1 Graduate School of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; and 2 Toxicology Laboratory, SRI International, Menlo Park, California 94025-3943
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ABSTRACT |
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Biliary excretion of several anionic compounds
was examined by assessing their ATP-dependent uptake in bile
canalicular membrane vesicles (CMV) prepared from six human liver
samples.
2,4-Dinitrophenyl-S-glutathione (DNP-SG), leukotriene C4
(LTC4), sulfobromophthalein
glutathione (BSP-SG), E3040 glucuronide (E-glu),
-estradiol 17-(
-D-glucuronide) (E2-17G), grepafloxacin glucuronide (GPFXG),
pravastatin, BQ-123, and methotrexate, which are known to be
substrates for the rat canalicular multispecific organic anion
transporter, and taurocholic acid (TCA), a substrate for the bile acid
transporter, were used as substrates. ATP-dependent and saturable
uptake of TCA, DNP-SG, LTC4,
E-glu, E2-17G, and GPFXG was observed in all human CMV
preparations examined, suggesting that these compounds are excreted in
the bile via a primary active transport system in humans. Primary active transport of the other substrates was also seen in some of CMV
preparations but was negligible in the others. The ATP-dependent uptake
of all the compounds exhibited a large inter-CMV variation, and there
was a significant correlation between the uptake of glutathione
conjugates (DNP-SG, LTC4, and
BSP-SG) and glucuronides (E-glu, E2-17G, and GPFXG). However,
there was no significant correlation between TCA and the other organic
anions, implying that the transporters for TCA and for organic anions
are different also in humans. When the average value for the
ATP-dependent uptake by each preparation of human CMVs was compared
with that of rat CMVs, the uptake of glutathione conjugates and
nonconjugated anions (pravastatin, BQ-123, and methotrexate) in humans
was ~3- to 76-fold lower than that in rats, whereas the uptake of
glucuronides was similar in the two species. Thus there is a species
difference in the primary active transport of organic anions across the
bile canalicular membrane that is less marked for glucuronides.
canalicular membrane vesicles; canalicular multispecific organic anion transporter; glutathione conjugates; glucuronides; species difference
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INTRODUCTION |
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SEVERAL TYPES of transporters located on the liver plasma membrane have recently been identified as being associated with the disposition and detoxification of xenobiotics. On sinusoidal membranes, many types of drugs and toxins are taken up in hepatocytes by active transport systems and then are subjected to metabolic conversion and/or biliary excretion. Also, many reports have described that several types of xenobiotics and their conjugated metabolites are excreted from hepatocytes in bile by primary active transporters (12, 34, 54). Recently, such transporters on the bile canalicular membrane have been shown to excrete several types of drugs, such as hydroxymethylglutaryl- CoA reductase inhibitors (53), angiotensin-converting enzyme inhibitors (16), renin inhibitors (44, 55), and endothelin antagonists (43), in bile. Biliary excretion is one of the major elimination pathways for those compounds (17, 43, 44, 53, 55). Therefore, as far as the development of new drugs is concerned, it is becoming increasingly important to be able to predict biliary excretion in humans. Nevertheless, there are still few reports of species differences in such transport systems.
So far, four kinds of primary active transport systems for xenobiotics
and endogenous substrates, which are driven directly by cellular ATP
hydrolysis, have been identified on the rat bile canalicular membrane
(12, 34, 54): P-glycoprotein (P-gp, mdr-1), which excretes amphipathic
compounds; canalicular bile acid transporter; canalicular multispecific
organic anion transporter (cMOAT), a hepatocyte-specific homologue of
the multidrug resistance-associated protein (MRP), for organic anions;
and mdr-2, which transports phospholipids. Moreover, the existence of
another transporter for organic anions, apart from cMOAT, has been
suggested (15, 36). The discovery of mutant rats such as the
TR (22) and EHBR
(13, 31) strains, which have an inherited deficiency in their biliary
excretion system for organic anions, including the glucuronide or
glutathione conjugates of xenobiotics, has led to the identification of
the primary active transporter for organic anions (13, 22, 31).
