3h taurocholate

Villus cells were isolated from the ileum of the experimental animals by a calcium chelation technique as previously described [19 (link)] with few modifications. Briefly, isolated distal ileal villus cells were washed twice in TMA-HEPES buffer (50 mM KCl, 0.1 mM MgSO₄, 50 mM Mannitol, 50 mM HEPES-Tris (pH 7.5), and 100 mM TMA-Cl) and suspended in 100 µL of the same buffer. Next, 10 μL of the cells from the suspension were added to 100 μL of reaction media containing 0.1 mM taurocholate (TCA; Sigma-Aldrich Corporation, St. Louis, MO, USA) with 100 μM ³H-taurocholate (PerkinElmer, Inc., Waltham, MA, USA), 50 mM KCl, 0.1 mM MgSO₄, 50mM Mannitol, 50 mM HEPES-Tris (pH 7.5), and either 100 mM TMA-Cl or 100 mM NaCl. The reaction was stopped at two minutes by adding 1 mL of ice-cold TMA-HEPES buffer. The stopped reaction mixture was filtered on 0.65 μm Millipore (HAWP) filter and washed twice with ice-cold stop solution. The reactions were carried out in triplicate for each of the two reaction mixtures. The filter was dissolved in 5 mL of scintillation fluid (Ecoscint A, National Diagnostics), and radioactivity was determined in a Beckman 6500 scintillation counter (Beckman Coulter Inc., Brea, CA, USA).

The active ingredient of MT921, cholic acid (CA), was synthesized from Medytox Inc. (Suwon, Korea). Cyclosporine A, diclofenac, bromsulphthalein, deoxycholate, chenodeoxycholic acid, and Dulbecco’s Phosphate Buffered Saline (DPBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). [³H]estrone-3-sulfate (45 Ci/mmol), [³H]estradiol-17β-d-glucuronide (34.3 Ci/mmol), and [³H]taurocholate (5 Ci/mmol) were purchased from Perkin Elmer (Boston, MA, USA). Fetal bovine serum (FBS), non-essential amino acids, penicillin, and streptomycin were purchased from Gibco BRL, Life Technologies (Grand Island, NY, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), poly-d-lysine coated 24-well plates, and poly-d-lysine coated 96-well plates were purchased from Corning-Gentest (Tewksbury, MA, USA). The acetonitrile used was analytical grade and purchased from Merck (Darmstadt, Germany).
The HEK 293-OATP1B1, -OATP1B3, -OAT3, -NTCP, and -ASBT stable cells and mock cells were purchased from Corning Life Science (Woburn, MA, USA).

Hygromycin (merckmillipore), anti-HA HRP (sigma), ^3HTaurocholate (Perkin Elmer), Taurocholate (Sigma), MG132 (Z-Leu-Leu-Leu-al) (sigma), Bafilomycin A1 (sigma), DMSO (VWR), Myrcludex B-FITC (Pepscan), Transferrin receptor (Invitrogen), anti-mouse-HRP(DAKO).