Characterization of bile acid transport mediated by multidrug resistance associated protein 2 and bile salt export pump

Abstract

Biliary excretion of certain bile acids is mediated by multidrug resistance associated protein 2 (Mrp2) and the bile salt export pump (Bsep). In the present study, the transport properties of several bile acids were characterized in canalicular membrane vesicles (CMVs) isolated from Sprague--Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBR) whose Mrp2 function is hereditarily defective and in membrane vesicles isolated from Sf9 cells infected with recombinant baculovirus containing cDNAs encoding Mrp2 and Bsep. ATP-dependent uptake of [(3)H]taurochenodeoxycholate sulfate (TCDC-S) (K(m)=8.8 microM) and [(3)H]taurolithocholate sulfate (TLC-S) (K(m)=1.5 microM) was observed in CMVs from SD rats, but not from EHBR. In addition, ATP-dependent uptake of [(3)H]TLC-S (K(m)=3.9 microM) and [(3)H]taurocholate (TC) (K(m)=7.5 microM) was also observed in Mrp2- and Bsep-expressing Sf9 membrane vesicles, respectively. TCDC-S and TLC-S inhibited the ATP-dependent TC uptake into CMVs from SD rats with IC(50) values of 4.6 microM and 1.2 microM, respectively. In contrast, the corresponding values for Sf9 cells expressing Bsep were 59 and 62 microM, respectively, which were similar to those determined in CMVs from EHBR (68 and 33 microM, respectively). By co-expressing Mrp2 with Bsep in Sf9 cells, IC(50) values for membrane vesicles from these cells shifted to values comparable with those in CMVs from SD rats (4.6 and 1.2 microM). Moreover, in membrane vesicles where both Mrp2 and Bsep are co-expressed, preincubation with the sulfated bile acids potentiated their inhibitory effect on Bsep-mediated TC transport. These results can be accounted for by assuming that the sulfated bile acids trans-inhibit the Bsep-mediated transport of TC.

Two common PFIC2 mutations are associated with the impaired membrane trafficking of BSEP/ABCB11

“…Reverse transcription was performed using Oligo dT primer and myeloblastosis virus reverse transcriptase (Takara Shuzo) as previously described. 14 To quantify the expression of mRNA of BSEP in wild-type and mutated BSEP-expressing MDCK II cells, real-time quantitative PCR was performed using a LightCycler and the appropriate software (version 3.53; Roche Diagnostics, Mannheim, Germany). Quantitative PCR was performed using a QuantiTect SYBR Green PCR Kit (Quiagen, Valencia, CA) with 5Ј-dAGTGGGGGAGCTGAATACAA-3Ј and 5Ј-dCCAATGGTGGCTGCTCCAAT-3Ј (BSEP) and 5Ј-dACTATCGGCAATGAGCGGTTC-3Ј and 5Ј-dAGAGCCACCAATCCACACAGA-3Ј (␤-actin) as primers.…”

“…To determine the transport function of BSEP, HEK 293 cells were seeded 72 hours before infection at a density of 4.0 ϫ 10 6 cells per 15-cm dish and were infected with recombinant human BSEP adenovirus at 25 MOI. Crude membrane fractions 16 and isolated membrane vesicles 14 were prepared 48 hours after infection as previously described. 14,16 Prepared crude membrane fractions and isolated membrane vesicles were subjected to Western blot analysis.…”

The bile salt export pump

“…Since the substrate binding site(s) of the various Bsep isoforms is unknown at present, it cannot be excluded that inhibitory drugs are also potential transport substrates of Bsep. Very interestingly, estradiol-17β-glucuronide, a cholestat-ic metabolite of estrogen, requires coexpression of Mrps for inhibition of Bsep [1,86]. This has been interpreted as so-called trans-inhibition since estradiol-17β-glucucronide needs to be transported into the canalicular lumen before it can exert its cholestatic action on Bsep.…”

Regulation of Plasma Membrane Localization of the Na+-taurocholate cotransporting polypeptide (Ntcp) by Hyperosmolarity and Tauroursodeoxycholate

“…It is therefore critical that hepatocellular uptake and secretion of bile acids are coordinately regulated. At the canalicular membrane, bile salt excretion is mediated by the bile salt export pump (Bsep) 2 (7) and to a lesser extent by the multidrug resistance-associated protein 2 (Mrp2) (8). The expression of Bsep and Mrp2 in the canalicular membrane of the hepatocyte is regulated by oxidative stress (9 -11), lipopolysaccharide (LPS) (12)(13)(14), drugs (15,16), and ambient osmolarity (1,10,(17)(18)(19).…”