Abstract
The efflux transporter responsible for the canalicular elimination of bile salts from the hepatocytes is the bile salt export pump (BSEP, ABCB11). Absence or inhibition of this transporter leads to bile salt retention in the hepatocyte and in turn can lead to cholestatic liver disease. We expressed the BSEP/Bsep protein from three species (human, rat, and mouse) in a baculovirus-infected Sf9 system. Vesicles prepared from these cells were used to evaluate bile salt transport of four conjugated bile salts. Because the Sf9 system contains less membrane cholesterol than the liver canalicular membrane, the effect of added cholesterol on the kinetics of BSEP/Bsep-mediated bile salt transport was also investigated. Cholesterol treatment increased the Vmax values in all the species, with the most pronounced effect observed in the rat transporter. In contrast, Km values, with the exception of glycochenodeoxycholate, remained largely unchanged. The species-specific bile salt transport inhibition potential of three compounds known to cause clinical cholestasis was investigated in vesicles containing BSEP/Bsep. Troglitazone and glibenclamide inhibited the BSEP/Bsep-mediated transport of different bile salts with similar affinities, whereas the potential of cyclosporine A to inhibit bile salt transport showed species- and bile salt-specific variations. In conclusion, the cholesterol-loaded Sf9 vesicles overexpressing BSEP/Bsep seem to be a useful system for the identification of potential cholestatic compounds and can also be used for the investigation of species specificity. We observed greater differences in IC50 values for inhibitors than in Km values for substrates between species.
Footnotes
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This work was supported in part by Asbóth [Grant XTTPSRT1]; Kozma [Grants TUDAS_06-HTShuMXR and NKFP-1A/041/04]; European Community [Grants BIOSIM-LSHB-CT-2004-005137 and LIINTOP-LSHB-CT-2006-037499]; and Memtrans [Grant LSBH-CT-2006-518246].
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.024778.
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ABBREVIATIONS: BSEP/Bsep, bile salt export pump, ABCB11; MRP, multidrug resistance-associated protein, ABCC; MDR, multidrug resistance protein, ABCB; ABCG, ATP-binding cassette protein subfamily G; ABC, ATP-binding cassette; P-gp, P-glycoprotein; CsA, cyclosporine A; SA, specific activity; TC, taurocholate; GC, glycocholate; TCDC, taurochenodeoxycholate; GCDC, glycochenodeoxycholate; HAM, high activity membrane; HRP, horseradish peroxidase; CMV, canalicular membrane vesicle.
- Accepted June 10, 2009.
- Received September 22, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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