Departments of 1 Internal Medicine and 2 Pathology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
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ABSTRACT |
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The rat and mouse organic anion-transporting polypeptides (oatp) subtype 3 (oatp3) were cloned to further define components of the intestinal bile acid transport system. In transfected COS cells, oatp3 mediated Na+-independent, DIDS-inhibited taurocholate uptake (Michaelis-Menten constant ~30 µM). The oatp3-mediated uptake rates and affinities were highest for glycine-conjugated dihydroxy bile acids. In stably transfected, polarized Madin-Darby canine kidney (MDCK) cells, oatp3 mediated only apical uptake of taurocholate. RT-PCR analysis revealed that rat oatp3, but not oatp1 or oatp2, was expressed in small intestine. By RNase protection assay, oatp3 mRNA was readily detected down the length of the small intestine as well as in brain, lung, and retina. An antibody directed to the carboxy terminus localized oatp3 to the apical brush-border membrane of rat jejunal enterocytes. The mouse oatp3 gene was localized to a region of mouse chromosome 6. This region is syntenic with human chromosome 12p12, where the human OATP-A gene was mapped, suggesting that rodent oatp3 is orthologous to the human OATP-A. These transport and expression properties suggest that rat oatp3 mediates the anion exchange-driven absorption of bile acids previously described for the proximal small intestine.
intestinal transport; brush-border membranes; organic anion transport; taurocholate
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INTRODUCTION |
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BILE ACIDS ARE SYNTHESIZED from cholesterol in the liver and secreted across the canalicular membrane. Along with other biliary constituents, bile acids enter the small intestine, where they facilitate the absorption of dietary lipids and fat-soluble vitamins. Bile acids are efficiently absorbed from the small intestine through a combination of passive absorption in the proximal small intestine and active absorption in the distal ileum. The absorbed bile acids are then returned to the liver in the portal circulation and resecreted into bile (20). The active absorption of bile acids in the terminal ileum is mediated by the well-characterized ileal apical Na+-bile acid transporter (ASBT) (31). This Na+- and potential-driven (47) transporter moves bile acids from the lumen of the small intestine across the apical brush-border membrane. The bile acids are then shuttled to the basolateral membrane and secreted by an anion exchange mechanism (46). Several observations suggest that the terminal ileum is the major site of bile acid reabsorption in humans and experimental animal models. These include the finding that there is little decrease in intraluminal bile acid concentration before the ileum (18) and the appearance of bile acid malabsorption after ileal resection (21). More recent studies using in situ perfused intestinal segments to measure bile acid absorption (32) have also demonstrated that ileal bile acid transport is a high-capacity system sufficient to account for the biliary output of bile acids. The consensus from these studies was that the ileal active transport system is the major route for conjugated bile acid uptake, whereas the passive or facilitative absorption down the length of the small intestine may be significant for unconjugated and some glycine-conjugated bile acids.
In contrast to the ileal absorption of bile acids, little is known regarding the mechanism for jejunal transport of bile acids. A fraction of the glycine conjugates and unconjugated bile acids are protonated and may be absorbed by passive diffusion across the apical brush-border membrane. However, there is emerging evidence for carrier-mediated transport of bile acids in the proximal small intestine in addition to membrane diffusion. In vivo uptake and cis-inhibition studies demonstrated bile acid specificity consistent with a facilitative carrier (3, 5). More recently, Amelsburg and co-workers (4) demonstrated an anion exchange mechanism for conjugated bile acid uptake in rat jejunal brush-border membranes. The properties of this transport system are similar to those ascribed to the family of organic anion-transporting polypeptides (oatp).
The rat oatp family includes the bile acid and organic solute transporters oatp1 (22), oatp2 (35), oatp3 (1), and lst-1/oatp4 (8, 23), the renal methotrexate transporters OAT-K1 (38), and the PG transporter PGT (25). Two human members of the family are LST-1 (2), the human ortholog of lst-1, and OATP-A (28), for which a rodent ortholog has not been identified. The mouse ortholog of rat oatp1 was recently identified (16). Although several members of this family transport bile acids, Northern blot and RT-PCR analysis indicate that they are not expressed in the small intestine (2, 22, 33, 38). To identify other bile acid transporters that may be involved in the apical uptake or basolateral secretion of bile acids, we employed low-stringency PCR and rapid amplification of cDNA ends (RACE) strategies to isolate a rat oatp3 cDNA from rat small intestine. The mRNA and protein expression properties, substrate specificity, and membrane localization determined in this work suggest that rat oatp3 is an intestinal apical transporter that participates in bile acid absorption down the length of the small intestine. The mouse oatp3 cDNA was also obtained by identification of mouse EST clones and subsequent 5'-RACE. Its chromosomal location suggests that oatp3 is the ortholog of human OATP-A.
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MATERIALS AND METHODS |
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Materials.
Male Sprague-Dawley rats were purchased from Zivic Miller Laboratories
(Zelienopole, PA). [3H]taurocholic acid (2.0-2.6
Ci/mmol), [2,4-3H]cholate (27.5 Ci/mmol), and
[3H(G)]digoxin (19 Ci/mmol) were purchased from NEN Life
Sciences Products (Boston, MA). Other tritiated bile acids (2-60
Ci/mmol) were obtained from Dr. Alan Hofmann (University of California, San Diego) and were synthesized as described previously (12, 37). Inulin [14C]carboxylic acid (2-10
mCi/mmol), [3',5',7,9-3H]folic acid (26.0 Ci/mmol), [3',5',7-3H]methotrexate (5.74 Ci/mmol),
[32P]dCTP (3,000 Ci/mmol), and [32P]UTP
(800 Ci/mmol) were purchased from Amersham (Arlington Heights, IL).
125I-labeled microcystin was kindly provided by Dr. Maria
Runnegar (University of Southern California). Unlabeled bile acids were purchased from Sigma Chemical (St. Louis, MO) and Calbiochem (San Diego, CA). DIDS was purchased from Sigma Chemical. COS-1 cells were
maintained in medium A, which consists of DMEM containing 4,500 mg/l D-glucose, 10% (vol/vol) FCS, 100 U/ml
penicillin, and 100 µg/ml streptomycin (Life Technologies,
Gaithersburg, MD). Madin-Darby canine kidney (MDCK) cells were obtained
from the American Type Culture Collection and maintained in
medium B, which consists of DMEM containing 1,000 mg/l
D-glucose, 10% (vol/vol) FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Sprague-Dawley rat hepatocytes were isolated by
collagenase perfusion (7) and kindly provided by Dr. Tom
Thuren (Dept. of Internal Medicine, Wake Forest University).
