Previous studies have shown that during normoxia the N-methyl-D-aspartate(NMDA) receptor ion channel complex is activated by glycine resulting in ion channel opening as measured by 3[H]MK-801 binding in the cerebral cortex of newborn piglets. During hypoxia, the degree of NMDA receptor activation increases as the energy metabolism of the cell decreases. The present study tests the hypothesis that the degree of glycine activation of the NMDA receptor ion channel during hypoxia is regulated by the decrease in cerebral energy metabolism induced by hypoxia. Studies were performed in 5 normoxic (Nx) and 7 hypoxic (Hx) ventilated piglets. In the Hx group varying degrees of cerebral energy metabolism were achieved using different concentrations of O2 (5-9%) and were documented biochemically by tissue ATP and phosphocreatine (PCr) levels. 3[H]MK-801 binding was performed in P2 membrane fractions in the presence of 100mM glutamate, 12.5nM3[H]MK-801 and increasing concentrations of glycine from 0.01-15mM. Nonspecific binding was determined in the presence of 10mM unlabeled MK-801. Percent activation at each glycine concentration and the concentrations of glycine resulting in 50% activation of the receptor (Act50) were determined. Receptor activation at 1.0mM glycine (%) and brain tissue ATP(mmol/g brain) concentrations were as follows: (6.3, 136); (5.3, 106); (5.2, 189); (4.9, 129); (4.9, 107); (3.2, 68); (3.2, 97); (3.1, 103); (3.0, 133);(2.8, 77); (2.6, 106) and (0.1, 87), respectively, demonstrating that receptor activation at 1.0mM glycine decreased linearly as ATP levels decreased(r=0.6). Receptor activation at 0.1mM glycine correlated with brain tissue levels of PCr, with% activation decreasing linearly as PCr levels decreased(r=0.5). Act50 also correlated with tissue PCr levels, with Act50 increasing as PCr levels decreased (r=0.5). The data demonstrate that as brain cell energy metabolism decreases, maximal receptor activation by glycine decreases in a linear relationship indicating that increasing concentrations of glycine are necessary to activate the receptor at low tissue levels of high energy phosphates. We speculate that dephosphorylation of the glutamate recognition site of the NMDA receptor during hypoxia leads to either a decrease in assessibility of glycine to its binding site or to a decrease in glycine-dependent activation of the NMDA receptor ion channel.
