Nitration as a mechanism of Na+, K+-ATPase modification during hypoxia in the cerebral cortex of the guinea pig fetus.

Previous studies have shown that hypoxia induces nitric oxide synthase-mediated generation of nitric oxide free radicals leading to peroxynitrite production. The present study tests the hypothesis that hypoxia results in NO-mediated modification of Na+, K+-ATPase in the fetal brain. Studies were conducted in guinea pig fetuses of 58-days gestation. The mothers were exposed to FiO2 of 0.07% for 1 hour. Brain tissue hypoxia in the fetus was confirmed biochemically by decreased ATP and phosphocreatine levels. P2 membrane fractions were prepared from normoxic and hypoxic fetuses and divided into untreated and treated groups. The membranes were treated with 0.5 mM peroxynitrite at pH 7.6. The Na+, K+-ATPase activity was determined at 37 degrees C for five minutes in a medium containing 100 mM NaCl, 20 mM KCl, 6.0 mM MgCl2, 50 mM Tris HCl buffer pH 7.4, 3.0 mM ATP with or without 10 mM ouabain. Ouabain sensitive activity was referred to as Na+, K+-ATPase activity. Following peroxynitrite exposure, the activity of Na+, K+-ATPase in guinea pig brain was reduced by 36% in normoxic membranes and further 29% in hypoxic membranes. Enzyme kinetics was determined at varying concentrations of ATP (0.5 mM-2.0 mM). The results indicate that peroxynitrite treatment alters the affinity of the active site of Na+, K+-ATPase for ATP and decreases the Vmax by 35% in hypoxic membranes. When compared to untreated normoxic membranes Vmax decreases by 35.6% in treated normoxic membranes and further to 52% in treated hypoxic membranes. The data show that peroxynitrite treatment induces modification of Na+, K+-ATPase. The results demonstrate that peroxynitrite decreased activity of Na+, K+-ATPase enzyme by altering the active sites as well as the microenvironment of the enzyme. We propose that nitric oxide synthase-mediated formation of peroxynitrite during hypoxia is a potential mechanism of hypoxia-induced decrease in Na+, K+-ATPase activity.

Post-hypoxic magnesium decreases nuclear oxidative damage in the fetal guinea pig brain.

This study was to determine if administration of MgSO(4) after the hypoxic insult (post-hypoxia) would attenuate neuronal damage in the fetal guinea pig brain. Pregnant guinea pigs (45-60 days gestation) were exposed to hypoxia (7% O2) for 1 h. Following hypoxia, one group recovered for 24 h with no additional treatment (post-hypoxia) and another group received MgSO(4), 300 mg/kg i.p., followed by 100 mg/kg i.p., each hour for three doses (post-hypoxia+Mg) and allowed to recover for 24 h. Fetal brain magnesium content was decreased (P<0.05) 4 h post-hypoxia which was prevented by treatment with MgSO(4). High energy phosphates were significantly lower (P<0.05) in the post-hypoxia group which was partially prevented by post-hypoxic magnesium. Na+,K+-ATPase activity was significantly lower (P<0.05) and nuclear membrane fluorescent compounds were significantly higher (P<0.05) in the post-hypoxia group but were not significantly changed in the post-hypoxia+Mg group compared with the normoxic control group. DNA fragmentation was observed to be lower in the Mg-treated post-hypoxic group. This study demonstrates that maternal MgSO(4) administration following in utero hypoxia prevents associated decreases in fetal brain magnesium and suppresses alterations in both the neuronal and nuclear membranes and genomic fragmentation in the fetal guinea pig brain.