From the Division of Clinical Pharmacology and
Toxicology, Department of Medicine, University Hospital, CH-8091
Zurich, Switzerland, the ¶ Institute of Anatomy, University of
Basel, CH-4056 Basel, Switzerland, the
Division of Cellular and
Molecular Pathology, Department of Pathology, University Hospital,
CH-8091 Zurich, Switzerland, and the ** Division of Gastroenterology,
Department of Medicine, University of California,
San Diego, California 92093-0813
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ABSTRACT |
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Canalicular secretion of bile salts is a vital
function of the vertebrate liver, yet the molecular identity of the
involved ATP-dependent carrier protein has not been
elucidated. We cloned the full-length cDNA of the sister of
P-glycoprotein (spgp; Mr ~160,000) of rat
liver and demonstrated that it functions as an ATP-dependent bile salt transporter in cRNA injected
Xenopus laevis oocytes and in vesicles isolated from
transfected Sf9 cells. The latter demonstrated a 5-fold
stimulation of ATP-dependent taurocholate transport as
compared with controls. This spgp-mediated taurocholate transport was
stimulated solely by ATP, was inhibited by vanadate, and exhibited
saturability with increasing concentrations of taurocholate (Km 5 µM). Furthermore,
spgp-mediated transport rates of various bile salts followed the same
order of magnitude as ATP-dependent transport in
canalicular rat liver plasma membrane vesicles, i.e.
taurochenodeoxycholate > tauroursodeoxycholate = taurocholate > glycocholate = cholate. Tissue distribution
assessed by Northern blotting revealed predominant, if not exclusive,
expression of spgp in the liver, where it was further localized to the
canalicular microvilli and to subcanalicular vesicles of the
hepatocytes by in situ immunofluorescence and immunogold
labeling studies. These results indicate that the sister of
P-glycoprotein is the major canalicular bile salt export pump of
mammalian liver.
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INTRODUCTION |
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Bile formation is an important function of vertebrate liver (1). It is mediated by hepatocytes that generate bile flow within bile canaliculi by continuous vectorial secretion of bile salts and other solutes across their canalicular (apical) membrane (2). Studies in isolated membrane vesicles of rat and human livers have shown that canalicular bile salt transport is an ATP-dependent process (3-7). However, the molecular identity of the primary active canalicular bile salt transporter or bile salt export pump (BSEP)1,2 has not yet been elucidated (8, 9). Although the canalicular ecto-ATPase has been proposed as a possible candidate (9, 10), other investigations have provided evidence that BSEP of mammalian liver is an ABC (ATP binding cassette)-type of membrane transporter (11, 12). This assumption has recently been further supported by the cloning of an ATP-dependent bile salt transporter from Saccharomyces cerevisiae (13). This yeast bile salt transporter (BAT1) belongs to a subgroup of ABC-type proteins that includes also the canalicular multiorganic anion transporter or multidrug resistance protein MRP2 (human)/mrp2 (rat) (14-16). Although MRP2/mrp2 mediates canalicular excretion of a broad range of divalent amphipathic anionic conjugates (1, 14, 17), it does not transport primary bile salts such as taurocholate or glycocholate (1, 18). Therefore, we designed degenerate oligonucleotide primers spanning the Walker A and B motifs of ABC proteins and performed reverse transcription-polymerase chain reactin with total rat liver mRNA. One of the amplified fragments revealed an 88% identity with the published pig liver cDNA-fragment of the so called "sister of P-glycoprotein" (spgp), a novel putative canalicular ABC transporter of unknown function (19). Here we report the full-length isolation of spgp from rat liver, demonstrate its function as an ATP-dependent bile salt transporter in cRNA-injected Xenopus laevis oocytes and in transfected Sf9 cells, and document its subcellular localization at the canalicular microvilli and at subcanalicular smooth membrane vesicles of rat hepatocytes using immunofluorescence and immunogold electron microscopy.
