The overall goal of this proposal is to determine the regulatory mechanisms controlling active potassium secretion and absorption in the mammalian colon. The working model is that the vacuolated columnar cells of the mammalian colonic crypt possess pathways for K, Cl and H exit across the apical membrane as well as K and Cl channels on the basolateral membrane. The hypothesis to be tested is that the coordinated, albeit differential regulation, of these processes by different hormones/modulators is responsible for the range of secretory behavior exhibited by the colonic epithelium. Thus epinephrine and aldosterone cause a low rate of fluid secretion that is proposed to be a modulatory type of regulation whereas prostaglandin E2 (PGE2) causes a high rate of fluid flow that is proposed to be more of a flushing type of secretion. A dysfunction in either of these regulatory cascades in the vacuolated columnar cells would form the basis of a variety of gastrointestinal diseases ranging from secretory diarrhea to inflammatory bowel disease. In the first two specific aims the regulatory mechanisms acting on K and Cl channels during the flushing and modulatory type of stimulation will be examined, largely through patch clamp methodology. These studies will allow the events at the apical and basolateral membranes to be studied separately. They will be complemented by measurements of intracellular Cl using a fluorescent probe, and by morphological (differential interference contrast microscopy) and biophysical (membrane capacitance) studies to track vacuole release. In the third specific aim, using fluorescent probes, the involvement of intracellular Ca and H in the regulation of the ion channels will be investigated. Preliminary data suggest the involvement of these two pathways in the actions of PGE2, aldosterone and epinephrine. In addition apical membrane H/K pump activity will be assessed and the presence of an H/K ATPase transcript determined by in situ hybridization.
