vasoactive-intestinal-peptide and 3--5-dichlorodiphenylamine-2-carboxylic-acid

The mechanism of regulated Cl- secretion was evaluated in the mucin-secreting cell line HT29-Cl.16E by transepithelial electrophysiology and fura 2 measurements of cytosolic Ca2+. Carbachol by itself was a weak secretagogue, but augmented adenosine 3',5'-cyclic monophosphate (cAMP)-mediated secretion more than twofold, consistent with activation of a rate-limiting K+ conductance. To characterize this conductance, monolayers were apically permeabilized with amphotericin B. At least two types of K+ conductances were identified. One type was activated by elevated cytosolic cAMP levels and inhibited by Ba2+ (inhibitor constant 0.3 mM) in the basolateral solution but was not affected by quinidine or elevated cytosolic Ca2+. The other type was activated by carbachol via cytosolic Ca2+ and was partially inhibited by quinidine (60% inhibition by 2.5 mM quinidine) but was not affected by Ba2+ up to 1 mM. Both conductances appear to be involved in active, transepithelial Cl- secretion in intact monolayers but under different conditions because 1) the cAMP-stimulated short-circuit current (Isc) can be partially inhibited by 1 mM Ba2+ (50%) but not quinidine, 2) the Ba2+ inhibition does not affect the carbachol-induced increase in Isc in cells with elevated cAMP levels, and 3) the carbachol-dependent Isc can be inhibited by quinidine. Therefore, the contribution of the cAMP-dependent K+ conductance appears important for maintaining the membrane potential and therewith Cl- secretion when cAMP is the only messenger for secretion signals, whereas the Ca(2+)-dependent K+ conductance is responsible for the carbachol-stimulated increase in Isc.

Topics: Amphotericin B; Barium; Bumetanide; Calcimycin; Calcium; Carbachol; Cell Line; Cell Membrane Permeability; Colforsin; Cyclic AMP; Digitonin; Diphenylamine; Electric Conductivity; Epithelial Cells; Epithelium; Fura-2; Humans; Intestinal Mucosa; Membrane Potentials; Models, Biological; Ouabain; Potassium; Potassium Channels; Quinidine; Tetradecanoylphorbol Acetate; Thionucleotides; Vasoactive Intestinal Peptide