![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION
Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina
Received September 5, 2002 ; accepted October 24, 2002.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
4-fold in
wild-type livers (65 ± 8% versus 15 ± 2% of dose over 2 h). In
livers from TR– rats, CDF was not excreted into bile and
probenecid decreased perfusate CDF concentrations in a concentration-dependent
manner, in part due to inhibition of Mrp3. Plasma membrane vesicles from rat
Mrp2- or Mrp3-transfected Sf9 cells were used to confirm that CDF is a
substrate for Mrp2 and Mrp3; probenecid inhibited the transport of CDF by Mrp2
and Mrp3 in a concentration-dependent manner. CDF uptake in collagen
sandwich-cultured rat hepatocytes was temperature-dependent and saturable
(Km = 22 ± 10 µM; Vmax = 97
± 9 pmol/min/mg protein). Uptake of CDF in sandwich-cultured rat
hepatocytes was impaired significantly by bromosulfophthalein, a substrate for
organic anion-transporting polypeptides (Oatps), but was not modulated by
specific Oatp2 or organic anion transporter (Oat) substrates. CDFDA uptake was
not saturable, temperature-dependent, or impaired by inhibitors. The
hydrolysis of CDFDA to CDF is mediated by basic pH and esterases in biological
media. CDFDA passively diffuses into hepatocytes where it is hydrolyzed to
CDF. In contrast, CDF appears to be taken up by Oatp-mediated transport into
rat hepatocytes and effluxed via Mrp2 into bile and via Mrp3 into sinusoidal
blood.
;
Kullak-Ublick et al., 2000;
Kusuhara and Sugiyama, 2002).
However, many compounds are substrates for more than one transport system, and
this adds to the complexity of studying hepatobiliary transport in the whole
cell and intact organ.
Probe compounds have been used to study alterations in hepatobiliary
transport systems and to investigate the transport properties of new agents
(Courtois et al., 1999;
Payen et al., 2000).
Fluorescent compounds can be assayed easily and with high sensitivity, thus
having advantages over nonfluorescent compounds. The fluorophore 5 (and
6)-carboxy-2',7'-dichlorofluorescein (CDF) is a multivalent
organic anion at physiological pH (Fig.
1). The plasma membrane of cells presents a diffusional barrier
for CDF. 5 (and 6)-Carboxy-2',7'-dichlorofluorescein diacetate
(CDFDA) carries only one negative charge
(Leonhardt et al., 1971) and
is permeable to cells. CDFDA, the diacetate promoiety used traditionally for
CDF delivery to cells, diffuses through plasma membranes and is hydrolyzed to
CDF by intracellular esterases (Breeuwer et
al., 1995).
|
CDF is not subject to hepatic metabolism and has been used as a substrate
for organic anion transport. The efflux of CDF was impaired in unpolarized
hepatocytes from Mrp2-deficient TR– rats (Jansen et al.,
1985,
1987) and Dubin-Johnson-like
golden lion tamarins (Schulman et al.,
1993), which was consistent with the recognition of CDF as an Mrp2
substrate (Kitamura et al.,
1990). CDF has been used as a model compound to evaluate the
biliary excretion of organic anions in sandwich-cultured rat hepatocytes
(Liu et al., 1999b).
Fluorescein also has been used extensively to study Mrp1 transport in a
variety of cell types (Huai-Yun et al.,
1998; Sun et al.,
2001).
Although CDF is eliminated from hepatocytes primarily via biliary excretion and, hence, appears to be a promising probe to study biliary excretion of organic anions, the hepatic transport of CDF has not been characterized fully. Alterations in hepatic disposition of CDF have been attributed traditionally to changes in biliary excretion, ignoring other relevant transport mechanisms, namely, uptake and basolateral efflux, where important interactions are likely to occur. In the present study, a multi-experimental approach was employed to examine the mechanisms of hepatic uptake, basolateral efflux, and canalicular excretion of CDF.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Animals. Male Wistar rats (275–300 g; Charles River Laboratories, Inc. (Raleigh, NC) or male Mrp2-deficient TR– rats bred in our animal facility (275–300 g; obtained from Dr. Mary Vore, University of Kentucky, Lexington, KY) were used as liver donors in isolated perfused liver studies and hepatocyte isolation. Retired male Wistar breeders (>400 g, Charles River Laboratories) were used as blood donors. Rats were maintained on a 12-h light/dark cycle with access to water and rodent chow ad libitum. Rats were allowed to acclimate for at least 5 days before experimentation. Anesthesia was induced with ketamine/xylazine (60/12 mg/kg i.p.). The Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill approved all procedures.
Isolated Perfused Rat Liver Studies. Recirculating isolated perfused
liver experiments were performed using standard techniques
(Brouwer and Thurman, 1996).
Livers were allowed to acclimate for 10 min before infusion of CDF or
CDFDA. Bile was collected continuously every 15 min. Perfusate samples
(0.75 ml), collected every 15 min, were centrifuged immediately, and the
supernatant was used for analysis. Measures of liver viability included portal
pressure (<15 cm of H2O) and initial bile flow (>0.8 and
>0.2 µl/min/g liver for wild-type and TR– rat livers,
respectively). Unless specified otherwise, taurocholate (15 mM in saline, 2
ml/h) was infused continuously into the reservoir to maintain bile flow. To
investigate the effects of taurocholate on CDF disposition, a set of
experiments was performed without taurocholate. In the probenecid inhibition
studies, probenecid (a bolus of 25 µmol followed by continuous infusion at
15.5 µmol/h) was administered to maintain steady-state perfusate
concentrations of probenecid at 260 µM
(Turner, 1996). In a separate
set of experiments in TR– livers, probenecid was administered
at a 10-fold higher dose. CDFDA or CDF (10 mM in dimethyl sulfoxide, 0.01
ml/min, 35 min) was infused into the reservoir. Dimethyl sulfoxide (<0.05%,
v/v) did not affect liver viability as determined by bile flow and the release
of lactate dehydrogenase from the liver.
