Polyspecific substrate uptake by the hepatic organic anion
transporter Oatp1 in stably transfected CHO cells
Uta
Eckhardt1,
Alice
Schroeder1,
Bruno
Stieger1,
Mathias
Höchli2,
Lukas
Landmann3,
Ronald
Tynes4,
Peter J.
Meier1, and
Bruno
Hagenbuch1
1 Division of Clinical
Pharmacology and Toxicology, Department of Medicine, University
Hospital, CH-8091 Zurich;
2 Central Laboratory for Electron
Microscopy, University of Zurich, CH-8028 Zurich;
3 Department of Anatomy,
University of Basel, CH-4000 Basel; and
4 Drug Metabolism and
Pharmakokinetics, Novartis Pharma, CH-4002 Basel, Switzerland
 |
ABSTRACT |
The rat liver organic anion transporting
polypeptide (Oatp1) has been extensively characterized mainly in the
Xenopus laevis expression system as a
polyspecific carrier transporting organic anions (bile salts), neutral
compounds, and even organic cations. In this study, we extended this
characterization using a mammalian expression system and confirm the
basolateral hepatic expression of Oatp1 with a new antibody. Besides
sulfobromophthalein [Michaelis-Menten constant
(Km) of ~3
µM], taurocholate
(Km of ~32
µM), and estradiol- 17
-glucuronide
(Km of ~4
µM), substrates previously shown to be transported by Oatp1 in
transfected HeLa cells, we determined the kinetic parameters for
cholate (Km of
~54 µM), glycocholate (Km of ~54
µM), estrone-3-sulfate
(Km of ~11
µM), CRC-220
(Km of ~57
µM), ouabain
(Km of ~3,000
µM), and ochratoxin A
(Km of ~29
µM) in stably transfected Chinese hamster ovary (CHO) cells. In
addition, three new substrates, taurochenodeoxycholate
(Km of ~7
µM), tauroursodeoxycholate
(Km of ~13
µM), and dehydroepiandrosterone sulfate
(Km of ~5
µM), were also investigated. The results establish the polyspecific
nature of Oatp1 in a mammalian expression system and definitely
identify conjugated dihydroxy bile salts and steroid conjugates as
high-affinity endogenous substrates of Oatp1.
sodium-independent organic anion transport; multispecificity; Chinese hamster ovary cells
 |
INTRODUCTION |
DETOXIFICATION OF endo- and xenobiotics is a major
function of the liver. Hepatic uptake of many of these amphipathic
compounds is mediated by polyspecific organic anion transporting
polypeptides (Oatp) that have been cloned from liver, brain, and kidney
(20). The first member of the Oatp
gene family of membrane transporters (Oatp1) has been isolated from rat
liver and shown to mediate Na+-independent saturable
transport of sulfobromophthalein (BSP) [Michaelis-Menten constant
(Km) of
1.5-3.3 µM] and taurocholate (Km of 19-50
µM) when expressed in Xenopus laevis
oocytes (11, 16) and in transfected HeLa cells (12, 30, 32). Oatp1 represents an ~80-kDa glycoprotein that in addition to the
basolateral plasma membrane of hepatocytes is also localized at the
apical membranes of kidney proximal tubule (S3 segment) (3) and choroid plexus epithelial cells (2). The endogenous substrates as well as the
transport mechanism are not definitively elucidated yet. In stably
transfected HeLa cells, Oatp1 has been shown to function as a
taurocholate/HCO
3 exchanger (30),
whereas in Xenopus laevis oocytes
Oatp1-mediated taurocholate transport was transstimulated by reduced
glutathione (19). Besides BSP and taurocholate, Oatp1 has been shown to
transport also estradiol-17
-glucuronide in transfected HeLa cells
(13, 30, 32). In addition, with the use of the Xenopus
laevis expression system, a wide variety of
structurally unrelated compounds have been suggested to be transported
by Oatp1, including the steroid conjugates estrone-3-sulfate (Km of ~4.5
µM) (5), the neutral steroids aldosterone
(Km of ~15 nM),
cortisol (Km of
~13 nM), and ouabain
(Km of 1,700 µM) (5), the thrombin inhibitor CRC-220
(Km of ~30
µM) (7), and the mycotoxin ochratoxin A
(Km of ~ 17 µM) (14). So far, the kinetic parameters of only three substrates
have been determined in a mammalian expression system (13, 30, 32).
