Substrate specificity of the rat liver
Na+-bile salt cotransporter in
Xenopus
laevis oocytes and in CHO
cells
Alice
Schroeder1,
Uta
Eckhardt1,
Bruno
Stieger1,
Ronald
Tynes2,
Claudio D.
Schteingart3,4,
Alan F.
Hofmann3,
Peter J.
Meier1, and
Bruno
Hagenbuch1
1 Division of Clinical
Pharmacology and Toxicology, Department of Medicine, University
Hospital, CH-8091 Zurich; 2 Drug
Metabolism and Pharmakokinetics, Novartis Pharma, CH-4002 Basel,
Switzerland; 3 Division of
Gastroenterology, Department of Medicine, University of California
San Diego, La Jolla 92093-0813; and
4 Ferring Research, La Jolla,
California 92037
 |
ABSTRACT |
It has been proposed that the hepatocellular
Na+-dependent bile salt uptake
system exhibits a broad substrate specificity in intact hepatocytes. In
contrast, recent expression studies in mammalian cell lines have
suggested that the cloned rat liver Na+-taurocholate cotransporting
polypeptide (Ntcp) may transport only taurocholate. To characterize its
substrate specificity Ntcp was stably transfected into Chinese hamster
ovary (CHO) cells. These cells exhibited saturable
Na+-dependent uptake of
[3H]taurocholate
[Michaelis constant
(Km) of ~34
µM] that was strongly inhibited by all major bile salts,
estrone 3-sulfate, bumetanide, and cyclosporin A. Ntcp cRNA-injected
Xenopus
laevis oocytes and the transfected CHO
cells exhibited saturable
Na+-dependent uptake of
[3H]taurochenodeoxycholate
(Km of ~5
µM),
[3H]tauroursodeoxycholate
(Km of ~14
µM), and
[14C]glycocholate
(Km of ~27
µM). After induction of gene expression by sodium butyrate,
Na+-dependent transport of
[3H]estrone 3-sulfate
(Km of ~27
µM) could also be detected in the transfected CHO cells. However,
there was no detectable
Na+-dependent uptake of
[3H]bumetanide or
[3H]cyclosporin A. These results show that the cloned Ntcp can mediate Na+-dependent uptake of all
physiological bile salts as well as of the steroid conjugate estrone
3-sulfate. Hence, Ntcp is a multispecific transporter with preference
for bile salts and other anionic steroidal compounds.
sodium-dependent bile salt transport; organic anion; multispecificity
 |
INTRODUCTION |
UPTAKE OF ORGANIC ANIONS such as bile salts from blood
into hepatocytes is mediated by
Na+-dependent as well as
Na+-independent transport systems
(7). Previous kinetic uptake and inhibition studies in several
experimental systems have indicated that the hepatocellular
Na+-dependent bile salt uptake
system exhibits a broad substrate specificity for a wide variety of
amphipathic organic molecules, including conjugated bile salts,
electroneutral steroids, cyclic oligopeptides, and a number of drugs,
such as bumetanide and cyclosporin A (3, 14, 22). In contrast, recent
expression studies in the liver-derived immortalized cell line HPCT-1E3
have suggested that the cloned rat liver
Na+-taurocholate cotransporting
polypeptide (Ntcp) may exclusively transport taurocholate and that
other transport systems, such as epoxide hydrolase, account for
Na+-dependent uptake of cholate
and glycocholate into rat hepatocytes (15, 21). Because Ntcp is thought
to represent a major bile salt transport system in mammalian liver (8)
and because only taurocholate has been used as a transport substrate in
previous Ntcp expression studies (15, 19), we have reexamined the
substrate specificity of Ntcp in more detail in cRNA-injected
Xenopus
laevis oocytes and in transfected
Chinese hamster ovary (CHO) cells. The results clearly demonstrate that
Ntcp transports typical natural di- and trihydroxy-conjugated bile
salts, as well as, albeit to a lesser degree, the steroid conjugate
estrone 3-sulfate.
 |
MATERIALS AND METHODS |
Materials.