Hepatocyte Culture in a Collagen Sandwich Configuration. Hepatocyte
isolation and culture were performed using standard techniques
(Liu et al., 1998). Cell
viability was measured by staining with trypan blue and was >90%.
Hepatocytes were seeded on 60-mm polystyrene dishes coated with gelled
collagen at a density of 3 million cells/dish. Hepatocytes were overlaid
with gelled collagen 24 h later. Hepatocytes were cultured for 4 days before
experimentation to allow the formation of canalicular networks between
cells.
Hepatocyte Uptake Studies. All culture dishes were rinsed with 3 ml
of Hanks' balanced salt solution (HBSS; 37°C or 4°C) prior to
experimentation. To determine the concentration dependence of CDF or CDFDA
uptake, cells were incubated in HBSS containing various concentrations of CDF
or CDFDA (1–1000 µM) at 37°C for 10 min; the effect of
temperature on uptake was examined by comparing uptake at 4°C or 37°C.
Cells used for uptake at 4°C were prechilled before the cells were
incubated in ice-cold HBSS containing 10 µM CDF or CDFDA. For inhibition
studies, cells were preincubated with 3 ml of HBSS containing either
inhibitors or vehicle at 37°C for 10 min, followed by incubation with 3 ml
of HBSS containing 10 µM CDF or CDFDA with inhibitors or vehicle. After the
incubation, dishes were rinsed with ice-cold HBSS and cells were lysed with 2
ml of lysing buffer [0.5% (v/v) Triton X-100 in phosphate-buffered saline].
Incubations in blank dishes coated with collagen were used to correct for
nonspecific binding. Uptake clearance was calculated in the linear range as
follows:
 |
Protein Binding Assay. Binding of CDF and CDFDA to the plasma proteins in perfusate was assessed with Centrifree Micro-partition Devices (Millipore Corp., Bedford, MA), following a 5-min incubation (37°C) of either CDF or CDFDA (5 and 50 µM) with the supernatant obtained after centrifugation of perfusate, in the presence of various concentrations of probenecid (0, 260 µM, or 4 mM). CDF binding to the device was negligible.
CDFDA Hydrolysis. The conversion rates of CDFDA to CDF in perfusate and buffer (pH = 7.4) were determined in vitro. The effect of probenecid (0, 260 µM or 4 mM) on the conversion of CDFDA to CDF in perfusate was investigated after a 5-min incubation (37°C; pH = 7.4). The hydrolysis of CDFDA (initial concentration of 35 µM) in male Wistar rat blood and hepatic cytosolic fractions, prepared by centrifugation of tissue homogenate at 9000g for 15 min, was determined in the presence or absence of probenecid (300 µM, pH 7.4; 37°C) over 4 h to determine the linear range for CDFDA hydrolysis. The reaction was stopped at designated times by protein precipitation with cold acetonitrile. To determine the concentration dependence of CDFDA hydrolysis in cytosol, initial hydrolysis rates were measured over a range of CDFDA concentrations (10 min; 2.5–500 µM; n = 4 per concentration).
Production of Recombinant Baculovirus. Recombinant pFASTBAC1 plasmids containing either rat Mrp2, Mrp3, or green fluorescence protein (GFP) encoding sequence were kindly provided by Dr. Yuichi Sugiyama (University of Tokyo, Tokyo, Japan). Recombinant baculovirus was generated with BAC-TO-BAC Baculovirus Expression System according to the manufacturer's instructions. Recombinant baculovirus stocks (>108 plaque-forming units/ml) were stored at 4°C. The viral titer of each stock was determined by plaque assays.
Viral Infection of Sf9 Insect Cells and Preparation of Plasma
Membrane. Sf9 insect cells were cultured in spinner flasks at 27°C
with Grace's insect cell medium supplemented with 5% fetal bovine serum, 3.33
g/l lactalbumin hydrolysate, 3.33 g/l yeastolate, and
antibiotics/antimycotics. Log phase Sf9 cells (1.0–1.5 x
106 cells/ml) were infected with recombinant baculovirus at a
multiplicity of infection between 3 and 5, and harvested 3 days later. Plasma
membranes were prepared as previously described
(Huang et al., 1998).
Plasma Membrane Vesicle Uptake Studies. Uptake of 10 µM CDF into
plasma membrane vesicles over 5 min was assessed in the presence or absence of
probenecid after determining that uptake was linear during this time period.
Substrate uptake into plasma membrane vesicles was measured by a quick
filtration technique (Xiong et al.,
2000). Briefly, aliquots of membrane suspensions (20 µl;
20–40 µg of protein) were preincubated for 5 min at 37°C, and
uptake was initiated by the addition of 80 µl of prewarmed incubation
buffer [20 mM HEPES (pH 7.5) 100 mM potassium nitrate, 100 mM sucrose, 5 mM
hemimagnesium gluconate, 0.5 mM hemicalcium gluconate, 10 mM phosphocreatine,
100 µg/ml creatine phosphokinase, 10 mM magnesium chloride, 4 mM ATP or
AMP] containing the substrate and the inhibitor to the membrane suspensions.
