Biochemical Journal

Research article

Transport of bile acids in multidrug-resistance-protein 3-overexpressing cells co-transfected with the ileal Na+-dependent bile-acid transporter

Noam ZELCER, Tohru SAEKI, Ilse BOT, Annemieke KUIL, Piet BORST

Abstract

Many of the transporters involved in the transport of bile acids in the enterohepatic circulation have been characterized. The basolateral bile-acid transporter of ileocytes and cholangiocytes remains an exception. It has been suggested that rat multidrug resistance protein 3 (Mrp3) fulfills this function. Here we analyse bile-salt transport by human MRP3. Membrane vesicles from insect (Spodoptera frugiperda) cells expressing MRP3 show time-dependent uptake of glycocholate and taurocholate. Furthermore, sulphated bile salts were high-affinity competitive inhibitors of etoposide glucuronide transport by MRP3 (IC5010μM). Taurochenodeoxycholate, taurocholate and glycocholate inhibited transport at higher concentrations (IC50100, 250 and 500μM respectively). We used mouse fibroblast-like cell lines derived from mice with disrupted Mdr1a, Mdr1b and Mrp1 genes to generate transfectants that express the murine apical Na+-dependent bile-salt transporter (Asbt) and MRP3. Uptake of glycocholate by these cells is Na+-dependent, with a Km and Vmax of 29±7μM and 660±63pmol/min per mg of protein respectively and is inhibited by several organic-aniontransport inhibitors. Expression of MRP3 in these cells limits the accumulation of glycocholate and increases the efflux from cells preloaded with taurocholate or glycocholate. In conclusion, we find that MRP3 transports both taurocholate and glycocholate, albeit with low affinity, in contrast with the high-affinity transport by rat Mrp3. Our results suggest that MRP3 is unlikely to be the principal basolateral bile-acid transporter of ileocytes and cholangiocytes, but that it may have a role in the removal of bile acids from the liver in cholestasis.

  • ASBT (ileal apical Na+-dependent bile-acid transporter)
  • cholestasis
  • MRP3 (multidrug-resistant protein 3)
  • transport

Footnotes

  • 1 Present address: Laboratory of Molecular Nutrition, Department of Biological Resource Chemistry, Faculty of Agricultural Science, Kyoto Prefectural University, Nakaragi, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan.

  • Abbreviations used: ASBT and Asbt, human and murine ileal apical Na+-dependent bile-acid transporter respectively; BSEP and Bsep, human and murine bile-salt export pump respectively; E217βG, 17β-oestradiol 17β-d-glucuronide; His6, hexahistidine tag; MDCK, Madin—Darby canine kidney; MRP3 and Mrp3, multidrug resistance protein 3 of human and rat or mouse respectively; Ntcp, Na+/taurocholate co-transporting polypeptide; OATP, organic anion transport protein; Sf9, Spodoptera frugiperda (fall armyworm); t-ASBT, truncated ASBT; TR/EHBR, Transport mutant/Eisai hyperbilirubinaemic rat; 4-MUS, 4-methylumbelliferone sulphate.