Transporters other than such primary active transport systems have also
been identified for endogenous and xenobiotic compounds on bile
canalicular membranes: cystic fibrosis transmembrane conductance
regulator, a chloride channel that also transports bromide, iodide, and
fluoride (1); ectonucleotidase, a purine-specific
Na+-nucleotide cotransporter (7);
canalicular sulfate anion transporter-1, which transports sulfate
anions (such as oxalate; see Ref. 3); and canalicular organic
cation/H+ exchanger, which
transports organic cations (such as 1-methyl-4-phenylpyridinium; see
Ref. 32). Recent rapid progress in research in this area has been due
to the development of an isolation technique for bile canalicular
membrane vesicles (CMVs). Unfortunately, the study of biliary excretion
in humans has been limited because of the restricted availability of
human CMVs (50).
In the present study, we prepared six CMV preparations from humans and performed a transport study using representative substrates for the transporters that have already been identified in rats. We proposed to discover if these compounds are also substrates for the primary active transporter in humans.
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METHODS |
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Materials.
[3H]taurocholate
([3H]TCA, 3.47 µCi/nmol),
-[3H]estradiol
17-(
-D-glucuronide)
([3H]E2-17G, 51.0 µCi/nmol),
[3H]leukotriene
C4
([3H]LTC4,
52.0 µCi/nmol), and
[3H]methotrexate
([3H]MTX, 38.3 µCi/nmol) with purities of 98.5, 99.0, >95, and 99.4%, respectively, were purchased from New England Nuclear (Boston, MA).
Unlabeled and
[3H]2,4-dinitrophenyl-S-glutathione
([3H]DNP-SG, 44.8 µCi/nmol) were synthesized by the method of Kobayashi and colleagues
(25). Unlabeled and
[3H]sulfobromophthalein
glutathione
([3H]BSP-SG, 4.48 µCi/nmol) were synthesized enzymatically as described previously
(42). The purity of
[3H]DNP-SG and
[3H]BSP-SG was checked
by TLC and was 84.9 and 91.4%, respectively. [Prolyl-3,4(n)-3H]BQ-123
(sodium
cyclo[D-Trp-D-Asp-L-Pro-D-Val-L-Leu],
37 µCi/nmol) and
[14C]grepafloxacin
(GPFX, 31.6 µCi/mol) with purity of 97.4 and 97.1%, respectively,
were obtained from Amersham International (Buckinghamshire, UK). The
glucuronide of
[14C]GPFX
([14C]GPFXG) with a
purity of >99% was prepared from the bile of rats given
[14C]GPFX by infusion
(41).
[2-14C]6-Hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl)benzothiazole dihydrochloride
([14C]E3040; specific
activity, 50.9 µCi/mol; purity, 98.7%) was kindly donated by Eisai
(Tsukuba, Japan). The glucuronide of
[14C]E3040 (E-glu;
specific activity, 50.9 µCi/mol; purity, >99%) was
prepared by incubating E3040 with rat liver microsomes as described
previously (45).
[3H]pravastatin
(specific activity, 62 µCi/nmol; purity, 97.2%) was kindly donated
by Sankyo (Tokyo, Japan). ATP, creatine phosphate, creatine
phosphokinase, p-nitrophenylthymidine
5'-monophosphate, acivicin, and glutathione
S-transferase were purchased from
Sigma Chemical (St. Louis, MO). All other chemicals used were
commercially available and of reagent grade.