Membrane vesicle uptake was terminated by addition of 3.5 ml of ice-cold
membrane suspension buffer [10 mM HEPES/Tris (pH 7.4) 250 mM sucrose, 0.2 mM
magnesium chloride]. Vesicle-associated substrate was separated from free
substrate by rapid filtration through a 0.45-µm filter. Filters were rinsed
twice with 3.5 ml of ice-cold membrane suspension buffer. Filters were washed
in 2 ml of lysis buffer (phosphate-buffered saline containing 0.5% Triton
X-100) for 20 min at room temperature. Nonspecific binding of substrates to
the filter was determined in the absence of membrane vesicles.
Analytical Methods. CDF concentrations in bile, perfusate,
hepatocyte lysate, and Sf9 cell plasma membrane vesicle lysate from filters
were determined by spectrofluorometry
(
ex/em, 505/523 nm) at pH 7.4 with a
PerkinElmer LS50B luminescence spectrophotometer (PerkinElmer Life Sciences,
Boston, MA). Samples were diluted in phosphate-buffered saline before
determination. Standard curves of CDF (0.5–100 nM) were prepared daily
and were linear (r2 > 0.999). Protein concentrations
were determined via a BCA protein assay kit (Pierce Chemical, Rockford, IL).
Standard curves with bovine serum albumin (0.2–2 mg/ml) were prepared
daily and were linear (r2 > 0.99).
Pharmacokinetic Modeling. A compartmental modeling approach was
employed to describe the hepatobiliary disposition of CDF and CDFDA in the
isolated perfused livers of wild-type and TR– rats. Various
models employing linear and nonlinear processes were fit to the data. The
goodness of fit of each model was assessed by visual examination of the
distribution of residuals, the condition number, and Akaike's Information
Criterion (Akaike, 1976).
Differential equations based on the concentration of CDF in the perfusate and
the amount of CDF appearing in bile per unit time were resolved simultaneously
by nonlinear least-squares regression with a weighting scheme of 1/Y and the
Gauss-Newton (Levenberg and Hartley) minimization method (WinNonlin 3.1;
Pharsight Corporation, Mountain View, CA). The two-compartment model that best
described the CDF infusion data in wild-type livers
(Fig. 2A) was fit to CDF
perfusate concentration-time and CDF biliary excretion rate data. The
equations generated based on the scheme presented in
Fig. 2A were as follows:
 |
|
The model was modified to describe CDF disposition during CDFDA infusion in TR– livers. CDF excretion into bile of TR– livers was negligible; k20 was thus set equal to zero. The estimated efficiencies of CDFDA conversion to CDF in perfusate in control, low-dose probenecid, and high-dose probenecid livers were set at 84%, 83%, and 63%, respectively. The estimated efficiencies were calculated from the ratio of CDF recovery from CDFDA and CDF in perfusate at a given probenecid concentration (see Table 2). The impact on the ratio of intercompartmental rate constants (k21/k12) was assessed for wild-type and TR– livers. The TR– data set, essentially perfusate concentrations for kinetic purposes, was not amenable to reliable estimation of uptake and basolateral efflux rate constants. The model could converge at numerous minima in the sum of squared error, whenever efflux and uptake were at an appropriate ratio, and thus only this ratio is reported and discussed for the TR– data set. WinNonlin also was used to generate Km and Vmax estimates for saturable kinetics of CDF uptake.
|
Statistical Analyses. The Student's two-tailed t test was used to assess statistical significance. Where variances were significantly different between two groups, Wilcoxon's rank sum test was used to assess statistical significance. The criterion for significance in all cases was p < 0.05, with the Bonferroni correction where appropriate. All data are presented as mean ± S.D. except for hepatocyte uptake clearances, where the data represent the mean ± (S.E.M.) of means from three different animal preparations (n = 5/animal).
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
Protein Binding of CDF and CDFDA. Binding of CDF and CDFDA to plasma proteins in perfusate (20% rat blood) was modest. The unbound fractions of CDF were 83 ± 4% and 87 ± 6% at 5 and 50 µM CDF, respectively, and 82 ± 7% and 86 ± 12% at 5 and 50 µM CDFDA, respectively. The protein binding of neither CDF nor CDFDA was altered by the presence of 260 µM or 4 mM probenecid in vitro.
CDFDA Hydrolysis Studies. The in vitro recovery of CDF in perfusate, administered as CDF, was not decreased in the presence of probenecid. In contrast, the in vitro recovery of CDF (Table 2) from CDFDA in perfusate was reduced by the presence of probenecid and was lower than that associated with free CDF at the same molar dose. The base hydrolysis of CDFDA to CDF was a first-order process with a half-life of 7.6 ± 0.1 h in phosphate-buffered saline at physiological pH and temperature (data not shown). In perfusate (20% rat blood), the rate of CDFDA hydrolysis to CDF was faster than detection permitted. Within 10 s after the addition of CDFDA to perfusate, the CDF concentration was the same when measured directly and when the sample was treated with base, which would hydrolyze the acetates on any remaining CDFDA. Hydrolysis of CDFDA by blood and hepatic cytosolic esterases was not inhibited in vitro by 300 µM and 4 mM probenecid.
