Hyperabsorption of sodium across CF airway epithelium was identified over twenty years ago and is a key factor in the progression of airway disease. Much has been learned about mechanisms regulating the function of the epithelial sodium channel (ENaC), but why ENaC function is specifically increased in CF and how its regulation relates to CFTR function has remained largely elusive. Earlier data suggests that the presence of functional CFTR inverted the cAMP-PKA regulation of ENaC from positive regulation to negative regulation. We have recently reported that the loss of CFTR function triggers a feedback response in epithelial cells leading to increased cAMP signaling that results in cholesterol accumulation. We demonstrated in this work that the cAMP binding competitor Rp-cAMPS reverses cholesterol accumulation to wt distribution. An increase in the expression of arrestin-3, a protein involved in G-protein coupled receptor (GPCR) internalization and other signaling events, is the initiating factor in Rp-cAMPS-sensitive cholesterol accumulation in CF. Other work in our laboratory not yet published demonstrates the ability of Rp-cAMPS to reverse many other aspects of CF biology to wt profiles including markers of inflammation, signaling proteins we have previously reported on, and over 145 proteins in proteomic analysis. Given the broad impact of Rp-cAMPS in CF epithelial cells and mouse models on CF cell biology, we were interested in determining if Rp-cAMPS could influence amiloride-sensitive transepithelial potential difference (TEPD) in CF mice. Preliminary data demonstrate that treatment of Cftr -/- mice for 12 days with 5 mg/kg/day Rp-cAMPS via osmotic pump reduces amiloride sensitive transepithelial potential difference to wt levels. In cellular studies, statin treatment of primary renal collecting duct epithelial cells significantly reduces amiloride- sensitive baseline Isc suggesting sodium transport is being influenced. Previous data demonstrating the impact of Rp-cAMPS on cholesterol processing in CF and the influence of statin treatment on amiloride-sensitive transport in polarized cells lead to the postulation that cholesterol processing contributes to hyperabsorption of sodium across CF airway epithelium. The hypothesis of this study is that chronically increased cAMP signaling in CF contributes to hyperabsorption of sodium across CF airway epithelium through modulation of cholesterol trafficking. Mechanistically, the hypothesis that NPC1- driven control of membrane cholesterol content and subsequent modulation of Na+/K+ ATPase function mediate Rp-cAMPS sensitive regulation of sodium transport will be tested. We know that in vivo treatment with Rp-cAMPS is effective in reverting sodium transport in CF mice to wt levels. The goal of this study is to identify the mechanism of this action. PUBLIC HEALTH RELEVANCE: A fundamental issue in CF airway disease is the hyperabsorption of sodium, which influences fluid levels and mucociliary clearance. A mechanism explaining why absorption is increased specifically in CF has been lacking. This proposal hypothesizes that alterations in chronic cAMP signaling in CF airway epithelial cells increases sodium transport through modulating membrane cholesterol levels.