Our previous studies have shown that hypoxia modifies the NMDA(N-methyl-D-aspartate) receptor in the brain, resulting in decreased Bmax(number of functional receptor ion-channels) and decreased Kd (apparent affinity) and that alteration of the NMDA receptor is related to brain tissue levels of high energy phosphates (ATP and phosphocreatine). The present study tests the hypothesis that the altered structure of the NMDA receptor during hypoxia is due to dephosphorylation. Guinea pig fetuses were obtained from anesthetized, normoxic (21% oxygen, n=6) and hypoxic (7% oxygen for 60 min, n=6) mothers. Brain tissue hypoxia in the fetus was documented biochemically by decreased levels of ATP and phosphocreatine. P2 membrane fractions were prepared from fetal cerebral cortical tissue. Both the normoxic and hypoxic membranes were incubated with 10 units of alkaline phosphatase for 30 min. at pH 9.0 NMDA receptor ion channel activity was measured using 3MK-801 binding as an index of receptor function. The results show that in normoxic membranes, enzyme treatment decreased the Bmax from 07.0±0.03 to 0.48±0.02 pmols/mg protein, (p<0.005). The Kd value decreased from 7.5±0.4 to 3.9±0.3 nM (p<0.005). In hypoxic membranes, enzyme treatment had no effect either on the Bmax (0.50±0.021 vs 0.50±0.020 pmols/mg protein, untreated vs treated) or the Kd(4.5±0.3 vs 4.3±0.2 nM, untreated vs treated). Bmax and Kd in untreated hypoxic brain were similar to the results obtained using enzyme treatment leading to dephosphorylation in normoxic brain. The data suggest that, during normoxia, NMDA receptor ion channel function may be regulated by phosphorylation/dephosphorylation and that hypoxia results in dephosphorylation of the NMDA receptor ion channel. We speculate that the increased affinity of the NMDA receptor ion-channel in the dephosphorylated state leads to the initiation of NMDA receptor-mediated events and may be a mechanism of neuronal injury during hypoxia. (Supported by NIH-20337)
