The pathogenesis of lung disease induced by Cystic Fibrosis (CF) is due in part to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-stimulated Cl (CFTR) channel. The inability of cAMP to stimulate Cl (CFTR) secretion and the increased rate of Na reabsorption in the airway of patients with CF contributes to the dehydration of the airway and to the pathogenesis of lung disease. Recently, it was shown that aerosol therapy with the sodium channel inhibitor amiloride improved mucociliary clearance and slowed the rate of decline in lung vital capacity. Aerosol therapy designed to correct defective Cl secretion in CF patients may also be beneficial for lung disease. However, an effective treatment to increase CAMP-stimulated Cl (CFTR) secretion by the airway of patients with CF has not been identified. The long-term objective of the research proposed in this application, therefore, is to develop a pharmacological approach to stimulate CAMP-activated Cl (CFTR) channels in CF patients using aerosol-based therapy. Our strategy to develop such an approach is based on our preliminary experiments demonstrating that heterotrimeric G proteins inhibit cAMP-activated Cl (CFTR) channels in human tracheal epithelial cells and that inactivating inhibitory G proteins with A1 adenosine receptor antagonists restores cAMP-activation of Cl (CFTR) channels. We propose to test the hypothesis that adenosine, produced and released by airway epithelial cells, activates an A1 adenosine receptor that stimulates inhibitory G proteins. The G proteins, in turn, inactivate cAMP-activated Cl (CFTR) channels. Phosphorylation of Cl (CFTR) channels by protein kinase A may relieve G protein inhibition in normal but not in CF cells.
Our specific aims are to: 1) Characterize G protein regulation of Cl (CFTR) channels using whole-cell and single channel patch clamp techniques. We will identify G proteins by Northern blot and Western blot analysis and their cellular location by confocal immunocytochemical microscopy: 2) Elucidate the signal transduction pathways involving G protein regulation of Cl (CFTR) channels using whole-cell and single channel patch clamp techniques. We will determine if G proteins inhibit Cl(CFTR) channels via phospholipase A2 and arachidonic acid and 3) Develop a therapeutic approach, based on inactivating inhibitory G proteins with A1 receptor antagonists, to increase Cl (CFTR) secretion by CF cells using patch clamp techniques and measurements of Cl transport across monolayers of human airway cells. Because A1 adenosine antagonists are nontoxic and are effective in nuM concentrations, A1 antagonists are promising candidates for aerosol pharmacotherapy for CF patients. We anticipate that our studies will increase our understanding of the signalling pathways regulating cAMP-activated Cl (CFTR) channels in airway epithelial cells and elucidate the mechanism of A1 adenosine receptor-G protein interaction in regulating Cl (CFTR) channels. Finally, we anticipate that our studies will lead to the development of an aerosol-based treatment for stimulating Cl (CFTR) secretion by CF airways.
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