Confocal Imaging with a Fluorescent Bile Acid Analogue Closely
Mimicking Hepatic Taurocholate Disposition
Tom De Bruyn,1 Wouter Sempels,2 Jan Snoeys,3 Nico Holmstock,1 Sagnik Chatterjee,1 Bruno Stieger,4 Patrick Augustijns,1 Johan
Hofkens,2 Hideaki Mizuno,5 Pieter Annaert1
1 Drug
Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, O&N2, Leuven 3000, Belgium
Division of Molecular Imaging and Photonics, KU Leuven Department of Chemistry, Leuven, Belgium
3
Drug Metabolism and Pharmacokinetics, Janssen Research and Development, Beerse, Belgium
4
Department of Clinical Pharmacology and Toxicology, Zürich, Switzerland
5 Biochemistry, Molecular and Structural Biology Section, Department of Chemistry, KU Leuven, Belgium
2
ABSTRACT: This study aimed to characterize the in vitro hepatic transport mechanisms in primary rat and human hepatocytes of the
fluorescent bile acid derivative N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3a,7a,12a-trihydroxy-27-nor-5�cholestan-26-oyl)-2t -aminoethanesulfonate (tauro-nor-THCA-24-DBD), previously synthesized to study the activity of the bile salt export pump
(BSEP). The fluorescent bile acid derivative exhibited saturable uptake kinetics in suspended rat hepatocytes. Hepatic uptake was inhibited
in the presence of substrates/inhibitors of the organic anion transporting polypeptide (Oatp) family and Na+ -taurocholate cotransporting
peptide (Ntcp). Concentration-dependent uptake of the fluorescent bile acid was also saturable in Chinese hamster ovary cells transfected with
rNtcp, hNTCP, OATP1B1, or OATP1B3. The fluorescent bile acid derivative was actively excreted in the bile canaliculi of sandwich-cultured rat and
human hepatocytes (SCRH and SCHH), with a biliary excretion index (BEI) of 26% and 32%, respectively. In SCRH, cyclosporin A significantly
decreased the BEI to 5%. Quantification by real-time confocal imaging further confirmed canalicular transport of the fluorescent bile acid
derivative (BEI = 75%). We conclude that tauro-nor-THCA-24-DBD is a useful probe to study interference of drugs with NTCP/Ntcp- and
BSEP/Bsep-mediated transport in fluorescence-based in vitro assays.
INTRODUCTIO
N
The sodium taurocholate cotransporting polypeptide [NTCP
(human), Ntcp (rat); SLC10A1, Slc10a1] and the bile salt export pump [BSEP (human), Bsep (rat); ABCB11, Abcb11] are
key transporters in the hepatobiliary disposition of bile acids.1
NTCP/Ntcp is expressed in the basolateral membrane of hepatocytes, transporting bile acids from the blood into hepatocytes,
whereas BSEP/Bsep is present at the canalicular membrane
of hepatocytes and mediates the efflux of bile acids from the
hepatocyte into the bile. Thus, NTCP/Ntcp and BSEP/Bsep act
together to facilitate polarized transport of bile acids from sinusoidal blood plasma to the bile and have therefore been shown to
be critical in maintaining bile acid homeostasis. Consequently,
reduced BSEP/Bsep or NTCP/Ntcp activity, resulting from inhibition or decreased expression levels, may cause cholestasis
or hypercholanemia, respectively.2,3
In addition to their role in bile acid homeostasis, both transporters mediate the hepatic disposition of certain xenobiotics;
recent studies revealed the role of NTCP/Ntcp in the
hep- atic
uptake of micafungin, rosuvastatin,
and
pitavastatin.4–6
BSEP/Bsep might participate in the biliary excretion of vinblastine and pravastatin.7,8
The importance of NTCP has further been demonstrated by
the effect of genetic polymorphisms on transporter activity. For
example, Ho et al.9 revealed that NTCP exhibits a domain critical for bile acid recognition, and they linked genetic modifications in this region with a significant decrease (or complete
loss) of bile acid transport. Consistently, polymorphic changes
in NTCP were also associated with decreased transport of rosuvastatin, suggesting that loss of NTCP activity may potentially
also affect drug disposition.10
These examples illustrate the need to develop preclinical tools to study NTCP/Ntcp-mediated drug–drug interaction potential at an early stage of drug development. Existing in vitro assays to study NTCP/Ntcp inhibition potential
often use radiolabeled bile acids as probe substrates (e.g., 3 Htaurocholic acid).11–13 In addition, several publications have reported on the development of fluorescent NTCP/Ntcp probes
by linking bile acids to a fluorescent dye (e.g., fluorescein).
Such fluorescent bile acid derivatives offer the advantage
that they can be visualized by microscopic/confocal imaging.
This was illustrated by Boyer et al.14 who measured the
uptake of the two fluorescent bile acid derivatives cholylglycyl-fluorescein and chenodeoxycholyl-lysyl-NBD in Ntcptransfected COS-7 cells using microscopic imaging. Consistently, OATP1B-mediated transport of the fluorescent bile acid
derivative chenodeoxycholyl-(Ng-NBD)–lysine was visualized
by confocal imaging.15
N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3",7",12"-trihydroxy-27-nor-5$-cholestan-26-oyl)2t -aminoethanesulfonate
(tauro-nor-THCA-24-DBD) is a
synthetic, fluorescent bile acid derivative that has previously
been used as a screening probe to evaluate the inhibitory
potential of various drugs on hBSEP-mediated transport.16
To use tauro-nor-THCA-24-DBD as a probe substrate in more
complex model systems like primary hepatocytes, expressing
multiple transporter families, detailed information on transporter selectivity is critical. The aim of this study was to clarify
the relative contribution of organic anion transporting polypeptides (OATPs/Oatps) and NTCP/Ntcp in the hepatic uptake
of tauro-nor-THCA-24-DBD in comparison to taurocholate.
