Patients with acute lung injury develop hypoxemia and gas exchange impairment which results in significant morbidity and mortality. Previous studies have demonstrated that hypoxia and lung injury impair the lung's ability to clear edema by inhibiting Na+ channels and Na,K-ATPase in the alveolar epithelium. The focus of this application is to determine whether hypoxia of 1.5 percent or 3 percent (-10 or 20 mm Hg) for up to 1 hour inhibits Na,K-ATPase by causing endocytosis of the Na+ pump into intracellular compartments via specific pathways activated by reactive oxygen species (ROS) and protein kinase C (PKC) signaling molecules. We will determine whether after a short time of hypoxia (1 hour) reoxygenation of the alveolar epithelial cells (AEC) modulates mechanisms to recruit Na+ pumps previously endocytosed back into the cell basolateral membranes (BLM). We will study if activation of the protein kinase A pathway by terbutaline and forskolin prevents and/or reverts the effects of hypoxia on AEC Na+ pump. We will also determine whether in AEC exposed to prolonged hypoxia (>12 hours) the Na,K-ATPase proteins undergo ubiquitination and degradation via proteosomal/lysosomal pathways. As such, we will study the effects of hypoxia on the alveolar epithelium by focusing on the mechanisms of Na,K-ATPase regulation by four interrelated aims: in Specific Aim # 1 we propose to determine the role of mitochondrial ROS generated during hypoxia on Na,K-ATPase function and protein endocytosis; in Specific Aim # 2 we propose to study whether the alveolar epithelial Na,K-ATPase is phosphorylated during hypoxia by PKC triggering the endocytosis of the Na+ pump; in Specific Aim # 3 we will study whether hypoxia-mediated inhibition of the Na,K-ATPase activity and endocytosis of Na+ pump proteins are reversible upon reoxygenation and whether activation of the PKA pathway by terbutaline and forskolin prevents and/or reverses the effects of hypoxia, and in Specific Aim #4 we propose to determine whether prolonged exposure of AEC to hypoxia leads to ubiquitination and degradation of the Na,K-ATPase proteins via the proteosomal/lysosomal pathways. Experiments have been conducted for each of the specific aims and the preliminary results support the feasibility of this proposal. Completion of the proposed studies will provide novel information on the effects of hypoxia, specifically as it pertains to mechanisms of inhibition and degradation of the Na+ pump as well as pathways of reversal of Na,K-ATPase inhibition, which may be of importance for the design of novel strategies to increase alveolar fluid clearance in patients with hypoxemic respiratory failure.