Bile salt export pump is highly conserved during vertebrate evolution and its expression is inhibited by PFIC type II mutations

Shi-Ying Cai*,1, Lin Wang*,1, Nazzareno Ballatori2,3, and James L. Boyer1,3

1 Liver Center, Yale University School of Medicine, New Haven, Connecticut 06520-8019; 2 Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, New York 14642, and 3 Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bile secretion is a fundamental function of the liver of all vertebrates and is generated by ATP-dependent transport proteins at the canalicular membrane of hepatocytes, particularly by the bile salt export pump BSEP. To determine the evolutionary origin and structure-function relationship of this transport mechanism, a liver cDNA library from the marine skate Raja erinacea, a 200 million-year-old vertebrate, was screened for BSEP orthologues. A full-length clone was isolated that encodes for 1,348 amino acids and shares 68.5% identity to human BSEP. Northern blot analysis revealed a 5-kb transcript only in skate liver. Expression of skate Bsep in Sf9 cells demonstrated a sixfold stimulation of ATP-dependent taurocholate transport compared with controls, with a Michaelis-Menten constant of 15 µM, which is comparable to rat Bsep. Sequences at the site of published mutations in human BSEP are also conserved in skate Bsep. When two of these mutations were introduced into the skate Bsep cDNA, this resulted in defective expression of the mutant proteins in Sf9 cells. These studies demonstrate that Bsep is a liver-specific ATP-dependent export pump that is highly conserved throughout evolution and provide insights into critical determinants for the function of this transporter in higher vertebrates.

ATP binding cassette transporters; bile secretion; cholestasis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

THE BILE SALT EXPORT PUMP, BSEP, originally described as the sister of P-glycoprotein, is the major if not sole transporting polypeptide for the biliary excretion of bile salts (8, 9). This member of the ATP binding cassette (ABC) superfamily is expressed only at the canalicular membrane of hepatocytes and is responsible for production of bile salt-dependent bile flow. The significance of this export pump to normal physiology is emphasized by the discovery that mutations in the human BSEP result in a form of progressive familial intrahepatic cholestasis (PFIC), known as PFIC type II (11, 18). Although full-length cDNAs have been cloned from several species, functional expression studies have been described only for rat and mouse (8, 9) and the mechanisms by which BSEP mutations impair the expression of this ABC transporter in humans are not yet known.

The evolutionary development of Bseps has also not been examined, although Bseps are most closely related to the P-glycoproteins and were initially designated as the "sister of P-glycoprotein" (5, 6). Partial sequences have been cloned from teleost liver in flounder (Pseudopleuronectes americanus) (6) and killifish (Fundulus heteroclitus). Analysis of bile from primitive elasmobranch vertebrates indicates that bile alcohols rather than bile salts are present (12), raising questions concerning the endogenous substrate. Nevertheless, elasmobranchs have the capacity to excrete bile salts into bile as demonstrated by studies in the dogfish shark (Squalus acanthius) and small skate (Raja erinacea), which are capable of excreting 3H-labeled taurocholate into bile in high concentrations, both in free-swimming fish (2, 3) and in isolated, perfused skate liver preparations (16). Furthermore, recent studies indicate that fluorescent bile salt analogs can be excreted into the lumen of isolated skate hepatocyte clusters (10, 13), whereas ATP-dependent taurocholate transport can be demonstrated in liver plasma membrane vesicle preparations from skate livers (1). All of these studies provide compelling evidence to suggest that a Bsep orthologue with functional properties quite similar to the mammalian bile salt export pump may have evolved early in vertebrate evolution.