Additionally, biliary excretion of tauro-nor-THCA-24-DBD
and taurocholate was compared and quantified in sandwichcultured hepatocytes. Finally, we visualized biliary excretion
of tauro-nor-THCA-24-DBD by confocal imaging. Our results
demonstrate that tauro-nor-THCA-24-DBD can be used as an
alternative to taurocholate as a probe substrate for exploring
bile acid disposition specifically mediated by NTCP/Ntcp and
BSEP/Bsep.
A (CsA), L-proline, ethylene glycol-bis(2-aminoethylether)N,N,Nt ,Nt -tetraacetic acid (EGTA), and sodium butyrate were
purchased from Sigma–Aldrich (Schnelldorf, Germany). Mineral oil was purchased from Acros organics (Geel, Belgium).
Human cryopreserved hepatocytes for sandwich culture were
provided by Kaly-Cell (Plobsheim, France). Standard buffer
consisted of HBSS containing 10 mM HEPES and adjusted
to pH 7.4. Ca2+ /Mg2+ -free buffer consisted of Ca2+ /Mg2+ -free
HBSS containing 10 mM HEPES, 1 mM EGTA, and pH
adjusted to 7.4.
MATERIALS
METHODS
Uptake Experiments with Suspended Hepatocytes
AND
Materials
N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3",7",12"-trihydroxy-27-nor-5$-cholestan-26-oyl)-2t aminoethanesulfonate was obtained from Genomembrane
Company, Ltd. (Yokohama, Japan). Ritonavir was donated
by Hetero Drugs Limited (Hyderabad, India). Dulbecco’s
modified Eagle medium (DMEM) and geneticin G418 were
purchased from Invitrogen (Paisley, UK). [3 H]Taurocholic
acid (specific activity, 4.6 Ci/mmol) and scintillation cocktail
were obtained from PerkinElmer Life Sciences Inc. (Boston,
Massachusetts). Williams’ Medium E (WEM), L-glutamine,
penicillin–streptomycin mixture (contains 10,000 IU potassium penicillin and 10,000 : g streptomycin sulfate per
milliliter in 0.85% saline), fetal bovine serum (FBS), trypsin
ethylenediaminetetraacetic acid, Hanks’ balanced salt solution (HBSS), and phosphate-buffered saline (PBS) were
purchased from Lonza SPRL (Verviers, Belgium). HEPES
[4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid] was purchased from MP Biochemical (Illkirch, France). Triton X-100,
collagenase (type IV), silicon oil, rifampicin, taurocholic acid,
5(6)-carboxy-2t ,7t -dichlorofluorescein (CDF),
5(6)-carboxy2t ,7t -dichlorofluorescein diacetate (CDFDA), ECM gel (from
Engelbreth–Holm–Swarm murine sarcoma), esteron-3-sulfate,
bromosulfophthalein, indomethacin, prazosin, cyclosporin
Methods
Animals
All rats were housed according to the Belgian and European
laws, guidelines, and policies for animal experiments, housing,
and care in the Central Animal Facilities of the university. The
Institutional Ethical Committee for Animal Experimentation
granted the approval for this project.
Isolation of Rat Hepatocytes
Hepatocytes were isolated from male Wistar (170–200 g)
rats using a two-step collagenase perfusion, as described
previously.17 After isolation, cells were centrifuged (50g) for 3
min at 4◦ C, and the pellet was resuspended in WEM containing
5% FBS, 2 mM L-glutamine, 100 IU/mL penicillin, 100 : g/mL
streptomycin, 4 : g/mL insulin, and 1 : M dexamethasone. Before seeding, cell viability (>90%) was determined by TrypanO
blue exclusion, and hepatocytes were diluted to a final concentration of 1 × 106 cells/mL. For uptake experiments in suspended hepatocytes, freshly isolated rat hepatocytes were resuspended in Krebs–Henseleit buffer (NaCl 130 mM, KCl 5.17
mM, CaCl2 1.2 mM, MgCl2 1.2 mM, HEPES 12.5 mM, glucose
11.1 mM, Na-pyruvate 5 mM; pH 7.4). For sodium depletion
experiments, NaCl and Na pyruvate were replaced by 130 mM
choline Cl and 5 mM pyruvic acid, respectively.
R
All experiments with suspended hepatocytes were conducted as
described previously.18 Briefly, 500 : L of a double-concentrated
cell suspension was preincubated with 250 : L of uptake buffer
(or 250 : L of a fourfold concentrated inhibitor solution) for
5 min at 37◦ C. Subsequently, 250 : L of a prewarmed fourfold
concentrated substrate solution was added to initiate the incubation. To determine the nonsaturable uptake component,
experiments were also conducted at 4◦ C. After an incubation
period of 30 s (tauro-nor-THCA-24-DBD) or 60 s (taurocholate),
triplicate 200 : L aliquots of the suspension were transferred
to 1.5 mL ice-cold Eppendorf tubes, containing 700 : L of an
oil layer (silicone/mineral oil mixture; density, 1.015) above
300 : L of 2 M NaCl or NaOH (for taurocholate) solution. Subsequently, the tubes were immediately centrifuged for 2 min at
16,000g using a tabletop centrifuge (Eppendorf 5415 C, Hamburg, Germany). After freezing Eppendorf tubes in dry ice, the
centrifuge tube bottoms were cut and the contents solubilized
in 400 : L 0.5% Triton X (in PBS). Tauro-nor-THCA-24-DBD
concentration was measured by fluorescence spectroscopy (excitation 450 nm, emission 570 nm) in a Tecan Infinite 200 plate
reader (Tecan Benelux, Mechelen, Belgium). For taurocholate,
centrifuge bottoms were placed in a scintillation vial containing
2 mL of scintillation cocktail, and radioactivity was quantified
using liquid scintillation spectrometry (Wallac 1410, Finland).