Sf9 Cell Plasma Membrane Vesicle Uptake Studies. The uptake of CDF into GFP-expressing Sf9 cell plasma membrane vesicles in the presence of AMP or ATP was minimal. In contrast, CDF uptake into rat Mrp2- or Mrp3-expressing Sf9 cell plasma membrane vesicles was significantly higher in the presence of ATP than in the presence of AMP (Fig. 4). Probenecid inhibited CDF uptake into both Mrp2- and Mrp3-expressing Sf9 cell plasma membrane vesicles in a concentration-dependent manner, and to a similar extent (Fig. 5).
|
|
Hepatocyte Uptake Studies. The uptake clearance of CDFDA in rat
hepatocytes cultured in a collagen sandwich configuration for 4 days was
significantly higher than that of CDF (4.9 ± 0.3 versus 1.9 ±
0.1 µl/min/mg protein; 10 µM). Low temperature (4°C) significantly
decreased (2-fold) CDF uptake clearance but had no effect on CDFDA uptake
(Fig. 6A). Cellular uptake of
CDF was saturable and well described by Michaelis-Menten kinetics
(Km = 22 ± 10 µM; Vmax = 97
± 9 pmol/min/mg protein) (Fig.
7A). Uptake of CDFDA (1–500 µM) was a first-order process
(Fig. 7B). Uptake of CDFDA at a
concentration of 1 mM was assessed but is not reported due to very high
binding to hepatocytes. CDF uptake was significantly inhibited by
bromosulfophthalein (100 µM), but not by digoxin (100 µM) or
p-aminohippurate (100 or 1000 µM)
(Fig. 6B). Bromosulfophthalein,
in combination with digoxin, did not inhibit CDF uptake further. Nonspecific
Oatp inhibitors, taurocholate (100 µM), rifampicin (100 µM), ouabain
(100 µM), and probenecid (260 µM), impaired the uptake of CDF by 41
± 18, 32 ± 12, 38 ± 7, and 65 ± 16% of control
(mean ± S.E.M.), respectively. In contrast to CDF, hepatic uptake of
CDFDA was not inhibited by bromosulfophthalein, digoxin, or
p-aminohippurate (data not shown).
|
|
Pharmacokinetic Modeling. Representative fits of the two-compartment model described in Fig. 2A to perfusate concentration and biliary excretion rate data are shown in Fig. 2B. First-order rate constants estimated for wild-type isolated perfused livers in the presence and absence of probenecid are summarized in Table 3. Probenecid significantly decreased the uptake and biliary excretion rate constants. Table 4 reports the ratios of basolateral efflux to uptake rate constants in wild-type and TR– livers. In wild-type livers, probenecid significantly increased this ratio, primarily due to the decrease in basolateral uptake. In TR– livers, probenecid decreased the basolateral efflux-to-uptake ratio in a concentration-dependent manner. Comparison of controls between the two groups indicated that TR– livers exhibited a significantly elevated ratio of basolateral efflux to uptake compared with livers from wild-type rats.
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
),
suggesting Mrp2-mediated excretion of CDF in hepatocytes. Probenecid inhibited
the excretion of CDF into bile. The concentration-dependent decrease in
perfusate CDF concentrations by probenecid in isolated perfused rat livers
from TR– rats could be explained by 1) inhibition of a
basolateral efflux mechanism, or 2) a decrease in CDF available from the
hydrolysis of CDFDA in the presence of probenecid. This second perturbation
was incorporated in the pharmacokinetic modeling, so that only transport
perturbations were estimated. The decreased perfusate concentrations of CDF in
perfused livers from TR– rats can be attributed in part to
probenecid inhibition of basolateral efflux of CDF by Mrp3, a major efflux
route for anionic xenobiotics in the absence of Mrp2
(Ortiz et al., 1999;
Ogawa et al., 2000). Uptake studies with plasma membrane vesicles prepared from rat Mrp2- or Mrp3-expressing Sf9 cells demonstrated that CDF is a substrate for both Mrp2 and Mrp3. Probenecid inhibited the transport of CDF by Mrp2 and Mrp3 in a concentration-dependent manner. Despite a similar extent of Mrp2 and Mrp3 inhibition by probenecid, the inhibition of basolateral efflux may not be as important as that of biliary excretion because the biliary excretion is the dominant pathway for CDF efflux out of wild-type hepatocytes.
Collagen sandwich-cultured hepatocytes were used for uptake experiments
because this culturing technique re-establishes canalicular networks and cell
polarity (Liu et al.,
1999a,c).
Based on hepatocyte uptake studies, CDFDA appears to be taken up by a passive
process, whereas CDF uptake is Oatp-mediated. CDFDA uptake was neither
saturable nor temperature-dependent; in contrast, CDF uptake was significantly
inhibited at 4°C and was saturable. CDF uptake does not appear to be
Oat-mediated, because CDF uptake was not inhibited by
p-aminohippurate. Recently, fluorescein accumulation in choroid
plexus tissue was shown to be inhibited 3-fold in the presence of 100
µM p-aminohippurate (Breen et
al., 2002). Interestingly, CDF uptake was not inhibited even by 1
mM p-aminohippurate, suggesting that addition of the carboxyl group
and two chlorines to the fluorescein molecule changes its substrate
specificity. Uptake of CDF was digoxin-independent, suggesting that CDF may
not be an Oatp2 substrate. However, recently, Meng et al.
(2002) demonstrated that
digoxin did not inhibit the uptake of an Oatp2 substrate,
sulfolithocholyltaurine. CDF appears to be an Oatp1 substrate based on
bromosulfophthalein inhibition of CDF uptake. Since bromosulfophthalein is
very highly protein-bound (Reyes et al.,
1969), to ensure that the decrease in intracellular accumulation
of CDF was due to inhibition of transport by Oatp1 and not a protein binding
effect, the results of the study were confirmed with nonspecific Oatp
substrates. Probenecid, a nonspecific organic anion transport inhibitor
(Sugiyama et al., 2001), also
inhibited the uptake of CDF. Recently, rat Oatp4 has been shown to mediate
hepatic uptake of xenobiotics (Cattori et
al., 2001). Identification of the specific Oatp isoforms
responsible for the hepatic uptake of CDF requires further investigation.