To examine this question in greater detail and to provide comparative models for examining the structural determinants for Bseps, we have cloned and characterized a Bsep orthologue from the liver of the small skate, a 200 million-year-old vertebrate. Functional expression studies and substrate specificity were compared with rat Bsep (rBsep) in Sf9 expression systems. Our results provide evidence that Bsep diverged from P-glycoproteins early in vertebrate evolution. Although bile alcohols were probably the original substrate, sequences of functional importance for this transporting polypeptide are highly conserved between this evolutionarily distant orthologue and humans.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials. [3H]taurocholic acid (2 Ci/mmol) was obtained from NEN Life Science Products. All other chemicals and reagents of analytical grade are purchased from Sigma, Merck, Pharmacia Amersham, or J. T. Baker.

cDNA library construction and screening. RNA was isolated from the liver of the small skate, Raja erinacea, on a cesium chloride gradient (17) and used for construction of a cDNA library in a ZAP expression vector by Stratagene. In the first-round library screening, 1.5 million independent plaques were screened under low stringency using two probes corresponding to the killifish Bsep's ABC region (a RT-PCR product kindly provided by Dr. Peter Cooper, National Center for Biotechnology Information, Bethesda, MD) and a flounder Bsep's COOH-terminal region (a genomic DNA fragment provided by Dr. Peter Davies, Queen's University, Kingston, ON, Canada). The final single clone was sequenced by the W. M. Keck sequencing facility at Yale University.

Skate beta -actin gene fragment cloning. Two degenerate oligonucleotide primers were designed based on the consensus region for beta -actin proteins of different species from the database and were used to amplify a skate beta -actin gene fragment by RT-PCR with 5 µg of skate liver total RNA: forward primer, 5' GARAARATGACNCARATHATGTT 3'; reverse primer, 5' RTTNCCDATNGTDATNACYTGNCC 3'. A 410-bp DNA fragment was produced by a touch-down PCR reaction. DNA sequencing confirmed that the PCR product was a unique sequence with 89%, 85%, and 84% identity to zebrafish, rat, and human actins, respectively.

Northern blot analysis. Total RNA was isolated from fresh skate tissues by using cesium chloride gradient centrifugation or an RNeasy kit (Qiagen). Fifteen micrograms of total RNA were loaded per lane on a 1% agarose gel. After electrophoresis, the gel was treated with 0.05 N NaOH to partially hydrolyze the RNA. The gel was neutralized with Tris buffer (pH 7.4) and transferred to GeneScreen (NEN Life Science Products) nylon membrane. The blot was hybridized with either a probe corresponding to a 5' 300-bp coding region of skate Bsep (sBsep) cDNA or the skate beta -actin cDNA fragment labeled with [alpha -32P]dCTP by random priming.

Mutagenesis of sBsep. To facilitate sBsep subcloning and mutant generation, we first deleted 300 bp from the 3' noncoding region of sBsep cDNA in a pFastBac-sBsep construct. Then we subcloned a fragment encoding the region amino acid 996 to amino acid 1289 of sBsep into pBlueScript-KS vector, which covers transmembrane domains 1 and 2, and the second ABC domain of sBsep. The Stratagene PCR mutagenesis system was used to introduce three mutations, G1009R, F1041A, and R1180C. All three mutated sBsep fragments in the pBlueScript-KS constructs were confirmed by sequencing before the wild-type region was replaced by the mutated fragment in the pFastBac-sBsep construct.

Expression of rBsep and sBsep in Sf9 insect cells. The Bac-to-Bac system (Life Technologies) was used to generate the recombinant baculovirus encoding sBsep and rBsep. The full-length rat Bsep cDNA was kindly provided by Dr. Peter Meier (University Hospital, Zurich, Switzerland). Sf9 cells were infected with virus and maintained in culture for 72 h (28°C, air). The Sf9 cells were then harvested from culture dishes, and a crude membrane fraction was prepared following a protocol similar to that described by Gerloff et al. (8). After high-speed centrifugation, the membrane pellet was finally resuspended in taurocholate uptake buffer consisting of (in mM) 50 sucrose, 100 KNO3, 12.5 Mg(NO3)2, and 10 HEPES-Tris (pH 7.4) as described (8), and the vesicles were stored in liquid nitrogen. The supernatant is defined as the cytosol fraction. Protein concentration was determined using the Bradford assay with bovine serum albumin as a standard (4).

