Hypoxia-mediated degradation of Na, K-ATPase via mitochondrial reactive oxygen species and the ubiquitin-conjugating system

We set out to determine whether cellular hypoxia, via mitochondrial reactive oxygen species, promotes Na, K-ATPase degradation via the ubiquitin-conjugating system. Cells exposed to 1.5% O-2 had a decrease in Na, K-ATPase activity and oxygen consumption. The total cell pool of alpha 1 Na, K-ATPase protein decreased on exposure to 1.5% O-2 for 30 hours, whereas the plasma membrane Na, K-ATPase was 50% degraded after 2 hours of hypoxia, which was prevented by lysosome and proteasome inhibitors. When Chinese hamster ovary cells that exhibit a temperature-sensitive defect in E1 ubiquitin conjugation enzyme were incubated at 40 degrees C and 1.5% O-2, the degradation of the alpha 1 Na, K-ATPase was prevented. Exogenous reactive oxygen species increased the plasma membrane Na, K-ATPase degradation, whereas, in mitochondrial DNA deficient rho(0) cells and in cells transfected with small interfering RNA against Rieske iron sulfur protein, the hypoxia-mediated Na, K-ATPase degradation was prevented. The catalase/superoxide dismutase (SOD) mimetic (EUK-134) and glutathione peroxidase overexpression prevented the hypoxia-mediated Na, KATPase degradation and overexpression of SOD1, but not SOD2, partially inhibited the Na+ pump degradation. Accordingly, we provide evidence that during hypoxia, mitochondrial reactive oxygen species are necessary to degrade the plasma membrane Na, K-ATPase via the ubiquitin- conjugating system.