Sprague-Dawley rat retinas were kindly provided by Dr. Elena Grigorenko
(Dept. of Neurosurgery, Wake Forest University). A mouse/hamster
radiation hybrid panel (catalog no. RH04.02) was purchased from
Research Genetics (Huntsville, AL). The oligonucleotide primers are
listed in Table 1.
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Isolation of the rat oatp3 cDNA. cDNA was synthesized from rat ileal poly(A) RNA (Superscript Kit, Life Technologies) and used with primers RBOATP3 and RBOATP4 to PCR amplify a 297-bp product. Sequence analysis revealed that the PCR product was distinct but related to the previously reported oatp1 and oatp2 cDNA clones. A larger 1,083-bp oatp3 fragment was PCR amplified using RBOATP4 and a degenerate primer corresponding to amino acids 187-193 of oatp1 (oligonucleotide OATP5). The RACE procedure was employed to obtain the remainder of the coding region, the 3'-untranslated region, and a portion of the 5'-untranslated region of rat oatp3. For 3'-RACE, rat ileal poly(A) RNA was reverse transcribed using the oligo(dT) adapter primer AP2. The primer pairs RBOATP3/UAP2 and RIOATP1/UAP2 were employed in the primary and secondary PCR amplifications, respectively. The PCR-amplified product was isolated from a 0.8% (wt/vol) agarose gel, subcloned into pBluescript II KS, and sequenced using a Perkin-Elmer ABI Prism 377 sequencer. For 5'-RACE, a rat ileal double-stranded, adapter-ligated cDNA library was generated using 1 µg of rat ileal poly(A) RNA (Marathon cDNA Amplification Kit, Clontech, Palo Alto, CA). The primer pairs AP1/RIOATP8 and NAP2/RIOATP10 were employed in the primary and secondary PCR amplifications, respectively, of the adapter-ligated cDNA. Both reactions were performed as suggested by the manufacturer. The product of this secondary amplification reaction was subcloned and yielded the oatp3 sequence corresponding to nt 244-861. A second 5'-RACE reaction was performed using primer pairs AP1/RIOATP16 and NAP2/RIOATP18 in the primary and secondary PCR amplifications, respectively. The amplified product of this reaction was sequenced and corresponded to oatp3 nt 1-361.
Construction of rat oatp and ASBT expression plasmids. A rat oatp3 expression plasmid was constructed as follows. First-strand cDNA was synthesized from rat ileal poly(A) RNA using a cDNA synthesis kit (Superscript Kit, Life Technologies). A pair of oligonucleotide primers, RIOATPK and RIOATP12, corresponding to rat oatp3 nt 238-263 and 2259-2283, was used for PCR amplification with the rat ileal cDNA. Long PCR amplification conditions (6) were employed using 2.5 U of Taq polymerase (Life Technologies) and 0.25 U of Pfu polymerase (Stratagene, La Jolla, CA) at an annealing temperature of 65°C. After PCR amplification, the 2,049-bp product was isolated from a 0.8% (wt/vol) agarose gel. The fragment was treated with T4 DNA polymerase, phosphorylated with T4 polynucleotide kinase, and ligated into Sma I-digested pCMV5. For the rat oatp1 and oatp2 expression vectors, the coding regions were amplified from rat liver cDNA using the long PCR conditions and oatp1-specific oligonucleotide primers RLOATP1 and RLOATP2 and oatp2-specific oligonucleotide primers RBOATP1 and RBOATP2. The 2,151-bp oatp1 product was subcloned into EcoR V-digested pcDNAI (Invitrogen), and the 2,139-bp oatp2 product was subcloned into EcoR V-digested pcDNA3. For the rat ASBT expression plasmid, an EcoR I-Xho I fragment from BS37C1 (41) encompassing the rat ASBT coding region, as well as 115 and 83 bp of 5'- and 3'-untranslated sequences, respectively, was subcloned into EcoR I- and Sal I-digested pCMV5. The cDNA inserts for all the transporter expression plasmids were sequenced on both strands using a Perkin-Elmer ABI Prism 377 sequencer.
Transport assays in transfected COS cells. COS-1 cells were maintained in monolayer at 37°C in an atmosphere of 5% CO2 in medium A. On day 0, 100-mm plates were seeded with 1.5 × 106 COS cells. On day 1, each plate of cells was transfected using 18 µl of the FuGENE 6 Transfection Reagent (Boehringer Mannheim, Indianapolis, IN) and 6 µg of the indicated plasmid. On day 2, the transfected cells were trypsinized, pooled, and replated at 2.2 × 105 cells/dish in 35-mm plates or at 7 × 104 cells/well in 24-well plates. On day 4, the cells were incubated at 37°C in a Hanks' balanced salt solution (HBSS) containing 137 mM Na+ or K+ in the presence of the indicated concentration of radiolabeled solute for the indicated times. For the DIDS inhibition experiment, the cells were preincubated in HBSS containing 137 mM NaCl and the indicated concentrations of DIDS for 30 min at 37°C. The cells were washed twice and then incubated for 30 min at 37°C in HBSS containing 10 µM [3H]taurocholate and the indicated concentrations of DIDS. The cells were then washed and harvested to determine cell-associated radioactivity and protein, as described previously (10).
Transport assays in stably transfected MDCK cells. Stable expression of the human ileal apical Na+-bile acid transporter and the rat oatp3 in MDCK cells was achieved as follows. On day 0, 100-mm plates were seeded with 5 × 105 MDCK cells. On day 3, the cells were transfected with 5.5 µg of the transporter expression plasmid plus 0.5 µg of pSV3Neo using the FuGENE 6 Transfection Reagent. On day 5, the cells were switched to medium B containing 700 µg/ml G-418. After selection for 15 days, individual colonies were picked, expanded in 24-well plates, and screened for [3H]taurocholate uptake. The colonies expressing the highest taurocholate uptake activity were selected; the MDCK-oatp3 cells were further isolated through one round of dilution cloning. For the dilution cloning, each colony was expanded and then replated at ~10 cells/well in a 24-well plate. These cells were expanded, transferred to duplicate plates, and assayed for taurocholate uptake activity. The final MDCK cell clones were maintained in medium B containing 350 µg/ml G-418.
