Introduction: Bile acids, classically known for their roles in facilitating lipid digestion, are now also appreciated as a family of enterocrine hormones that modulate many aspects of intestinal and metabolic function. We have previously shown lithocholic acid (LCA), a secondary bile acid formed in the colon, to be protective against colonic inflammation. Here, we sought to investigate if LCA might also regulate colonic epithelial fluid and electrolyte transport.

Methods: T₈₄ cell monolayers were mounted in Ussing chambers for measurements of Cl^– secretion, the primary driving force for colonic fluid secretion. qRT-PCR and Western blotting were used to analyze mRNA and protein expression. To assess the effects of LCA on CFTR promoter activity, we used a luciferase promoter/reporter system in HEK293. 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.

Results: Pretreatment of T₈₄ cell monolayers with LCA inhibited subsequent Cl^– secretory responses to the cAMP-dependent agonist, forskolin (FSK; 10 mM), 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 (n = 7; p < 0.01) and  0.43 ± 0.06 (n = 6; p < 0.001) fold of controls, 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 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.

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 regulatory actions of LCA in the colon and its potential role as a target for the treatment of intestinal disorders.