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EXPERIMENTAL PROCEDURES |
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Materials-- [3H]Taurocholic acid (2.1-4.6 Ci/mmol), [3H]cholic acid (13.2 Ci/mmol), and [4C]glycocholic acid (44.6 mCi/mmol) were obtained from NEN Life Science Products. 3H-Labeled taurochenodeoxycholic and tauroursodeoxycholic acids of high specific activity (2-60 Ci/mmol) were prepared as described previously (20-22). All other chemicals and reagents were of analytical grade and were readily available from commercial sources.
cDNA Cloning of Rat Liver spgp-- A cDNA probe spanning the Walker A and B motifs of ABC proteins was constructed by the reverse transcription-polymerase chain reaction of rat liver poly(A)+ RNA using the following primers: forward primers, GGCGGATCCTCIGGIKSIGGIAARAGYAC and GGCGGATCCTCIGGIKSIGGIAARTCIIC; reverse primers, CGGGAATTCTCIARIGCRCTIACIGSYTCRTC, CGGGAATTCTCIARIGCRCTIGTIGSYTCRTC, CGGGAATTCTCIARIGCICAIACIGSYTCRTC, and CGGGAATTCTCIARIGCIGAIGTIGSYTCRTC. After cloning and sequencing a single polymerase chain reaction product of 430 bp was identified that revealed an 88% identity to the published portion of the pig liver spgp cDNA (19). It was used to sequentially isolate the full-length spgp cDNA from two cDNA libraries constructed from total rat liver mRNA with the Superscript Plasmid System (Life Technologies, Inc.) and from size-fractionated poly(A+) RNA (4.0-6.0 kb) using the ZAP ExpressTM cDNA Synthesis and ZAP ExpressTM cDNA Gigapack III Gold Cloning kits (Stratagene), respectively. The final single clone was sequenced by Microsynth Gmbh (Balgach, Switzerland).
Functional Expression of spgp in X. laevis Oocytes-- Oocytes were prepared, injected with cRNAs, cultured, and preloaded with 28.5 pmol of [3H]taurocholate (60 nl of 475 µM [3H]taurocholate (2.1 Ci/mmol)) as described elsewhere (23, 24). [3H]Taurocholate efflux from individual oocytes was determined at 25 °C as described previously (18, 24) using a Na+-free incubation medium of 100 mM choline chloride, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES/Tris, pH 7.5.
Expression and Functional Characterization of spgp in Sf9 Insect Cells-- The Bac-to-Bac system (Life Technologies, Inc.) was used to generate the recombinant baculovirus. Sf9 cells were infected with virus and kept in culture (27 °C, air) for 72 h. Thereafter, Sf9 cells were scraped from culture dishes, centrifuged at 1400 × gav, and homogenized with a glass-Teflon tissue homogenizer in 50 mM mannitol, 2 mM EGTA, 50 mM Tris/HCl, pH 7.0, 1 µg/ml leupeptin/antipain, and 0.5 mM phenylmethylsulfonyl fluoride (25). Undisrupted cells, nuclear debris, and large mitochondria were pelleted at 500 × gav for 10 min. The supernatant was centrifuged for 60 min at 100,000 × gav. The resulting pellet was resuspended in taurocholate uptake buffer consisting of 50 mM sucrose, 100 mM KNO3, 12.5 mM Mg(NO3)2, and 10 mM HEPES/Tris, pH 7.4. The vesicles were stored frozen in liquid nitrogen. Protein was determined using the BCA assay with bovine serum albumin as a standard (26).
Western and Northern Blotting--
A polyclonal antibody was
raised in rabbits against an oligopeptide containing the C-terminal 13 amino acids coupled via cysteine to keyhole limpet hemocyanin
(Neosystem, Strasbourg, France) and used for Western blotting as
described previously (27). A multiple tissue Northern blot
(CLONTECH) (~2 µg of poly(A+) RNA
per lane) was hybridized with the full-length spgp cDNA clone or
with a -actin probe labeled with [
-32P]dCTP by
random priming. Hybridization was performed at 65 °C overnight in
Amersham hybridization buffer. The blot was washed at 68 °C in 0.2×
SSC, 0.1% SDS.
Immunofluorescence and Immunogold Electron Microscopy-- Immunofluorescence studies (27) and immunogold electron microscopy studies on ultrathin frozen-thawed sections of perfusion-fixed (3% formaldehyde, 0.1% glutaraldehyde) liver were performed as described previously (28, 29).