For the DIDS inhibition, bile acid uptake kinetics, and non-bile acid substrate uptake experiments, the MDCK and MDCK-oatp3 cells were seeded on day 0 in 24-well plates at 7 × 104 cells/well, induced with 10 mM sodium butyrate on day 2, and assayed for activity on day 3. The DIDS sensitivity of taurocholate transport was assayed as described for the transiently transfected COS cells, except the MDCK cells were incubated for 2.5 min at 37°C in HBSS containing 10 µM [3H]taurocholate. To determine the kinetics of bile acid transport, the cells were incubated with increasing concentrations of the radiolabeled bile acids in HBSS containing 137 mM choline for 5 min at 37°C. The cells were incubated in duplicate with six different concentrations of bile acid from 1 to 100 µM. The Michaelis-Menten constants (Km) and maximal velocities (Vmax) were determined by Eadie-Hofstee analysis. A similar procedure was used to examine the uptake of the non-bile acid substrates (folic acid, methotrexate, digoxin, and microcystin). For membrane localization transport assays, untransfected MDCK, stably transfected MDCK-oatp3, or stably transfected MDCK-ASBT cells were seeded onto 12-mm Transwell filter inserts (Costar, Cambridge, MA) at 8.8 × 104 cells/insert. The media in the apical and basolateral chambers were replaced every 2 days; on day 5, expression of the transfected cDNAs was induced by addition of media containing 10 mM sodium butyrate. Formation of a tight seal between the apical and basolateral chambers was monitored by transepithelial transport of inulin [14C]carboxylic acid (~50 µM). After incubation for 30 min at 37°C, the diffusion of radiolabeled inulin across the cell monolayer from the apical and basolateral chambers was <1.5 and 0.3%, respectively. On day 6, the cells were assayed for [3H]taurocholate uptake. The cell monolayers were washed with warm PBS, and each well was incubated with the indicated volumes of HBSS plus 10 µM [3H]taurocholate added to the apical (0.5 ml) or basolateral (1.0 ml) chamber. After 30 min at 37°C, the medium was removed, and the cells were washed in ice-cold PBS and harvested to determine cell-associated radioactivity and protein (10).oatp3 mRNA tissue expression. RT-PCR analysis was used to compare the liver and small intestinal expression of oatp1, oatp2, and oatp3 mRNA. Total cellular RNA was isolated using TRIzol Reagent (Life Technologies) as suggested by the manufacturer. Poly(A) RNA was prepared by oligo(dT)-cellulose chromatography using a MicroPoly(A) Pure Kit (Ambion, Austin, TX). The RNA was then reverse transcribed using a random-hexamer primer and a cDNA synthesis kit (Superscript Kit, Life Technologies). For the PCR amplification of oatp2 and oatp3 (50 µl; 30 cycles at 94°C for 45 s, 65°C for 1 min, and 72°C for 2 min), the reactions contained 1 µl of cDNA, 0.7 µM primers, 0.2 mM dNTPs, 2 mM MgCl2, and 0.5 U of Taq polymerase. Oligonucleotide primers specific for oatp2 (RBOATP1 and RBOATP4) and oatp3 (RIOATP1 and RIOATP6) were used to PCR amplify the 1,726- and 972-bp products, respectively. The oligonucleotide primers RLOATP1 and RLOATP6 and an annealing temperature of 60°C were used to amplify a 531-bp fragment for oatp1. For each primer pair, a reaction containing no cDNA template was included as a control for reagent contamination. Rat liver cDNA, small intestine cDNA, and mock cDNA reactions, to which no reverse transcriptase was added, were also PCR amplified using primers CYCF and CYCR for cyclophilin as a control for cDNA load and genomic DNA contamination. The amplified products were separated on a 1% (wt/vol) agarose gel and visualized with ethidium bromide.
RNase protection assays (RPAs) were performed using an RPA II Kit (Ambion) and the indicated amount of RNA isolated from rat tissues. The antisense 32P-labeled riboprobes were transcribed using [Antibody preparation.
The cDNA coding for the carboxy-terminal 47 amino acids of rat oatp3
was PCR amplified, subcloned into pGEX 3X (Pharmacia), and sequenced.
The glutathione S-transferase (GST)-oatp3 fusion protein was
purified from Escherichia coli cytosol by glutathione affinity chromatography, as described elsewhere (42).
Three New Zealand White rabbits were immunized with 500 µg of the
GST-oatp3 fusion protein in Freund's complete adjuvant, and an
Ig-enriched fraction was prepared from immune serum by precipitation
with 50% ammonium sulfate. Affinity purification of the anti-oatp3 antibody was performed by sequential affinity chromatography using GST-coupled agarose to remove GST-specific antibodies followed by
GST-oatp3-coupled agarose according to the manufacturer's instructions (AminoLink Immobilization Kit, Pierce, Rockford, IL). The
affinity-purified antibody was stored at 70°C and subjected to only
one freeze-thaw cycle.
Immunolocalization. Sections (6 µm) of rat intestine or 35-mm dishes of COS cells were fixed in 3.7% formaldehyde, washed in PBS, and incubated in blocking solution (PBS containing 1% BSA and 0.1% saponin) for 10 min at room temperature. The sections were then incubated with 2.9 µg/ml affinity-purified anti-oatp3-GST fusion protein antibody or preimmune Ig (50% ammonium sulfate-precipitated fraction) in the blocking solution for 0.5-1.5 h at room temperature. The sections were washed four times in PBS and once in the blocking solution. The sections were then incubated with 25 µg/ml rhodamine-conjugated goat anti-rabbit IgG secondary antibody (Jackson ImmunoResearch Laboratories) in blocking solution for 0.5-1.5 h at room temperature. After four washes with PBS, the sections were fixed in 3.7% formaldehyde for 10 min and viewed using a Zeiss Axioplane 2 fluorescence microscope equipped with rhodamine epifluorescence optics. Images were captured using a Dage 300 charge-coupled device digital camera. For each experiment, a section or dish was incubated with the secondary antibody alone to determine background fluorescence.
Identification of the mouse oatp3 cDNA. Oligonucleotide primers Moatp3-6 and Moatp3-4 were designed on the basis of the 3'-untranslated sequence of two mouse EST clones (GenBank accession nos. AA690580 and AA612385) and used to obtain the 5'-untranslated sequence and complete coding region sequence of the mouse oatp3 cDNA by 5'-RACE (Smart Race Kit, Clontech). Briefly, first-strand cDNA was synthesized from 1 µg of mouse brain poly(A) RNA (Clontech) using an oligo(dT) primer. During the first-strand synthesis, a 5'-adapter sequence was added to the cDNA. The primer pairs UPM/Moatp3-6 and NUP/Moatp3-4 were employed in the primary and secondary PCR amplifications, respectively, of the cDNA. The PCR-amplified product was isolated from a 0.8% (wt/vol) agarose gel, subcloned into pBluescript II KS, and sequenced using a Perkin-Elmer ABI Prism 377 sequencer.
