The alveolar epithelium in normal lungs is comprised of two morphologically distinct types of cells (type 1 and type 2) that are responsible for maintaining lung fluid balance. Tight regulation of alveolar fluid clearance is essential for maintaining a dry breathing space, and hence, proper gas exchange. It has been established that net ion transport through amiloride-sensitive epithelial sodium channels (ENaC), located on the apical surface of alveolar epithelial cells, play a critical role in fluid clearance in normal lung. However, the specific mechanisms regulating ENaC function are not completely understood. Within the alveoli, complex regulatory mechanisms must also be in place to balance the redox state of type 1 and type 2 cells, since inspired oxygen is converted to superoxide anions (O2-). Excessive O2- production, caused by high oxygen tensions, can lead to tissue damage and cell death, whereas insufficient oxygenation can result in anything from fatigue to life threatening conditions. In vivo, superoxides react quickly and irreversibly with nitric oxide (NO) to form peroxynitrite. We hypothesize that endogenous 02- binding to NO limits nitric oxide inhibition of ENaC function, thereby enhancing alveolar fluid clearance. Indeed, we have preliminary data suggesting that nitric oxide-unresponsive AT1 cells may have elevated levels of O2-, and that increasing O2- enhances Na transport. To investigate the role of redox signaling in alveolar fluid clearance, and more specifically, ENaC function, I will utilize single channel patch clamp analysis to examine ion transport in lung slice preparations, bio-molecular techniques to examine redox signaling in pneumocytes, and perform whole lung studies in vivo.
My first aim, performed during the mentored phase, directly examines the role of O2- in alveolar fluid clearance. Successful completion of this aim will naturally transition into aims 2 and 3, which utilizes several protocols that will be established in the mentored phase, as well as incorporate new approaches to studying type 1 and type 2 cells.
The second aim will determine the role of NO in lung function, and lastly, the third aim examines the putative reciprocal relationship between O2- and NO regulation of lung fluid balance. The studies proposed have real clinical relevance, and the potential for a very productive independent research career.
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|Thomas, Sheela V; Kathpalia, Paru P; Rajagopal, Madhumitha et al. (2011) Epithelial sodium channel regulation by cell surface-associated serum- and glucocorticoid-regulated kinase 1. J Biol Chem 286:32074-85|