Uptake of CDFDA was not inhibited by bromosulfophthalein, digoxin,
p-aminohippurate, or probenecid, which further confirms that uptake
of the promoiety is a passive process.
Pharmacokinetic analysis yielded further insight into CDF transport.
Fitting the two-compartment model to the data from isolated perfused livers
from wild-type rats in the presence or absence of probenecid elucidated the
sites of inhibition. Uptake and biliary excretion were inhibited significantly
(10- and 2-fold, respectively) by probenecid in wild-type livers. The two
sites of inhibition indicate that both hepatic uptake and biliary excretion
must be considered in examining mechanisms of inhibition, especially in cases
where uptake is the rate-limiting step in hepatic elimination. Traditionally,
interactions at the site of biliary excretion have been emphasized.
Interestingly, probenecid coadministration did not decrease the basolateral
efflux rate constant despite the ability of probenecid to inhibit Mrp3 in
vitro. The absence of the anticipated decrease in this rate constant may be
due to increased intrahepatic CDF concentrations secondary to a decrease in
biliary excretion. Clearly, basolateral efflux plays an important role when
biliary excretion is compromised. In fact, in livers from TR–
rats in the absence of probenecid, where biliary excretion is negligible due
to the absence of Mrp2, the ratio of basolateral efflux to uptake was
significantly increased 100-fold (15.3 ± 6.8 versus 0.13 ±
0.17), just as when probenecid was administered to isolated perfused livers
from wild-type rats where the ratio increased 30-fold (3.4 ± 1.6
versus 0.13 ± 0.17) relative to wild-type controls. In
TR– rats, probenecid decreased the basolateral
efflux-to-uptake ratio in a concentration-dependent manner. Pharmacokinetic
modeling suggests that when basolateral efflux is the only route for CDF
elimination, basolateral transport proteins such as Mrp3 may be inhibited to a
greater extent than Oatp-mediated uptake. Mrp3 expression in
TR– rats is much greater than in wild-type rats
(Xiong et al., 2002),
consistent with the observation that the basolateral efflux-to-uptake ratio
for Mrp3 substrates is much greater in these Mrp2-deficient mutants, even with
probenecid coadministration.
The CDF promoiety, CDFDA, traditionally has been used as a means of hepatic
delivery of CDF, the disposition of which has been used to draw conclusions
about Mrp-mediated transport. The current study demonstrates that there are no
real advantages to using the promoiety instead of the parent compound for
delivery, and, in fact, using CDFDA complicates experiments by adding the
ester hydrolysis step. CDFDA has been used in transport experiments with the
assumption that it diffuses freely across membranes, whereas CDF is not taken
up extensively (Courtois et al.,
1999). In fact, CDF uptake in hepatocytes is only one-half CDFDA
uptake at the dosing concentrations most commonly used (1–10 µM).
Attempting CDFDA delivery in a biological matrix, such as perfusate of the
isolated perfused liver, essentially is no different from delivering CDF,
because CDFDA will be hydrolyzed to CDF almost instantaneously by esterases in
the biological medium. Furthermore, in perfusate, hydrolysis of CDFDA to CDF
is not 100% efficient, which complicates data analysis. Although the
hydrolysis of CDFDA in buffer at physiological temperature and pH is not
rapid, it must be taken into consideration in experiments lasting longer than
several minutes, and where buffer capacity is low and pH is variable. In
conclusion, for hepatic transport experiments where esterases are present in
the media, it is advantageous to use CDF instead of CDFDA, because use of CDF
circumvents the complications of the hydrolysis step while not greatly
compromising the uptake rate.
Probenecid is used routinely as a nonspecific inhibitor of organic anion
transport. However, sites and mechanisms of action have not been elucidated
fully. Recently, several studies demonstrated that probenecid could inhibit
human MRP1 and MRP2 (Hooijberg et al.,
1999; Bakos et al.,
2000). In the present study, probenecid significantly reduced the
biliary excretion of CDF in isolated perfused livers from Wistar wild-type
rats, which may be achieved by inhibiting either hepatic uptake or biliary
excretion of CDF. Pharmacokinetic modeling showed that probenecid
significantly inhibited both uptake and biliary excretion. This dual
inhibition was further confirmed in Sf9 cell plasma membrane vesicles where
probenecid inhibited Mrp2-mediated transport of CDF, and in hepatocytes where
probenecid inhibited Oatp-mediated uptake of the probe. This finding
illustrates the problems associated with attributing kinetic alterations in
whole organs, or in vivo, to inhibition of a single transporter, oftentimes
assumed to mediate efflux, when in fact multiple transport processes may
exist. This is especially true for compounds such as CDF, the uptake of which
is the rate-limiting step. As demonstrated here, hepatobiliary transport can
be mediated by several transporters, and to fully understand the hepatic
transport of a compound, all processes, not merely excretion into bile, must
be considered.
In summary, hepatobiliary transport of CDF has been characterized (Fig. 8). CDF is taken up into hepatocytes by a saturable, temperature-dependent Oatp-mediated mechanism. In contrast, the diacetate promoiety, CDFDA, is taken up by passive diffusion and instantaneously hydrolyzed by intracellular esterases to CDF. The disadvantage of CDFDA is its instability in biological media. CDF is excreted from liver into bile by Mrp2 and from liver into sinusoidal blood by Mrp3. When Mrp2-mediated excretion of CDF is impaired, basolateral efflux by Mrp3 increases.
|
|
Acknowledgements |
|---|
|
Footnotes |
|---|
ABBREVIATIONS: CDF, 5 (and 6)-carboxy-2',7'-dichlorofluorescein; CDFDA, 5 (and 6)-carboxy-2',7'-dichlorofluorescein diacetate; Mrp, multidrug resistance-related protein; TR– rats, Mrp2-deficient rats; HBSS, Hanks' balanced salt solution; GFP, green fluorescence protein; Oatp, organic anion-transporting polypeptide; Oat, organic anion transporter.