[3H]taurocholic acid
(2.6 Ci/mmol),
[3H]cholic acid (13.2 Ci/mmol),
[14C]glycocholic acid
(44.6 mCi/mmol), and
[3H]estrone 3-sulfate
(49.0 Ci/mmol) were obtained from DuPont-New England Nuclear (Boston,
MA). [3H]cyclosporin A
(11.1 Ci/mmol) was purchased from Amersham International (Little
Chalfont, Buckinghamshire, UK).
[3H]taurochenodeoxycholic
acid (0.5 Ci/mmol) and
[3H]tauroursodeoxycholic
acid (0.5 Ci/mmol) were conjugated as described previously (11).
[3H]bumetanide was
kindly provided by E. Petzinger of Justus Liebig-Universität (Giessen, Germany). 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.
Expression of Ntcp in Xenopus
laevis oocytes.
Xenopus
laevis oocytes were prepared as
described previously (8). After an overnight incubation at 18°C,
healthy oocytes were injected with 2.5 ng of Ntcp cRNA or water. After
3 days in culture, uptake of the indicated substrates was measured at 25°C in a medium containing 100 mM NaCl or choline chloride,
2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and
10 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid/tris(hydroxymethyl)aminomethane, pH 7.5, as described previously (6).
Stable transfection of CHO cells with Ntcp.
Wild-type CHO cells (CHO-K1) were stably transfected with the coding
region of the rat Ntcp (9) subcloned into vector pCMV vector-1 as
previously described (18). The vector pCMV vector-1 contains the strong
cytomegalovirus promoter-enhancer unit excised from pcDNA1/NEO
(Invitrogen) and subcloned for user convenience into pBluescript
(Stratagene, La Jolla, CA), which also contained a
Xenopus-rabbit 5'-untranslated leader, the
rabbit
-globin poly(A) signal, and a gastrin gene transcription stop
signal. From the resulting transfected cell pool, single
clones were isolated using cloning cylinders and tested for
Na+-dependent transport of
taurocholate. The best transporting clone (CHO 9-6) was selected and
used for all further experiments.
Cell culture.
CHO cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, 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 media contained an additional 400 µg/ml G418-sulfate (Geneticin).
Uptake studies in CHO cells.
For the determination of
Na+-dependent uptake of potential
substrates for Ntcp, the cells were grown to confluency on 35-mm dishes
and rinsed three times with prewarmed (37°C) sodium or choline
containing Earle's balanced saline solution (16) supplemented with 5.5 mM D-glucose. The cells were
then incubated at 37°C in the presence of radiolabeled substrate.
After the indicated time interval, the transport was stopped with 3 ml
of ice-cold Na+-containing Earle's solution. After two
additional washing steps with the same stop solution, the cells were
solubilized in 1 ml of 1% Triton X-100 and mixed with 5 ml of
scintillation fluid (Ultima Gold; Canberra Packard International,
Zurich, Switzerland). Radioactivity was determined in a Packard
Tri-Carb 2200 CA liquid scintillation counter (Canberra Packard). For
some experiments, expression of Ntcp was induced by incubating the
cells for 24 h with culture medium supplemented with 5 mM butyrate as
described previously (13).
 |
RESULTS |
To evaluate the proposed broad substrate specificity of Ntcp in a
stably transfected cell line, we constructed an expression vector by
inserting the coding region of Ntcp into pCMV vector-1 (18) and
electroporated the resulting construct into CHO cells. Several
G418-resistant cell clones were isolated using cloning cylinders and
tested for Na+-dependent
taurocholate uptake. Clone CHO 9-6 exhibited the highest transport
activity.
Time-dependent uptakes of taurocholate in wild-type and Ntcp-expressing
CHO cells are compared in Fig. 1. Wild-type
cells (Fig. 1A) showed no
significant Na+-dependent
transport of taurocholate, reflecting the absence of Ntcp in these
cells. In contrast, Ntcp-expressing CHO 9-6 cells (Fig.