Uptake rates were normalized for the cell density during the
incubation and expressed as pmol/(million cells × min).
Culture of OATP-Transfected Chinese Hamster Ovary
Cells
Wild-type, hNTCP-, rNtcp-, OATP1B1-, and 1B3-transfected
Chinese hamster ovary (CHO) cells were cultured at passage
45–65, as described previously.19 Briefly, CHO cells were grown
in 75 cm2 T-flasks in DMEM containing 1 g/L D-glucose, 1 mM
L-glutamine, 25 mM HEPES, and 110 mg/L sodium pyruvate,
supplemented with 10% FBS, 50 : g/mL L-proline, 100 IU/mL
penicillin, 100 : g/mL streptomycin. The culture media of the
transfected cell lines additionally contained 500 : g/mL geneticin. Cells were incubated at 5% CO2 and 37◦ C. For uptake
experiments, wild-type CHO cells were seeded in 24-well cell
culture plates (Greiner-Bio-One, Wemmel, Belgium) at a density of 20,000 cells/well, whereas CHO-transfected cells were
seeded at a density of 25,000 cells/well. Culture medium was
replaced every other day, and uptake experiments were performed on day 5 after seeding when cells were confluent. One
day before the experiment, all cells were additionally treated
with 5 mM sodium butyrate to induce gene expression.
Uptake Experiments with Transfected CHO Cells
Cells were washed twice with 0.5 mL/well prewarmed uptake
buffer (HBSS with 10 mM HEPES, pH 7.4) and preincubated for
10 min at 37◦ C. In experiments wherein inhibitors were investigated, cells were preincubated (for 10 min) with 0.5 mL/well
uptake buffer containing the inhibitor at the desired concentration. After the preincubation, uptake buffer was aspirated and
0.5 mL/well of uptake buffer containing the desired substrate
(and inhibitor) concentration was added. After incubation for
90 s, medium was quickly removed, and cells were rinsed three
times with ice-cold uptake buffer. Subsequently, cells were lysed
with 0.3 mL of 0.5% Triton X solution (in PBS) and placed in a
plate shaker for 30 min at room temperature. Cell lysates were
analyzed for tauro-nor-THCA-24-DBD or taurocholate content
by fluorescence spectroscopy and liquid scintillation spectrometry, respectively (as described above). Uptake rates were normalized for protein content, which was measured using a BCA
Protein assay kit (Pierce Chemical, Rockford, Illinois).
Culture of Sandwich-Cultured Hepatocytes
Freshly isolated rat hepatocytes were seeded in sterile plastic
culture dishes (MatTek, Ashland, Massachusetts) or six-well
culture plates (Greiner-Bio-One). Culture plates and dishes
were precoated 12 h before cell seeding with 100 : L/well gelled
collagen and 50 : g/mL rigid collagen diluted in 0.02 N acetic
acid, respectively. Gelled collagen was prepared by neutralizing 4 mL of rat tail type I collagen, 4 mL of sterile water, and
1 mL of 10× DMEM with 1 mL of 0.2 N NaOH. Several hours
before seeding, each dish or well was rinsed three times with
warm PBS to hydrate the collagen. Freshly isolated rat hepatocytes were seeded at 2 × 106 cells/well. Unattached cells were
removed 2 h after plating by shaking and aspirating the cell
medium. Subsequently, cultures were overlaid with gelled collagen solution to obtain a sandwich configuration. Cell medium
was replaced 24 h after plating by WEM containing 1% (v/v)
TM
ITS+ Premix, 100 IU/mL penicillin, 100 : g/mL streptomycin,
and 0.1 : M dexamethasone (day-1 medium). Medium was replaced every day with fresh day-1 medium.
Plateable cryopreserved human hepatocytes (lot number
S2406A) were provided by Kaly-Cell. Human hepatocytes were
seeded in 48-well plates at a density of 0.25 × 106 cell/well.
Thawing and seeding were carried out following the protocols
provided by Kaly-Cell. Briefly, culture plates were coated 1 day
before seeding with 50 : g/mL rigid collagen diluted in 0.02 N
acetic acid, and were placed overnight at 37◦ C in humidified atmosphere 5% CO2 . Just before seeding, the plates were washed
twice with warm PBS and once with thawing medium. Thawing medium consisted of DMEM, 10% (v/v) FBS, 100 IU/mL
penicillin, 100 : g/mL streptomycin, 4 : g/mL insulin, and 1 : M
dexamethasone. After quickly thawing the cells at 37◦ C, they
were suspended in a mixture of PercollO and thawing medium
and centrifuged (168g) for 20 min at room temperature. Subsequently, the pellet was resuspended in thawing medium, and
cells were centrifuged again for 5 min (100g) at room temperature. The pellet was then resuspended in seeding medium,
which consisted of WEM supplemented with components of
thawing medium. Hepatocytes were counted using a hemocytometer, and cell viability was determined using TrypanO
blue. The minimum viability obtained for all the batches was
90%. 24 h after seeding, the hepatocytes were overlaid with
ECM gel solution (0.25 mg/mL) in ice-cold WEM containing 1%
TM
(v/v) ITS+ Premix, 100 IU/mL penicillin, 100 : g/mL streptomycin, and 0.1 : M dexamethasone (day-1 medium). Medium
was changed daily with day-1 medium.
