with expression increasing as tissue high energy phosphates decreased during hypoxia. The results demonstrate that as cerebral energy metabolism decreases, expression of the NR1, NR2A and NR2B subunits of the NMDA receptor increases in a linear relationship. We speculate that as cerebral energy metabolism decreases, altered phosphorylation of the NMDA receptor results in increased intracellular Ca++ flux which increases NMDA receptor subunit expression and may increase NMDA receptor recycling during hypoxia.
