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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29DK048816-05
Application #
2905690
Study Section
General Medicine A Subcommittee 2 (GMA)
Program Officer
Mckeon, Catherine T
Project Start
1995-08-01
Project End
2000-11-30
Budget Start
1999-08-01
Budget End
2000-11-30
Support Year
5
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Missouri-Columbia
Department
Type
Organized Research Units
DUNS #
112205955
City
Columbia
State
MO
Country
United States
Zip Code
65211
Walker, Nancy M; Liu, Jinghua; Stein, Sydney R et al. (2016) Cellular chloride and bicarbonate retention alters intracellular pH regulation in Cftr KO crypt epithelium. Am J Physiol Gastrointest Liver Physiol 310:G70-80
Liu, Jinghua; Walker, Nancy M; Ootani, Akifumi et al. (2015) Defective goblet cell exocytosis contributes to murine cystic fibrosis-associated intestinal disease. J Clin Invest 125:1056-68
Liu, Jinghua; Walker, Nancy M; Cook, Matthew T et al. (2012) Functional Cftr in crypt epithelium of organotypic enteroid cultures from murine small intestine. Am J Physiol Cell Physiol 302:C1492-503
Walker, N M; Simpson, J E; Hoover, E E et al. (2011) Functional activity of Pat-1 (Slc26a6) Cl(?)/HCO?(?) exchange in the lower villus epithelium of murine duodenum. Acta Physiol (Oxf) 201:21-31
Alper, Seth L; Stewart, Andrew K; Vandorpe, David H et al. (2011) Native and recombinant Slc26a3 (downregulated in adenoma, Dra) do not exhibit properties of 2Cl-/1HCO3- exchange. Am J Physiol Cell Physiol 300:C276-86
Hug, Martin J; Clarke, Lane L; Gray, Michael A (2011) How to measure CFTR-dependent bicarbonate transport: from single channels to the intact epithelium. Methods Mol Biol 741:489-509
Simpson, Janet E; Walker, Nancy M; Supuran, Claudiu T et al. (2010) Putative anion transporter-1 (Pat-1, Slc26a6) contributes to intracellular pH regulation during H+-dipeptide transport in duodenal villous epithelium. Am J Physiol Gastrointest Liver Physiol 298:G683-91
Catalan, Marcelo A; Nakamoto, Tetsuji; Gonzalez-Begne, Mireya et al. (2010) Cftr and ENaC ion channels mediate NaCl absorption in the mouse submandibular gland. J Physiol 588:713-24
Clarke, Lane L (2009) A guide to Ussing chamber studies of mouse intestine. Am J Physiol Gastrointest Liver Physiol 296:G1151-66
Walker, Nancy M; Simpson, Janet E; Brazill, Jennifer M et al. (2009) Role of down-regulated in adenoma anion exchanger in HCO3- secretion across murine duodenum. Gastroenterology 136:893-901

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