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 alpha1 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 degrees C and 1.5% O2, the degradation of the alpha1 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,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.
Comellas
Alejandro P
AP
Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
Dada
Laura A
LA
Lecuona
Emilia
E
Pesce
Liuska M
LM
Chandel
Navdeep S
NS
Quesada
Nancy
N
Budinger
G R Scott
GR
Strous
Ger J
GJ
Ciechanover
Aaron
A
Sznajder
Jacob I
JI
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D000860:Hypoxia / Q000201:enzymology / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D008928:Mitochondria / Q000378:metabolism*; D010101:Oxygen Consumption; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*
D017382:Reactive Oxygen Species; D044763:Ubiquitin-Conjugating Enzymes; D046988:Proteasome Endopeptidase Complex; D000254:Sodium-Potassium-Exchanging ATPase