Address correspondence to: Dr. Kim L. R. Brouwer, Division of Drug Delivery and Disposition, School of Pharmacy, CB# 7360, Kerr Hall, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360. E-mail: kbrouwer{at}unc.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Akaike H (1976) An information criterion.
Math Sci 14:
5–9.
Bakos E, Evers R, Sinko E, Varadi A, Borst P, and Sarkadi B
(2000) Interactions of the human multidrug resistance proteins
MRP1 and MRP2 with organic anions. Mol Pharmacol
57:
760–768.
Breen CM, Sykes DB, Fricker G, and Miller DS (2002)
Confocal imaging of organic anion transport in intact rat choroid plexus.
Am J Physiol 282:F
877–F885.
Breeuwer P, Drocourt JL, Bunschoten N, Zwietering MH, Rombouts FM,
and Abee T (1995) Characterization of uptake and hydrolysis of
fluorescein diacetate by intracellular esterases in Saccharomyces cerevisiae,
which result in accumulation of fluorescent product. Appl Environ
Microbiol 61:
1614–1619.[Abstract]
Brouwer KLR and Thurman RG (1996) Isolated perfused
liver, in Models for Assessing Drug Absorption and
Metabolism (Borchardt RT, Smith PL, and Wilson G, eds) pp
161–192, Plenum Press, New York.
Cattori V, van Montfoort JE, Stieger B, Landmann L, Meijer DK,
Winterhalter KH, Meier PJ, and Hagenbuch B (2001) Localization of
organic anion transporting polypeptide 4 (Oatp4) in rat liver and comparison
of its substrate specificity with Oatp1, Oatp2 and Oatp3. Pflueg
Arch Eur J Physiol 443:
188–195.[CrossRef][Medline]
Courtois A, Payen L, Lagadic D, Guillouzo A, and Fardel O
(1999) Evidence for a multidrug resistance-associated protein 1
(MRP1)-related transport system in cultured rat liver biliary epithelial
cells. Life Sci 64:
763–774.[CrossRef][Medline]
Hooijberg JH, Broxterman HJ, Kool M, Assaraf YG, Peters GJ,
Noordhuis P, Scheper RJ, Borst P, Pinedo HM, and Jansen G (1999)
Antifolate resistance mediated by the multidrug resistance proteins MRP1 and
MRP2. Cancer Res 59:
2532–2535.
Huai-Yun H, Secrest DT, Mark KS, Carney D, Brandquist C, Elmquist
WF, and Miller DW (1998) Expression of multidrug
resistance-associated protein (MRP) in brain microvessel endothelial cells.
Biochem Biophys Res Commun
243:
816–820.[CrossRef][Medline]
Huang L, Hoffman T, and Vore M (1998) Adenosine
triphosphate-dependent transport of estradiol-17
(-D-glucuronide)
in membrane vesicles by MDR1 expressed in insect cells.
Hepatology 28:
1371–1377.[CrossRef][Medline]
Jansen PL, Groothuis GM, Peters WH, and Meijer DF
(1987) Selective hepatobiliary transport defect for organic
anions and neutral steroids in mutant rats with hereditary-conjugated
hyperbilirubinemia. Hepatology
7:
71–76.[Medline]
Jansen PL, Peters WH, and Lamer WH (1985) Hereditary
chronic conjugated hyperbilirubinemia in mutant rats caused be defective
hepatic anion transport. Hepatology
5:
573–579.[Medline]
Kitamura T, Jansen P, Hardenbrook C, Kamimoto Y, Gatmaitan Z, and
Arias IM (1990) Defective ATP-dependent bile canalicular
transport of organic anions in mutant (TR–) rats with conjugated
hyperbilirubinemia. Proc Natl Acad Sci USA
87:
3557–3561.
Konig J, Nies AT, Cui Y, Leier I, and Keppler D (1999)
Conjugate export pumps of the multidrug resistance protein (MRP) family:
localization, substrate specificity and Mrp2-mediated drug resistance.
Biochim Biopys Acta
1461:
377–394.[Medline]
Kullak-Ublick GA, Beuers U, and Paumgartner G (2000)
Hepatobiliary transport. J Hepatol
32:
3–18.[Medline]
Kusuhara H and Sugiyama Y (2002) Role of transporters
in the tissue-selective distribution and elimination of drugs: transporters in
the liver, small intestine, brain and kidney. J Control
Release 78:
43–54.[CrossRef][Medline]
Leonhardt H, Gordon L, and Livingston R (1971)
Acid-base equilibria of fluorescein and 2',7'-dichlorofluorescein
in their ground and fluorescent states. J Phys Chem
75:
245–249.[CrossRef]
Liu X, Brouwer KLR, Liang-Sheng LG, Brouwer KR, Stieger B, Meier
PJ, Audus KL, and LeCluyse EL (1998) Partial maintenance of
taurocholate uptake by adult rat hepatocytes cultured in a collagen sandwich
configuration. Pharm Res (NY)
15:
1533–1539.[CrossRef][Medline]
Liu X, Chism JP, LeCluyse EL, Brouwer KR, and Brouwer KLR
(1999a) Correlation of biliary excretion in sandwich-cultured rat
hepatocytes and in vivo in rats. Drug Metab Dispos
27:
637–644.
