Transport by vesicles of glycine- and taurine-conjugated bile salts and taurolithocholate 3-sulfate: A comparison of human BSEP with rat Bsep

https://doi.org/10.1016/j.bbalip.2005.10.006Get rights and content

Abstract

The bile salt export pump (BSEP) of hepatocyte secretes conjugated bile salts across the canalicular membrane in an ATP-dependent manner. The biliary bile salts of human differ from those of rat in containing a greater proportion of glycine conjugates and taurolithocholate 3-sulfate (TLC-S). In the present study, the transport properties of hBSEP and rBsep were investigated using membrane vesicles from HEK293 cells infected with recombinant adenoviruses containing hBSEP or rBsep cDNA. ATP-dependent uptake of radiolabeled glycine-, taurine-conjugated bile salts, and [3H]cholate was observed when hBSEP or rBsep was expressed. Comparison of initial uptake rates indicated that for both transporters, taurine-conjugated bile salts were transported more rapidly than glycine-conjugated bile salts, however, hBSEP transported glycine conjugates to an extent that was approximately 2-fold greater than rBsep. In addition, [3H]TLC-S was significantly transported by hBSEP, and hardly transported by rBsep. The mean Km value for the uptake of [3H]TLC-S by hBSEP was 9.5 ± 1.5 μM, a value similar to that for hMRP2 (8.2 ± 1.3 μM). In conclusion, both hBSEP and rBsep transport taurine-conjugated bile salts better than glycine-conjugated bile salts, but hBSEP transports glycine conjugates to a greater extent as compared to rBsep. TLC-S, which is present in human bile but not rodent bile, is more avidly transported by hBSEP compared with rBsep.

Introduction

Bile formation is one of important functions of the liver. Vectorial transport of bile salts from the sinusoidal space to the canaliculus via hepatocytes provides an osmotic driving force for bile formation [1], [2]. Transport of bile salts across the sinusoidal membrane is mediated at least in part by both Na+-taurocholate co-transporting polypeptide (human NTCP/SLC10A1 and rat Ntcp/Slc10a1) [3], [4] and Na+-independent organic anion transporting polypeptides (human OATP/SLCO and rat Oatp/Slco) [1], [2]. After reaching the canalicular membrane, monovalent taurine- and glycine-conjugated bile salts are secreted into bile by the bile salt export pump (human BSEP/ABCB11 and rat Bsep/Abcb11) [5], [6], [7], [8], [9], [10], whereas sulfated bile salt amidates and bile salt ethereal glucuronides (divalent) are excreted by the multidrug resistance associated protein 2 (human MRP2/ABCC2 and rat Mrp2/Abcc2) [11], [12], [13].

The transport properties of hBSEP/rBsep and hMRP2/rMrp2 have been clarified recently. The function of hBSEP/rBsep has been characterized by examining the ATP-dependent transport of taurine- and glycine-conjugated bile salts in isolated bile canalicular membrane vesicles (CMVs) and/or membrane vesicles isolated from hBSEP/rBsep-expressing cells [5], [6], [7], [8]. For hMRP2/rMrp2, the transport characteristics have been mainly investigated by comparing transport across the bile canalicular membrane in normal rats with that in Mrp2-deficient rats [8], [14], [15], [16]. Mutations in BSEP gene are now known to be the cause of progressive familial intrahepatic cholestasis type 2 (PFIC2) [17], [18], [19], [20], [21], whereas mutations in MRP2 are the cause of Dubin–Johnson syndrome (DJS) [22], [23], [24].

Major bile salts in mammals, i.e., ionized forms of C24-bile acids, are synthesized from cholesterol in the liver and then conjugated (N-acylamidated) with glycine or taurine. In health, sulfation of primary bile salts does not occur in most mammals, but in cholestasis at least in some species, bile salts are extensively sulfated and eliminated into urine. The biliary bile salt composition in the human significantly differs from that in the rat. In humans, the majority of bile salts are conjugated with glycine, whereas in rats, most bile salts are conjugated with taurine. Human bile, but not rat bile, contains sulfated and unsulfated amidates of lithocholate (LCA) [25], [26].

Two groups have attempted to relate the transport properties of hBSEP to biliary bile salt composition in humans as compared to that in rats [5], [7]. Noe et al. concluded that the transport property of hBSEP/rBsep does not explain the differences in steady state biliary bile salt composition between the two species, because of the similarity of Km value and intrinsic clearance value for TC, GC, TCDC and TUDC [7]. On the other hand, Byrne et al. concluded that the transport properties of hBSEP/rBsep do in fact correlate with the different bile salt pools in human and rat due to the difference of the relative affinities for TC, GC, TCDC and GCDC between hBSEP and rBsep [5]. In the present study, we have characterized the transport function of hBSEP and rBsep for twelve physiologic bile salts including several kinds of bile salts untested in the previous reports, such as taurolithocholate 3-sulfate. The transport function was determined using membrane vesicles from HEK293 cells infected with recombinant adenoviruses containing hBSEP and rBsep cDNA. We were able to confirm that the transport properties of hBSEP/rBsep reflect the difference in bile salt composition between human and rat. Kinetic analyses to hBSEP and hMRP2 were also performed for [3H]TLC-S, since the initial uptake study demonstrated that this sulfated bile salt is good substrate for hBSEP, not good substrate for rBsep.

