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-medi ated generation of nitric oxide free radicals leading to peroxynitrite prod uction. The present study tests the hypothesis that hypoxia results in NO-m ediated modification of Na+, K+-ATPase in the fetal brain. Studies were con ducted in guinea pig fetuses of 58-days gestation. The mothers were exposed to FiO(2) of 0.07% for 1 hour. Brain tissue hypoxia in the fetus was confi rmed biochemically by decreased ATP and phosphocreatine levels. P-2 membran e fractions were prepared from normoxic and hypoxic fetuses and divided int o untreated and treated groups. The membranes were treated with 0.5 mM pero xynitrite at pH 7.6. The Na+, K+-ATPase activity was determined at 37 degre esC 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 ouaba in. 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 hypox ic membranes. Enzyme kinetics was determined at varying concentrations of A TP (0.5 mM-2.0 mM). The results indicate that peroxynitrite treatment alter s the affinity of the active site of Na+, K+-ATPase for ATP and decreases t he Vmax by 35% in hypoxic membranes. When compared to untreated normoxic me mbranes Vmax decreases by 35.6% in treated normoxic membranes and further t o 52% in treated hypoxic membranes. The data show that peroxynitrite treatm ent induces modification of Na+, K+-ATPase. The results demonstrate that pe roxynitrite decreased activity of Na+, K+-ATPase enzyme by altering the act ive sites as well as the microenvironment of the enzyme. We propose that ni tric oxide synthase-mediated formation of peroxynitrite during hypoxia is a potential mechanism of hypoxia-induced decrease in Na+, K+-ATPase activity .