Furthermore, the kinetics of Oatp1-mediated uptake of bile salts has
only been determined for taurocholate (16). This gap of knowledge is
closed in this study with the use of stably transfected Chinese hamster ovary (CHO) cells to determine the kinetic parameters of several bile
salts and various nonbile acid endo- and xenobiotics in a mammalian
expression system. The results demonstrate that similar to the
Na+-taurocholate cotransporting
polypeptide (Ntcp) (31), Oatp1 also exhibits the highest affinity for
the dihydroxy bile salt taurochenodeoxycholate among all bile salts
tested. Furthermore, the steroid conjugates, including the newly
characterized dehydroepiandrosterone sulfate (DHEAS), have been
identified as substrates with the highest affinities for Oatp1,
supporting the concept that these conjugates represent important
endogenous substrates of Oatp1.
 |
MATERIALS AND METHODS |
Materials
[3H]DHEAS (16 Ci/mmol),
[3H]taurocholic acid
(2.6 Ci/mmol),
[3H]cholic acid (13.2 Ci/mmol),
[3H]estrone-3-sulfate
(49.0 Ci/mmol),
[3H]estradiol-17
-D-glucuronide
(49 Ci/mmol), and
[3H]ouabain (20.5 Ci/mmol) were obtained from DuPont NEN (Boston, MA).
[35S]BSP (4.1 Ci/mmol)
was kindly provided by A. W. Wolkoff of Albert Einstein College of
Medicine (Bronx, NY), CRC-220 by W. Stüber of Behringwerke
(Marburg, Germany),
[3H]ochratoxin A by E. Petzinger of Justus Liebig-Universität (Giessen, Germany), and
[2-3H]taurochenodeoxycholate
(0.5 Ci/mmol) and
[2-3H]tauroursodeoxycholate
(0.5 Ci/mmol) by A. W. Hofmann and C. D. Schteingart of University of
California at San Diego (La Jolla, CA). All cell culture media and
reagents were obtained from Life Technologies (Paisley, UK). All other
chemicals and reagents were of analytical grade and were readily
available from commercial sources.
Antibody production, immunofluorescence, and Western blotting.
The cDNA coding for the last 40 amino acids of Oatp1 (11) was PCR
amplified using the following primers:
5'-GACATTGACTCTTCAGCAACTG-3' (corresponding to nucleotides
1977-1998) and 5'-CTGTTCATGGCCTTGAACAGG-3' (corresponding to nucleotides 2135-2115). The blunted PCR product was cloned into the Asp700-cut pMAL-c2. After
sequencing was performed to verify the correct in-frame subcloning, the
fusion protein between Oatp1 and the maltose binding protein of
E. coli was isolated and a rabbit was
immunized as described (34). The antibody raised did not cross-react
with Oatp2 (21) as verified in separate in vitro expression and in vivo
localization experiments (data not shown). Immunofluorescence was
performed as described previously (34). SDS-PAGE and Western blotting
were performed according to standard procedures (17, 26).
Stable transfection of Chinese hamster ovary cells with Oatp1.
The complete coding region of Oatp1 was cut out from the original
plasmid (11) using Sal I and
Hind III. This region was blunt ended
and then subcloned into the Stu
I-digested and dephosphorylated pCMV vector-1 (31). This construct was
introduced into CHO cells by electroporation, and stably transfected
cells were selected by adding G418 to the culture medium. From the
resulting transfected cell pool, single clones were isolated with the
use of cloning cylinders and tested for
Na+-independent taurocholate
uptake. Clone CHO-03 exhibited the highest transport activity of
taurocholate and was selected for use in all further experiments.
Cell culture.
CHO cells were grown in DMEM supplemented with 10% FCS, 2 mM
L-glutamine, 50 µg/ml
L-proline, 100 U/ml penicillin,
100 µg/ml streptomycin, and 0.5 µg/ml Fungizone (amphotericin B) at
37°C with 5% CO2 and 95%
humidity. Selective medium contained additional 400 µg/ml G418
sulfate (Geneticin).
Uptake studies in CHO cells.
