In patients with lung injury and pulmonary edema, alveolar gas exchange is impaired which results in hypoxemia. We have previously reported that hypoxia impairs the lung's ability to clear edema by inhibiting the alveolar epithelial Na,K-ATPase. The focus of this application is to determine the mechanisms regulating the effects of severe hypoxia: 1.5%, 3% or 5% (pO2~10 to 40 mm Hg) on alveolar epithelial function focusing on the regulation of Na,K-ATPase endocytosis and degradation. In the previous cycle of the grant, we reported that in alveolar epithelial cells (AEC) exposed to hypoxia the plasma membrane Na,K-ATPase was rapidly degraded, while the degradation of the intracellular Na,K-ATPase molecules was kept at steady state. We now have preliminary data to suggest that this apparent discrepancy is related to the "acute versus chronic" effects of hypoxia and cell adaptation via the hypoxia inducible factor a (HIF1a). Thus, we propose to dissect the mechanisms that regulate the effects of acute and prolonged hypoxia. We will determine whether a brief exposure of AEC to hypoxia results in mitochondrial reactive oxygen species mediated phosphorylation of the AMP Kinase leading to activation of protein kinase C zeta (PKCQ and Na,K-ATPase downregulation. We reason that prolonged exposure to hypoxia results in cell adaptation via a HIF1amediated mechanism in which the downregulation of the PKC? prevents further endocytosis/degradation of the Na,K-ATPase. We will study the effects of hypoxia on the alveolar epithelium via three interrelated aims:
in Specific Aim # 1 we propose to determine whether hypoxia activates AMPK and its role in the regulation of alveolar epithelial Na,K-ATPase and fluid reabsorption;
in Specific Aim # 2 we will study whether the HIF1a ubiquitin ligase, von Hippel Lindau protein (pVHL), regulates Na,K-ATPase endocytosis/degradation during hypoxia;and in Specific Aim # 3 we will determine whether HIF1a stabilization leads to PKC? ubiquitination and degradation as a mechanism of regulating total cell Na,K-ATPase levels and thus, alveolar epithelial function. Experiments have been conducted for each of the specific aims and the preliminary results support the feasibility and importance of this proposal.
Completion of the proposed studies will provide novel information on the effects of hypoxia on the alveolar epithelium, specifically as it pertains to mechanisms of inhibition of the Na,K-ATPase, impairment of alveolar epithelial function and cell adaptation to hypoxia which may be of importance for the understanding and design of novel approaches to improve alveolar epithelial function in patients with pulmonary edema.
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