Acute hypoxemic respiratory failure (AHRF) is a life-threatening condition with high morbidity and mortality. In the initial events of AHRF, lung edema accumulates and impairs alveolar oxygen exchange until mechanisms for edema clearance become effective. Lung edema is cleared predominantly by active Na+ transport; recent evidence suggests that accelerated edema clearance is associated with reduced mortality in patients with AHRF. It has been recently reported that alveolar epithelial type II (ATII) cells contain apical amiloride-sensitive Na+ channels and basolaterally located Na,K-ATPase (the alpha-1,beta-1 heterodimer); both are important contributors to Na+ transport across the alveolar epithelium. Until now Na,K-ATPase has not been localized to alveolar epithelial type I (ATI) cells. In this application we propose to study the role and regulation of the Na,K-ATPase in alveolar epithelial cells (AFC) which contributes to active Na+transport and thus lung edema clearance, by five specific aims. Specific aim # 1. To determine whether as ATII cells in different culture conditions differentiate phenotypically into ATII cells in different culture conditions differentiate phenotypically into ATI-like cells, they shift from predominantly expressing the Na,K-ATPase alpha-1,beta-1 heterodimer to the alpha-2,beta-1 heterodimer. Specific aim # 2. To determine whether this shift in isoform expression in cultured AEC is transcriptionally regulated. Specific aim #3. To determine mechanisms of Na,K-ATPase regulation in cultured ATII cells as they differentiate. Specific aim #4: To determine whether rat ATII and ATI cells express both Na,K-ATPase isozymes in situ, and if the ATI cells express predominantly the alpha-2, beta-1 Na,K-ATPase. Specific aim #5: To determine whether AEC Na,K-ATPase abundance and function can be reduced by transfecting AEC with isoform specific antisense constructs, or increased by overexpressing isoform specific sense constructs. Further, to determine whether these modulations will alter Na+ transport and lung liquid clearance. Preliminary studies have been conducted for each of the specific aims and the preliminary results support our hypotheses and the feasibility of the proposed studies. Completion of the proposed experiments will provide new information on the role and regulation of the NaK-ATPase in alveolar epithelial cells and eventually help with the design of new strategies to enhance lung edema clearance in patients with hypoxemic respiratory failure.