1B) showed a strong intracellular
accumulation of taurocholate in the presence, but not in the absence,
of Na+. As shown in Fig.
2, the
Na+-dependent taurocholate uptake
portion was strongly inhibited by cholate, taurodeoxycholate,
taurochenodeoxycholate (TCDC), tauroursodeoxycholate (TUDC), the
sulfate-conjugated steroids estrone 3-sulfate and
17
-estradiol 3-sulfate, and the drugs bumetanide and cyclosporin
A. Inhibition by the unconjugated steroids such as
testosterone and progesterone was less pronounced.

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Fig. 1.
Time course of Na+-dependent
taurocholate uptake into Chinese hamster ovary (CHO) cells. Wild-type
(CHO-K1; A) or
Na+-taurocholate cotransporting
polypeptide (Ntcp)-expressing (CHO 9-6;
B) CHO cells were
incubated with 2.5 µM
[3H]taurocholate at
37°C for the indicated time periods in the presence ( ) and
absence ( ) of an inwardly directed NaCl gradient. Data points
represent means ± SD of triplicate determinations.
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Fig. 2.
Cis-inhibition of
Na+-dependent taurocholate
transport in Ntcp-expressing CHO 9-6 cells. CHO cells were incubated
for 1 min at 37°C with 2.5 µM
[3H]taurocholate and
indicated concentrations of inhibitors in the presence and absence of
an inwardly directed NaCl gradient. Net
Na+-dependent uptake (uptake in
the presence minus the absence of NaCl) in the absence of inhibitors is
expressed as 100% (control). Results represent means ± SE and are
given as %control. Differences were evaluated using Student's
t-test.
* P < 0.01, ** P < 0.001 vs. control. TDC,
taurodeoxycholate; TCDC, taurochenodeoxycholate; TUDC,
tauroursodeoxycholate.
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To test whether the cis-inhibiting
substrates are also transported by Ntcp, we performed uptake studies in
the Xenopus laevis oocyte expression
system. As summarized in Table 1,
Na+-dependent transport of all
tested bile salts was stimulated between 10- and 100-fold in
cRNA-injected oocytes. In contrast,
Na+-dependent uptake of
radiolabeled estrone 3-sulfate was stimulated only twofold, and no
Na+-dependent uptake was observed
for bumetanide and cyclosporin A. Similar results were also obtained in
stably transfected CHO 9-6 cells. As depicted in Fig.
3, both TCDC and TUDC accumulated in an
Na+-dependent manner in
Ntcp-expressing CHO 9-6 cells, whereas no significant
Na+-dependent uptake was seen in
wild-type CHO-K1 cells. Because Na+-dependent uptake of
glycocholate was only minimally stimulated in uninduced CHO 9-6 cells
(Fig. 4), we induced gene expression by 24 h
preincubation with 5 mM sodium butyrate (13). As can be seen in Fig. 4,
butyrate induction resulted in an ~10-fold increase in the expressed
Na+-dependent glycocholate uptake
rate, whereas no effect of butyrate was seen on
Na+-independent uptake. In
contrast, wild-type CHO-K1 cells did not show any
Na+-dependent accumulation of
glycocholate even after butyrate induction (Fig. 4).

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Fig. 3.
Time course of Na+-dependent
taurochenodeoxycholate (A,
B) and tauroursodeoxycholate
(C,
D) uptake into CHO cells. Wild-type
(CHO-K1) (A,
C) or Ntcp-expressing (CHO 9-6) CHO
cells (B,
D) were incubated with 2.5 µM
[3H]taurochenodeoxycholate
(A,
B) or 2.5 µM
[3H]tauroursodeoxycholate
(C,
D) at 37°C for the indicated
time periods in the presence ( ) and absence ( ) of an inwardly
directed NaCl gradient. Data points represent means ± SD of
triplicate determinations.