Electrophoresis and immunoblotting. Sf9 cell membranes were subjected to SDS-PAGE. The separated polypeptides were electrotransferred to polyvinylidene difluoride membranes (Bio-Rad) overnight at 30 mA. The membranes were blocked for 1 h with 5% nonfat milk and then incubated for 2 h at room temperature with the rabbit polyclonal antibody raised against the COOH-terminal 13 amino acids of rBsep (kindly provided by Dr. Bruno Stieger, University Hospital, Zurich, Switzerland). The immunoreactive proteins were visualized using an enhanced chemiluminescence kit (Amersham).

Vesicle transport assays. Uptake of [3H]taurocholate was measured by rapid filtration on Millipore 0.45-µm filters under vacuum essentially as described previously (8). Briefly, Sf9 cell membrane vesicles (50-100 µg) were diluted in 20 µl of taurocholate uptake buffer. Transport was started by mixing the diluted membrane vesicles with 80 µl of uptake buffer containing substrate with or without 5 mM ATP. Transport was carried out at 28°C for timed intervals. Taurocholate uptake was stopped by adding 3.5 ml of ice-cold uptake buffer, vesicles were collected by applying the quenched reaction solution to prewetted filters under vacuum, and filters were washed with an additional 7 ml of ice-cold uptake buffer. Filters were collected and dissolved in 5 ml of Opti-Fluor (Packard Instrument, Meriden, CT), and vesicle radioactivity was measured by liquid scintillation counting. Controls for nonspecific binding were assessed by determining the amount of radioactivity retained on the filter after simultaneous additions of isotope and 4°C cold stop solutions.

Statistical analysis. Kinetic data from experiments measuring uptake of radiolabeled substrate in membrane vesicles were fit to the Michaelis-Menten equation. Maximal velocity (Vmax) and Michaelis-Menten constant (Km) values were derived from a Lineweaver-Burk plot, and inhibition constant (Ki) values were calculated using the Dixon plot analysis. Comparison of data measuring initial rates of uptake of [3H]taurocholate in the presence and absence of inhibitors was performed by unpaired Student's t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A full-length Bsep cDNA containing 5.1 kb was isolated from a cDNA library from the liver of a primitive vertebrate, R. erinacea. This cDNA encodes for a polypeptide of 1,348 amino acids (Fig. 1A) and contains 580 bp of untranslated region at the 3' end and 480 bp of untranslated region at the 5' end (Genbank no. AF367243). Sequence analysis indicates that there are 12 transmembrane domains, 2 ATP binding sites, and 3 predicted N-glycosylation sites in the first extracellular loop, features characteristic of ABC transporting polypeptides (Fig. 1). Comparisons of the full-length amino acid sequences with other closely related members of the ABC transporter superfamily indicate that this evolutionarily primitive gene product is 67% and 68% identical with rBsep and human BSEP, 50% and 51% identical with rat and human P-glycoprotein [multidrug resistance protein 1 (Mdr1/MDR1)], and only 16% identical with rat and human multidrug resistance-associated protein 2 (Mrp2/MRP2). sBsep contains an additional 27 amino acids at the NH2-terminal cytoplasmic region compared with its rat and human orthologues. Interestingly, the identified human PFIC II mutation sites (18) are all conserved in sBsep (Fig. 1B). A phylogenic analysis (Fig. 1C) indicates that this primitive sBsep is closely related to mammalian Bseps, which as a group are evolutionarily closest to the more ubiquitous P-glycoproteins.


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Fig. 1.   A: deduced amino acid sequence of skate bile salt export pump (Bsep) derived from a full-length cDNA clone. The predicted 12 transmembrane domains are bold and underlined, the Walker A and B motifs are underlined, and the potential extracellular N-glycosylation sites are bold. B: the mutation sites from human progressive familial intrahepatic cholestasis type II (PFIC-II) (18) are conserved in skate Bsep (sBsep). Alignment of mutation sites in Bseps from human, rat, rabbit, mouse, skate, and Fundulus. C: phylogenetic tree of Bseps and multidrug resistance protein 1 (Mdr1).

A Northern blot analysis of RNA extracted from multiple tissues of the skate indicates that this 5.1-kb sBsep transcript is exclusively expressed in skate liver (Fig. 2A). The skate actin gene is shown as control in Fig. 2B. A Southern blot analysis demonstrates that sBsep is a single-copy gene (data not shown). As previously demonstrated (1), antibodies to rBsep, which are directed to the conserved COOH-terminal region of this protein, specifically stain the canalicular membrane in both rat and skate livers, consistent with its functional properties as a bile export pump.


