Activation of T84 cell chloride channels by carbachol involves a phosphoinositide-coupled muscarinic M3 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 [3H]N-methylscopolamine ([3H]NMS). [3H]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 Kd = 278 (+/- 44) pM and Bmax = 40 (+/- 6) fmol/mg protein (n = 4). Binding was reversible (diss. t1/2 = 18 +/- 3 min) and stereoselective (dexetimide Ki = 0.3 nM) much greater than levetimide (Ki = 8300 nM). Antagonists exhibited the following rank order of potencies and Ki values (nM): atropine (0.54) greater than 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) (0.84) greater than dicyclomine (14) = hexahydrosiladifenidol (18) greater than pirenzepine (136) greater than AF-DX 116 (3610). The same sequence was observed for inhibition of carbachol-induced 125I efflux from T84 monolayers. This is indicative of an M3 'glandular' muscarinic receptor. Coupling to second messenger systems was examined by labelling monolayers with [14C]arachidonic acid (AA) or [3H]inositol. Carbachol (0.3 mM) did not release [14C]AA from labelled lipids, but ionomycin produced a dose-dependent increase in media [14C]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.
Dickinson
K E
KE
Division of Gastroenterology, University of Alabama, Birmingham 35294.
Frizzell
R A
RA
Sekar
M C
MC
D016718:Arachidonic Acid / Q000378:metabolism; D001285:Atropine / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology*; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism*; D003110:Colonic Neoplasms; D006801:Humans; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism; D010276:Parasympatholytics / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D018118:Chloride Channels; D002712:Chlorides; D007473:Ion Channels; D008565:Membrane Proteins; D010276:Parasympatholytics; D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D016718:Arachidonic Acid; D015759:Ionomycin; D001285:Atropine; D002217:Carbachol