R
R
Biliary
Excretion
Hepatocytes
Studies
in
Sandwich-Cultured
After aspirating the culture medium, sandwich-cultured hepatocytes at day 4 (rat) or day 7 (human) were rinsed twice with
warm standard HBSS buffer. To maintain or disrupt bile canaliculi, cells were then preincubated with standard or Ca2+ /Mg2+ free HBSS buffer for 10 min at 37◦ C, respectively. Next, hepatocytes were incubated with 10 : M of tauro-nor-THCA-24-DBD
for 25 min or 1 : M of taurocholate for 10 min in standard or
Ca2+ -free HBSS buffer. At the end of the incubation period,
substrate solution was aspirated, and the cells were washed
four times with ice-cold standard HBBS buffer. Subsequently,
cells were lysed with 1.5 mL of 0.5% Triton X solution (in PBS)
and placed in a plate shaker for 30 min at room temperature.
Substrate concentrations were determined as describe above.
Uptake rates were normalized for protein content, which was
measured using a BCA Protein assay kit (Pierce Chemical).
Microscopic Imaging
Biliary excretion of nor-THCA-24-DBD was studied by live
cell confocal imaging with a laser-scanning microscope (Fluoview FV1000; Olympus, Tokyo, Japan) equipped with a 488nm DPSS laser (Spectra Physics, Santa Clara, CA) for live
cell confocal imaging of canalicular excretion of tauro-norTHCA-24-DBD (20 : M) and carboxy dichlorofluorescein diacetate (10 : M). We employed a UPLSAPO 60X (1.20 NA) waterimmersion objective (Olympus) to match the refractive index
of the medium with the immersion media. The dichroic and
band pass filters were DM405/488 and BP505-540, respectively
(Olympus). The image size was adjusted to 1024 × 1024 or 512
× 512 pixels with a pixel dwell time of 12 or 20 : s/pixel, respectively. Z-stack images were taken at every 1 : m over a total
sample thickness of 10 : m. Although measuring, a 4× line
Kalman filter was applied to reduce noise in the image. The
entire system was under tight humidity and temperature control (37 ± 0.5◦ C) via a box incubator covering the microscope.
The three-dimensional (3D) confocal Z-stacks were transposed on top of each other in a two-dimensional image, allowing image analysis and processing time-dependent changes in
fluorescence. After calibration, the experiment was initiated by
adding tauro-nor-THCA-24-DBD to the buffer. Time-lapse images were overlaid of both the DIC and the fluorescence channel.
Regions of interest (ROI) were selected to monitor fluorescence intensity in the bile ducts and in the cells. Multiple regions were selected of the bile ducts, the intracellular regions,
and the background (no cells present). After subtraction of the
background, the intensities are normalized with respect to the
maximal intracellular signal in time for an experiment. The obtained signals are averaged out over a large relevant region and
subsequently averaged out over multiple comparable regions to
remove outliers.
Figure 1. Structure of (a) tauro-nor-THCA-24-DBD [N-(24-[7-(4N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3a,7a,12atrihydroxy-27-nor-5b-cholestan-26-oyl)-2t -aminoethanesulfonate] and
(b) taurocholate.
Data Analysis
Net uptake values were obtained by subtracting tauro-norTHCA-24-DBD uptake in wild-type CHO cells or hepatocytes
at 4◦ C from total uptake at 37◦ C in transfected cells or hepatocytes, respectively. Uptake kinetics was determined by fitting
the Michaelis–Menten equation to net uptake values:
v=
Vmax × C
Km + C
with Vmax and Km representing the kinetic parameters for saturable accumulation.
The biliary excretion index (BEI) in sandwich-cultured hepatocytes was determined using B-CLEARO technology (Qualyst,
Inc., Durham, North Carolina) by dividing the difference in accumulation in standard and Ca2+ /Mg2+ -free uptake buffer by
the accumulation in the standard buffer according to the following equation20 :
R
BEI =
Figure 2. Concentration-dependent uptake of tauro-nor-THCA-24DBD in suspended rat hepatocytes. Saturable tauro-nor-THCA-24DBD uptake was obtained by subtracting uptake at 4◦ C from total
uptake at 37◦ C. The data shown were obtained from a representative
batch of suspended rat hepatocytes. Points represent experimental data
(±SD, n = 4). Line represent best fit to experimental data as described
in the Methods section.
AccumulationStandard − AccumulationCa2+ /Mg2+ −free
AccumulationStandard
×100
Statistics
ANOVA (Dunnett’s test) was used to evaluate statistical differences (GraphPad Prism v. 5.00 for Windows; GraphPad Prism
software, California) between tauro-nor-THCA-24-DBD uptake
in the presence of inhibitors and uptake under control condition.
RESULT
S
Uptake Kinetics of Tauro-Nor-THCA-24-DBD in Suspended
Rat
Hepatocytes
The chemical structures of tauro-nor-THCA-24-DBD and taurocholate are shown in Figures 1a and 1b, respectively. Initial
uptake rates (30 s) of tauro-nor-THCA-24-DBD in suspended
rat hepatocytes were concentration dependent and followed
Michaelis–Menten type kinetics (Fig. 2). Kinetic parameters
for net active uptake were 13.7 ± 4.2 : M for Km and 3.2 ±
corresponds to an uptake clearance value of 233 : L/(million
cells × min).
