Gastroenterology

Gastroenterology

Volume 118, Issue 2, February 2000, Pages 422-430
Gastroenterology

Liver, Pancreas, and Biliary Tract
Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver,☆☆

https://doi.org/10.1016/S0016-5085(00)70224-1Get rights and content

Abstract

Background & Aims: Drug-induced cholestasis is a frequent form of acquired liver disease. To elucidate the molecular pathogenesis of drug-induced cholestasis, we investigated the effects of prototypic cholestatic drugs on the canalicular bile salt export pump (Bsep) of rat liver. Methods: Vesicles were isolated from Bsep-, Mrp2-, and Bsep/Mrp2-expressing Sf9 cells. Canalicular plasma membrane (cLPM) vesicles from rat liver and Sf9 cell vesicles were used to study adenosine triphosphate (ATP)-dependent solute uptake by a rapid filtration technique. Results: Bsep-expressing Sf9 cell vesicles showed ATP-dependent transport of numerous monoanionic bile salts with similar Michaelis constant values as in cLPM vesicles, whereas several known substrates of the multispecific organic anion transporter Mrp2 were not transported by Bsep. Cyclosporin A, rifamycin SV, rifampicin, and glibenclamide cis-inhibited Bsep-mediated bile salt transport to similar extents as ATP-dependent taurocholate transport in cLPM vesicles. In contrast, the cholestatic estrogen metabolite estradiol-17β-glucuronide inhibited ATP-dependent taurocholate transport only in normal cLPM and in Bsep/Mrp2-coexpressing Sf9 cell vesicles, but not in Mrp2-deficient cLPM or in selectively Bsep-expressing Sf9 cell vesicles, indicating that it trans-inhibits Bsep only after its secretion into bile canaliculi by Mrp2. Conclusions: These results provide a molecular basis for previous in vivo observations and identify Bsep as an important target for induction of drug- and estrogen-induced cholestasis in mammalian liver.

GASTROENTEROLOGY 2000;118:422-430

Section snippets

Cell culture

Sf9 cells were obtained from Pharmingen (San Diego, CA) and kept at 27°C under standard conditions. Infections with baculoviruses encoding Bsep8 were performed with a multiplicity of infection (MOI) of 10 and 3 days of incubation before subcellular fractionation. Mrp2 recombinant baculovirus11 was generated with the Bac-to-Bac system (Life Technologies Inc., Gaithersburg, MD). Sf9 cells were infected with Mrp2-encoding baculovirus at MOI of 7.5 and incubated for 2 days before subcellular

Functional expression of Bsep in baculovirus-infected Sf9 cells

In mammalian (including human) liver, the canalicular secretion of anionic bile salts and nonbile salt organic anions is mediated by the 2 separate ABC transporters Bsep (BSEP) and Mrp2 (MRP2).17 These complementary transport functions were confirmed by the separate expression of rat liver Bsep and Mrp2 in baculovirus-infected Sf9 cells (Table 1). Although membrane vesicles from Sf9 cells infected with the wild-type baculovirus did not exhibit any ATP-dependent transport activities, infection

Discussion

Canalicular bile salt secretion is the major driving force of hepatic bile formation. In our previous study, the so-called sister of P-glycoprotein32 was identified as a canalicular ATP-dependent Bsep in rat liver.8 In parallel, the coding region of the human BSEP gene has been elucidated, and several mutations predicted to disrupt BSEP gene function have been identified in patients with type 2 of PFIC (PFIC-2).2 The phenotype of PFIC-2 is entirely consistent with an isolated defect in

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    Supported in part by grants 3100-045536.95 and 3200-052190.97 from the Swiss National Science Foundation and a SCORE-A clinical research development award of the Swiss National Science Foundation (to K.F.).

    ☆☆

    Address requests for reprints to: Peter J. Meier-Abt, Division of Clinical Pharmacology and Toxicology, Department of Medicine, University Hospital, CH-8091 Zurich, Switzerland. e-mail: [email protected]; fax: (41) 1-255-4411.

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