Preparation of CMV from human and rat
liver. Human liver samples were obtained from six
people (H1, a Hispanic male aged 34 yr; H2, a Caucasian female aged 21 yr; H3, a Caucasian female aged 10 yr; H4, a Hispanic female aged 47 yr; H5, a Caucasian male aged 35 yr; H6, a Caucasian female aged 45 yr;
H1 died from anoxic brain injury, and the others died from head trauma;
1 subject smoked, and none of the subjects drank alcohol). All of the
human CMV preparations originated from frozen livers except when the use of fresh liver was mentioned in the text. Male Sprague-Dawley rats
(250-300 g body wt) from Charles River Japan (Kanagawa, Japan) were used. CMV were prepared from human and Sprague-Dawley rat liver as
described previously (25) except that human liver was homogenized in a
Polytron homogenizer (Brinkmann Instruments, Westbury, NY) for 30 s
before the Dounce homogenizer step. Each human CMV preparation
originated from one individual human liver, whereas each rat CMV
preparation came from a pool of five to eight rat livers. Thus
R1-R17 represents the number of preparations. Next, 0.1 mM
phenylmethylsulfonyl fluoride was added to the homogenate. After
suspension in 50 mM Tris buffer (pH 7.4) containing 250 mM sucrose, the
CMV were frozen in liquid N2 and
stored at 100°C until used.
Determination of enzymatic activity of
CMV. To check the purity of the prepared CMV, the
activities of alkaline phosphatase (ALP), leucine aminopeptidase (LAP),
and -glutamyltranspeptidase (
-GTP) were determined by,
respectively, the method of Yachi et al. (51) and assay kits for LAP
and
-GTP (Wako Pure Chemical Industries, Osaka, Japan). Vesicle
"sidedness" was determined by measuring nucleotide
pyrophosphatase activity in the presence and absence of detergent (4).
The activity of CMV used in the present study was also checked by
measuring the ATP-dependent uptake of standard substrates,
[3H]TCA (1 µM) and
[3H]DNP-SG (1 µM),
in a 2-min incubation performed at 37°C. Protein concentrations
were determined as described previously (5), using the Bio-Rad protein
assay kit with BSA as a standard.
Uptake of ligands by CMV. The uptake study of ligands was studied as reported previously (8, 36). The transport medium (10 mM Tris, 250 mM sucrose, and 10 mM MgCl2 · 6H2O, pH 7.4) contained the ligands, 5 mM ATP, and an ATP-regenerating system (10 mM creatine phosphate and 100 µg/ml of creatine phosphokinase). Similar incubation without ATP, AMP, or the ATP regeneration system served as "the uptake in the absence of ATP." An aliquot of transport medium (16-18 µl) was mixed rapidly with the vesicle suspension (10 µg protein in 2-4 µl). The uptake study with GPFXG was performed using double these quantities. The transport reaction was stopped by the addition of 1 ml ice-cold stop solution containing 250 mM sucrose, 0.1 M NaCl, and 10 mM Tris · HCl (pH 7.4). The stopped reaction mixture was filtered through a 0.45-µm HA filter (Millipore, Bedford, MA) and then was washed two times with 5 ml ice-cold stop solution. The radioactivity retained on the filter and reaction mixture was combined with scintillation cocktail (Clear-sol I; Nacarai Tesque, Tokyo, Japan) and measured in a liquid scintillation counter (LS 6000SE; Beckman Instruments, Fullerton, CA). To ensure reliability in the determination of transport activity, the data were used only if the observed count in each sample was at least 10 times higher than the background count. The uptake of ligands was normalized in terms of both ligand concentrations in the medium and amount of membrane protein.
Effect of acivicin treatment on -GTP
activity of CMV. After pretreatment of the vesicle
suspension (10 µg protein in 4 µl) with or without acivicin (4 µl) at 25°C, the reaction was started by the addition of
transport medium (12 µl) containing glutathione conjugates
([3H]DNP-SG and
[3H]BSP-SG), ATP, and
the ATP-regenerating system at 37°C. The reaction was stopped by
the addition of 80 µl ice-cold ethanol, and the sample was extracted
by vortex mixing for 10 s. After sitting on ice for >5 min, the
sample was centrifuged for 30 s, and a 20-µl aliquot of supernatant
was spotted on a TLC plate (Silicagel LK6DF; Whatman, Clifton,
NJ). The plate was developed at a distance of ~10 cm using a
mobile phase of n-propyl
alcohol, water, and glacial acetic acid, 10:5:1 (vol/vol/vol). The zone
of interest was confirmed by irradiating the unlabeled compound with a
254-nm ultraviolet lamp [DNP-SG, retardation factor
(Rf) = 0.67; BSP-SG, Rf = 0.63]. Each zone was
scraped off, and the radioactivity was quantified. The ratio of intact
form to the total form was calculated by dividing the radioactivity of
the intact zone by that of the total zone.