Inhibition of Tauro-Nor-THCA-24-DBD Uptake by Known
Substrates/Inhibitors of Hepatic Drug Transporters in Suspended
0.4 nmol/(million cells x min) for Vmax , respectively (n = 3). This
Rat Hepatocytes
Different diagnostic inhibitors/substrates were used to identify
the predominant transporters involved in the hepatic uptake
of tauro-nor-THCA-24-DBD in suspended rat hepatocytes. The
uptake of tauro-nor-THCA-24-DBD (2 : M) in the presence of
rifampicin (50 : M) decreased by 32% (Fig. 3a). When extracellular sodium was replaced by choline, tauro-nor-THCA-24-DBD
uptake decreased by 65%, with no additional decrease when
rifampicin was present. In comparison, rifampicin and sodium
depletion decreased the uptake of the Ntcp reference probe substrate taurocholate by 55% and 78%, respectively. Coincubation
with CsA (10 : M), bromosulfophtalein (20 : M), indomethacin
(50 : M), and taurocholate (100 : M) resulted in a significant
decrease of tauro-nor-THCA-24-DBD uptake (Fig. 3b) by 40%,
59%, 20%, and 51%, respectively. On the contrary, the Oatp
substrate estrone-3-sulfate (ES; 25 : M), the Oat substrate
p-aminohippuric acid (PAH; 25 : M), and the organic cation
proximately constant, with a relative NTCP-mediated uptake
of 0.7.
Inhibition of Taurocholate and Tauro-Nor-THCA-24-DBD
Uptake in hNTCP/rNtcp-Transfected CHO Cells
Figure 5 shows the effect of the NTCP/Ntcp substrate taurocholate and the NTCP/Ntcp inhibitors CsA and ritonavir
on the uptake of taurocholate and tauro-nor-THCA-24-DBD
in rNtcp- (panel a) and hNTCP- (panel b) transfected CHO
cells. NTCP/Ntcp-mediated uptake of tauro-nor-THCA-24-DBD
(2 : M) decreased to 25%/9%, 49%/13%, and 46/32% of the
control uptake in the presence of taurocholate (50 : M), CsA
(10 : M), and ritonavir (20 : M), respectively (Fig. 5a). Consistently, coincubation with these compounds inhibited the uptake
of taurocholate in hNTCP/rNtcp-transfected CHO cells to 42%/
33%, 35%/28%, and 27%/28% of the control uptake (Fig. 5b).
Biliary Excretion of Taurocholate and Tauro-Nor-THCA-24DBD
in Sandwich-Cultured Hepatocytes
Figure 3. Inhibition of tauro-nor-THCA-24-DBD uptake (2 : M) and
taurocholate (2 : M) by inhibitors/substrates of different hepatic uptake transporter proteins in suspended rat hepatocytes. (a) The effect
of rifampicin (50 : M) on tauro-nor-THCA-24-DBD (open bars) and taurocholate (black bars) uptake in the presence and absence of sodium.
(b) Inhibition of tauro-nor-THCA-24-DBD (2 : M) uptake by different
transporter inhibitors/substrates: ES (25 : M), CsA (10 : M), bromosulfophthalein (20 : M), taurocholate (100 : M), indomethacin (50 : M),
PAH (25 : M), corticosterone (10 : M), and prazosin (10 : M). *p < 0.05
(ANOVA, Dunnett’s test), compared with the control net uptake. Bars
represent mean (±SD) of values obtained in three batches of rat hepatocytes.
transporter (Oct) inhibitors prazosin and corticosterone
(10 : M) had no significant effect on tauro-nor-THCA-24-DBD
uptake (Fig. 3b).
Uptake Kinetics of Taurocholate and Tauro-Nor-THCA-24-DBD
in
Transfected CHO Cells
Time-dependent uptake of taurocholate and tauro-nor-THCA24-DBD in OATP1B1-, OATP1B3-, rNtcp-, and hNTCP transfected CHO cells was linear for incubation times up to
2 min (data not shown). Initial uptake rates of tauro-nor-THCA24-DBD and taurocholate in transfected CHO cells were concentration dependent and followed saturable kinetics (Figs. 4a
and 4b) with kinetic parameters summarized in Table 1. To
compare the relative contribution of both OATP1B isoforms and
NTCP in the overall hepatic uptake of taurocholate/tauro-norTHCA-24-DBD, the ratio of the uptake by one specific transporter isoform to the sum of the uptake by all transporters was
calculated in function of the substrate concentration (Figs. 4c
and 4d). For tauro-nor-THCA-24-DBD, the relative contribution changes in function of the concentration, with a maximal
NTCP-mediated uptake of 0.7 at a concentration of greater than
10 : M. In comparison, the relative uptake of taurocholate is ap-
Accumulation of taurocholate and tauro-nor-THCA-24-DBD
was measured in standard buffer (representing accumulation
in cell and bile compartments) and in Ca2+ -free buffer (representing accumulation in cell compartment only). The difference
in accumulation measured in both conditions represents biliary
excretion, as previously validated.17,21
Biliary excretion of tauro-nor-THCA-24-DBD (10 : M) and
taurocholate (1 : M) was determined in sandwich-cultured day
4 rat and day 7 human hepatocytes (Table 2). Both substrates
were actively secreted in the bile network; the BEI of taurocholate was 52.5 ± 6.9% and 65.0% in sandwich-cultured
human hepatocytes (SCRH) and sandwich-cultured human
hepatocytes (SCHH), respectively. In comparison, the BEI of
tauro-nor-THCA-24-DBD was lower and amounted to 26.2
± 5.0% and 32.4%. The BEI of tauro-nor-THCA-24-DBD decreased to 5.0 ± 3.2% in the presence of CsA (10 : M) in SCRH
(Table 2). In contrary, the MRP inhibitor MK571 (10 : M) did
not significantly alter the BEI of tauro-nor-THCA-24-DBD in
SCRH (data not shown).