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Fig. 2.   Northern blot and Western blot analysis of sBsep expression. The lanes correspond to the following tissues: 1, brain; 2, heart; 3, kidney; 4, intestine; 5, liver; 6, pancreas; 7, rectal gland; 8, spleen; 9, stomach; 10, testes. A: the blot was probed with a 32P-labeled skate Bsep 5' 300-bp coding region cDNA. B: the blot was hybridized with 32P-labeled skate actin gene fragment. C: Western blot analysis of membrane vesicles isolated from Sf9 cells expressing rat Bsep (rBsep) and sBsep and mock-treated Sf9 cells (50 µg/lane). Mr, relative molecular weight.

To analyze the function of the Bsep skate orthologue, both skate and rat Bsep cDNAs were expressed in Sf9 cells. When sBsep was expressed in Sf9 cells, a 143-kDa protein was detected (Fig. 2C). The small molecular shift in mobility between skate and rat Bseps expressed in Sf9 cells is consistent with the known difference in their size. The immunoreactive 143-kDa protein is only present in vesicles isolated from Bsep cDNA-transfected Sf9 cells but not in vesicles isolated from wild-type cells or in vesicles isolated from Sf9 cells infected with wild-type or with beta -galactosidase-containing baculovirus (data not shown).

Crude membrane fractions were prepared from the infected Sf9 cells and analyzed for functional expression. As can be seen in Fig. 3A, an approximately eightfold increase in [3H]taurocholate uptake was observed in membrane vesicles isolated from skate Bsep cDNA-infected Sf9 cells compared with control vesicles from wild-type cells or cells infected with a beta -galactosidase-containing baculovirus (data not shown). Initial rates of sBsep-mediated, ATP-dependent [3H]taurocholate uptake exhibited saturability with increasing concentrations of taurocholate (Fig. 3B). The apparent Km for taurocholate was 15 µM. Little or no stimulation of [3H]taurocholate uptake into sBsep-expressing Sf9 vesicles was observed with the nonhydrolyzable ATP analog 5'-adenylylimidodiphosphate (Table 1).


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Fig. 3.   ATP-dependent [3H]taurocholate transport in membrane vesicles isolated from sBsep- and rBsep-expressing Sf9 cells. A: time course. Membrane vesicles isolated from wild-type (mock) and Sf9 cells expressing either rBsep or sBsep were incubated in (mM) 50 sucrose, 100 KNO3, 12.5 Mg(NO3)2, and 10 HEPES-Tris (pH 7.4) supplemented with 2 µM [3H]taurocholate. Vesicle uptake of [3H]taurocholate was determined in the presence and absence of ATP (5 mM) at 28°C. Data represent the means ± SD of 3 determinations with the variability either shown or smaller than the symbols. B: saturation of sBsep-mediated ATP-dependent taurocholate uptake. Initial uptake rates were determined at 60 s in the presence and absence of ATP (5 mM) with increasing taurocholate concentrations and corrected for values obtained in the absence of ATP. The values are the means ± SE of triplicate determinations in 1 of 2 separate experiments. Km, Michaelis-Menten constant.


                              
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Table 1.   Effects of substrates and inhibitors of ABC transporters on ATP-dependent uptake of 2 µM [3H]taurocholate in membrane vesicles expressing either rat or skate Bsep

ATP-dependent [3H]taurocholate uptake was competitively inhibited by scymnol sulfate, its presumed natural substrate (7, 12), with a Ki of 14 µM (Fig. 4). ATP-dependent taurocholate uptake was also inhibited by other primary bile acids and bile acid derivatives (Table 1). Lithocholate, glycolithocholate, and ursodeoxycholate had small inhibitory effects (Table 1).