Determination of kinetic parameters. The kinetic parameters for ligand uptake were estimated from the following equation
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(1) |
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RESULTS |
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Effect of acivicin on -GTP activity in
CMVs. In the present study, we pretreated human CMVs
with acivicin to avoid the degradation of glutathione conjugates by
-GTP. Acivicin pretreatment inhibited the degradation of DNP-SG
during incubation with human CMVs, and the maximum inhibitory effect
was observed when CMVs were pretreated with 1 mM acivicin for 30 min
(Fig. 1). After such pretreatment of CMVs,
81 and 90% of DNP-SG and BSP-SG remained intact after 2 min of
incubation with human CMVs, whereas only 37 and 44%, respectively,
remained intact without such pretreatment with acivicin (Table
1). Degradation of these glutathione
conjugates was much less in rat CMVs where ~90% remained intact
without acivicin pretreatment (Table 1). When the incubation period was
increased, the fraction of the intact form was considerably reduced in
human CMVs, and approximately one-half of the total amount of DNP-SG
and BSP-SG applied was degraded during 30 min of incubation (Table 1).
The uptake of BSP-SG over 2 min by human CMVs pretreated with acivicin was not very different from that without acivicin pretreatment, although degradation of BSP-SG was arrested by such acivicin
pretreatment (Table 1). This finding led us to consider the possibility
that the uptake of BSP-SG in human CMVs can be affected by degradation products if the CMVs are not treated with acivicin. In rat CMVs, the
uptake of DNP-SG in the absence of acivicin was lower than in its
presence (Table 1). Such a reduction was ~30% in R17 (Table 1) and
10% in R12 (data not shown). Therefore, even if the uptake study for
DNP-SG in rats was conducted in the absence of acivicin, the absolute
value for its uptake may be underestimated by, at most, 10-30%.
From these results, we decided to pretreat human CMVs with 1 mM
acivicin for 30 min and then determine the uptake over a 2-min period
in the presence of acivicin in human CMVs and in its absence in rat
CMVs, when the initial uptake of glutathione conjugates (DNP-SG,
BSP-SG, and LTC4) was measured.
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Enzymatic activity of each CMV
preparation. The activity of marker enzymes in CMVs
prepared from human and rat liver samples is summarized in Table
2. The average values for relative
enrichment (ratio of specific activity in membranes to the specific
activity in the homogenate) of marker enzymes for bile canalicular
membrane, ALP, and LAP were relatively comparable between human and rat CMVs, whereas that for -GTP was up to twofold higher in rat CMVs than in human CMVs (Table 2). The ratio of inside-out CMVs (IO) was
lower in rat CMVs (35%) compared with human CMVs (56%).
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Transport activity of human and rat
CMVs. Time profiles for the uptake of TCA, E-glu,
E2-17G, and LTC4 by human and
rat CMVs were examined (Fig. 2). In rat
CMVs, an ATP dependence was observed for all four compounds, and
overshoot phenomena were observed in the presence of ATP (Fig. 2). On
the other hand, in human CMVs, although ATP dependence was observed,
there was no overshoot phenomenon for any of these compounds (Fig. 2).
