Cystic fibrosis (CF) is a disease of exocrine epithelia. Not only is a lack of cyclic AMP-activated chloride (C1-) channel activity observed in CF epithelial cells, but dysregulation of other epithelial C1- and Na+ channels, epithelial vesicle trafficking, and epithelial immune function are also observed in CF. The PI has shown recently that there is a loss of ATP release across the apical membrane of CF epithelia under basal or isotonic and hypotonic conditions and that extracellular ATP signaling may be lost in CF. Without basal or stimulated ATP release in CF epithelia, we hypothesize that the pathogenesis of CF epithelial dysfunction may lie in defective ATP signaling and defective cell volume regulation by the CF epithelia. An ionic imbalance in the airway surface liquid has been observed; we hypothesize that this reflects an osmotic imbalance due to an inherent defect in autocrine ATP control of epithelial cell volume regulation. As such, this completely re-constructed proposal focuses on one concise, central hypothesis: ATP release and signaling is essential for autocrine control of cell volume regulation.
Two aims derive from this hypothesis: (1) Test the hypothesis that RVD requires ATP release, ATP receptors, and CFTR expression in the plasma membrane, and (2) Test the hypothesis that ATP release occurs via transport- and exocytotic-driven mechanisms. To fulfill these Aims, parental CF epithelial cells will be compared to CF cells complemented transiently or stably with wild-type CFTR. A synergistic combination of cell volume regulation assays, bioluminescence detection assays of ATP released from cells, patch-clamp electrophysiological assays of ATP whole cell current and membrane capacitance, and exocytosis assays will be used to address each Aim. These assays will identify the ATP release mechanisms, the dependence of these mechanisms upon CFTR expression, and the purinergic receptors involved in autocrine ATP control of cell volume regulation. We are confident that novel results will be found concerning the cellular and molecular mechanisms of autocrine ATP release, signaling, and regulation of cell volume.
