title: Role of the small GTPase RhoA in the hypoxia-induced decrease of plasma membrane Na,K-ATPase in A549 cells.
abstract: Hypoxia impairs alveolar fluid reabsorption by promoting Na,K-ATPase endocytosis, from the plasma membrane of alveolar epithelial cells. The present study was designed to determine whether hypoxia induces Na,K-ATPase endocytosis via reactive oxygen species (ROS)-mediated RhoA activation. In A549 cells, RhoA activation occurred within 15 minutes of cells exposure to hypoxia. This activation was inhibited in cells infected with adenovirus coding for gluthatione peroxidase (an H2O2 scavenger), in mitochondria depleted (rho0) cells or cells expressing decreased levels of the Rieske iron-sulfur protein (inhibitor of mitochondrial complex III), which suggests a role for mitochondrial ROS. Moreover, exogenous H2O2 treatment during normoxia mimicked the effects of hypoxia on RhoA, further supporting a role for ROS. Cells expressing dominant negative RhoA failed to endocytose the Na,K-ATPase during hypoxia or after H2O2 treatment. Na,K-ATPase endocytosis was also prevented in cells treated with Y-27632, a Rho-associated kinase (ROCK) inhibitor, and in cells expressing dominant negative ROCK. In summary, we provide evidence that in human alveolar epithelial cells exposed to hypoxia, RhoA/ROCK activation is necessary for Na,K-ATPase endocytosis via a mechanism that requires mitochondrial ROS.
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA. lauradada@northwestern.edu
authors.forename: Laura A
authors.initials: LA
authors.lastname: Dada
authors.forename: Eva
authors.initials: E
authors.lastname: Novoa
authors.forename: Emilia
authors.initials: E
authors.lastname: Lecuona
authors.forename: Haiying
authors.initials: H
authors.lastname: Sun
authors.forename: Jacob I
authors.initials: JI
authors.lastname: Sznajder
mesh_terms: D000256:Adenoviridae; D000577:Amides / Q000494:pharmacology; D001992:Bronchoalveolar Lavage Fluid; D015687:Cell Hypoxia; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D014450:Electron Transport Complex III / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000378:metabolism; D005799:Genes, Dominant; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D020545:Respiratory Mucosa / Q000201:enzymology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000577:Amides; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D007506:Iron-Sulfur Proteins; D011725:Pyridines; C037989:Rieske iron-sulfur protein; C108830:Y 27632; D006861:Hydrogen Peroxide; D005979:Glutathione Peroxidase; D020742:rhoA GTP-Binding Protein; D014450:Electron Transport Complex III; D000254:Sodium-Potassium-Exchanging ATPase