Gastrointestinal disease manifestations in the human genetic disease, cystic fibrosis (CF), are indicative of the central role that the cystic fibrosis transmembrane conductance regulator (CFTR) plays in gastrointestinal electrolyte transport physiology. Meconium ileus/distal obstruction syndrome, maldigestion/malabsorption disorders, and an increased risk of gastroduodenal ulcer disease and neoplasia have been reported for patients with mutations in the cftr gene and, hence, its defective epithelial protein product, CFTR. Although it is now widely recognized that CFTR can function as a cAMP-activated Cl- channel protein, numerous reports have described significant effects of CFTR mutations on other epithelial transport processes, including Na+ absorptive mechanisms, transcellular bicarbonate movements and sulfate transport. Our preliminary findings in a CFTR """"""""knockout"""""""" mouse model of CF demonstrate that CFTR is critical for intestinal transcellular pH regulation by being required for 1) cAMP-stimulated HCO3- secretion in the proximal duodenum, and 2) cAMP-induced inhibition of coupled Na+/H+, CL0/HCO3- exchange mechanisms in the jejunum. Furthermore, the reported location of these transport mechanisms suggest that CFTR expression is not only localized to the intestinal crypts but uniquely functions in villous epithelium. The long-term goal of this laboratory is to uncover the cellular and molecular mechanisms involved in the modulation of intestinal acid/base transporters by CFTR. Littermates of the CFTR ~knockout"""""""" mouse model will be used to study intestinal epithelia with normal [cftr(+/+) mice], reduced [cftr(+/-) mice], or absent [cftr(-/-) mice] CFTR protein expression.
In Specific Aim 1, we will identify the role of CFTR in cAMP-induced HCO3- transport in the proximal duodenal mucosa.
This aim will be accomplished using voltage-clamp measurement of isolated transepithelial ion currents and immunodetection of CFTR protein in epithelial cell lysates.
In Specific Aim 2, we will localize CFTR (or other anion conductances) and relevant intracellular anion driving forces along the crypt-villus axis using intracellular ion- selective microelectrode recordings. Isolated intact villous epithelium will be used for immunodetection of CFTR and to localize relevant ionic currents.
In Specific Aim 3, we will identify the role of CFTR in cAMP- mediated inhibition of coupled Na+/H+, Cl-/HCO3- exchange mechanisms in the intestine. Isotopic flux and cell volume measurements will be used to test the hypothesis that CFTR-dependent changes in cell volume inhibit the coupled Na+/H+, Cl-/HCO3- absorptive process. Reconstitution of CFTR-negative alimentary epithelial cell lines with wild-type CFTR will be used to verify or disprove the hypothesis.
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