To further illustrate the biliary excretion of tauro-norTHCA-24-DBD in rat, SCRH were further examined via direct imaging by confocal microscopy after incubation with
tauro-nor-THCA-24-DBD for 25 min. The functional activity
of canalicular transporters was first validated with the fluorescent multidrug resistance-associated protein-2 (ABCC2)
(Mrp2) substrate CDF. It has previously been demonstrated
that after passive diffusion into hepatocytes, the nonfluorescent CDFDA is rapidly hydrolyzed by intracellular esterases to
the fluorescent dye CDF, which is subsequently actively transported into the canalicular compartments by Mrp2.22,23 The
overlay of confocal and transmission images of SCRH (Fig. 6)
showed that CDF is indeed predominantly located in the
canalicular networks after 15 min of incubation, confirming
the functional activity of canalicular transport in SCRH.
Biliary excretion of tauro-nor-THCA-24-DBD (20 : M) in
function of incubation time was studied in the absence or
presence of CsA (10 : M). Microscopic real-time quantification
showed that in the control condition (Fig. 7, panel a), normalized fluorescence in the canalicular compartments rapidly increased in function of incubation time. In contrast, in the presence of the Bsep inhibitor CsA, normalized fluorescence in the
bile canaliculi increased only slightly in function of time and
Figure 4. Concentration-dependent uptake of (a) tauro-nor-THCA-24-DBD and (b) taurocholate in OATP1B1- (squares), OATP1B3- (diamonds),
hNTCP- (circles), or rNtcp- (triangles) transfected CHO cells. CHO cells were incubated with 1–30 : M tauro-nor-THCA-24-DBD or 1–50 : M
taurocholate. Net uptake was obtained by subtracting linearized accumulation in wild type from total uptake in transfected cells, followed by
applying the Michaels–Menten equation. Graphs show data obtained from a representative batch of transfected CHO cells. Points represent
mean (±SD; n = 3). The relative contribution of OATP1B1, OATP1B3, and hNTCP in the uptake of tauro-nor-THCA-24-DBD (c) and taurocholate
(d). Relative uptake was calculated by dividing the uptake by the isoform by the sum of the uptake rates mediated by all transporter isoforms.
Table 1. Kinetic Parameters Describing Concentration-Dependent Uptake of Taurocholate and Tauro-Nor-THCA-24-DBD in Transfected
CHO Cells
OATP1B1
Taurocholate
Tauro-nor-THCA-24-DBD
OATP1B3
rNtcp
hNTCP
Km
Vmax
Km
Vmax
Km
Vmax
Km
Vmax
7.5
0.2
42.9
156.4
18.8
0.4
500.0
57.9
46.6
13.8
2535.2
736.9
18.2
49.9
2358.2
811.9
Km and Vmax values are expressed in : M and pmol / (mg protein × min), respectively.
was lower than the intracellular fluorescence (panel b), demonstrating the inhibition of Ntcp- and Bsep-mediated transport
of tauro-nor-THCA-24-DBD by CsA. The BEI of tauro-norTHCA-24-DBD transport in the absence and presence of CsA
amounted to approximately 75% and 40%, respectively. Representative pictures at the end of the incubation further illustrate the visualization of biliary excretion of tauro-nor-THCA24-DBD in SCRH.
DISCUSSION
The aim of this in vitro work was to elucidate the in vitro transport mechanisms determining the hepatic uptake of the fluorescent bile acid derivative, tauro-nor-THCA-24-DBD, in rats
and humans. Specific attention was given to the relative role of
OATP/Oatp isoforms and NTCP/Ntcp in the hepatic uptake of
nor-tauro-THCA-24-DBD, in comparison with endogenous reference substrate taurocholate. Additionally, we determined the
biliary excretion of both compounds in sandwich-cultured rat
and human hepatocytes.
Our results revealed saturable uptake of tauro-nor-THCA24-DBD in suspended rat hepatocytes, with a Km value of 13.7
± 4.2 : M (Fig. 2). This affinity constant is slightly lower than
the reported Km values of taurocholate uptake by suspended rat
hepatocytes (ranging from 19 to 26 : M).24–26 Consistently, the
affinity constant of tauro-nor-THCA-24-DBD uptake by rNtcptransfected CHO cells (Km = 13.8 : M, Table 1) was lower than
that of taurocholate uptake (Km = 46.6 : M, Table 1). This indicates that linking taurocholate to the fluorophore DBD results in a higher affinity to the Ntcp transporter. In contrast
to tauro-nor-THCA-24-DBD, the hepatic uptake of another
Figure 6. Overlay of confocal and transmission image of hepatocytes
cultured in a sandwich configuration (day 4) after incubation with
CDFDA (10 : M) for 15 min. The fluorescence bands (green) in between the cells indicate that fluorescent CDF is excreted into the bile
canalicular spaces.
Figure 5. Inhibition of taurocholate (2 : M, black bars) and tauronor-THCA-24-DBD (2 : M, open bars) uptake by taurocholate (50 : M),
CsA (10 : M), or ritonavir (20 : M) in rNtcp- (panel a) or hNTCP- (panel
b) expressing CHO cells. Net accumulation values were obtained by
subtracting accumulation in wild-type CHO cells from total uptake
in transfected cells. Uptake in the presence of inhibitors is expressed
relatively to the uptake in the control condition. *p < 0.05 (ANOVA,
Dunnett’s test), compared with the control net uptake. Bars represent
mean (±SD; n = 3).