Determination of Na+-independent
uptake of potential substrates for Oatp1 was performed as described
(31). For some experiments, expression of Oatp1 was induced by
incubation of the cells for 24 h with culture medium supplemented with
5 mM butyrate as described (22). For determination of the kinetic
parameters, the linear range of uptake was first determined for each
substrate individually. Transport was then measured at a time point
well within this linear range (usually 20-30 s), and net uptake
values used for the calculation of the kinetic parameters were obtained
by subtracting the uptake values obtained with wild-type CHO-K1 cells
from values obtained with stably transfected CHO-03 cells. Because
preliminary experiments did not demonstrate any
Na+ dependency, all uptake
experiments were performed in choline chloride-containing solutions.
Determination of protein concentration.
Protein concentrations were determined using the bicinchoninic acid
protein assay kit (Pierce, Rockford, IL) (33).
 |
RESULTS |
The antiserum generated against the fusion protein of the COOH-terminal
40 amino acids of Oatp1 and the maltose binding protein of
E. coli was tested by Western blot
analysis with rat liver basolateral and canalicular membrane vesicles.
As shown in Fig. 1 (lane
1), the antiserum reacted with an antigen in the
basolateral membrane fraction, yielding a single broad band with an
apparent molecular mass of 81 ± 6 kDa (mean ± SD of 6 independent determinations). The signal was virtually absent in the
canalicular lane (Fig. 1, lane 2),
confirming the selective localization of Oatp1 to the basolateral
plasma membrane domain of hepatocytes (3). The specificity of the
signal was tested by preincubation of the antiserum with the fusion
protein (Fig. 1, lanes 3 and
4). The results demonstrate that the
raised antiserum is specific for a single 81-kDa basolateral liver
plasma membrane protein.

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Fig. 1.
Characterization of antiserum raised against organic anion transporting
polypeptide (Oatp1) in rat liver basolateral and canalicular membrane
vesicles. Seventy-five micrograms of rat liver basolateral
(lanes 1 and
3) or canalicular
(lanes 2 and
4) membrane vesicles were separated
using 7.5% SDS-PAGE and subsequently transferred to nitrocellulose.
Western blot was incubated with a 1:2,000 dilution of the Oatp1
antiserum (lanes 1 and
2) or with Oatp1 antiserum
preabsorbed with the fusion protein used to raise the antiserum
(lanes 3 and
4). Bound antibodies were visualized
using 125I-labeled protein A. Molecular mass standards are indicated on the
right (in kDa).
|
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To localize the native Oatp1 in intact rat liver, the antiserum was
used on cryosections for immunofluorescence studies. As demonstrated in
Fig. 2 and supporting the Western blot
results from Fig. 1, Oatp1 immunoreactivity is restricted to the
basolateral plasma membrane of hepatocytes. No immunostaining could be
detected at the canalicular domain (Fig. 2). In addition, biliary
epithelial cells were immunonegative for Oatp1 (data not shown).

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Fig. 2.
Immunofluorescent localization of Oatp1 in intact rat liver. The 0.5- to 1.0-µm cryosections were incubated as described (35).
Immunoreactivity is restricted to the basolateral membrane, whereas the
canalicular domain (arrows) is immunonegative. Bar = 25 µm.
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To prove that the immunopositive basolateral hepatocyte antigen indeed
represents the native Oatp1, we next investigated whether expression of
cell surface immunopositivity is associated with Na+-independent transport of BSP.
As demonstrated in Fig. 3, CHO cells stably
transfected with Oatp1 cDNA (CHO-03) exhibited immunopositive surface
staining as well as
Na+-independent BSP uptake. In
contrast, wild-type CHO-K1 cells were immunonegative and showed only
minimal BSP uptake. These results demonstrate that the immunopositive
protein represents the functionally active rat liver Oatp1.

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Fig. 3.