Liu X, LeCluyse EL, Brouwer KR, Liang-Sheng LG, Lemasters JJ,
Stieger B, Meier PJ, and Brouwer KLR (1999b) Biliary excretion in
primary rat hepatocytes cultured in a collagen-sandwich configuration.
Am J Physiol 277:G
12–G21.
Liu X, LeCluyse EL, Brouwer KR, Lightfoot RM, Lee JI, and Brouwer
KLR (1999c) Use of Ca2+ modulation to
evaluate biliary excretion in sandwich-cultured rat hepatocytes. J
Pharmacol Exp Ther 289:
1592–1599.
Meng LJ, Wang P, Wolkoff AW, Kim RB, Tirona RG, Hofmann AF, and
Pang KS (2002) Transport of the sulfated, amidated bile acid,
sulfolithocholyltaurine, into rat hepatocytes is mediated by Oatp1 and Oatp2.
Hepatology 35:
1031–1040.[CrossRef][Medline]
Ogawa K, Suzuki H, Hirohashi T, Ishikawa T, Meier PJ, Hirose K,
Akizawa T, Yoshioka M, and Sugiyama Y (2000) Characterization of
inducible nature of MRP3 in rat liver. Am J Physiol
278:G
438–G446.
Ortiz DF, Li S, Iyer R, Zhang X, Novikoff P, and Arias IM
(1999) MRP3, a new ATP-binding cassette protein localized to the
canalicular domain of the hepatocyte. Am J Physiol
276:G
1493–G1500.
Payen L, Courtois A, Campion JP, Guillouzo A, and Fardel O
(2000) Characterization and inhibition by a wide range of
xenobiotics of organic anion excretion by primary human hepatocytes.
Biochem Pharmacol 60:
1967–1975.[CrossRef][Medline]
Reyes H, Levi AJ, Gatmaitan Z, and Arias IM (1969)
Organic anion-binding protein in rat liver: drug induction and its physiologic
consequence. Proc Natl Acad Sci USA
64:
168–170.
Schulman FY, Montali RJ, Bush M, Citino SB, Tell LA, Ballou JD,
Hutson TL, St. Pierre M, Dufour JF, and Gatmaitan Z (1993)
Dubin-Johnson-like syndrome in golden lion tamarins (Leontopithecus rosalia
rosalia). Vet Pathol 30:
491–498.[Abstract]
Sugiyama D, Kusuhara H, Shitara Y, Abe T, Meier PJ, Sekine T, Endou
H, Suzuki H, and Sugiyama Y (2001) Characterization of the efflux
transport of 17-estradiol-D-17-glucuronide from the brain across
the blood-brain barrier. J Pharmacol Exp Ther
298:
316–321.
Sun H, Johnson DR, Finch RA, Sartorelli AC, Miller DW, and Elmquist
WF (2001) Transport of fluorescein in MDCKII-MRP1 transfected
cells and mrp1-knockout mice. Biochem Biophys Res
Commun 284:
863–869.[CrossRef][Medline]
Turner KC (1996) Mechanism of
Probenecid-Associated Perturbations in the Hepatobiliary Disposition of
Acetaminophen. Doctoral dissertation, University of North
Carolina at Chapel Hill, Chapel Hill, NC.
Xiong H, Suzuki H, Sugiyama Y, Meier PJ, Pollack GM, and Brouwer
KLR (2002) Mechanisms of impaired biliary excretion of
acetaminophen glucuronide after acute phenobarbital treatment or phenobarbital
pretreatment. Drug Metab Dispos
30:
962–969.
Xiong H, Turner KC, Ward ES, Jansen PM, and Brouwer KLR
(2000) Altered hepatobiliary disposition of acetaminophen
glucuronide in isolated perfused livers from multidrug resistance-associated
protein 2-deficient TR– rats. J Pharmacol Exp
Ther 295:
512–518.
This article has been cited by other articles:
|
|
 |
|
  K. Howe, G. G. Gibson, T. Coleman, and N. Plant In Silico and in Vitro Modeling of Hepatocyte Drug Transport Processes: Importance of ABCC2 Expression Levels in the Disposition of Carboxydichlorofluroscein Drug Metab. Dispos., February 1, 2009; 37(2): 391 - 399. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Tian, B. Swift, M. J. Zamek-Gliszczynski, M. G. Belinsky, G. D. Kruh, and K. L. R. Brouwer Impact of Basolateral Multidrug Resistance-Associated Protein (Mrp) 3 and Mrp4 on the Hepatobiliary Disposition of Fexofenadine in Perfused Mouse Livers Drug Metab. Dispos., May 1, 2008; 36(5): 911 - 915. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Heredi-Szabo, E. Kis, E. Molnar, A. Gyorfi, and P. Krajcsi Characterization of 5(6)-Carboxy-2,'7'-Dichlorofluorescein Transport by MRP2 and Utilization of this Substrate as a Fluorescent Surrogate for LTC4 J Biomol Screen, April 1, 2008; 13(4): 295 - 301. [Abstract] [PDF]
|
|
|
|
|
|