Chromosomal localization of the mouse oatp3 gene. The oatp3-specific oligonucleotide primers Moatp3-3 and Moatp3-5 were designed on the basis of the untranslated sequence of the mouse oatp3 and used to screen a whole mouse genome/hamster radiation hybrid panel (Research Genetics) by PCR amplification. The reactions contained 18.65 ng of the panel DNA, 50 mM KCl, 10 mM Tris · HCl, pH 8.2, 1.5 mM MgCl2, 0.2 mM dNTPs, each oligo at 0.4 µM, and 0.5 U of Taq polymerase. Reactions containing mouse or hamster genomic DNA or no template were also included as controls. The reactions underwent 35 cycles in a GeneAmp PCR System 9700 (PE Applied Biosystems, Foster City, CA) at 94°C for 40 s, 59°C for 40 s, and 72°C for 40 s. The PCR products were analyzed on 1% TAE agarose gels. The hybrid panel DNA samples were scored as positive or negative for the 216-bp mouse oatp3 product, and the retention pattern was submitted to the Jackson Laboratories Mouse Radiation Hybrid Database (http://www.jax.org/resources/documents/cmdata/rhmap/rhsubmit.html) for analysis.
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RESULTS |
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Isolation of oatp3 cDNA from rat intestine. To isolate additional components of the intestinal bile acid transport system, a 297-bp fragment of a novel oatp was identified from rat ileal cDNA using oatp2-specific oligonucleotide primers and low-stringency PCR. A combination of RACE and PCR amplification was then used to obtain the complete 2,765-bp cDNA, designated oatp3 (GenBank accession no. AF083469). This complete intestinal oatp cDNA includes a 237-bp 5'-untranslated region, a 2,010-bp open reading frame, and a 518-bp 3'-untranslated region. The 3'-untranslated sequence contains a canonical polyadenylation signal (AATAAA) located at position 2747 and is followed by a 17-nt poly(A) tail. The initiator methionine lies in an appropriate consensus for initiation of translation (26) and is preceded by an in-frame stop codon. The three upstream ATG codons located in the predicted 5'-untranslated region are closely followed by in-frame stop codons. The 2,010-bp open reading frame encodes a 670-amino acid polypeptide with a calculated molecular mass of 74,496 Da. The intestinal oatp cDNA sequence was nearly identical to the oatp3 that was identified from rat retina (1) (GenBank accession no. AF041105), except for five nucleotide changes at positions 334, 401, 445, 1864, and 1865. These nucleotide substitutions result in four amino acid differences between the retinal and ileal oatp3 clones (retinal clone residue listed first: Q33K, I55T, F70L, and K543E). These differences may be due to PCR errors, sequencing errors, or polymorphisms; in each case, the nucleotide sequence reported here changed the predicted amino acid to a residue that matched the oatp1/2 consensus. A search of available nucleic acid databases revealed that the rat oatp3 protein sequence is 81, 82, 77, 40, and 36% identical to that of rat oatp1 (22) (GenBank accession no. L19031), oatp2 (35) (GenBank accession no. U88036), OAT-K1 (38), (GenBank accession no. D79981), lst-1 (23) (GenBank accession no. AF147740), and PGT (25) (GenBank accession no. M64862), respectively. Hidden Markov model analysis (http://www.enzim.hu/hmmtop) of the protein sequence predicted a topology with 12 transmembrane domains, similar to other members of the oatp family (45).
Transport properties of oatp3.
To examine the kinetics of oatp3-mediated bile acid uptake, transfected
COS cells were incubated with increasing concentrations of
[3H]taurocholate for 2.5 min at 37°C. Previous studies
had shown that transport of [3H]taurocholate by oatp3 was
linear up to 5 min (Fig. 1,
inset). The transport of [3H]taurocholate by
oatp3-transfected COS cells was saturable (Fig. 1), with an apparent
Km of 30 µM and a Vmax
of 240 pmol · min1 · mg
protein
1. To compare the bile acid substrate specificity
of the rat oatp3 and ASBT, transfected COS cells were incubated for 2.5 min with 10 µM radiolabeled bile acid, and uptake was quantitated.
Rat oatp3 and ASBT transport all the major species of bile acids, but
ASBT transported them at a greater rate (Fig.
2). Oatp3 transported glycine-conjugated
bile acids more rapidly than taurine conjugates. This preference is
particularly evident for the glycine conjugates of deoxycholate and
ursodeoxycholate, which exhibited initial uptake rates that were 3.6- and 2.6-fold, respectively, greater than the corresponding taurine
conjugates. Also, oatp3 transported the dihydroxy bile acids more
rapidly than the trihydroxy bile acids for glycine and taurine
conjugates (3.5-, 2-, and 3.9-fold over glycocholate for
glycodeoxycholate, glycochenodeoxycholate, and
glycoursodeoxycholate, respectively; 1.4-, 2.3-, and 2.2-fold over
taurocholate for taurodeoxycholate, taurochenodeoxycholate, and
tauroursodeoxycholate, respectively). In contrast, ASBT showed neither
of these trends for bile acid transport rate.
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Analysis of oatp3 membrane expression.
The membrane localization of oatp3 was examined in the stably
transfected MDCK cells. Figure 4 shows
the cellular accumulation of [3H]taurocholate from the
apical and basolateral chambers in monolayers of the parental MDCK
cells and MDCK cells stably transfected with the human ASBT (MDCK-ASBT)
and the rat oatp3 (MDCK-oatp3). The parental MDCK and MDCK-ASBT cells
were examined as a negative and positive control, respectively.
Taurocholate uptake was expressed only on the apical surface for the
human ASBT-transfected MDCK cells (Fig. 4A), in agreement
with previous studies of the rat ASBT (43). These findings
indicate that the transfected MDCK cells reproduced the normal
trafficking for this bile acid transporter. Cellular taurocholate
accumulation from the apical chamber was 26-fold greater for the
MDCK-oatp3 cells than for the parental MDCK cells (Fig. 4B).
In contrast, taurocholate uptake from the basolateral chamber was
similar in the parental MDCK and oatp3 stably transfected MDCK cells.
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Analysis of rat oatp3 tissue expression.
RT-PCR and RPA were used to investigate the expression of oatp3 in
liver and intestine. As shown in Fig.