Correlation of Oatp1 expression and
Na+-independent
sulfobromophthalein (BSP) uptake in stably transfected Chinese
hamster ovary (CHO) cells. Control CHO cells (CHO-K1,
A) or Oatp1-expressing CHO cells
(CHO-03, B) were grown to confluency
on coverslips and treated as described in MATERIALS
AND METHODS. On separate dishes, 2 µM
[35S]BSP uptake was
measured, after a 24-h incubation in 5 mM sodium butyrate, for 5 min in
a choline chloride medium. Uptake values,
bottom, represent means ± SD of
triplicate determinations.
|
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In a previous study, CHO cells stably expressing Ntcp showed a
~10-fold stimulation of
Na+-dependent uptake of bile salts
when gene expression was induced by sodium butyrate (31). As
illustrated in Fig. 4, sodium butyrate exerted a similar inducing effect on the expression of Oatp1 in stably
transfected CHO-03 but not in wild-type CHO-K1 cells. Thus sodium
butyrate increased the maximal velocity
(Vmax) value
for Oatp1-mediated estrone-3-sulfate uptake ~10-fold, whereas the Km value remained
unchanged (Table 1). Because high level
expression is important for the correct delineation of the substrate
specificity of a given transport system, we performed all subsequent
transport studies under butyrate-induced conditions. On the basis of
previous initial uptake activities (20), we next determined the
kinetics of a variety of established and new Oatp1 substrates. As
demonstrated in Table 1, BSP and estradiol-17
-glucuronide exerted
the highest affinities for Oatp1
(Km of ~3 µM)
among all substrates tested. These results are similar to previous
studies in Xenopus laevis oocytes (11,
16) and in transfected HeLa cells (13), respectively. Second were
taurocheno- and tauroursodeoxycholate, DHEAS, and estrone-3-sulfate
(Km values of
5-13 µM) (Table 1). The identification of DHEAS as a new
substrate of Oatp1 (Fig. 5) supports the
concept that endogenous steroid conjugates are important physiological high-affinity substrates of Oatp1 (13). A third group of substrates with Km values
between 29 and 57 µM included ochratoxin A, taurocholate, cholate,
glycocholate, and CRC-220 (Table 1). Finally, the studies in stably
transfected CHO-03 cells confirmed the low affinity of Oatp1 for
ouabain (Km of
~3,000 µM) (5). These studies in stably transfected CHO-03 cells
confirm the polyspecific substrate spectrum of Oatp1 and identify
dihydroxy bile salts and DHEAS as new high-affinity Oatp1 substrates.

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Fig. 4.
Butyrate induction of Oatp1-mediated estrone-3-sulfate uptake into CHO
cells. Wild-type CHO-K1 cells (A)
and Oatp1-expressing CHO-03 cells
(B) were incubated for 24 h in the
presence ( ) or absence ( ) of 5 mM sodium butyrate. For uptake
measurements, cells were incubated with 15 µM
[3H]estrone-3-sulfate
at 37°C for the indicated time periods in a choline chloride
medium. Data points represent means ± SD of
triplicate determinations.
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Fig. 5.
Kinetics of Oatp1-mediated dehydroepiandrosterone sulfate (DHEAS)
uptake in stably transfected CHO-03 cells. Wild-type CHO-K1 cells ( )
or Oatp1-expressing CHO-03 cells ( ) were incubated with increasing
concentrations of
[3H]DHEAS at 37°C
for 20 s in a choline chloride medium. Kinetic parameters were
calculated using a nonlinear curve fitting computer program according
to the Michaelis-Menten equation with the net Oatp1-mediated uptake
values ( ) (values obtained with wild-type CHO-K1 cells subtracted
from values obtained with stably transfected CHO-03 cells). Means ± SD of triplicate determinations are given. Curve represents the fitted
model. Km,
Michaelis-Menten constant.
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 |
DISCUSSION |
Previous studies in Oatp1-expressing Xenopus
laevis oocytes and in transfected HeLa cells have
provided evidence that Oatp1 mediates transport of a wide variety of
amphipathic compounds (5, 11-13, 16, 20, 32). Among several
anionic, neutral, and even cationic substrates identified, only the
anions BSP, taurocholate, and estradiol-17
-glucuronide have been
characterized in a mammalian expression system (12, 13, 32). In this
study, we directly correlate surface expression of Oatp1 and
multispecific transport of several anionic as well as neutral compounds
in a stably transfected mammalian CHO cell line (Figs. 3-5).
