Published online before print April 13, 2006, doi: 10.1161/01.RES.0000222418.99976.1d
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Submitted on February 7, 2006
Revised on March 22, 2006
Accepted on April 4, 2006
Hypoxia-Mediated Degradation of Na,K-ATPase via Mitochondrial Reactive Oxygen Species and the Ubiquitin-Conjugating System
Alejandro P. Comellas ;
From the Division of Pulmonary and Critical Care Medicine (A.P.C., L.A.D., E.L., L.M.P., N.S.C., N.Q., G.R.S.B., A.C., J.I.S.), Feinberg School of Medicine, Northwestern University, Chicago, Ill; The Vascular and Tumor Biology Research Center (A.C.), Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel; and University Medical Center Utrecht and Institute of Biomembranes (G.J.S.), Utrecht, The Netherlands.
* To whom correspondence should be addressed. E-mail: j-sznajder{at}northwestern.edu.
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 
1 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 1 Na,K-ATPase was prevented. Exogenous reactive oxygen species increased the plasma membrane Na,K-ATPase degradation, whereas, in mitochondrial DNA deficient 
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,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.
Key words: ATP • oxygen • proteasome • antioxidants • cell adaptation
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