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Finally, we measured uptake of the
cis-inhibiting substrate estrone
3-sulfate and determined the kinetics of substrate uptake in
butyrate-induced Ntcp-expressing CHO 9-6 cells. As in oocytes (Table 1)
there was again a clear
Na+-dependent estrone 3-sulfate
transport in the CHO 9-6 cells (Fig. 5), whereas wild-type CHO-K1
cells showed no Na+-dependent
transport signal. Furthermore,
Na+-dependent uptake of estrone
3-sulfate and various bile salts exhibited clear saturability with the
highest affinity for TCDC, followed by TUDC > glycocholate = estrone
3-sulfate = taurocholate (Table 2). In
contrast, several attempts failed to demonstrate Na+-dependent uptake of bumetanide
and cyclosporin A in butyrate-induced Ntcp-expressing CHO 9-6 cells. No
difference in bumetanide (1 µM) uptake was found in the presence
(1.42 ± 0.17 pmol · min
1 · mg
protein
1) and absence
(1.30 ± 0.18 pmol · min
1 · mg
protein
1) of
Na+ in transfected CHO cells. For
cyclosporin A (1 µM) similar uptakes were found in transfected (175 ± 23 and 126 ± 9 pmol · min
1 · mg
protein
1 for NaCl and
choline chloride, respectively) and in wild-type (172 ± 21 and 128 ± 9 pmol · min
1 · mg
protein
1 for NaCl and
choline chloride, respectively) CHO cells. These results are similar to
the data obtained in injected Xenopus
laevis oocytes (Table 1). They prove the universal
nature of Ntcp as a bile salt carrier and document its relative broad
substrate specificity. However, the data also indicate that not all
cis-inhibiting substrates are also
transport substrates of Ntcp, thus emphasizing the need for direct
transport studies rather than kinetic inhibition studies for the
correct determination of the substrate specificity of a single carrier
protein.

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Fig. 4.
Na+-dependent uptake of
glycocholate into CHO cells with and without butyrate induction. Before
uptake measurements wild-type (CHO-K1) or Ntcp-expressing (CHO 9-6) CHO
cells were incubated for 24 h without or with 5 mM sodium butyrate
(butyrate). For uptake measurements cells were incubated with 2.2 µM
[14C]glycocholate at
37°C for 5 and 30 min in the presence (solid bars) and absence
(open bars) of an inwardly directed NaCl gradient. Values represent
means ± SD of triplicate determinations.
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Fig. 5.
Time course of Na+-dependent
estrone 3-sulfate uptake into CHO cells. Wild-type (CHO-K1;
A) or Ntcp-expressing (CHO 9-6;
B) CHO cells were incubated for 24 h
in presence of 5 mM sodium butyrate. For uptake measurements cells were
incubated at 37°C for the indicated time periods in the presence
( ) and absence ( ) of an inwardly directed NaCl gradient with 10 µM [3H]estrone
3-sulfate. Data points represent means ± SD of triplicate
determinations.
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 |
DISCUSSION |
Previous kinetic inhibition studies with hepatocytes and isolated rat
liver basolateral membrane vesicles have provided evidence for a rather
broad substrate specificity of the hepatocellular Na+-dependent bile salt uptake
system (3, 14, 22). However, these transport studies in intact cells
and isolated membrane vesicles could not distinguish between the
presence of a single multispecific or several monospecific transport
systems. To discriminate between these two possibilities, the involved
transporting polypeptides must be isolated and transfected into a cell
line that does not constitutively express the respective transport
function. Although functional expression of Ntcp in
Xenopus laevis oocytes (9) as well as
in various mammalian cell lines (2, 15, 18, 19) has clearly established
its function as an Na+-dependent
taurocholate uptake system, recent experiments in stably transfected
HPCT-1E3 cells have suggested that Ntcp might not transport other bile
salts such as glycocholate and cholate (15). In fact, additional
Na+-dependent bile salt
transporting polypeptides, such as microsomal epoxide hydrolase (21),
have been postulated to contribute to the obvious multispecificity of
the Na+-dependent bile salt uptake
function of rat hepatocytes (20).