The antibody generated against the COOH-terminal end of Oatp1
recognized an ~81-kDa antigen at the basolateral membrane of hepatocytes (Figs. 1 and 2), which is similar to previous studies using
a different antibody raised against a 13-amino acid peptide close to
the COOH-terminal end (3). The antigen used to generate our new
antibody encompasses the last 40 amino acids of Oatp1. The specificity
for Oatp1 is indicated by an amino acid identity of the epitope of 70%
to Oatp3 (1), 61% to OAT-K1 (29), and only 56% to Oatp2
(21). Furthermore, the specificity of the antibody for Oatp1 is
supported by the observation of 1)
basolateral immunopositivity in hepatocytes (Fig. 2), which do not
express Oatp3 (1), and 2) a distinct
distribution of Oatp2 in liver (24) and choroid plexus (8). The
positive correlation between immunostaining of the plasma membrane and
transport of BSP in the CHO-03 cells (Fig. 3) definitively shows that
the basolateral protein recognized by the antiserum indeed represents
functionally active Oatp1.
Because high level expression of a transport protein is required for
correct delineation of its substrate specificity and kinetic transport
parameters (31), we treated the stably transfected CHO-03 cells with 5 mM sodium butyrate. Similar to Ntcp-expressing CHO 9-6 cells (31),
butyrate induction also resulted in a 10-fold increase of
Oatp1-mediated estrone-3-sulfate uptake (Fig. 4; Table 1). This
butyrate-induced expression level of Oatp1 in CHO-03 cells is about
fivefold higher than in hepatocytes, as estimated on the basis of the
apparent Vmax
values for Na+-independent cholate
uptake (Table 1; Ref. 4). A similar increase in Oatp1 expression is
also evident in comparison to stably transfected HeLa cells (30, 32).
Hence, butyrate-induced CHO-03 cells were routinely used to determine
the kinetics of a variety of presumptive Oatp1 substrates not
previously tested in a mammalian cell system. On the basis of the ratio
of Vmax to
Km, the best transport substrate of Oatp1 was BSP followed by the dihydroxylated bile salts taurourso- and taurochenodeoxycholate, the steroid conjugates estradiol-17
-glucuronide, estrone-3-sulfate, and DHEAS, the mycotoxin ochratoxin A, the trihydroxylated bile salts cholate, glycocholate, and taurocholate, and the thrombin inhibitor CRC-220 (Table 1). Similar to previous studies in Xenopus
laevis oocytes, the cardiac glycoside ouabain exhibited
by far the lowest affinity for Oatp1 among all substrates tested (Table
1). Our results indicate that dihydroxy bile salts and steroid 3 and 17 conjugates represent important endogenous substrates of Oatp1. This
conclusion is further supported by the identification of DHEAS as a new
high-affinity endogenous substrate of Oatp1 (Fig. 5; Table 1). DHEAS is
also transported by the human OATP with an apparent
Km value of ~7 µM (15). Because the concentration of DHEAS in human blood plasma reaches 10 µmol/l and because OATP is widely distributed in the human
brain, it is possible that OATP plays an important role in the
intracerebral distribution and action of DHEAS in humans (15). In the
rat, the physiological role of Oatp1 in the disposition of DHEAS and
other steroid conjugates is most probably concentrated in the liver and
kidney (3, 25), since cerebral expression of Oatp1 is confined to the
apical portion of choroid plexus epithelial cells only (2).
Besides endogenous substrates, Oatp1 also mediates transport of the
mycotoxin ochratoxin A (Table 1, Ref. 14), which is a frequent
contaminant of food and animal chow (14, 27). Its principal mechanism
of action is inhibition of protein synthesis by competition with
phenylalanine. The main target organ for ochratoxin A toxicity is the
kidney, whereas the liver is less frequently affected (27), most likely
because in the liver the toxin is rapidly glucuronidated, sulfated, and
excreted into bile (28). In the kidney, ochratoxin A is reabsorbed in
the proximal straight tubule, resulting in toxic intracellular
concentrations of the mycotoxin in kidney epithelial cells (6).
Interestingly, renal reabsorption of ochratoxin A can be partially
inhibited by BSP (6), which represents a classical high-affinity
substrate of Oatp1 (11) (Table 1). Moreover, a significant portion of
peritubular uptake of ochratoxin A is probenecid sensitive and
p-aminohippurate insensitive (9).
These characteristics of renal ochratoxin A transport could be
explained by Oatp1-mediated transport (16), since Oatp1 is also
localized at the brush-border membrane of the late proximal straight
tubule (S3) (3). Thus, in addition to uptake into hepatocytes, Oatp1
might play a role in the renal reabsorption of ochratoxin A and thus
significantly contribute to the overall nephrotoxicity of this
mycotoxin. Whether Oatp1 and/or other members of the
Oatp gene family of membrane
transporters are also involved in hepatic and/or renal transport of
other mycotoxins such as microcystin is not yet known and remains to be investigated.