Table 2. Biliary Excretion Index of Tauro-Nor-THCA-24-DBD,
Taurocholate, and CDFDA in Sandwich-Cultured Rat or Human
Hepatocytes
Tauro-nor-THCA-24-DBD
SCRH (n = 3)
SCHH (lot #S2406A)
Control
+CsA 10 : M
26.2 ± 5.0
32.4 ± 5.5
5.0 ± 3.2
ND
Taurocholate
52.5 ± 6.9
65.0 ± 16.0
ND, not determined.
fluorescent bile acid (CGamF) is mainly mediated by Oatps
rather than Ntcp.27 This difference is consistent with the 3D
pharmacophore model by Baringhaus and coworkers,28,29 which
indicated that the negatively charged side chain, acting as a hydrogen bond acceptor, is an important feature for affinity to the
ileal Na+ /bile acid cotransporter. Substitution of this negatively
charged side chain by fluorescein (as for CGamF) may lead to
a decreased affinity to Ntcp, whereas tauro-nor-THCA-24-DBD
still has a negatively charged side chain and is therefore expected to retain its Ntcp affinity.
The involvement of sodium-dependent transport by Ntcp
in the hepatic uptake of tauro-nor-THCA-24-DBD was confirmed by the decreased uptake when extracellular sodium was
replaced by choline (Fig. 3a). Indeed, even though the effect
of sodium depletion on tauro-nor-THCA-24-DBD uptake (65%
decrease) was slightly lower compared with the effect on taurocholate uptake (78% decrease), decreased uptake in the absence
of the driving force for Ntcp transporter suggests that Ntcp is
important in the hepatic uptake of the fluorescent bile acid
derivative tauro-nor-THCA-24-DBD. An additional evidence
for the role of Ntcp was obtained via inhibition experiments
(Fig. 3b); the Ntcp probe substrate taurocholate and the inhibitors CsA and bromosulfophthalein significantly decreased
the uptake of tauro-nor-THCA-24-DBD.
In contrast to the pronounced effect by sodium depletion, the
potent Oatp inhibitor rifampicin only decreased the uptake of
tauro-nor-THCA-24-DBD and taurocholate by 32% and 54%,
respectively. Surprisingly, the uptake of tauro-nor-THCA-24DBD in the presence of the Oatp substrate ES was comparable
to the control condition. The presence of multiple binding sites
on Oatps may explain this observation.30
In order to further assess the selectivity of tauro-nor-THCA24-DBD toward Ntcp in suspended rat hepatocytes, we evaluated the effect of probe substrates/inhibitors of the Oat and Oct
transporter families (Fig. 3b). The Oct inhibitors prazosin and
corticosterone had no effect on tauro-nor-THCA-24-DBD uptake, excluding the involvement of Oct-mediated transport. No
uptake inhibition in the presence of the Oat substrate PAH and
the limited inhibitory effect of the Oat inhibitor indomethacin
suggested a minor role for Oat-mediated transport. As indomethacin has been reported to inhibit both Oat and Oatp,
it is likely that the inhibition of tauro-nor-THCA-24-DBD uptake in the presence of indomethacin can be attributed to Oatp
rather than Oat inhibition.
Together, these results indicate that tauro-nor-THCA-24DBD is mainly transported across the basolateral membrane
Figure 7. Microscopic quantification of biliary excretion of tauro-nor-THCA-24-DBD in SCRH (day 4) in the absence (panel a) or presence
(panel b) of CsA (10 : M). Normalized fluorescence in the intracellular compartment (open circles) or bile canaliculi (closed circles) in function
of incubation time is plotted on the left y-axis. The calculated BEI (diamonds) is plotted on the right y-axis. The fluorescence images are
representative overlaid confocal images at the end of the incubation period in the absence (panel c) or presence (panel d) of CsA.
by Ntcp with a minor role for Oatps. Thus, these data
support comparable profiles of taurocholate and tauro-norTHCA-24-DBD with respect to Ntcp/Oatp-mediated uptake
transport. This contrasts with the limited role of Ntcp and
the predominant role of Oatps in the hepatic uptake of
other fluorescent bile acid derivatives such as CGamF and
CLF.27,31
Although human hepatocytes would have been useful to
study the affinity to human isoforms, these cells are scarce and
often show high interdonor variability in their transporter activities, which can challenge data interpretation.18 Therefore,
we examined the involvement of human hepatic transporter
isoforms in the disposition of tauro-nor-THCA-24-DBD in CHO
cells transfected with hNTCP, OATP1B1, or OATP1B3 (Fig. 4a,
Table 1). Tauro-nor-THCA-24-DBD showed saturable uptake
kinetics in all cell lines with a much lower affinity constant for
both OATP1B isoforms (Km = 0.2/0.4 : M) compared with the
Km value for hNTCP (Km = 49.9 : M). Additionally, the transport capacity (presented as the Vmax value) was 5–14 times
higher in hNTCP-transfected CHO compared with OATP1Btransfected cells. These results suggest that hNTCP-mediated
uptake of tauro-nor-THCA-24-DBD can be seen as a low-affinity
and high-capacity process, retaining its ability to transport the
fluorescent bile acid derivative at high substrate concentrations (Fig. 4c). In comparison with tauro-nor-THCA-24-DBD,
unlabeled taurocholate exhibited a somewhat higher affinity
to hNTCP (Km = 18.2 : M), consistent with the pivotal role
of hNTCP in the hepatic disposition of taurocholate in human
hepatocytes.
