ACTIVATION OF T84 CELL CHLORIDE CHANNELS BY CARBACHOL INVOLVES A PHOSPHOINOSITIDE-COUPLED MUSCARINIC-M(3) RECEPTOR

Muscarinic agonists stimulate Cl- secretion across monolayers of the colon tumor epithelial cell line, T84. The muscarinic receptor has been characterized in T84 cell homogenates by radioligand binding using [H-3]N-methylscopolamine ([H-3]NMS). [H-3]NMS bound to a single population of sites at 25-degrees-C in 100 mM NaCl, 20 mM HEPES, 10 mM MgCl2, pH 7.4 buffer, with calculated K(d) = 278 (+/- 44) pM and B(max) = 40 (+/- 6) fmol/mg protein (n = 4). Binding was reversible (diss. t1/2 = 18 +/- 3 min) and stereoselective (dexetimide K(i) = 0.3 nM) much greater than levetimide (K(i) = 8300 nM). Antagonists exhibited the following rank order of potencies and K(i) values (nM): atropine (0.54)>4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) (0.84) > dicyclomine (14) = hexahydrosiladifenidol (18) > pirenzepine (136) > AF-DX 116 (3610). The same sequence was observed for inhibition of carbachol-induced I-125 efflux from T84 monolayers. This is indicative of an M3 'glandular' muscarinic receptor. Coupling to second messenger systems was examined by labelling monolayers with [C-14]arachidonic acid (AA) or [H-3]inositol. Carbachol (0.3 mM) did not release [C-14]AA from labelled lipids, but ionomycin produced a dose-dependent increase in media [C-14]AA. Carbachol (0.3 mM) elevated inositol monophosphate 14-fold. The results suggest that muscarinic agonists stimulate Cl- secretion by interacting with an M3 receptor coupled to inositide lipid hydrolysis.