In conclusion, the present study proves the multispecific nature of
Oatp1-mediated amphipathic substrate transport in a mammalian cell
system. In addition to the substrates identified previously and in this
study, recent evidence indicates that Oatp1 can also transport
glutathione conjugates, leukotriene
C4, and certain dipeptidic drugs
(10, 19, 23). In one of these studies, leukotriene
C4 exhibited a very high affinity
for Oatp1 (Km of ~0.27 µM), indicating that Oatp1 mediates leukotriene
C4 uptake into hepatocytes under
physiological conditions (18, 19). Furthermore, reduced glutathione
efflux has been proposed as a new driving force for Oatp1-mediated
substrate uptake into hepatocytes (19). Hence, the importance of Oatp1
as a polyspecific organic anion and drug transporter is increasingly
recognized. Furthermore, additional members of the
Oatp gene family of membrane
transporters have been cloned and shown to exhibit partially
overlapping substrate specificities with Oatp1 (1, 20). Thus exact
delineation of the transport characteristics of each Oatp in high
expression mammalian cell systems is important in defining the
physiological and pathophysiological roles of individual Oatps in the
normal and diseased body.
 |
ACKNOWLEDGEMENTS |
This study was supported by Swiss National Science Foundation Grants
31-45536.95 and 31-45677.95 (to P. J. Meier and B. Hagenbuch). B. Hagenbuch is a recipient of a Cloëtta Foundation Fellowship.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: B. Hagenbuch,
Division of Clinical Pharmacology and Toxicology, Dept. of Medicine,
Univ. Hospital, CH-8091 Zürich, Switzerland (E-mail:
Bruno.Hagenbuch{at}access.unizh.ch).
Received 9 June 1998; accepted in final form 22 January 1999.
 |
REFERENCES |
1.
Abe, T.,
M. Kakyo,
H. Sakagami,
T. Tokui,
T. Nishio,
M. Tanemoto,
H. Nomura,
S. C. Hebert,
S. Matsuno,
H. Kondo,
and
H. Yawo.
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[Abstract/Free Full Text].
2.
Angeletti, R. H.,
P. M. Novikoff,
S. R. Juvvadi,
J. M. Fritschy,
P. J. Meier,
and
A. W. Wolkoff.
The choroid plexus epithelium is the site of the organic anion transport protein in the brain.
Proc. Natl. Acad. Sci. USA
94:
283-286,
1997[Abstract/Free Full Text].
3.
Bergwerk, A. J.,
X. Y. Shi,
A. C. Ford,
N. Kanai,
E. Jacquemin,
R. D. Burk,
S. Bai,
P. M. Novikoff,
B. Stieger,
P. J. Meier,
V. L. Schuster,
and
A. W. Wolkoff.
Immunologic distribution of an organic anion transport protein in rat liver and kidney.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G231-G238,
1996[Abstract/Free Full Text].
4.
Boelsterli, U. A.,
B. Zimmerli,
and
P. J. Meier.
Identification and characterization of a basolateral dicarboxylate/cholate antiport system in rat hepatocytes.
Am. J. Physiol.
268 (Gastrointest. Liver Physiol. 31):
G797-G805,
1995[Abstract/Free Full Text].
5.
Bossuyt, X.,
M. Müller,
B. Hagenbuch,
and
P. J. Meier.
Polyspecific drug and steroid clearance by an organic anion transporter of mammalian liver.
J. Pharmacol. Exp. Ther.
276:
891-896,
1996[Abstract].
6.
Dahlmann, A.,
W. H. Dantzler,
S. Silbernagl,
and
M. Gekle.
Detailed mapping of ochratoxin A reabsorption along the rat nephron in vivo: the nephrotoxin can be reabsorbed in all nephron segments by different mechanisms.
J. Pharmacol. Exp. Ther.
286:
157-162,
1998[Abstract/Free Full Text].
7.
Eckhardt, U.,
J. A. Horz,
E. Petzinger,
W. Stüber,
M. Reers,
G. Dickneite,
H. Daniel,
M. Wagener,
B. Hagenbuch,
B. Stieger,
and
P. J. Meier.
The peptide-based thrombin inhibitor CRC 220 is a new substrate of the basolateral rat-liver organic anion-transporting polypeptide.