5A, oatp1 and oatp2 mRNAs were
detected in rat liver, but not rat small intestine, cDNA. In contrast,
oatp3 was readily detected in small intestine, with only a faint but
reproducible signal amplified from rat liver cDNA. RPA analysis of the
horizontal gradient of oatp3 mRNA expression down the length of the
small intestine is shown in Fig. 5B. The assay employed a
32P-labeled oatp3 probe containing 199 nt of coding
sequence and 150 nt of 3'-untranslated region. Oatp3 mRNA was present
at ~0.04 pg/µg total RNA and was expressed at similar levels down
the length of the small intestine. This oatp3 probe could potentially
cross-hybridize with other members of the family. However, no smaller
cross-hybridizing products were detected with the oatp3 probe, nor was
a protected fragment detected with an oatp1-specific probe in the RPA
analysis of the intestinal RNAs (data not shown). The absence of
cross-hybridizing related oatp transcripts in small intestine agrees
with the RT-PCR analysis (Fig. 5A) and previous studies of
OAT-K1 mRNA expression (38).
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Isolation of oatp3 cDNA from mouse brain.
A search of the EST database yielded two overlapping mouse EST clones,
one from skin and one from mammary gland (GenBank accession nos.
AA690580 and AA612385), with significant identity to rat oatp3. A
contig of the two clones encompassed 499 bp of coding region and 434 bp
of 3'-untranslated sequence. At the nucleotide level, the coding region
of this contig shares 92% identity with the rat oatp3 coding region
and <86% identity with other members of the family. In addition, the
contig and the rat oatp3 share ~81% identity along the length of
their 3'-untranslated regions, whereas the 3'-untranslated regions of
the other oatp family members shares <50% identity. At the amino acid
level, the contig shares 92% identity with the rat oatp3 and <78%
identity with other members of the oatp family. This very high degree
of sequence identity indicates that the EST clones represent the mouse
ortholog of the rat oatp3. The sequence of the complete mouse oatp3
cDNA (GenBank accession no. AF240694) was obtained by 5'-RACE. The cDNA
includes 94 bp of 5'-untranslated sequence, a 2,010-bp coding region,
and 434 bp of 3'-untranslated region. The 3'-untranslated sequence included a canonical polyadenylation signal (AATAAA) located at position 2519 and was followed by a 22-nt poly(A) tail. The initiator methionine lies in an appropriate consensus for initiation of translation (26) and is preceded by an in-frame stop
codon. An upstream ATG codon is closely followed by an in-frame stop codon. The 2,010-bp open reading frame encodes a 670-amino acid polypeptide with a calculated molecular mass of 74,758 Da and a
putative topology of 12 transmembrane domains (45). The
predicted mouse oatp3 protein sequence is 90, 80, 82, 78, 43, and 35%
identical to the rat oatp3, oatp1 (22), oatp2
(35), oatk1 (38), lst-1 (23),
and PGT (25), respectively (Table
3).
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Chromosomal localization of the mouse oatp3 gene.
To determine the chromosomal location of the mouse oatp3
gene, two mouse oatp3 3'-untranslated region-specific oligonucleotide primers were used to screen a mouse/hamster radiation hybrid panel by
PCR. The retention patterns were submitted to the Jackson Laboratories Mouse Radiation Hybrid Database, where linkage analysis was performed. An optimal logarithm of odds score of 13.0 was obtained with marker D6Mit58. This localizes the mouse oatp3 gene (locus symbol
Slc21a7) to a region of mouse chromosome 6 syntenic with human
chromosome 12p12. The human OATP-A gene has been localized
to this region of chromosome 12 by radiation hybrid analysis
(28) and fluorescent in situ hybridization
(30), suggesting that OATP-A is the human ortholog of
oatp3. Interestingly, the human LST-1 gene (aliases: OATP-C, OATP2, and SLC21A6) is encoded by a BAC
clone (GenBank accession no. AC022335) also mapping to human chromosome
12p (www.hgsc.bcm.tmc.edu/seq_data/project-table.cgi?submit=Run&maplocation=Human+12p). This observation raises the possibility that a cluster of
oatp-like genes is encoded in this chromosomal region and
that another human ortholog may exist for oatp3. A comparison of the
amino acid sequences for OATP-A and rat and mouse oatp3 (Fig.
9) reveals identities of 72% shared with
both rodent polypeptides. This high degree of identity suggests that
oatp3 and OATP-A are orthologous.
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DISCUSSION |
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Bile acid absorption in the jejunum was originally considered to occur only by passive diffusion across the brush-border membrane (11). However, recent studies in a variety of species indicate that facilitated transport is present in jejunum (3-5). This study reports the cloning and analysis of a strong candidate for the facilitative intestinal bile acid transporter oatp3.
The rat oatp3 cDNA shares considerable identity with other members of the rat oatp family, including oatp1, oatp2, and OAT-K1. The sequence identity extends throughout the coding region and ends abruptly 23-53 nt upstream of the initiator methionine and 41-50 nt downstream of the stop codon. The divergent 3'- and 5'-untranslated regions indicate that oatp3 is a distinct gene product and not an alternately spliced isoform of a previously identified oatp. The 74-kDa protein encoded by this oatp3 cDNA also shares considerable structural identity with the other known rat oatps. It is predicted to be a polytopic membrane protein with a cytoplasmic amino terminus, 12 putative transmembrane segments, and a cytoplasmic carboxy terminus. The predicted protein contains four potential glycosylation sites, three of which are conserved among the rat oatps.
When expressed in transfected COS cells, rat oatp3 mediates
Na+-independent, saturable taurocholate uptake with an
apparent Km of 30 µM and a
Vmax of 240 pmol · min1 · mg
protein
1. This Km is
similar to that reported for the retinal library-derived oatp3 clone
expressed in Xenopus oocytes (18 µM) (1), for
oatp1 expressed in Xenopus oocytes (50 µM)
(29), HeLa cells (27 µM) (24), and Chinese
hamster ovary (CHO) cells (32 µM) (13), and for oatp2
(34 µM) (35) and human OATP (60 µM) (28)
expressed in Xenopus oocytes. In addition, the apparent
Km is very similar to the value of 54 µM
determined for the taurocholate transport by rat jejunal brush-border
membrane vesicles (4).
In the rat jejunal brush-border membrane vesicle study, the dihydroxy bile acid taurochenodeoxycholate was transported at twice the rate of the trihydroxy bile acid taurocholate. Also, the Km of taurocholate uptake by the vesicles was approximately twofold greater than the Km of taurochenodeoxycholate uptake (4). Previous studies of jejunal absorption have also shown a preference for glycine conjugates and dihydroxy bile acids in humans (19, 40), rabbits (3), and guinea pigs (5). These substrate specificities and relative affinities are similar to those exhibited by rat oatp3 in transfected cells. The greater influence of steroid nucleus hydroxylation than the conjugation on relative affinities as measured in the MDCK-oatp3 stable cells is similar to observations of oatp1-mediated uptake in stably transfected CHO cells (13). The rat ASBT did not exhibit the trends in substrate specificity observed for rat oatp3. ASBT transported taurochenodeoxycholate and taurocholate at the same rate, in contrast to the observations of the rat jejunal brush-border membrane vesicles. These results support the hypothesis that rat jejunal bile acid uptake is mediated by a distinct transporter and is not due to low levels of ASBT expression in the proximal small intestine (4, 5).