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RESULTS |
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The isolated full-length spgp cDNA3 contained 5,036 base pairs and encoded a polypeptide of 1321 amino acids with 12 putative membrane-spanning domains, four predicted N-glycosylation sites in the first extracellular loop, and the typical structural features of ABC-transporting polypeptides (Fig. 1). Comparison of the full-length amino acid sequence with other members of the ABC-transporter superfamily revealed the following similarities/identities to spgp: mdr1a (mouse) 71%/50%, mdr1b (rat) 70%/49%, mdr2 (rat) 69%/48%, MRP1 (human) 51%/26%, mrp2 (rat) 49%/25%, BAT1 48%/23%, and cystic fibrosis conductance regulator (mouse) 48%/22%. These data confirm the close homology of spgp to the MDR/mdr gene family (19).
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To elucidate its function, spgp was expressed in X. laevis
oocytes (18) and in Sf9 cells. As illustrated in Fig.
2, an approximately 1.5-fold increase in
[3H]taurocholate efflux was observed in spgp
cRNA-injected oocytes as compared with water- and mrp2 cRNA-injected
oocytes (18). In addition, membrane vesicles isolated from spgp
cDNA-infected Sf9 cells (see "Experimental Procedures")
demonstrated marked ATP-dependent uptake of taurocholate,
whereas control vesicles from wild type cells (or cells infected with
wild type or with -galactosidase-containing baculovirus; data not
shown) did not (Fig. 3A).
Initial rates of this spgp-mediated, ATP-dependent [3H]taurocholate uptake exhibited saturability with
increasing concentrations of taurocholate (Km value
~5.3 µM) (Fig. 3B). Furthermore, only
minimal or no stimulation of [3H]taurocholate uptake into
spgp expressing Sf9 vesicles was observed with other nucleotides
(UTP, CTP, and GTP), ATP degradation products (ADP, AMP, and
adenosine), or nonhydrolyzable ATP analogs
(adenosine-5'-[
-thio]triphosphate and
adenosine-5'-[
,
-imido]triphosphate) (data not shown). Also, increasing concentrations of vanadate between 5 µM and
100 µM inhibited ATP-dependent
[3H]taurocholate uptake between 32% and 63%. And
finally, comparison of initial ATP-dependent uptake rates
(linear time-dependent uptake phase) of various bile salt
derivatives in spgp-expressing Sf9 cell vesicles and in isolated
canalicular liver plasma membrane vesicles (6) showed the highest
ATP-dependent uptake rate for taurochenodeoxycholate
followed by tauroursodeoxycholate and taurocholate in both vesicle
preparations (Table I). In contrast,
glycocholate and cholate were less efficiently transported in
canalicular vesicles, and no ATP-dependent transport of
these bile salt derivatives could be detected in spgp-expressing
Sf9 cell vesicles. These latter results most probably resulted
from the overall lower expression of ATP-dependent bile
salt transport activities in spgp-expressing Sf9 cells compared
with canalicular vesicles (Table I), thus preventing the detection of
low residual spgp-mediated cholate and glycocholate transport.
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To directly correlate transport activity with protein expression in
Sf9 cells, a polyclonal antiserum against the C-terminal 13 amino acids of BSEP/spgp was raised in rabbits. The antiserum recognized a predominant protein band with an apparent molecular mass
of ~140 kDa (140.5 ± 2.7; mean ± SD, n = 3) in vesicles isolated from BSEP/spgp cDNA-transfected Sf9
cells (Fig. 4A, lane
2), but not in vesicles isolated from wild type cells (Fig.
4A, lane 1) nor in vesicles isolated from
Sf9 cells infected with wild type or with -galactosidase
containing baculovirus (data not shown). Thus, the expression of
BSEP/spgp correlates with ATP-dependent taurocholate
transport. An immunopositive protein band was also observed in
canalicular, but not in basolateral plasma membrane vesicles isolated
from rat liver (Fig. 4A, lanes 3 and
4). This canalicular form of BSEP/spgp exhibited a slightly
higher molecular mass of ~160 kDa (157.4 ± 3.1; mean ± SD, n = 3), which most probably reflects a different
N-linked glycosylation pattern of hepatocytes as compared
with Sf9 cells (25). Based on Northern blot analysis of various
rat tissue mRNAs, BSEP/spgp is predominantly, if not exclusively,
expressed in the liver (Fig. 4B). When tested for its
presence in other animal species the BSEP/spgp cDNA yielded positive hybridization reactions also with mRNAs of mouse (~5.0 kb), chicken (~2.0 kb), and turtle (~4.0 kb) livers (data not shown), indicating the occurrence of BSEP/spgp-related proteins also in
lower vertebrates.