Hepatology
24:
380-384,
1996[Medline].
8.
Gao, B., B. Stieger, and P. J. Meier. Organ and
cellular distribution of the organic anion transporting polypeptide 2 (oatp2) in rat (Abstract). J. Hepatol.
28, Suppl.: 124, 1998.
9.
Groves, C. E.,
M. Morales,
and
S. H. Wright.
Peritubular transport of ochratoxin A in rabbit renal proximal tubules.
J. Pharmacol. Exp. Ther.
284:
943-948,
1998[Abstract/Free Full Text].
10.
Ishizuka, H.,
K. Konno,
H. Naganuma,
K. Nishimura,
H. Kouzuki,
H. Suzuki,
B. Stieger,
P. J. Meier,
and
Y. Sugiyama.
Transport of temocaprilat into rat hepatocytes: role of organic anion transporting polypeptide.
J. Pharmacol. Exp. Ther.
287:
37-42,
1998[Abstract/Free Full Text].
11.
Jacquemin, E.,
B. Hagenbuch,
B. Stieger,
A. W. Wolkoff,
and
P. J. Meier.
Expression cloning of a rat liver Na+-independent organic anion transporter.
Proc. Natl. Acad. Sci. USA
91:
133-137,
1994[Abstract].
12.
Kanai, N.,
R. Lu,
Y. Bao,
A. W. Wolkoff,
and
V. L. Schuster.
Transient expression of oatp organic anion transporter in mammalian cells: identification of candidate substrates.
Am. J. Physiol.
270 (Renal Fluid Electrolyte Physiol. 39):
F319-F325,
1996[Abstract/Free Full Text].
13.
Kanai, N.,
R. Lu,
Y. Bao,
A. W. Wolkoff,
M. Vore,
and
V. L. Schuster.
Estradiol 17
-D-glucuronide is a high-affinity substrate for oatp organic anion transporter.
Am. J. Physiol.
270 (Renal Fluid Electrolyte Physiol. 39):
F326-F331,
1996[Abstract/Free Full Text].
14.
Kontaxi, M.,
U. Eckhardt,
B. Hagenbuch,
B. Stieger,
P. J. Meier,
and
E. Petzinger.
Uptake of the mycotoxin ochratoxin A in liver cells occurs via the cloned organic anion transporting polypeptide.
J. Pharmacol. Exp. Ther.
279:
1507-1513,
1996[Abstract].
15.
Kullak-Ublick, G. A.,
T. Fisch,
M. Oswald,
B. Hagenbuch,
P. J. Meier,
U. Beuers,
and
G. Paumgartner.
Dehydroepiandrosterone sulfate (DHEAS): identification of a carrier protein in human liver and brain.
FEBS Lett.
424:
173-176,
1998[Medline].
16.
Kullak-Ublick, G.-A.,
B. Hagenbuch,
B. Stieger,
A. W. Wolkoff,
and
P. J. Meier.
Functional characterization of the basolateral rat liver organic anion transporting polypeptide.
Hepatology
20:
411-416,
1994[Medline].
17.
Laemmli, U. K.
Cleavage of strucural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1979.
18.
Leier, I.,
M. Müller,
G. Jedlitschky,
and
D. Keppler.
Leukotriene uptake by hepatocytes and hepatoma cells.
Eur. J. Biochem.
209:
281-289,
1992[Abstract].
19.
Li, L. Q.,
T. K. Lee,
P. J. Meier,
and
N. Ballatori.
Identification of glutathione as a driving force and leukotriene C-4 as a substrate for Oatp1, the hepatic sinusoidal organic solute transporter.
J. Biol. Chem.
273:
16184-16191,
1998[Abstract/Free Full Text].
20.
Meier, P. J.,
U. Eckhardt,
A. Schroeder,
B. Hagenbuch,
and
B. Stieger.
Substrate specificity of sinusoidal bile acid and organic anion uptake systems in rat and human liver.
Hepatology
26:
1667-1677,
1997[Medline].
21.
Noé, B.,
B. Hagenbuch,
B. Stieger,
and
P. J. Meier.
Isolation of a multispecific organic anion and cardiac glycoside transporter from rat brain.