One of the possible explanations may be the slower level of ATP
consumption by human CMVs compared with rat CMVs. The other possibility
is that the contribution of nonspecific binding on CMVs to the apparent
uptake may be greater in the presence of ATP compared with that in its
absence, since there should be a difference in the osmolarity of the
extravesicular medium under the different conditions, resulting in the
smaller intravesicular volume in the presence of ATP. The binding
and/or incorporation of radiolabeled substrates to CMV may interfere with the detection of the equilibrium state. This may also result in no
overshoot phenomenon being observed in human CMVs. Because the uptake
of TCA, E-glu, and E2-17G was linear up to 1 min in rat CMVs (Fig.
2), the initial uptake of these compounds was determined at 1 min in subsequent studies. In the same way, the initial uptake of
LTC4 was determined at 2 min in
subsequent studies.
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The initial uptake rate data obtained in this way for several compounds
in various human and rat CMV preparations are summarized in Table 2.
The uptake is expressed as the clearance
(µl · min1 · mg
protein
1) by dividing the
uptake rate by the substrate concentration in the medium so that the
uptake abilities can be compared easily among the substrates. The
ATP-dependent uptake of TCA, glutathione conjugates (DNP-SG,
LTC4, and BSP-SG), and
glucuronides (E-glu, E2-17G, and GPFXG) was observed in
almost all human and rat CMV preparations examined (Table 2). For these
compounds, the ATP-dependent uptake was comparable or higher than the
ATP-independent uptake in both humans and rats (Table 2). On the other
hand, the ATP-dependent uptake of the other nonconjugated organic
anions, pravastatin, BQ-123, and MTX was much less than the
ATP-independent uptake and was not observed in some human CMV
preparations (Table 2). For pravastatin, the ATP-dependent and
-independent uptakes were about the same in three of six human CMV
preparations (Table 2). The absolute values for the ATP-dependent TCA
uptake in human CMV (14.8 ± 2.7 pmol · min
1 · mg
1
at 1 µM TCA, Table 2) were comparable with the value (9.0 ± 1.3 pmol · min
1 · mg
1
at 1 µM TCA) reported by Wolters and colleagues (49).
The average values of the ATP-dependent uptake rates by human and rat
CMVs are plotted in Fig. 3. The
ATP-dependent uptake of several compounds, other than glucuronides, in
human CMVs was ~ to 1/76 that in rat CMVs. A relatively
small difference was observed between human and rat CMVs as far as the ATP-dependent uptake of glucuronides (E-glu, E2-17G, and GPFXG) was concerned. Each plot of the ATP-dependent uptake of the
glucuronides appears to be located relatively higher than that of the
uptake of other compounds in terms of the relationship between humans and rats (P < 0.05; analysis of
covariance; Fig. 3).
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The concentration dependence of TCA, DNP-SG, and E-glu uptake was
examined, and typical Eadie-Hofstee plots are shown in Fig. 4. Saturable uptake was observed for each
compound both in human and rat CMVs, and the kinetic parameters
obtained are summarized in Table 3. There
was at most a twofold difference in the
Km and
Vmax of TCA
uptake between human and rat CMVs (Table 3). There was only a small
difference in the
Vmax for the
DNP-SG uptake between both types of CMVs, whereas the
Km was nine times
higher (P < 0.05) in CMVs from
humans than from rats (Table 3). On the other hand, the
Km and
Vmax of E-glu
uptake were four times and two times as high in human CMVs,
respectively, compared with rat CMVs, with the result that the
difference in the
Vmax/Km
was less than twofold between human and rat CMVs (Table 3).
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Effect of freezing on the enzymatic and
drug-transporting activity of human CMVs. Table
4 shows a comparison of the enzymatic and
uptake activity exhibited by CMVs prepared from fresh (not frozen)
human liver and from the same liver after freezing. Most of the
enzymatic activities and transport activities were approximately twofold lower in CMVs prepared from frozen human liver compared with
that in CMVs from fresh (not frozen) human liver (Table
4). In Table 4, the activity is also compared using CMVs
prepared from frozen human liver and 9 mo later from the same frozen
liver. No appreciable difference in enzymatic and transport activity was observed for the human CMVs treated in these different ways (Table
4).
