The Secondary Bile acid, Lithocholic Acid, Inhibits Cl− Secretion Across Colonic Epithelial Cells in Vitro

Abstract

Lithocholic acid (LCA) is a secondary bile acid formed in the colon by bacterial metabolism of chenodeoxycholic acid. Our previous studies have shown the bile acid to be protective under conditions of colonic inflammation. Here, we sought to determine if LCA might also play a role in regulating fluid and electrolyte transport. T84 cell monolayers were mounted in Ussing chambers for measurements of Cl− secretion, the primary driving force for fluid secretion into the colon. qRT-PCR and Western blotting were used to analyze mRNA and protein expression. Results were expressed as mean ± SEM and data were analyzed by one way ANOVA and Tukey’s post hoc test or by mixed-effects analysis and Dunnett's post hoc test. To assess the effects of LCA on CFTR promoter activity, we used a luciferase promoter/reporter system, where HEK293 cells were transfected with a plasmid containing the firefly luciferase gene under control of the CFTR promoter. Co-transfections were carried out with 2 additional plasmids expressing either the nuclear bile acid receptor, FXR, or its dimerization partner, RXR. Pretreatment of T84 cell monolayers with LCA inhibited subsequent Cl− secretory responses to the cAMP-dependent agonist, forskolin (FSK; 10 μM), in a concentration (1 – 10 μM) and time-dependent (3 - 24 hrs) manner. Maximal effects of LCA were observed at a concentration of 10 μM after treatment for 24 hrs, when responses to FSK were reduced to 50.9 ± 8.5% of those in controls (n = 6; p < 0.01). LCA (10 μM; 24 hrs) also inhibited responses to the Ca2+-dependent secretagogues, thapsigargin (2 μM) and histamine (100 μM), by 59.4 ± 2.4% (n = 4; p < 0.001) and 52.2 ± 1.9% (n = 5; p < 0.001), respectively. In further experiments, using nystatin-permeabilized T84 monolayers to isolate apical Cl− conductances, LCA (10 μM; 24 hrs) reduced FSK-stimulated responses to 72.7 ± 6.6% (n = 17; p < 0.001) of those in control cells. Analysis of CFTR expression, the primary exit pathway for Cl− in colonic epithelial cells, revealed that LCA treatment reduced mRNA and protein expression of the channel to 0.65 ± 0.05 fold (n = 7; p < 0.01) and 0.43 ± 0.06 fold (n = 6; p < 0.001), respectively. In CFTR promoter assays, LCA (10 μM) reduced CFTR promoter activity to 0.7 ± 0.02 fold of that in control cells (n = 5; p < 0.01), with co-expression of FXR being required for this effect to be observed. Finally, while LCA activated both FXR and the vitamin D receptor (VDR) in T84 cells, its effects in downregulating CFTR expression and Cl− conductances were mimicked by the FXR agonist, GW4064, but not by the VDR agonist, calcitriol. In conclusion, LCA, at physiologically relevant concentrations, inhibits Cl− secretion across colonic epithelial cells, likely through a mechanism involving FXR activation and inhibition of CFTR expression. These data add to the growing pool of knowledge regarding important regulatory actions of LCA in the colon and highlight its potential role as a target for the treatment of intestinal disorders. This work was supported by a grant from Science Foundation Ireland. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.