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The cellular and subcellular distribution of BSEP/spgp in rat liver was further investigated by immunofluorescence and immunogold electron microscopy. At low magnification positive immunoreactivity was confined homogenously to bile canaliculi of periportal and perivenous hepatocytes (data not shown). No immunoreactivity was detected in bile ductular epithelial cells. Within bile canaliculi the immunoreactivity for BSEP/spgp was predominantly associated with the canalicular microvilli, while the smooth intermicrovillar plasma membrane regions were essentially free of gold particle labeling (Fig. 4, C and D). No labeling was found along the basolateral plasma membrane of hepatocytes. Intracellularly, gold particle labeling was associated with subplasmalemmal smooth membrane vesicles, lucent vacuoles probably representing endosomes (Fig. 4, E and F) and the Golgi apparatus.
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DISCUSSION |
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This study has identified spgp (19) as a canalicular ATP-dependent BSEP of mammalian liver. This conclusion is derived from the functional expression of the full-length BSEP/spgp cDNA in X. laevis oocytes (Fig. 2) and in Sf9 insect cells (Figs. 3 and 4A). Vesicles isolated from BSEP/spgp-overexpressing Sf9 cells exhibited similar high affinity ATP-dependent taurocholate uptake (Km = ~5.3 µM) (Fig. 3B) as previously determined under identical conditions in canalicular rat liver plasma membrane vesicles (Km = ~2.1 µM) (6). Since canalicular secretion represents the rate-limiting step in the overall transport of bile salts from sinusoidal blood plasma into bile (2, 8), its high affinity for taurocholate indicates that BSEP/spgp represents the major canalicular bile salt transporter of rat liver. Based on Northern blot analysis, BSEP/spgp is predominantly, if not exclusively expressed in the liver (Fig. 4B) (19). This organ-selective expression of BSEP/spgp is not astonishing considering its bile salt transport function. However, BSEP/spgp-related proteins appear also to occur in the livers of other vertebrates including chicken and turtle (this study) as well as the winter flounder (34), supporting the concept that the origin of the BSEP/spgp gene predates the divergence of fish and mammals (19) and that BSEP/spgp mediates the transport of C27 bile acids (present in turtle) as well as C24 bile acids (present in most mammals and chickens). Finally, Western blot analysis as well as immunofluorescence and immunogold labeling studies demonstrated the selective localization of BSEP/spgp at the canalicular microvilli (Fig. 4, A, C, and D) and at subcanalicular smooth membrane vesicles (Fig. 4, E and F) of rat hepatocytes. Since infusion of taurocholate into intact animals has been shown to increase the canalicular bile salt transport capacity (2, 35), the labeled subcanalicular endosomes might represent a regulatory carrier pool that could be rapidly inserted into the canalicular plasma membrane under high bile salt load conditions.
The full length of the rat liver BSEP/spgp represents a canalicular protein with a molecular mass of ~160 kDa (Fig. 4A). Its homology to the MDR/mdr gene family is in contrast to the closer relatedness of the recently cloned yeast BAT1 with the MRP/mrp gene family of ABC transporters (13). Since mammalian MRP/mrp analogues do not mediate transport of monovalent bile salt derivatives (1, 4, 17, 18), and since BAT1 has been shown to exhibit an approximately 12-fold lower affinity for taurocholate (Km = ~63 µM) (13) as compared with BSEP/spgp (Fig. 3B), the participation of BAT1-related gene products in bile salt transport in mammalian liver seems unlikely. Furthermore, since fungi are thought to be devoid of bile salts, the true endogenous substrate(s) of BAT1 as well as the functional significance of bile salt transport in yeast remain unclear. Nevertheless, ATP-dependent taurocholate transport has also been demonstrated in plant vacuoles despite the absence of typical bile salts in plant tissue (36). Hence, amphipathic bile salt transport might be a nonspecific "bystander" function of several multispecific ABC-type transporters in a variety of biological tissues. In contrast, ongoing bile salt secretion serves an essential function in mammalian liver, where it mediates phospholipid and cholesterol excretion and water flow into bile canaliculi. Furthermore, in vertebrates bile salts are 100-1000-fold concentrated in bile as compared with plasma, stimulate absorption of lipids from the intestine and undergo efficient enterohepatic circulation (2, 8, 9). Therefore, it is conceivable that vertebrates have developed a separate high affinity bile salt export pump within the large superfamily of ABC-type proteins.
