Acid - base transport by airway epithelial cells likely play key roles in cystic fibrosis (CF) due to effects on mucus hydration and rheology, ciliary transport, bacterial binding and anti-microbial factors. Airway surface liquid (ASL) appears to be slightly acidic (pH 6.9), and acidity increases in CF. Cells in the submucosal glands secrete mucus, and secretion of (primarily) HCO3 and (some) Cl along with Na through tight junctions (to maintain electroneutrality) and fluid sweeps the mucus out of the glands. Permeability of tight junctions between submucosal gland cells is critical for the glands' ability to secrete ions. In contrast, surface cells seem to have tight junctions that are impermeant to ions, and this assures that these cells operate only to absorb Na (through ENaC), Cl (through CFTR) and water. According to these proposals, the absence of CFTR in CF will cause: (i) decreased HCO3, Na and water secretion by submucosal gland cells (leading to mucus clogging); (ii) increased acidity of ASL (due to reduced HCO3 secretion); (iii) thicker and stickier mucus (due to reversal of fluid secretion to absorption and increased acidity of ASL). According to these hypotheses, it is crucial to determine mechanisms of H and HCO3 transport across the membranes and tight junctions of WT and CF submucosal gland and surface epithelial cells, including the potential role of mucus secretion to the acid-base balance of the ASL.
The specific aims of this proposal are: (i) Use fluorescence microscopic imaging, immunomicroscopy and western blots to identify mechanisms for HCO3 and H transport across apical and basolateral membranes of submucosal gland and surface cells of WT and CF airway monolayers. (ii) Using genetic targeting of pH-sensitive dyes and imaging microscopy, measure pH (and regulation) in mucus granules and at the lumenal aspect of WT and CF airway epithelia. (iii) Using isotopic methods, measure secretion of mucus and of constitutive and regulated Golgi-derived vesicles and correlate to H/HCO3 transport. (iv) Using electrophysiological, isotopic flux and immunomicroscopic methods, determine the permeability/conductance properties of the tight junctions.
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