The demonstration of Na+-independent but bicarbonate trans-stimulated taurocholate uptake by rat jejunal brush-border membrane vesicles (4) also supports the presence of a bile acid transporter distinct from the ASBT. Like other oatps (22, 25, 28, 35, 38), rat oatp3-mediated transport is Na+ independent. The oatp3 may also be trans-stimulated by bicarbonate. Oatp3 shares extensive similarity with oatp1, which has been shown to function as a taurocholate/bicarbonate exchanger (39). Taurocholate uptake by the rat jejunal brush-border membrane vesicles (4) and by oatp3 is sensitive to the general anion transport inhibitor DIDS. Preincubation with 250 µM DIDS inhibited rat brush-border membrane taurocholate uptake by 81% (4). In rat oatp3-transfected COS and MDCK cells, the same concentration of DIDS inhibited taurocholate uptake to a comparable degree (59%).
The similar bile acid transport kinetics, substrate specificity, Na+ independence, and DIDS sensitivity shared by jejunal brush-border membrane vesicles and oatp3 suggest that oatp3 is the carrier responsible for the jejunal absorption of bile acids described by Amelsberg and colleagues (4, 5). Furthermore, the apical localization of oatp3 in MDCK cells, a model system for epithelial cell polarity, suggested that the transporter is expressed on the appropriate membrane to act as the brush-border membrane Na+-independent bile acid transporter.
The expression of oatp3 mRNA down the length of the small intestine is consistent with the hypothesis that oatp3 is the facilitative carrier responsible for intestinal passive absorption of bile acids. The other known bile acid-transporting rat oatps, oatp1 and oatp2, are not candidates, because they are not detected in the small intestine by Northern blot analysis (22), RT-PCR (38), or RPA. Staining of the apical membranes of jejunal epithelial cells by the oatp3 antibody confirmed that the protein is expressed in the small intestine on the jejunal apical brush-border membrane. The weak but reproducible staining by the anti-oatp3 antibody as well as the low level of oatp3 mRNA expression in the small intestine compared with ASBT may explain the previous difficulty in distinguishing Na+-independent facilitated transport from membrane diffusion of bile acids (3, 5, 11). Further studies to quantitate the oatp3 protein and activity in the small intestine are required to estimate the potential capacity of this system in humans and experimental animal models. The expression of oatp3 mRNA in rat brain, lung, and retina in addition to small intestine indicates that oatp3 must transport other solutes in addition to bile acids. This is not surprising given the broad substrate specificity demonstrated for the related oatps (13, 34). Oatp3 expressed in Xenopus oocytes has been shown to transport the triiodothyronine and thyroxine forms of thyroid hormone (1). Although other substrates for oatp3 remain to be determined, its intestinal expression and potentially broad substrate specificity suggest that oatp3 may play an important role in the absorption of other nutrients and drugs.
In a previously reported Northern blot analysis (1), the rat oatp3 cDNA hybridized with abundant transcripts in rat kidney and retina. In contrast, this study employing an RPA showed that oatp3 mRNA was present in retina at levels much lower than in brain and was not detected in kidney. It is likely that the discrepancy is due to cross-hybridization of the oatp3 probe with related oatp transcripts expressed in these tissues; the Sma I fragment used previously as a probe for the Northern blot analysis encompassed 260 nt of coding sequence that is highly conserved between the oatp family members. In contrast, the RNase protection probe used in this study was derived solely from divergent 3'-untranslated region sequence. Unfortunately, it has not been possible to directly compare the sequences of the probes used in these studies, since the untranslated region sequence of the retinal library oatp3 cDNA remains unpublished (1). The oatp3 expression pattern differences could also be explained by differences in the rat strains used in the two studies.
The high degree of sequence identity, particularly in the 3'-untranslated region, argues that the novel mouse cDNA in this study is orthologous to rat oatp3 and human OATP-A. The assignment of mouse and rat oatp3 as the rodent orthologs of human OATP-A was further supported by localization of the mouse oatp3 to a region of chromosome 6 syntenic with human chromosome 12p12 (28, 30). Kullak-Ublick et al. (30) showed by Northern blot analysis that OATP-A, like oatp3, is expressed at relatively high levels in brain and lung. Lower levels detected in the kidney and liver may represent cross-hybridization of the coding region probe with other members of the family or species differences. No OATP-A RNA was detected in small intestine. However, the low levels of oatp3 measured in rat intestine by RPA may be below the level of detection by Northern blot analysis. The uptake values of only six substrates are available for rat oatp3 and human OATP-A. Both proteins mediate transport of cholate, taurocholate, and tauroursodeoxycholate, although the Km of cholate varies greatly for the two transporters (8.8 µM for oatp3 and 93 µM for OATP-A). The cyanobacterial toxin microcystin is transported by OATP-A (14) but not by rat oatp3, and neither protein transports digoxin or methotrexate (35). The different transport properties may represent real species differences or differences between experimental systems.
It is widely accepted that active Na+-dependent transport is the predominant mechanism for intestinal bile acid absorption and maintenance of the enterohepatic circulation under normal physiological conditions (20). However, the question of what fraction of the bile acid pool is absorbed by mechanisms other than the ASBT has not been fully resolved in humans or in experimental animal models. The identification of a second apical intestinal carrier for bile acids provides a molecular mechanism for the non-ASBT-mediated absorption and has important physiological and therapeutic implications. In pathophysiological states in which ASBT function is compromised, such as ileal resection (9), primary bile acid malabsorption (36), and ileal inflammation (44), induction of oatp3 may slow bile acid loss. In contrast, induction of oatp3-mediated intestinal bile acid absorption would compromise the efficiency of specific ASBT inhibitors being developed to treat hypercholesterolemia (27). Identification of the oatp3 in small intestine will facilitate studies, including knockout mouse models, to define the quantitative significance of this pathway for the absorption of bile acids, other nutrients, and drugs under physiological and pathophysiological conditions.
