Regulation of electrolyte and water transport is a major function of the intestinal epithelium. Pathological alterations in anion secretion and coupled NaCI absorption play a significant role in the intestinal manifestations of cystic fibrosis, diarrheaI disease and duodenal ulcer disease. In this proposal, the processes of transepithelial HCO3- secretion across the duodenum and electroneutral NaCI absorption (via coupled Na+/H+ - CI-/HCO3- exchange) across the jejunum will be specifically addressed. Studies have shown that CFTR, the major anion conductance, and NHE3, the major non-nutrient Na+ absorptive protein, in the apical membrane of small intestinal epithelia are essential to normal function and regulation of both processes. However, our current understanding of these transport processes requires extension to include interactions with the apical membrane CI-/HCO3- exchangers. The identity of the apical membrane anion exchangers has been elucidated in recent years with the discovery of members in the multifunctional anion exchanger family Slc26a, primarily Slc26a3 (down-regulated in adenoma, DRA) and Slc26a6 (putative anion transporter-1, PAT-1). The interaction of these CI-/HCO3- exchangers with CFTR and NHE3 occur most extensively at the villous epithelium, but there are reciprocal gradients for CFTR (crypt to villus) and NHE3 (villus to crypt) expression along the villous axis. Therefore, we have developed techniques to measure the activity of transporters in epithelial cells in intact villi using BCECF ratio microfluorimetry to measure intracellular pH (pHi), single-barreled microelectrodes to measure apical membrane potential, and laser capture dissection coupled with quantitative real-time PCR and immunohistochemistry to measure mRNA and protein expression. Definitive results will be achieved through the opportunities afforded by studies using mice with single and double gene-targeted deletions of PAT-1, DRA, CFTR or NHE3. Based on our Preliminary Studies, we hypothesize in Specific Aim 1 that PAT-1 is the major CI-/HCO3- exchanger in the upper villous epithelium of the duodenum where it provides HCO3- secretion and regulates pHi during acid challenge. It is postulated that PAT-1 acts independently and has indirect associations with CFTR and NHE3.
In Specific Aim 2, we hypothesize that DRA is the major CI-/HCO3- exchanger in the lower villous epithelium of the duodenum where it is critical to stimulated HCO3- secretion and is closely regulated by CFTR.
In Specific Aim 3, we hypothesize that DRA is the major CI-/HCO3- exchanger in the upper villous epithelium of the jejunum where it provides CI- absorption and is closely regulated by NHE3. Completion of these aims will establish the roles of the apical membrane CI-/HCO3- exchangers in the functions of HCO3- secretion and NaCI absorption by native intestinal mucosa. Understanding these physiological processes has therapeutic implications for diseases of intestinal electrolyte and water transport.
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