Modification of the Glutamate Binding Site of the NMDA Receptor During Graded Hypoxia in the Cerebral Cortex of Newborn Piglets. † 183

Abstract

We have previously shown that during hypoxia the glutamate binding site of the N-methyl-D-aspartate (NMDA) receptor is altered in newborn piglet brain. The present study tests the hypothesis that hypoxia-induced alteration of the glutamate site of the NMDA receptor is progressive and correlates with the decrease in cerebral energy metabolism induced by hypoxia. Studies were performed in 8 ventilated piglets, 4 normoxic and 4 hypoxic. Varying degrees of hypoxia were achieved by exposure to decreased oxygen at different concentrations and confirmed by brain tissue ATP and phosphocreatine (PCr) levels. 3[H]glutamate binding was performed in P2 membrane fractions at concentrations of 25-1000 nM. Specific NMDA-displaceable3[H]-glutamate binding was determined with 100μM NMDA. Non-specific binding was determined with 1mM unlabelled glutamate. Bmax (receptor number) and Kd (dissociation constant) were calculated from Scatchard plots. ATP(μmol/g brain), PCr (μmol/g brain) and Bmax (fmol/mg protein) were as follows: (5.86, 2.77, 402), (4.77, 2.96, 388), (4.50, 2.05, 364), (4.03, 2.01, 332), (3.37, 0.96, 276), (2.49, 0.24, 253), (1.96, 0.77, 322), and (0.24, 0.46, 174). Bmax decreased in a linear relationship as both ATP (r=0.92) and PCr (r=0.83) decreased, but Kd did not. The data show that the number of glutamate binding sites decreased in a linear relationship as oxidative phosphorylation decreased during hypoxia. A decline in oxidative phosphorylation may lead to a reduction in glutamate binding sites by several mechanisms. Since phosphorylation regulates NMDA receptor distribution in neuronal membranes, we speculate that a decrease in oxidative phosphorylation as seen during hypoxia may lead to a loss in number of glutamate binding sites through changing the location and density of NMDA receptors in brain cell membranes. Alternatively, we speculate that dephosphorylation of the receptor may cause an allosteric or conformational change in the receptor preventing glutamate binding and may lead to lipid peroxidation and cell death.