Contrary to these suggestions, the present study demonstrates that
besides taurocholate, Ntcp also mediates transport of cholate, glycocholate, TCDC, and TUDC (Tables 1 and 2; Figs. 3 and 4). These
results indicate that Ntcp can transport all
cis-inhibiting bile salt derivatives
(Fig. 2), although the extent of Ntcp-mediated transport is different
for various bile salts. Thus, based on the ratio of the maximum
velocity (Vmax)
to the Michaelis constant, the best transport substrate of Ntcp was
TCDC followed by TUDC, taurocholate, estrone 3-sulfate, and
glycocholate (Table 2). These data are consistent with the previously
observed higher affinity of
Na+-dependent dihydroxy-conjugated
bile salt uptake compared with trihydroxy-conjugated bile salt uptake
in isolated hepatocytes (1, 20). Furthermore, the kinetic data
presented in Table 2 demonstrate that maximal
Na+-dependent bile salt uptake in
butyrate-induced Ntcp-expressing CHO 9-6 cells was as high as in
short-term cultured hepatocytes (19), indicating that the adopted CHO
cell system represents a suitable model for the correct delineation of
the substrate specificity of Ntcp.
In addition to various physiological bile salts, the
cis-inhibiting compound estrone
3-sulfate (Fig. 2) was also transported by Ntcp with an affinity
similar to that of bile salts (Table 2) in butyrate-induced CHO 9-6 cells (Fig. 5), indicating that the substrate specificity of Ntcp
indeed extends beyond bile salts as previously suggested in functional
transport studies in isolated basolateral membrane vesicles (22).
However, in comparison with bile salts, the Ntcp-mediated
Na+-dependent uptake portion of
estrone 3-sulfate is small (Table 1). In transfected CHO 9-6 cells it
could only be detected after butyrate-induced gene expression,
indicating that the level of Ntcp expression is a critical factor for
correct delineation of the true substrate specificity of Ntcp. In this
regard, it is important to realize that even after butyrate induction,
the transfected CHO 9-6 cells still expressed an approximately
threefold lower Vmax value for
Na+-dependent taurocholate uptake
compared with freshly isolated hepatocytes (1). Hence, it cannot be
definitely excluded that at higher expression levels Ntcp might also
transport the cis-inhibiting substrates bumetanide and cyclosporin A (Fig. 2). However, this possibility appears unlikely, since bumetanide transport is encoded by
a different rat liver mRNA species (10) and cyclosporin A exhibited
noncompetitive inhibition of
Na+-dependent taurocholate uptake
in isolated rat liver basolateral plasma membrane vesicles (22). The
present study nevertheless demonstrates that the achieved expression
level in butyrate-induced CHO 9-6 cells was sufficient to extend the
substrate specificity of Ntcp to various physiological bile salts and
to the estrogen conjugate estrone 3-sulfate. Because Ntcp expression is
decreased in various forms of cholestatic liver disease (4, 5, 17), its
downregulation may be associated with a continuous narrowing of the
spectrum of transported substrates also in vivo. Our results indicate
that even at low expression levels Ntcp would still maintain its
transport preference for taurine-conjugated bile salts, whereas its
less well-transported substrates could still be transported across the
sinusoidal membrane of cholestatic hepatocytes by
Na+-independent systems, including
members of the organic anion transporting polypeptide gene
(oatp) family of transporters (12).
 |
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). Work at the University of California San Diego was
supported in part by National Institute of Diabetes and Digestive and
Kidney Diseases Grant DK-21506 as well as by a Grant-in-Aid from the Falk Foundation (Freiburg, Germany). B. Hagenbuch is a recipient of a
Cloëtta Foundation Fellowship.
 |
FOOTNOTES |
A preliminary report of this study was presented at the annual meeting
of the American Gastroenterological Association in New Orleans, in May
1994, and was published previously in abstract form
(Gastroenterology 106: A979, 1994).
Address for reprint requests: B. Hagenbuch, Division of Clinical
Pharmacology and Toxicology, Dept. of Medicine, Univ. Hospital, CH-8091
Zurich, Switzerland.
Received 16 June 1997; accepted in final form 30 October 1997.
 |
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