Mammalian cells detect decreases in oxygen concentrations to activate a variety of responses that help cells adapt to low oxygen levels (hypoxia). One such response is stabilization of the protein HIF-1 alpha, a component of the transcription factor HIF-1. Here we show that a small interfering RNA (siRNA) against the Rieske iron-sulfur protein of mitochondrial complex III prevents the hypoxic stabilization of HIF-1 alpha protein. Fibroblasts from a patient with Leigh's syndrome, which display residual levels of electron transport activity and are incompetent in oxidative phosphorylation, stabilize HIF-1 alpha during hypoxia. The expression of glutathione peroxidase or catalase, but not superoxide dismutase 1 or 2, prevents the hypoxic stabilization of HIF-1 alpha. These findings provide genetic evidence that oxygen sensing is dependent on mitochondrial-generated reactive oxygen species (ROS) but independent of oxidative phosphorylation.

Abstract-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% O 2 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% O 2 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% O 2 , 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,KATPase degradation and overexpression of SOD1, but not SOD2, partially inhibited the Na ϩ 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 Ⅲ oxygen Ⅲ proteasome Ⅲ antioxidants Ⅲ cell adaptation A daptation to hypoxia represents a well-defined means to improve ischemic tolerance. Unfortunately, there is no definitive understanding of the mechanisms associated with these phenomena. 1 As mammalian cells encounter lower oxygen levels, they develop mechanisms to prevent depletion of oxygen to anoxia that might result in cell death. 2 Cells respond to hypoxia through the stabilization of the transcription factor hypoxia-inducible factor (HIF)-1␣. In normoxic conditions, prolyl hydroxylases hydroxylate conserved proline residues in HIF-1␣. 3,4 This substrate modification is recognized by a ubiquitin ligase enzyme (Von-Hippel-Lindau protein [VHL]) that ubiquitinates and targets HIF-1␣ to the proteasome. During hypoxia, VHL-mediated degradation of HIF-1␣ is suppressed, allowing its transcriptional activation. 4,5 The intracellular mechanisms by which cells sense hypoxia to stabilize HIF-1␣ are not fully understood. The generation of mitochondrial reactive oxygen species (mROS) during hypoxia has been proposed as part of an oxygen sensing pathway for the hypoxic stabilization of HIF-1␣. 6 Another mechanism to prevent the depletion of oxygen during hypoxia is to decrease the cellular demand for oxygen by upregulating anaerobic ATP-producing pathways and downregulating ATP-consuming processes. 2 This regulation allows ATP levels to remain constant, even while ATP turnover rates greatly decline. The ATP requirements of ion pumping are downregulated by generalized "channel" arrest in hepatocytes and by the arrest of specific ion channels in neurons. 7 The Na,...

A 79-year-old man with a history of diastolic left ventricular dysfunction, atrial fibrillation, and low back pain underwent vertebroplasty. The procedure was uneventful except for the development of low-grade fever 1 day after the procedure. The patient denied any cough, dyspnea, or chest pain. Initial physical examination revealed that he was not in respiratory distress. His temperature was 38.3°C, respiratory rate was 20/min, blood pressure was 125/72 mm Hg, and pulse was 92 bpm and irregularly irregular. Auscultation of the lungs revealed normal breath sounds except for decreased air entry at both bases. Findings of the cardiac examination were normal, without distention of the jugular veins. He had 2ϩ pitting edema of the lower extremities. Initial work-up included a chest radiograph that revealed high-density linear shadows bilaterally (Figure 1). There were no previous chest radiographs for comparison. Chest computed tomography scan confirmed the presence of bilateral, multiple, linear hyperdensities within the pulmonary arteries, as well as a peripheral, wedge-shaped defect in the right middle lobe (Figure 2). Transthoracic echocardiograms did not show any evidence of elevation in the pulmonary arteries. In view of the patient's recent vertebroplasty, these radiographic findings were attributed to pulmonary embolization of the acrylic cement (polymethylmethacrylate) used during the surgical procedure, which contains barium sulfate in the preparation, thus giving its radiopaque properties (Figure 3). The right middle lobe findings likely represented a pulmonary infarct. The patient did well without any long-term sequelae consistent with other reported cases of cement embolization in the literature.

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