Fluoresceinamine‐tagged Chenodeoxycholic Acid (CDCA‐FA) causes Epithelial Barrier Dysfunction and Moves Paracellularly in Human Colonic T84 Cells

Abstract

Bile acid (BA)‐induced diarrhea affects one‐third of patients with chronic diarrhea, but the underlying mechanism is not clear. We recently reported that exposure to CDCA (500μM) altered tight junction (TJ) pore function by decreasing transepithelial resistance (TER) and leak function by increasing paracellular permeability across T84 monolayers; this involved release of reactive oxygen species (ROS) and inflammatory cytokines. The secondary BA lithocholic acid (LCA) and ROS inhibitors attenuate leak, but not pore, TJ function (Physiol Reports, ‘17, 5: e13294). We also showed that CDCA alters TJs only when added to the apical side (AS) of the monolayer and increases Cl− secretion only when added to the basolateral side (BLS) (AJP Cell, 2013, 305: C447‐56). We hypothesize that luminal BAs alter TJ permeability and travel paracellularly to reach the BLS.To test this, we synthesized a novel fluorescent BA, CDCA‐FA, by converting the carboxylic acid of CDCA to an acid chloride and then adding the FA tag. This was purified via column chromatography and the structure, molecular formula and mass (C45 H53N O7; 719.38) confirmed by NMR and mass spectroscopy. Functional studies were performed on confluent T84 cells grown on Transwells (TER >1000Ωcm2), treated AS with DMSO (CTRL), 500μM CDCA or CDCA‐FA ± 50μM LCA for 0.5–18 hours (H). Cell viability was measured by propidium iodide staining, fluorescence microscopy and Image J analysis. CDCA‐FA effects on TJs were assessed by examining: i) pore function measured as TER; ii) leak function measured as apparent permeability of CDCA‐FA (PappCDCA‐FA) compared to CDCA‐induced Fluorescein Isothiocyanate‐10 kDa dextran (F10D) flux across the monolayer. The effect of ROS scavenger, N‐acetyl cysteine (NAC; 1mM), and LCA (50μM) on CDCA‐FA's effects on TJ functions were also assessed.Overnight exposure to 500μM CDCA‐FA, like CDCA, caused a small (~18%) decrease in cell viability that was not altered by LCA (n=6). Again, similar to CDCA, CDCA‐FA showed a time‐dependent decrease in TER (in Ωcm2; t=0 H: CTRL: 927 ± 130; CDCA‐FA: 926 ± 154; t =0.5 H: CTRL: 836 ± 54; CDCA‐FA: 222 ± 28*; t=18 H; CTRL: 731 ± 10; CDCA‐FA: 107 ± 18*; n=4; *p<0.05). Furthermore, CDCA‐FA's effect on TER was neither altered by NAC nor LCA (% decrease vs. CTRL; 18 H CDCA‐FA: 85±2; CDCA‐FA+NAC: 88±5; CDCA‐FA+LCA: 87±3; CDCA‐FA+LCA+NAC: 85±6, n=3). In contrast, CDCA‐FA permeability increased over time; this increase was attenuated by LCA and further decreased by NAC (18 H, Papp CDCA‐FA, cm/sec, CDCA‐FA: 57±2; CDCA‐FA+LCA: 24±3; CDCA‐FA+NAC: 20±4; CDCA‐FA+LCA+NAC: 8±2; p<0.05, n≥3). This action of CDCA‐FA on the leak function is similar to that of CDCA on F10D flux (18 H, Papp F10D, cm/sec: CTRL: 8±1; CDCA: 165±12; p<0.05, n=6). Thus, using a novel fluorescent BA probe, CDCA‐FA, we demonstrate that, like CDCA, it increases TJ pore and leak functions in T84 cells. We conclude that CDCA from the apical side traverses to the basolateral side by a ROS‐dependent increase in paracellular permeability and that LCA limits the movement of CDCA through the TJs. Identifying ways to prevent BA's access to the BLS may be an effective therapeutic tool for the treatment of BA‐induced diarrhea.Support or Funding InformationNSF ‐ MRI: DBI‐1427937 to JS and Institutional Funds, Ben. U. to JS and DMR; Institutional Funds, UIC to MCRThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.