Received for publication September 22, 2008.
Revised June 7, 2009.
Accepted for publication June 10, 2009.

Effect of membrane cholesterol on BSEP/Bsep activity - species specificity studies for substrates and inhibitors

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 baculovirus infected Sf9 system. Vesicles prepared from these cells were used to evaluate bile salt transport of four conjugated bile salts. As 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 V_max values in all species with the most pronounced effect observed in case of the rat transporter. In contrast, K_M values with the exception of GCDC 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 appears 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 K_M values for substrates between species.

Key words: ABC transporters, adverse drug reactions, bile acid transport, biliary excretion, drug-induced hepatotoxicity, hepatic elimination, hepatobiliary transport, hepatotoxicity, high throughput screening, liver toxicity