Mechanism of Hypoxia Induced Modification of Na + , K + - ATPase in the Cerebral Cortex of the Guinea Pig Fetus † 224

Previous studies have shown that cerebral hypoxia results in decreased Na+,K+-ATPase activity which is associated with increased lipid peroxidation. We have also shown that hypoxia induces nitric oxide synthase-mediated generation of nitric oxide leading to production of the oxidant peroxynitrite. The present study tests the hypothesis that in vitro treatment with peroxynitrite alters Na+,K+-ATPase enzyme activity during hypoxia in the fetal brain. Studies were conducted on fetal guinea pigs (n=6) at 58-days gestation (term). The mothers were exposed to FiO2 of 0.07 for 1 hour. Fetal brain tissue was removed and immediately placed in liquid nitrogen. Tissue hypoxia in the fetus was confirmed biochemically by decreased ATP and phosphocreatine levels. P2 membrane fractions were prepared and separated into two groups, untreated and treated with peroxynitrite (0.5 mM at pH 7.6). Na+, K+-ATPase enzyme activity was determined at 37°C for five minutes in a medium containing 100mM NaCl, 20mM KCl, 6.0mM MgCl2, 50mM Tris HCl buffer pH 7.4 and 3.0 mM ATP, with and without 1mM ouabain. Ouabain sensitive activity was referred to as Na+,K+-ATPase activity. Enzyme kinetics were determined at varying concentrations of ATP (0.5mM-2.0mM). Na+,K+-ATPase activity was 35.42 ± 6.91 [um]moles/mg protein/hr in untreated membranes, and 25.81 ± 7.99 μmoles/mg protein/hr in membrane treated with peroxynitrite (p<0.05). Lineweaver Burke plots were prepared and the Km (enzyme dissociation constant) for ATP and the Vmax (maximal enzyme velocity) calculated. The results show that Km for untreated samples was 0.85 ± 0.13 vs 0.58 ± 0.21 mM for treated samples. Vmax for untreated was 30.90 ± 3.39 vs 20.25 ± 2.05 μmole/mg protein/hr for treated samples. In vitro peroxynitrite treatment increased the affinity of the active site of Na+,K+-ATPase for ATP (decreases Km by 31%) and the Vmax by 34.4%. It is likely that peroxynitrite decreased the activity of Na+,K+-ATPase enzyme by altering the active sites as well as the microenviroment of the enzyme. We speculate that nitric oxide synthase-mediated formation of peroxynitrite during hypoxia is a potential mechanism of hypoxia-induced decrease in activity and increased affinity of the enzyme Na+,K+-ATPase for ATP.