This application proposes continuation of experiments that were initiated in 1971 to further understanding of the regulation of sodium and chloride transport in the mammalian large intestine. Current concepts of colonic function include the spatial distribution of absorptive and secretory processes to surface and crypt epithelial cells, respectively. We have recently established complementary methods to study fluid and ion transport in crypts, independent from surface cells. These methods - 1) isolation and perfusion of colonic crypts (using technology identical to that employed for renal tubules); 2) fluorescent video imaging microscopy to determine intracellular pH (pHi) in individual polar crypt cells with intact tight junctions; and 3) apical (AP) and basolateral (BL) membrane vesicles prepared from isolated crypts - will permit the initial direct examination of crypt cell fluid and electrolyte transport. As our preliminary studies have established that the crypt is capable of both fluid absorption, a hitherto unappreciated event, and fluid secretion, which has been considered the primary crypt function, detailed examination of the cellular mechanism of both absorptive and secretory processes in the crypt will be undertaken. Other preliminary studies of Na and C1 transport with crypt AP membranes indicate that crypt Na-H exchange (NHE) is C1-dependent which is unlike surface cell AP membrane NHE; we propose that crypt Na transport is either the NHE-2 isoform or a previously undescribed Na transport process and that planned functional and molecular studies will establish the nature of crypt AP membrane Na transport. We have also established in perfused crypts that vasoactive intestinal peptide and acetylcholine induce active bicarbonate secretion which, despite its importance in diarrheal diseases, has been but minimally studied due to the lack of experimental models. We plan to establish the cellular model and the regulatory control of agonist-induced colonic bicarbonate secretion. These studies should define the cellular mechanisms of Na/C1/HCO/3 absorption and secretion in the colonic crypt.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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General Medicine A Subcommittee 2 (GMA)
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May, Michael K
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Yale University
Internal Medicine/Medicine
Schools of Medicine
New Haven
United States
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