To illustrate the utility of tauro-nor-THCA-24-DBD to assess drug interactions at the uptake level, the inhibitory potential of the known NTCP/Ntcp substrate taurocholate and
the known NTCP/Ntcp inhibitors CsA and ritonavir were studied at concentrations higher than the reported Ki values.32,33
Taurocholate, ritonavir, and CsA significantly inhibited uptake of tauro-nor-THCA-24-DBD in both rNtcp- and hNTCPtransfected CHO cells (Fig. 5). In general, decreased uptake
was consistent with the effects of the NTCP/Ntcp inhibitors on
taurocholate uptake. These results illustrate that tauro-norTHCA-24-DBD can be used as an alternative probe substrate to
study interference with NTCP/Ntcp function in a fluorescencebased in vitro assay.
The hepatic transport properties of tauro-nor-THCA-24DBD have previously been determined in membrane vesicles
obtained from hBSEP-expressing Sf9 cells.16 These data revealed comparable kinetic parameters of the fluorescent bile
acid derivative with those of taurocholate, which is a probe
substrate for BSEP/Bsep often used to evaluate the risk of
drug-induced cholestasis by impaired bile acid transport.34,35
To further illustrate the applicability of the fluorescent bile
acid derivative, we evaluated the biliary excretion of tauronor-THCA-24-DBD in sandwich-cultured rat and human hepatocytes. Sandwich-cultured hepatocytes are considered an
excellent in vitro model system to study hepatobiliary drug
disposition.36–38 As shown in Table 2, tauro-nor-THCA-24-DBD
was actively excreted in the bile canaliculi of both sandwichcultured rat and human hepatocytes, with a BEI value of
26% and 32%, respectively. In comparison, taurocholate showed
higher BEI values (BEI = 53% and 65%, respectively), which
were consistent with those published previously.17,21,39,40 The
relatively lower biliary excretion of tauro-nor-THCA-24-DBD
compared with unlabeled taurocholate can be explained by
lower intracellular concentrations and/or lower Bsep affinity.
Nevertheless, the decreased biliary excretion in the presence
of the Bsep inhibitor CsA confirms that Bsep is involved in the
canalicular transport of tauro-nor-THCA-24-DBD as previously
shown for taurocholate.41 On the contrary, the Mrp2 inhibitor
MK571 did not significantly inhibit biliary excretion of tauronor-THCA-24-DBD, excluding the involvement of Mrp2.
We further quantified the biliary excretion of tauro-norTHCA-24-DBD by real-time confocal imaging. This technique
has extensively been used to image single cells or even whole
organisms and allows to obtain high spatial and temporal information of the transport system studied.42,43 This was illustrated
by Yamaguchi15 who showed accumulation of the fluorescent
bile acid derivative CDCA-NBD in the cytosol of OATP1B1and OATP1B3-expressing HepG2 cells. To confirm the functional activity of canalicular transporters in SCRH, we first visualized the biliary excretion of the fluorescent Mrp2 substrate
CDF, as established previously.23 As shown in Figure 6, CDF
was mainly localized in the canalicular networks of SCRH. Subsequently, real-time quantification by confocal imaging showed
pronounced biliary excretion of tauro-nor-THCA-24-DBD
(Fig. 7a). As the BEI is a relative number that compares the
intracellular and canalicular substrate accumulation, it can be
quantified by the relative fluorescence intensities measured by
confocal imaging. Interestingly, the BEI of tauro-nor-THCA24-DBD excretion in SCRH determined by real-time confocal
imaging was higher than the BEI determined by the conventional B-CLEARO technology (75% vs. 26%). This discrepancy
may be explained by the fact that background fluorescence (in
dead cells, see Fig. 7) can be largely eliminated when imaging
is used. Indeed, when the BEI is measured by a conventional
B-CLEARO assay, background fluorescence (which is a result
of nonspecific binding to dead cells) is incorrectly measured as
“intracellular accumulation”. On the contrary, determination
of the BEI by imaging techniques is based on an accurate selection of viable cells and specific regions of interest and will
therefore exclude background fluorescence. Consequently, relatively more weight is given to relevant accumulation in the
bile compartments, leading to higher BEI values. This illustrates one of the major advantages of imaging techniques over
R
R
conventional assays. Consistent with the B-CLEARO results
in SCRH, the potent Bsep inhibitor CsA decreased the biliary
excretion of tauro-nor-THCA-24-DBD (Fig. 7b).
R
CONCLUSIONS
We elucidated the in vitro transport mechanisms determining the hepatic disposition of the fluorescent bile acid derivative, nor-tauro-THCA-24-DBD, in rats and humans. Our work
clearly showed that nor-tauro-THCA-24-DBD is efficiently
transported by NTCP/Ntcp with a minor role for OATP/Oatp
isoforms. Therefore, nor-tauro-THCA-24-DBD is a useful tool to
study interference with NTCP/Ntcp function in a fluorescencebased in vitro assay. In addition, real-time confocal microscopy
unambiguously confirmed that biliary excretion of nor-tauro-
ACKNOWLEDGMEN
TS
Tom De Bruyn and Wouter Sempels received a PhD scholarship from the Agency for Innovation by Science and Technology,
Flanders. This study was supported by grants from “Fonds voor
Wetenschappelijk Onderzoek,” Flanders; “Onderzoeksfonds” of
the KU Leuven, Belgium; the European Research Council under the European Union’s Seventh Framework Programme; the
Flemish government in the form of long-term structural funding “Methusalem” grant; and the Hercules Foundation. The
authors would like to thank Janssen Research and Development for providing the fluorescent bile acid derivative tauronor-THCA-24-DBD.
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