euk-134 and Hypoxia

AMP-activated protein kinase (AMPK) is a sensor of cellular energy status found in metazoans that is known to be activated by stimuli that increase the cellular AMP/ATP ratio. Full activation of AMPK requires specific phosphorylation within the activation loop of the catalytic domain of the alpha-subunit by upstream kinases such as the serine/threonine protein kinase LKB1. Here we show that hypoxia activates AMPK through LKB1 without an increase in the AMP/ATP ratio. Hypoxia increased reactive oxygen species (ROS) levels and the antioxidant EUK-134 abolished the hypoxic activation of AMPK. Cells deficient in mitochondrial DNA (rho(0) cells) failed to activate AMPK during hypoxia but are able to in the presence of exogenous H(2)O(2). Furthermore, we provide genetic evidence that ROS generated within the mitochondrial electron transport chain and not oxidative phosphorylation is required for hypoxic activation of AMPK. Collectively, these data indicate that oxidative stress and not an increase in the AMP/ATP ratio is required for hypoxic activation of AMPK.

Topics: Adenosine Triphosphate; AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Animals; Antioxidants; Catalytic Domain; Cell Line; Electron Transport Complex III; Fibroblasts; Hydrogen Peroxide; Hypoxia; Mice; Mitochondria; Mutation; Organometallic Compounds; Oxidative Phosphorylation; Protein Serine-Threonine Kinases; Reactive Oxygen Species; Salicylates

Hypoxia inhibits Na-K-ATPase activity and leads to its degradation in mammalian cells. Von Hippel Lindau protein (pVHL) and hypoxia inducible factor (HIF) are key mediators in cellular adaptation to hypoxia; thus, we set out to investigate whether pVHL and HIF participate in the hypoxia-mediated degradation of plasma membrane Na-K-ATPase. We found that in the presence of pVHL hypoxia decreased Na-K-ATPase activity and promoted the degradation of plasma membrane Na-K-ATPase. In pVHL-deficient cells, hypoxia did not decrease the Na-K-ATPase activity and the degradation of plasma membrane Na-K-ATPase was prevented. pVHL-mediated degradation of Na-K-ATPase required the functional pVHL E3 ligase and Ubc5 since pVHL mutants and dominant-negative Ubc5 prevented Na-K-ATPase from degradation. The generation of reactive oxygen species was necessary for pVHL-mediated Na-K-ATPase degradation during hypoxia. Desferrioxamine, which stabilizes HIF1/2alpha, did not affect the half-life of plasma membrane Na-K-ATPase. In addition, stabilizing HIF1/2alpha by infecting mammalian cells with adenoviruses containing the oxygen-dependent degradation domain of HIF1alpha did not affect the plasma membrane Na-K-ATPase degradation. In cells with suppression of pVHL by short hairpin RNA, the Na-K-ATPase was not degraded during hypoxia, whereas cells with knockdown of HIF1/2alpha retained the ability to degrade plasma membrane Na-K-ATPase. These findings suggest that pVHL participates in the hypoxia-mediated degradation of plasma membrane Na-K-ATPase in a HIF-independent manner.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Membrane; Cells, Cultured; Chlorocebus aethiops; COS Cells; Deferoxamine; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Organometallic Compounds; Salicylates; Sodium-Potassium-Exchanging ATPase; Von Hippel-Lindau Tumor Suppressor Protein

Exposure of animals to hyperoxia results in respiratory failure and death within 72 h. Histologic evaluation of the lungs of these animals demonstrates epithelial apoptosis and necrosis. Although the generation of reactive oxygen species (ROS) is widely thought to be responsible for the cell death observed following exposure to hyperoxia, it is not clear whether they act upstream of activation of the cell death pathway or whether they are generated as a result of mitochondrial membrane permeabilization and caspase activation. We hypothesized that the generation of ROS was required for hyperoxia-induced cell death upstream of Bax activation. In primary rat alveolar epithelial cells, we found that exposure to hyperoxia resulted in the generation of ROS that was completely prevented by the administration of the combined superoxide dismutase/catalase mimetic EUK-134 (Eukarion, Inc., Bedford, MA). Exposure to hyperoxia resulted in the activation of Bax at the mitochondrial membrane, cytochrome c release, and cell death. The administration of EUK-134 prevented Bax activation, cytochrome c release, and cell death. In a mouse lung epithelial cell line (MLE-12), the overexpression of Bcl-XL protected cells against hyperoxia by preventing the activation of Bax at the mitochondrial membrane. We conclude that exposure to hyperoxia results in Bax activation at the mitochondrial membrane and subsequent cytochrome c release. Bax activation at the mitochondrial membrane requires the generation of ROS and can be prevented by the overexpression of Bcl-XL.

Topics: Animals; bcl-2-Associated X Protein; bcl-X Protein; Caspases; Cell Death; Cell Line; Cell Nucleus; Cells, Cultured; Cytochromes c; Enzyme Activation; Epithelial Cells; Glutathione; Hypoxia; Immunoblotting; Intracellular Membranes; L-Lactate Dehydrogenase; Lung; Mice; Microscopy, Confocal; Mitochondria; Models, Biological; Organometallic Compounds; Oxygen; Plasmids; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Retroviridae; Salicylates; Superoxide Dismutase; Time Factors