Proc. Natl. Acad. Sci. USA
94:
10346-10350,
1997[Abstract/Free Full Text].
22.
Palermo, D. P.,
M. E. DeGraaf,
D. R. Marotti,
E. Rehberg,
and
L. E. Post.
Production of analytical quantities of recombinant proteins in chinese hamster ovary cells using sodium butyrate to elevate gene expression.
J. Biotechnol.
19:
35-48,
1991[Medline].
23.
Pang, K. S.,
P. J. Wang,
A. Chung,
and
A. W. Wolkoff.
The modified dipeptide, enalapril, an angiotensin-converting enzyme inhibitor, is transported by the rat liver organic anion transport protein.
Hepatology
28:
1341-1346,
1998[Medline].
24.
Reichel, C.,
B. Gao,
V. Cattori,
L. Landmann,
Y. Sugiyama,
B. Stieger,
B. Hagenbuch,
and
P. J. Meier.
Heterogeneous expression of the polyspecific organic anion transporter oatp2 in rat liver and its identification as a cyclic peptide transporter (Abstract).
Hepatology
28:
425A,
1998.
25.
Reuter, S.,
and
D. Mayer.
Transport of dehydroepiandrosterone and dehydroepiandrosterone sulphate into rat hepatocytes.
J. Steroid Biochem. Mol. Biol.
54:
227-235,
1995[Medline].
26.
Roman, L. M.,
and
A. L. Hubbard.
A domain specific marker for the hepatocyte plasma membrane: localization of leucine aminopeptidase to the bile canalicular domain.
J. Cell Biol.
96:
1548-1558,
1983[Abstract].
27.
Röschenthaler, R.,
E. E. Creppy,
and
G. Dirheimer.
Ochratoxin A: on the mode of action of a ubiquitous mycotoxin.
J. Toxicol. Sci.
3:
53-86,
1984.
28.
Roth, A.,
K. Chakor,
E. E. Creppy,
A. Kane,
R. Roschenthaler,
and
G. Dirheimer.
Evidence for an enterohepatic circulation of ochratoxin A in mice.
Toxicology
48:
293-308,
1988[Medline].
29.
Saito, H.,
S. Masuda,
and
K. Inui.
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[Abstract/Free Full Text].
30.
Satlin, L. M.,
V. Amin,
and
A. W. Wolkoff.
Organic anion transporting polypeptide mediates organic anion/HCO
3 exchange.
J. Biol. Chem.
272:
26340-26345,
1997[Abstract/Free Full Text].
31.
Schroeder, A.,
U. Eckhardt,
B. Stieger,
R. Tynes,
C. D. Schteingart,
A. F. Hofmann,
P. J. Meier,
and
B. Hagenbuch.
Substrate specificity of the rat liver Na+-bile salt cotransporter in Xenopus laevis oocytes and in CHO cells.
Am. J. Physiol.
274 (Gastrointest. Liver Physiol. 37):
G370-G375,
1998[Abstract/Free Full Text].
32.
Shi, X. Y.,
S. Bai,
A. C. Ford,
R. D. Burk,
E. Jacquemin,
B. Hagenbuch,
P. J. Meier,
and
A. W. Wolkoff.
Stable inducible expression of a functional rat liver organic anion transport protein in HeLa cells.
J. Biol. Chem.
270:
25591-25595,
1995[Abstract/Free Full Text].
33.
Smith, P. K.,
R. I. Krohn,
G. T. Hermanson,
A. K. Mallia,
F. H. Gartner,
M. D. Provenzano,
E. K. Fujimoto,
N. M. Goeke,
B. J. Olson,
and
D. C. Klenk.
Measurement of protein using bicinchoninic acid.
Anal. Biochem.
150:
76-85,
1985[Medline].
34.
Stieger, B.,
B. Hagenbuch,
L. Landmann,
M. Höchli,
A. Schroeder,
and
P. J. Meier.
In situ localization of the hepatocytic Na+/taurocholate cotransporting polypeptide (Ntcp) in rat liver.
Gastroenterology
107:
1781-1787,
1994[Medline].
35.
Stieger, B.,
P. J. Meier,
and
L. Landmann.
Effect of obstructive cholestasis on membrane traffic and domain-specific expression of plasma membrane proteins in rat liver parenchymal cells.
Hepatology
20:
201-212,
1994[Medline].
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