D. A. J. Bow, J. L. Perry, D. S. Miller, J. B. Pritchard, and K. L. R. Brouwer Localization of P-gp (Abcb1) and Mrp2 (Abcc2) in Freshly Isolated Rat Hepatocytes Drug Metab. Dispos., January 1, 2008; 36(1): 198 - 202. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Naud, J. Michaud, C. Boisvert, K. Desbiens, F. A. Leblond, A. Mitchell, C. Jones, A. Bonnardeaux, and V. Pichette Down-Regulation of Intestinal Drug Transporters in Chronic Renal Failure in Rats J. Pharmacol. Exp. Ther., March 1, 2007; 320(3): 978 - 985. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Zamek-Gliszczynski, K.-i. Nezasa, X. Tian, A. S. Bridges, K. Lee, M. G. Belinsky, G. D. Kruh, and K. L. R. Brouwer Evaluation of the Role of Multidrug Resistance-Associated Protein (Mrp) 3 and Mrp4 in Hepatic Basolateral Excretion of Sulfate and Glucuronide Metabolites of Acetaminophen, 4-Methylumbelliferone, and Harmol in Abcc3-/- and Abcc4-/- Mice J. Pharmacol. Exp. Ther., December 1, 2006; 319(3): 1485 - 1491. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Franco and J. A. Cidlowski SLCO/OATP-like Transport of Glutathione in FasL-induced Apoptosis: GLUTATHIONE EFFLUX IS COUPLED TO AN ORGANIC ANION EXCHANGE AND IS NECESSARY FOR THE PROGRESSION OF THE EXECUTION PHASE OF APOPTOSIS J. Biol. Chem., October 6, 2006; 281(40): 29542 - 29557. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Zamek-Gliszczynski, K. A. Hoffmaster, J. E. Humphreys, X. Tian, K.-i. Nezasa, and K. L. R. Brouwer Differential Involvement of Mrp2 (Abcc2) and Bcrp (Abcg2) in Biliary Excretion of 4-Methylumbelliferyl Glucuronide and Sulfate in the Rat J. Pharmacol. Exp. Ther., October 1, 2006; 319(1): 459 - 467. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-i. Nezasa, X. Tian, M. J. Zamek-Gliszczynski, N. J. Patel, T. J. Raub, and K. L. R. Brouwer ALTERED HEPATOBILIARY DISPOSITION OF 5 (AND 6)-CARBOXY-2',7'-DICHLOROFLUORESCEIN IN Abcg2 (Bcrp1) AND Abcc2 (Mrp2) KNOCKOUT MICE Drug Metab. Dispos., April 1, 2006; 34(4): 718 - 723. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Zamek-Gliszczynski, K. A. Hoffmaster, X. Tian, R. Zhao, J. W. Polli, J. E. Humphreys, L. O. Webster, A. S. Bridges, J. C. Kalvass, and K. L. R. Brouwer MULTIPLE MECHANISMS ARE INVOLVED IN THE BILIARY EXCRETION OF ACETAMINOPHEN SULFATE IN THE RAT: ROLE OF MRP2 AND BCRP1 Drug Metab. Dispos., August 1, 2005; 33(8): 1158 - 1165. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Chandra, B. M. Johnson, P. Zhang, G. M. Pollack, and K. L. R. Brouwer MODULATION OF HEPATIC CANALICULAR OR BASOLATERAL TRANSPORT PROTEINS ALTERS HEPATOBILIARY DISPOSITION OF A MODEL ORGANIC ANION IN THE ISOLATED PERFUSED RAT LIVER Drug Metab. Dispos., August 1, 2005; 33(8): 1238 - 1243. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Chandra, P. Zhang, and K. L. R. Brouwer Short-term regulation of multidrug resistance-associated protein 3 in rat and human hepatocytes Am J Physiol Gastrointest Liver Physiol, June 1, 2005; 288(6): G1252 - G1258. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Zhang, X. Tian, P. Chandra, and K. L. R. Brouwer Role of Glycosylation in Trafficking of Mrp2 in Sandwich-Cultured Rat Hepatocytes Mol. Pharmacol., April 1, 2005; 67(4): 1334 - 1341. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J Wolf, T Stranzl, M Filipits, G Pohl, R Pirker, B Leeb, and J S Smolen Expression of resistance markers to methotrexate predicts clinical improvement in patients with rheumatoid arthritis Ann Rheum Dis, April 1, 2005; 64(4): 564 - 568. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. C. Kemp, M. J. Zamek-Gliszczynski, and K. L. R. Brouwer Xenobiotics Inhibit Hepatic Uptake and Biliary Excretion of Taurocholate in Rat Hepatocytes Toxicol. Sci., February 1, 2005; 83(2): 207 - 214. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Tian, M. J. Zamek-Gliszczynski, P. Zhang, and K. L. R. Brouwer Modulation of Multidrug Resistance-Associated Protein 2 (Mrp2) and Mrp3 Expression and Function with Small Interfering RNA in Sandwich-Cultured Rat Hepatocytes Mol. Pharmacol., October 1, 2004; 66(4): 1004 - 1010. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Z. Turncliff, P. J. Meier, and K. L. R. Brouwer EFFECT OF DEXAMETHASONE TREATMENT ON THE EXPRESSION AND FUNCTION OF TRANSPORT PROTEINS IN SANDWICH-CULTURED RAT HEPATOCYTES Drug Metab. Dispos., August 1, 2004; 32(8): 834 - 839. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P.-L. Tsai and T.-H. Tsai HEPATOBILIARY EXCRETION OF BERBERINE Drug Metab. Dispos., April 1, 2004; 32(4): 405 - 412. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
) profiles for CDF in
a recirculating isolated perfused liver from a wild-type rat following CDF
infusion (0.1 µmol/min, 35 min) into the perfusate reservoir. Note the
parallel decline in concentration and excretion rate. Lines represent the best
fit of the model to the data.
) or presence
([UNK]) of probenecid, and in TR– rats (
) in the absence
of probenecid (A). Corresponding perfusate CDF concentrations in isolated
perfused livers from wild-type (B) and TR– (C) rats in the
absence (
