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% O2 had a decrease in Na,K-ATPase activity and oxygen consumption. The total cell pool of α1 Na,K-ATPase protein decreased on exposure to 1.5% O2 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°C and 1.5% O2, the degradation of the α1 Na,K-ATPase was prevented. Exogenous reactive oxygen species increased the plasma membrane Na,K-ATPase degradation, whereas, in mitochondrial DNA deficient ρ 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,K-ATPase 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.