title: Chenodeoxycholic acid stimulates Cl(-) secretion via cAMP signaling and increases cystic fibrosis transmembrane conductance regulator phosphorylation in T84 cells.
abstract: High levels of chenodeoxycholic acid (CDCA) and deoxycholic acid stimulate Cl(-) secretion in mammalian colonic epithelia. While different second messengers have been implicated in this action, the specific signaling pathway has not been fully delineated. Using human colon carcinoma T84 cells, we elucidated this cascade assessing Cl(-) transport by measuring I(-) efflux and short-circuit current (Isc). CDCA (500 μM) rapidly increases I(-) efflux, and we confirmed by Isc that it elicits a larger response when added to the basolateral vs. apical surface. However, preincubation with cytokines increases the monolayer responsiveness to apical addition by 55%. Nystatin permeabilization studies demonstrate that CDCA stimulates an eletrogenic apical Cl(-) but not a basolateral K(+) current. Furthermore, CDCA-induced Isc was inhibited (≥67%) by bumetanide, BaCl2, and the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh-172. CDCA-stimulated Isc was decreased 43% by the adenylate cyclase inhibitor MDL12330A and CDCA increases intracellular cAMP concentration. The protein kinase A inhibitor H89 and the microtubule disrupting agent nocodazole, respectively, cause 94 and 47% reductions in CDCA-stimulated Isc. Immunoprecipitation with CFTR antibodies, followed by sequential immunoblotting with Pan-phospho and CFTR antibodies, shows that CDCA increases CFTR phosphorylation by approximately twofold. The rapidity and side specificity of the response to CDCA imply a membrane-mediated process. While CDCA effects are not blocked by the muscarinic receptor antagonist atropine, T84 cells possess transcript and protein for the bile acid G protein-coupled receptor TGR5. These results demonstrate for the first time that CDCA activates CFTR via a cAMP-PKA pathway involving microtubules and imply that this occurs via a basolateral membrane receptor.
authors.affiliation: Department of Physiology and Biophysics, University of Illinois, Chicago, Illinois;
authors.forename: Mei
authors.initials: M
authors.lastname: Ao
authors.forename: Jayashree
authors.initials: J
authors.lastname: Sarathy
authors.forename: Jada
authors.initials: J
authors.lastname: Domingue
authors.forename: Waddah A
authors.initials: WA
authors.lastname: Alrefai
authors.forename: Mrinalini C
authors.initials: MC
authors.lastname: Rao
mesh_terms: D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D002463:Cell Membrane Permeability; D016764:Cell Polarity; D002635:Chenodeoxycholic Acid / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D003110:Colonic Neoplasms / Q000276:immunology / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000187:drug effects / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D007413:Intestinal Mucosa / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D008564:Membrane Potentials; D008870:Microtubules / Q000378:metabolism; D010766:Phosphorylation; D015290:Second Messenger Systems / Q000187:drug effects; D013997:Time Factors; D015854:Up-Regulation
chemical_list: C505032:CFTR protein, human; D002712:Chlorides; D016207:Cytokines; D018836:Inflammation Mediators; D002635:Chenodeoxycholic Acid; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases
keywords: CDCA; CFTR; T84 cells; cAMP signaling