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ACKNOWLEDGEMENTS |
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We thank Dr. Greg Shelness for advice and critical reading of the manuscript and Sally Ann Fossey and Dr. Don Bowden for assistance with the chromosomal localization.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants DK-47987 and HL-49373. H. C. Walters was supported by National Institutes of Health Institutional National Service Training Award HL-07868. P. A. Dawson is an Established Investigator of the American Heart Association.
The nucleotide sequences reported in this study have been submitted to the GenBank database under GenBank accession nos. AF083469 for rat oatp3 and AF240694 for mouse oatp3. The mouse sequence reported in this study has been submitted to the Mouse Genome Database ( www.informatics.jax.org/support/nomen/) under the locus symbol Slc21a7.
Address for reprint requests and other correspondence: P. A. Dawson, Dept. of Internal Medicine, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157 (E-mail: pdawson{at}wfubmc.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 8 March 2000; accepted in final form 29 June 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abe, T,
Kakyo M,
Sakagami H,
Tokui T,
Nishio T,
Tanemoto M,
Nomura H,
Hebert SC,
Matsuno S,
Kondo H,
and
Yawo H.
Molecular characterization and tissue distribution of a new organic anion transporter subtype (oatp3) that transports thyroid hormones and taurocholate and comparison with oatp2.
J Biol Chem
273:
22395-22401,
1998
2.
Abe, T,
Kakyo M,
Tokui T,
Nakagomi R,
Nishio T,
Nakai D,
Nomura H,
Unno M,
Suzuki M,
Naitoh T,
Matsuno S,
and
Yawo H.
Identification of a novel gene family encoding human liver-specific organic anion transporter LST-1.
J Biol Chem
274:
17159-17163,
1999
3.
Aldini, R,
Montagnani M,
Roda A,
Hrelia S,
Biagi PL,
and
Roda E.
Intestinal absorption of bile acids in the rabbit: different transport rates in jejunum and ileum.
Gastroenterology
110:
459-468,
1996[ISI][Medline].
4.
Amelsburg, A,
Jochims C,
Richter CP,
Nitsche R,
and
Fölsch U.
Evidence for an anion exchange mechanism for uptake of conjugated bile acid from rat jejunum.
Am J Physiol Gastrointest Liver Physiol
276:
G737-G742,
1999
5.
Amelsburg, A,
Schteingart CD,
Ton-Nu HT,
and
Hofmann AF.
Carrier-mediated jejunal absorption of conjugated bile acids in the guinea pig.
Gastroenterology
110:
1098-1106,
1996[ISI][Medline].
6.
Barnes, WM.
PCR amplification of up to 35 kb with high fidelity and high yield from lambda bacteriophage templates.
Proc Natl Acad Sci USA
91:
2216-2220,
1994[Abstract].
7.
Berry, MN,
and
Friend DS.
High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study.
J Cell Biol
43:
506-520,
1969
8.
Cattori, V,
Hagenbuch B,
Hagenbuch N,
Stieger B,
Ha R,
Winterhalter KE,
and
Meier PJ.
Identification of organic anion transporting polypeptide (Oatp4) as a major full-length isoform of the rat-liver specific transporter-1 (rlst-1) in rat liver.
FEBS Lett
474:
242-245,
2000[ISI][Medline].
9.
Coppola, CP,
Gosche JR,
Arrese M,
Ancowitz B,
Madsen J,
Vanderhoof J,
and
Shneider BL.
Molecular analysis of the adaptive response of intestinal bile acid transport after ileal resection in the rat.
Gastroenterology
115:
1172-1178,
1998[ISI][Medline].
10.
Craddock, AL,
Love MW,
Daniel RW,
Kirby LC,
Walters HC,
Wong MH,
and
Dawson PA.
Expression and transport properties of the human ileal and renal sodium-dependent bile acid transporter.
Am J Physiol Gastrointest Liver Physiol
274:
G157-G169,
1998
11.
Dietschy, JM.
Mechanisms for the intestinal absorption of bile acids.
J Lipid Res
9:
297-309,
1968
12.
Duane, WC,
Schteingart CD,
Ton-Nu H-T,
and
Hofmann AF.
Validation of [22,23-3H]cholic acid as a stable tracer through conversion to deoxycholic acid in human subjects.
J Lipid Res
37:
431-436,
1996[Abstract].
13.
Eckhardt, U,
Schroeder A,
Stieger B,
Höchli M,
Landmann L,
Tynes R,
Meier PJ,
and
Hagenbuch B.
Polyspecific substrate uptake by the hepatic organic anion transporter oatp1 in stably transfected CHO cells.
Am J Physiol Gastrointest Liver Physiol
276:
G1037-G1042,
1999
14.
Fischer, WJ,
Hagenbuch B,
Meier PJ,
and
Dietrich DR.
The cyclic heptapeptide microcystin, a cyanobacterial toxin, is transported by the human OATP (Abstract).
Hepatology
30:
465A,
1999[ISI].
15.
Haendler, B,
Hofer-Warbinek R,
and
Hofer E.
Complementary DNA for human T-cell cyclophilin.
EMBO J
6:
947-950,
1987[Abstract].
16.
Hagenbuch, B,
Adler ID,
and
Schmid TE.
Molecular cloning and functional characterization of the mouse organic anion-transporting polypeptide 1 (oatp1) and mapping of the gene to chromosome X.
Biochem J
345:
115-120,
2000[ISI][Medline].
17.
Henikoff, S,
and
Henikoff JG.
Amino acid substitution matrices from protein blocks.
Proc Natl Acad Sci USA
89:
10915-10919,
1992[Abstract].
18.
Hepner, GW,
and
Demers LM.
Dynamics of the enterohepatic circulations of glycine conjugates of cholic, chenodeoxycholic, deoxycholic, and sulpholithocholic acid in man.
Gastroenterology
72:
499-501,
1977[ISI][Medline].
19.
Hislop, I,
Hofmann A,
and
Schoenfield L.
Determinants of the rate and site of bile acid absorption in man (Abstract).
J Clin Invest
46:
1070,
1967[ISI].
20.
Hofmann, AF.
Intestinal absorption of bile acids and biliary constituents: the intestinal component of the enterohepatic circulation and the integrated system.
In: Physiology of the Gastrointestinal Tract, edited by Johnson LR.. New York: Raven, 1994, p. 1845-1865.
21.
Hofmann, AF,
and
Poley JR.
Role of bile acid malabsorption in pathogenesis of diarrhea and steatorrhea in patients with ileal resection.
Gastroenterology
62:
918-934,
1972[ISI][Medline].
22.
Jacquemin, E,
Hagenbuch B,
Stieger B,
Wolkoff AW,
and
Meier PJ.