Several observations from this and other recent studies strongly
indicate that BSEP/spgp represents the major, if not the only bile salt
export pump in mammalian liver. First, BSEP/spgp-mediated bile salt
transport exhibits similar kinetics (see above) and similar substrate
preference (Table I) as ATP-dependent bile salt transport
in canalicular liver plasma membrane vesicles (6). Second, preferential
BSEP/spgp-mediated transport of the dihydroxy-conjugated bile salt
taurochenodeoxycholate is consistent with the preferential secretion
and transport of this bile salt in the isolated perfused liver and in
canalicular plasma membrane vesicles (37). Third, in contrast to the
ecto-ATPase canalicular expression of BSEP/spgp is decreased in various
forms of cholestatic liver disease (38-40), and its level of
expression correlates well with the activity of
ATP-dependent canalicular bile salt transport (37, 38). Fourth, overexpression of BSEP/spgp is associated with increased bile
salt secretion in a gallstone susceptible mouse strain (41). And fifth,
the BSEP/spgp gene is localized on the same chromosome (chromosome 2) as the locus for progressive familial intrahepatic cholestasis PFIC2 (41-43), which is characterized by
defective canalicular bile salt secretion in the presence of normal
serum cholesterol and normal plasma -glutamyltranspeptidase (44). These patients secrete exceptionally low levels of conjugated primary
bile salts into bile and have particularly low levels of biliary
chenodeoxycholic acid (45, 46), which represents the preferred
substrate of BSEP/spgp (Table I). In fact, and most importantly, in
preliminary studies missense mutations of the human liver SPGP
gene have been identified as the most probable cause of PFIC
linked to the PFIC2 locus
(42).4 These very recent
developments corroborate the findings presented in this study and
further support the conclusion that BSEP/spgp represents the major, if
not the only canalicular bile salt transporter of mammalian liver.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft (to T. G.), the Swiss National Science Foundation (to B. H., B. S., L. L., J. R. and P. J. M.), and the Cloetta Foundation Zurich (to B. H.). Work in San Diego was supported in part by National Institutes of Health Grant Dk 21506 (to A. F. H.), as well as a grant in aid from the Falk Foundation e.V., Freiburg, Germany.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This study has been presented in part at the 48th Annual Meeting of the American Association for the Study of Liver Diseases in Chicago, November 7-11, 1997, and published in abstract form (49).
§ To whom correspondence should be addressed. Tel.: 0041-1-255- 3652; Fax: 0041-1-255-4411; E-mail: bstieger{at}kpt.unizh.ch.
1 The abbreviations use are: BSEP, bile salt export pump of mammalian liver; ABC, ATP binding cassette; BAT1, bile acid transporter of S. cerevisiae; mdr1a/1b, rodent isoforms of the multidrug resistance P-glycoprotein; mdr2, phospholipid-transporting mdr isoform of rodent liver; MRP1, human multidrug resistance protein 1; MRP2/mrp2, human/rat canalicular multidrug resistance protein; spgp, sister of P-glycoprotein; BSEP/spgp, identity of spgp and BSEP; PFIC, progressive familial intrahepatic cholestasis; bp base pair(s); kb, kilobase pair(s).
2 BSEP rather than cBAT (9) or cBST (8) is chosen as abbreviation, since BAT has already been reserved for "basic amino acid transporter" (47) and BST for the sphingosine-phosphate lyase gene (48). Furthermore, the term "export pump" points to ATP as the primary driving force of the canalicular bile salt transporter.
3 The nucleotide sequence of the full-length spgp cDNA clone coding for the deduced amino acid sequence is deposited in the GenBankTM under accession number U69487.
4 R. J. Thompson, personal communication.
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REFERENCES |
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