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.

Lithocholic acid attenuates cAMP-dependent Cl−secretion in human colonic epithelial T84 cells

“…It is well recognized that primary (CDCA) and secondary (DCA) dihydroxy bile acids regulate epithelial ion transport processes and, in particular, stimulate colonic Cl Ϫ secretion (4,31,64,75). In contrast, neither the ␤-epimer of CDCA (UDCA) the primary trihydroxy bile acid (CA), nor the secondary monohydroxy bile acid (LCA) (4,41) (42) while inhibiting it in the rat colon (77).…”

“…In contrast, neither the ␤-epimer of CDCA (UDCA) the primary trihydroxy bile acid (CA), nor the secondary monohydroxy bile acid (LCA) (4,41) (42) while inhibiting it in the rat colon (77). Given the importance of LCA as a metabolite in the colon, in this paper, we focused on exploring further the cellular and ionic basis of the modulatory role of LCA in Cl Ϫ secretion using T84 cells as a model system.…”

“…Malfunctioning of one or more of the transporters and signaling proteins underlying this intricately regulated process can lead to disease (3). For example, ileal bile acid malabsorption leads to excess bile acids in the colon where it is well established that DCA and CDCA, but not UDCA, CA, or LCA, stimulate Cl Ϫ secretion leading to fluid loss and diarrhea (4,9,23,39,41,58,75).…”

“…We recently demonstrated that CDCA acts via an adenylate cyclase/PKA-dependent mechanism to stimulate Cl Ϫ secretion via cystic fibrosis transmembrane conductance regulator (CFTR) in the human colon carcinoma cell line T84 (4). Furthermore, at increasing concentrations, LCA did not stimulate Cl Ϫ secretion (4).…”

Key discoveries in bile acid chemistry and biology and their clinical applications: history of the last eight decades

“…Details of the biochemical mechanism(s) for bile acid-induced intestinal secretion are being elucidated by several laboratories, among these being that of Stephen Keely in Dublin ( 448 ), and that of Mrinalini Rao in Chicago ( 450 ). Bile acid malabsorption causes diarrhea, and this can be treated by sequestrant administration ( 451,452 ).…”