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Correlation of transport activity in human
CMVs. In Table 5, the
correlation between transport activity in each human CMV preparation
was examined. The correlation in the ATP-dependent uptake of several
organic anions, glucuronides, and glutathione conjugates (DNP-SG vs.
LTC4, BSP-SG, and GPFXG; E-glu vs.
BSP-SG, E2-17G, and GPFXG), which are substrates for rat cMOAT
(13, 16, 19, 22, 25, 29, 30, 36, 41, 42, 45-47), was significant,
whereas that for the ATP-dependent uptake of TCA was not significantly
correlated with the ATP-dependent uptake of any other organic anions
(Table 5).
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DISCUSSION |
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Little information has been published on the mechanism governing the biliary excretion of endogenous and xenobiotic compounds across the bile canalicular membrane in humans. Wolters and colleagues (49) have provided evidence for the existence of a bile acid transporter for TCA when they found that TCA is taken up by human CMVs in an ATP-dependent manner. However, the transport mechanism for other organic anions has remained unidentified. In the present study, as shown in Figs. 2 and 4 and Table 2, the ATP-dependent and saturable uptake of anionic compounds such as DNP-SG and E-glu, both known to be predominantly transported via cMOAT in rats (19, 25, 36, 42, 45), also occurs in human CMVs. The uptake of other organic anions, such as glucuronides (E2-17G and GPFXG) and glutathione conjugate (BSP-SG and LTC4), also exhibits ATP dependence in human CMVs (Table 2 and Fig. 2). These results demonstrate the existence of primary active transporters for these organic anions in the human bile canalicular membrane.
A significant correlation was observed in the ATP-dependent uptake of
several organic anions by human CMVs. This was especially true for
glutathione conjugates (DNP-SG,
LTC4, and BSP-SG) and also
glucuronides (E-glu, E2-17G, and GPFXG; Table 5). One possible reason for such a correlation is that these compounds are transported, at least partially, via the common transporter, such as cMOAT, in
humans. On the other hand, there was no significant correlation between
the ATP-dependent uptake of TCA and that of any other organic anions
(Table 5). If the transporter for TCA and such organic anions is a
common one in humans, a good correlation should be clearly apparent. In
rats, the bile acid transporter is known to be different from cMOAT,
since primary active transport of TCA can also be observed in CMVs from
cMOAT-deficient rats, TR
(37), and EHBR (45). Thus the transporter for TCA seems likely to be
different from that for organic anions in humans too.
The primary active transport of glucuronides is comparable in humans and rats, whereas that of the other anionic compounds is greatly reduced in humans compared with rats (Fig. 3). In addition, the difference in kinetic parameters for the uptake between human and rat CMV is small for E-glu compared with DNP-SG (Fig. 4 and Table 3). Thus the species difference in primary active transport on the bile canalicular membrane depends on the type of substrate and is more marked for glutathione conjugates and other nonconjugated anionic compounds than glucuronides. In particular, ATP-dependent uptake for nonconjugated anions (pravastatin, BQ-123, and MTX) was observed in almost all of the rat CMV preparations examined, whereas there was no clear ATP dependence in some human CMV preparations (Table 2). Thus the species difference in the biliary excretion activity for these nonconjugated compounds was much more marked between humans and rats than was the case for glucuronides.
There are at least two possibilities to explain the present finding that the species difference in transport activity is much more marked for organic anions other than glucuronides. The first is that both glucuronides and other organic anions are transported by the common transporter in both humans and rats, and the substrate specificity of the human transporter favors glucuronides, whereas the rat transporter (cMOAT) is equally effective for both glucuronides and other compounds. The homology in the amino acid sequence of human cMOAT with that of rat cMOAT is 77.6% (48). Although there is such a high degree of homology, we should note the possibility that a minute difference in the amino acid sequence may result in the large difference in substrate specificity. For instance, in the case of 5-hydroxytryptamine (5-HT) receptors, although the human receptor gene shares 93% identity of the deduced amino acid sequence with rodent 5-HT1B receptors (38), it differs profoundly in terms of the affinity for many drugs. The replacement of a single amino acid in the human receptor with a corresponding asparagine found in the rodent 5-HT1B receptor renders the pharmacology of the receptors essentially identical (38). Thus minute sequence differences between homologues of the same receptor from different species can cause large pharmacological variations.