Expression cloning of a rat liver Na+-independent organic anion transporter.
Proc Natl Acad Sci USA
91:
133-137,
1994[Abstract].
23.
Kakyo, M,
Unno M,
Yokui T,
Nakagomi R,
Nishio T,
Iwasashi H,
Nankai D,
Seki M,
Suzuki M,
Naitoh T,
Matsuno S,
Yawo H,
and
Abe T.
Molecular characterization and functional regulation of a novel rat liver-specific organic anion transporter rlst-1.
Gastroenterology
117:
770-775,
1999[ISI][Medline].
24.
Kanai, N,
Lu R,
Bao Y,
Wolkoff AW,
and
Schuster VL.
Transient expression of oatp organic anion transporter in mammalian cells: identification of candidate substrates.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F319-F325,
1996
25.
Kanai, N,
Lu R,
Satriano JA,
Bao Y,
Wolkoff AW,
and
Schuster VL.
Identification and characterization of a prostaglandin transporter.
Science
268:
866-869,
1995[ISI][Medline].
26.
Kozak, M.
An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs.
Nucleic Acids Res
15:
8125-8148,
1987[Abstract].
27.
Kramer, W,
and
Wess G.
Bile acid transport systems as pharmaceutical targets.
Eur J Clin Invest
26:
715-732,
1996[ISI][Medline].
28.
Kullak-Ublick, GA,
Hagenbuch B,
Stieger B,
Schteingart CD,
Hofmann AF,
and
Meier PJ.
Molecular and functional characterization of an organic anion transporting polypeptide cloned from human liver.
Gastroenterology
109:
1274-1282,
1995[ISI][Medline].
29.
Kullak-Ublick, GA,
Hagenbuch B,
Stieger B,
Wolkoff AW,
and
Meier PJ.
Functional characterization of the basolateral rat liver organic anion transporting polypeptide.
Hepatology
20:
411-416,
1994[ISI][Medline].
30.
Kullak-Ublick, GA,
Ulrich B,
Meier PJ,
Domdey H,
and
Paumgartner G.
Assignment of the human organic anion transporting polypeptide (OATP) gene to chromosome 12p12 by fluorescence in situ hybridization.
J Hepatol
25:
985-987,
1996[ISI][Medline].
31.
Love, M,
and
Dawson PA.
New insights into bile acid transport.
Curr Opin Lipidol
9:
225-229,
1998[ISI][Medline].
32.
Marcus, SN,
Schteingart CD,
Marquez ML,
Hofmann AF,
Xia Y,
Steinbach JH,
Ton-Nu HT,
Lillienau J,
Angellotti MA,
and
Schmassmann A.
Active absorption of conjugated bile acids in vivo.
Gastroenterology
100:
212-221,
1991[ISI][Medline].
33.
Masuda, S,
Ibaramoto K,
Takeuchi A,
Saito H,
Hashinoto Y,
and
Inui K-I.
Cloning and functional characterization of a multispecific organic anion transporter, OAT-K2, in rat kidney.
Mol Pharmacol
55:
743-752,
1999
34.
Meier, PJ,
Eckhardt U,
Schroeder A,
Hagenbuch B,
and
Stieger B.
Substrate specificity of sinusoidal bile acid and organic anion systems in rat and human liver.
Hepatology
26:
1667-1677,
1997[ISI][Medline].
35.
Noé, B,
Hagenbuch B,
Stieger B,
and
Meier PJ.
Isolation of a multispecific organic anion and cardiac glycoside transporter from rat brain.
Proc Natl Acad Sci USA
94:
10346-10350,
1997
36.
Oelkers, P,
Kirby LC,
Heubi JE,
and
Dawson PA.
Primary bile acid malabsorption caused by mutations in the ileal sodium-dependent bile acid transporter gene (SLC10A2).
J Clin Invest
99:
1880-1887,
1997
37.
Rossi, SS,
Converse JL,
and
Hofmann AF.
High-pressure liquid chromatographic analysis of conjugated bile acids in human bile: simultaneous resolution of sulfated and unsulfated lithocholyl amidates and the common conjugated bile acids.
J Lipid Res
28:
589-595,
1987[Abstract].
38.
Saito, H,
Masuda S,
and
Inui K.
Cloning and functional characterization of a novel rat organic anion transporter mediating basolateral uptake of methotrexate in the kidney.
J Biol Chem
271:
20719-20725,
1996
39.
Satlin, LM,
Amin V,
and
Wolkoff AW.
Organic anion transporting polypeptide mediates organic anion/HCO3 exchange.
J Biol Chem
272:
26340-26345,
1997
40.
Schalm, S,
LaRusso N,
Hofmann A,
Hoffman N,
van Berge-Henegouwen G,
and
Korman M.
Diurnal serum levels of primary conjugated bile acids. Assessment by specific radioimmunoassays for conjugates of cholic and chenodeoxycholic acid.
Gut
19:
1006-1014,
1978[ISI][Medline].
41.
Shneider, BL,
Dawson PA,
Christie DM,
Hardikar W,
Wong MH,
and
Suchy FJ.
Cloning and molecular characterization of the ontogeny of a rat ileal sodium-dependent bile acid transporter.
J Clin Invest
95:
745-754,
1995[ISI][Medline].
42.
Smith, DB,
and
Johnson KS.
Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase.
Gene
67:
31-40,
1988[ISI][Medline].
43.
Sun, AQ,
Ananthanarayanan M,
Soroka CJ,
Thevananther S,
Shneider BL,
and
Suchy FJ.
Sorting of rat liver and ileal sodium-dependent bile acid transporters in polarized epithelial cells.
Am J Physiol Gastrointest Liver Physiol
275:
G1045-G1055,
1998
44.
Sundaram, U,
Wisel S,
Stengelin S,
Kramer W,
and
Rajendran V.
Mechanism of inhibition of Na+-bile acid cotransport during chronic ileal inflammation in rabbits.
Am J Physiol Gastrointest Liver Physiol
275:
G1259-G1265,
1998
45.
Tusnady, GE,
and
Simon I.
Principles governing amino acid composition of integral membrane proteins: application to topology prediction.
J Mol Biol
283:
489-506,
1998[ISI][Medline].
46.
Weinberg, SL,
Burckhardt G,
and
Wilson FA.
Taurocholate transport by rat intestinal basolateral membrane vesicles.
J Clin Invest
78:
44-50,
1986[ISI][Medline].
47.
Weinman, SA,
Carruth MW,
and
Dawson PA.
Bile acid uptake via the human apical sodium-bile acid cotransporter is electrogenic.
J Biol Chem
273:
34691-34695,
1998