Section snippets

Materials and cell culture

[3H]cholate (CA) (24.5 Ci/mmol), [3H]taurocholate (TC) (2 Ci/mmol), [14C]chenodeoxycholate (CDCA) (48.6 mCi/mmol) and [2-3H]taurine (30.3 Ci/mmol) were purchased from NEN Life Sciences Products (Boston, MA). [14C]glycocholate (GC) (57.3 mCi/mmol) and [14C]lithocholate (LCA) (57.3 mCi/mmol) were purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). [3H]ursodeoxycholate (UDCA) (20 Ci/mmol), [3H]tauroursodeoxycholate (TUDC) (10 Ci/mmol), [3H]glycoursodeoxycholate (GUDC) (11

Uptake of [3H]TC into membrane vesicles

The expression of hBSEP or rBsep in the membrane vesicles prepared from the transfected HEK293 cells was confirmed by Western blot analysis (Fig. 1). As shown in Fig. 1, hBSEP and rBsep were detected as an approximately 170 kDa form in the fraction of membrane vesicles prepared from hBSEP- and rBsep-transfected HEK 293 cells. No expression of hBSEP or rBsep was detected in the control membrane vesicles prepared from GFP-transfected HEK293 cells.

The time-profiles for the uptake of [3H]TC into

Discussion

In the present study, we compared the transport properties of hBSEP and rBsep using membrane vesicles from HEK293 cells infected with adenoviruses containing these cDNAs. Our aim was to examine the correlation between the function of hBSEP/rBsep and the biliary bile salt composition.

Initially, transport studies were performed by measuring the ATP-dependent uptake of [3H]TC into the hBSEP- and rBsep-expressing membrane vesicles to confirm the transport function of hBSEP and rBsep. The Km values

Acknowledgements

We thank Mr. Masakazu Hirouchi for construction of hMRP2 expression system, Dr. Hidetaka Akita and Ms. Sachiko Mita for construction of rBsep expression system. This work was supported by Grant-in-Aid for Scientific Research on Priority Areas Epithelial Vectorial Transport 12144201 and Grant-in-Aid for Center of Excellence (COE) from The Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. Work in the laboratory of Alan F. Hofmann is supported by NIH Grant DK 64891.

References (43)

  • K. Hashimoto et al.

    Trafficking and functional defects by mutations of the ATP-binding domains in MRP2 in patients with Dubin–Johnson syndrome

    Hepatology

    (2002)
  • V. Keitel et al.

    Impaired protein maturation of the conjugate export pump multidrug resistance protein 2 as a consequence of a deletion mutation in Dubin–Johnson syndrome

    Hepatology

    (2000)
  • Y. Siow et al.

    Diabetes-induced bile acid composition changes in rat bile determined by high performance liquid chromatography

    Life Sci.

    (1991)
  • S. Sorscher et al.

    Conjugated bile acid uptake by Xenopus laevis oocytes induced by microinjection with ileal Poly A+ mRNA

    Biochem. Biophys. Res. Commun.

    (1992)
  • W.C. Duane et al.

    Validation of [22,23-3H]cholic acid as a stable tracer through conversion to deoxycholic acid in human subjects

    J. Lipid Res.

    (1996)
  • M. Sasaki et al.

    Transcellular transport of organic anions across a double-transfected Madin–Darby canine kidney II cell monolayer expressing both human organic anion-transporting polypeptide (OATP2/SLC21A6) and Multidrug resistance-associated protein 2 (MRP2/ABCC2)

    J. Biol. Chem.

    (2002)
  • N.E. Hoffman et al.

    Measurement of bile and acid kinetics by isotope dilution in man

    Gastroenterology

    (1974)
  • N.E. Hoffman et al.

    Metabolism of steroid and amino acid moieties of conjugated bile acids in man: V. Equations for the perturbed enterohepatic circulation and their application

    Gastroenterology

    (1977)
  • U. Bolder et al.

    Sulindac is excreted into bile by a canalicular bile salt pump and undergoes a cholehepatic circulation in rats

    Gastroenterology

    (1999)
  • P.J. Meier et al.

    Bile salt transporters

    Annu. Rev. Physiol.

    (2002)
  • M. Trauner et al.

    Bile salt transporters: molecular characterization, function, and regulation

    Physiol. Rev.

    (2003)
  • Cited by (96)

    • Research progress in the application of bile acid-drug conjugates: A “trojan horse” strategy

      2021, Steroids
      Citation Excerpt :

      Organic solution transporter alpha–beta (Ostα-Ostβ) is known as the major basolateral transporter of lipophilic BAs and conjugated steroids such as taurocholate, estrone-3 sulfate, dehydroepiandrosterone sulfate (DHEAS), which increases their absorption in corresponding sites [28,29]. Bile salt export pump (BSEP) takes endogenous hydrophilic bile salts and a small number of drugs (pravastin, vinblastine, fexofenadine) as substrates, which has important physiological significance for maintaining low concentration of bile salts in hepatocytes and accelerating drug efflux, because bile salts are highly cytotoxic detergent [30–33]. Fatty acid bile acid conjugate (FABAC) is a new type of synthetic lipid molecule, which is used to treat cholesterol gallstones.

    View all citing articles on Scopus
    View full text