Previous studies have shown that hypoxia induces nitric oxide synthase-mediated generation of nitric oxide leading to peroxynitrite production. The present study tests the hypothesis that peroxynitrite alters N-methyl-D-aspartate (NMDA) receptor ion-channel characteristics during hypoxia in the fetal brain. Studies were conducted in normoxic and hypoxic guinea pig fetuses at term, whose mothers were exposed to an FiO2 of 21% or 7% for 1 hr. Brain tissue hypoxia in the fetuses was confirmed biochemically by decreased ATP and phosphocreatine levels. P2 membrane fractions were prepared and treated with 0.5 mM peroxynitrite at pH 7.6. Following treatment, the membranes were washed and 3H-MK-801 binding performed at 32° C in the presence of 100 μM glutamate, 100 μM glycine and increasing concentrations of 3H-MK-801 (0.5 to 50 nM). Nonspecific binding was determined in the presence 10 μM MK-801. Peroxynitrite treatment increased the Bmax from 1.02 ± 0.09 to 1.41 ± 0.22 pmoles/mg protein in the normoxic group (p<0.05) and from 1.25 ± 0.18 to 1.48 ± 0.26 pmoles/mg protein in the hypoxic group (p=0.05). The Kd value increased from 3.07 ± 0.28 to 4.29 ± 1.07 pmoles/mg protein in the normoxic group (p<0.05) and from 4.94 ± 0.84 to 6.28 ± 0.71 pmoles/mg protein in the hypoxic group (p=0.05). The results demonstrate that peroxynitrite increases the number of ion channel binding sites and decreases receptor affinity, during normoxia and hypoxia. We speculate that peroxynitrite regulates NMDA receptor ion-channel activity by exposing additional ion channel sites and that peroxynitrite-induced modification via nitration of tyrosine residues may be a mechanism of regulation of NMDA receptor function.
(Funded by NIH#HD-20337, MOD 6-FY94-0135)
