The long term goal of this research is to understand the regulation of fluid and electrolyte transport by the epithelium of mammalian intestine. Here, we will be concerned with the effects of parasympathetic stimulation of the intestinal epithelium. This is a dominant excitatory stimulus for intestinal function during a meal. Parasympathetic stimulation causes Cl- secretion by the intestinal epithelium, by the cellular mechanisms remain largely undefined. This proposal is focused on the mechanisms by which the parasympathetic neurotransmitter, acetylcholine, regulates Cl- secretion. The central hypothesis is that parasympathetic stimulation of Cl- secretion is mediated by basolateral membrane K+ channels provide the driving force for Cl- efflux through apical membrane channels. The techniques of whole-cell voltage-clamp, single-channel recording and intracellular Ca2+ indicator will be used carry-out the proposed research. The research is based on preliminary studies carried out on cultured human colonic epithelial cells (T84 cell line) and has the following Specific Aims: (1) Ionic basis and gating mechanism: We will determine the ionic basis of muscarinic cholinergic agonist-induced whole-cell membrane current oscillations in isolated cells. We will establish whether the underlying channel is gated by membrane voltage, directly by the ligand, or by intracellular Ca2+ release, or both is/are necessary for the Ca2+ oscillations. (3) Membrane location of ion channels: We will determine whether the oscillating ion current is located in the apical or basolateral membrane, since the loci of ion channels determines their role in secretion or absorption. We will determine the sites and properties (eg., voltage dependence, selectivity) of cholinergic agonist-induced K+ and Cl- channels in cultured monolayers of T84 cells, and in the basolateral membrane of acutely dissociated crypts from rabbit distal colon. (4) Integrated response: We will determine whether a hyperpolarization of membrane voltage and intracellular Ca2+ oscillations are part of the Cl- secretory response of intact tissues during cholinergic stimulation.
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