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Fig. 4.   Scymnol sulfate competitively inhibits ATP-dependent taurocholate uptake with an inhibition constant (Ki) of 14 µM. ATP-dependent taurocholate uptake was measured in membrane vesicles (50 µg of protein) from Sf9 cells infected with baculovirus encoding sBsep. Vesicles were incubated in a buffer containing ATP in the presence and absence of, 5, 15, or 50 µM scymnol sulfate. Each data point represents mean of 2 experiments, each performed in triplicate.

In contrast to bile salts, ATP-dependent taurocholate uptake was not significantly inhibited by the substrates and inhibitors of the canalicular multidrug resistance-associated protein (Mrp2) (Table 1) or by the Mdr1 substrate valinomycin. However, cyclosporin A and vincristine inhibited ATP-dependent taurocholate uptake mediated by sBsep. Table 1 compares the pattern of substrate inhibition of sBsep with rBsep. Overall, the inhibitory profile between rat and skate was remarkably similar with the exception of rifampicin, which inhibited ATP-dependent [3H]taurocholate uptake mediated by rBsep but had little effect on the taurocholate uptake mediated by sBsep.

We next took advantage of the highly conserved nature of the sBsep orthologue to characterize the impact of the mutations reported in human BSEP that result in a form of PFIC, PFIC type II. Two single-point mutation constructs (R1180C, G1009R) were made based on sequence alignment, and a third mutation construct (F1041) was made in a region that is conserved throughout all known Bseps and Mdrs and was substituted with alanine. The recombinant baculovirus encoding these sBsep mutants was generated, and the expression of these mutants in Sf9 cells was examined in crude membranes and cytosol. As seen in Fig. 5A, the membrane expression level of all three sBsep mutants is significantly reduced compared with the wild-type skate protein. Interestingly, a small ~60-kDa fragment was detected by the Bsep antibody in the cytosol fraction of R1180C.


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Fig. 5.   Expression of sBsep mutants in Sf9 cells. A: Western blot analysis of membrane vesicles isolated from Sf9 cells infected with baculovirus encoding 3 mutants, G1009R (GR), F1041A (FA), and R1180C (RC). The infected Sf9 cells were fractionated as described in EXPERIMENTAL PROCEDURES into crude membranes and cytosol. Cytosolic proteins were precipitated with trichloric acid. One hundred micrograms of crude membranes and cytosolic proteins were analyzed on the gel. B: [3H]taurocholate uptake from membrane vesicles isolated from Sf9 cells infected with baculovirus encoding the 3 mutations, G1009R, F1041A, and R1180C. Vesicle uptake of [3H]taurocholate was determined in the presence of ATP (5 mM) at 28°C. Data represent the means ± SD of 3 determinations. WT, wild type; beta -gal, beta -galactosidase.

We then investigated whether the Sf9 cell membranes expressing these sBsep mutants retained activity for [3H]taurocholate uptake. As noted in Fig. 5B, the skate wild-type Bsep has a fourfold higher [3H]taurocholate uptake than either the membranes from mock-treated cells or the membranes from the cells infected with beta -galactosidase-containing baculovirus. In contrast, [3H]taurocholate uptake activity in membrane vesicles expressing F1041A or R1180C or G1009R was not significantly different from the level of uptake observed in the Sf9 cell membranes from mock-treated cells or the membranes from the cells infected with beta -galactosidase-containing baculovirus.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study we present molecular and functional evidence that the bile salt export pump Bsep evolved early in vertebrate evolution with functional properties that have remained essentially unchanged in their mammalian orthologues despite ~30% sequence divergence over a 200 million-year period of evolution. The Bsep cDNA isolated from a skate liver library demonstrated 67%, 67%, and 68% identity at the amino acid level with rat, mouse, and human Bsep, respectively. The cDNA codes for 1,348 amino acids, 27 amino acids larger than its mammalian counterparts. The protein is expressed exclusively in the liver, and antibodies developed to the COOH-terminal 13-amino acid peptide of rBsep, which differs from this region by only 1 amino acid in the skate, were reported previously to demonstrate strong immunofluorescence staining at the skate apical canalicular membrane domain, where it is functionally active (1). Expression studies in Sf9 cells indicate that the skate orthologue is highly expressed and functions as a bile salt export pump in this primitive vertebrate. Our data also confirm previous functional studies that expressed rBsep in Sf9 cells (8). In our study, the skate orthologue demonstrated higher initial ATP-dependent transport rates compared with the rat construct, consistent with previous in vivo measurements in free-swimming fish that have demonstrated very high bile-to-plasma [3H]taurocholate ratios of 1,000:1 (3). Differences in the native environment and Sf9 cells that might affect Bsep functional expression cannot be excluded. Kinetic studies indicate that sBsep has an affinity for taurocholate (15 µM) similar to that described for rat and mouse Bsep (5 and 11 µM, respectively) but four times higher than yeast BAT1 (63 µM) (8, 9, 14). In addition, substrate inhibition studies demonstrated nearly identical effects of bile acids and other organic solutes on inhibition of [3H]taurocholate transport in Sf9 cells as observed in Sf9 cells expressing rBsep. The only exception was the lack of cis-inhibitory effects of the cholestatic drug rifampicin in Sf9 cells expressing sBsep compared with rBsep. Together these findings indicate that the structural determinants for transport function have been highly conserved throughout more than 200 million years of vertebrate evolution.

Mass spectrometric analysis of bile samples from the skate indicate that scymnol sulfate, a bile alcohol, is the major component and that only trace amounts of cholic acid can be detected (12). Scymnol sulfate competitively inhibited [3H]taurocholate transport in Sf9 cells that expressed sBsep and also inhibited transport in Sf9 cells that expressed rBsep, suggesting that Bsep originally evolved as a bile alcohol transporter. As a sulfate conjugate, scymnol sulfate is also likely to be a substrate for Mrp2, which is also expressed in skate hepatocytes (Ref. 15; unpublished observations).

If the molecular determinants that regulate transport function in Bseps are highly conserved as the present findings suggest, we would predict that mutations in human BSEP that result in PFIC-II in infancy might reside in sequences common to sBsep. Indeed, as illustrated in Fig. 1B, when the sBsep cDNA sequence is compared with other known orthologues including human, all six published single-nucleotide deletions or substitutions that lead to progressive cholestatic liver disease in infancy (PFIC-II) as well as a number of unpublished mutations (R. Thompson, personal communication) are fully conserved between sBsep and human BSEP. These findings suggest that these conserved regions must be critical determinants of Bsep function. We confirmed this hypothesis in the present study by generating mutants R1180C, F1041A, and G1009R and demonstrating that these mutations significantly reduce the expression of sBsep in Sf9 cells. These results are consistent with previous findings that demonstrated absence of canalicular membrane immunostaining for BSEP in patients with PFIC-II (11). Together these results suggest that these mutations impair either the synthesis or membrane insertion of sBsep or the stability of the protein in the membrane. The possibility that sBsep is misfolded and prone to degradation is suggested by the detection of a COOH-terminal sBsep fragment in the Sf9 cell cytosol with the mutant construct R1180C.

Because 32% of the sBsep sequence is not conserved in human BSEP, yet functional studies suggest that sBsep and mammalian orthologues have similar if not identical properties, it is likely that these nonconserved sequences may not be essential for targeting of Bsep to its functional domain or for transport activity. Deletion and or chimerical constructs should allow a comparative functional genomics approach to further our understanding of Bsep's critical structural determinants.


    ACKNOWLEDGEMENTS

Dr. Peter Cooper, Dr. Peter Davies, and Dr. Bruno Stieger generously provided critical molecular reagents.


    FOOTNOTES

* S.-Y. Cai and L. Wang contributed equally to this work.

This project was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-25636 to J. L. Boyer, the Molecular Biology and Morphology Cores of the Yale Liver Center, P30-DK-34989, and National Institute of Environmental Health Sciences Marine and Freshwater Biomedical Science Center Grant P30-ES-03828 at the Mount Desert Island Biological Laboratory.

Address for reprint requests and other correspondence: J. L. Boyer, Box 208019, 333 Cedar St., New Haven, CT 06520-8019 (E-mail: james.boyer{at}yale.edu).

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.

Received 28 February 2001; accepted in final form 30 March 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 281(2):G316-G322
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