The other possibility involves the multiplicity in human organic anion transporters. If we consider that there are two transport systems, one preferentially recognizing glucuronides and the other recognizing both glucuronides and other anionic compounds, and bearing in mind that the transport efficiency and/or expression level of the latter transporter is much lower in humans than in rats, this might also explain the present findings. In rats, there are several reports that describe multiplicity in the organic anion transport system: DNP-SG is recognized predominantly by cMOAT, whereas E-glu can be recognized by another transporter in addition to cMOAT, because the uptake of E-glu by rat CMVs cannot be inhibited completely by DNP-SG, and ATP-dependent uptake of E-glu can also be observed in CMVs from EHBR (36). In rats, the primary active transport of several organic anions such as GPFXG (41), pravastatin (53), BQ-123 (43), the carboxylate form of irinotecan (CPT-11) (8, 9), and the carboxylate and lactone forms of SN-38 glucuronide (8, 9) cannot be completely attributed to cMOAT, since ATP-dependent uptake of these compounds can also be observed in CMVs from EHBR. The ATP-dependent uptake of SN-38 glucuronide by rat CMVs consists of, at least, two saturable components, the high-affinity component, which is deficient in EHBR, and the low-affinity component, which is present in EHBR (9). In humans, we recently identified two saturable components in the ATP-dependent uptake of the carboxylate forms of CPT-11 and SN-38 glucuronide by CMVs, implying that such multiplicity in organic anion transport systems is also present on the bile canalicular membrane in humans (10). Further research is necessary to clarify which of these two hypotheses may be true.
Recently, the cDNA cloning of cMOAT from humans (48) and from Wistar and Sprague-Dawley rats has been successfully achieved (6, 18, 40). The substrate specificity of rat cMOAT is similar to that of human MRP, which was found by Cole and colleagues (11) in a non-P-gp multidrug resistance cell line, the H69AR small-cell lung carcinoma line. MRP, P-gp, and cMOAT belong to the ATP-binding cassette (ABC) superfamily of transporter proteins (12, 34, 54), and they are able to act as plasma membrane pumps extruding drugs. P-gp recognizes predominantly amphipathic cationic and neutral compounds, whereas MRP recognizes anionic compounds such as LTC4 (23, 27, 33), DNP-SG (23, 33), and glutathione disulfide (27). In addition, we have recently identified two cDNA fragments encoding the carboxy terminal ABC region, which was amplified by RT-PCR from EHBR liver based on the homology with human MRP (15). The cloned full-length cDNA of the two fragments, designated MRP-like proteins (MLP-1 and MLP-2), exhibits amino acid sequences homologous with both rat cMOAT and human MRP and has characteristics of ABC transporters (15). The sequence alignment suggested that rat MLP-2 is a homologue of human MRP-3, the partial sequence of which has been reported recently (26). Although the presence of additional two kinds of MRP homologue (MRP-4 and -5) was reported (26), further research may lead to the discovery of novel ABC transporters in humans that are essential to clarify the molecular mechanism for the multiplicity and species differences in organic anion transport systems.
The enzymatic activity and its enrichment in each of the human CMV samples exhibited at most 2- to 4- and 2- to 10-fold intersample difference, respectively, whereas the transport activity of, for example, LTC4 exhibited a 48-fold difference (Table 2). Therefore, such a difference in LTC4 transport activity cannot simply be explained by the difference in membrane preparation but may also include the interindividual variability in its biliary excretion. However, by the present analysis, we cannot further discriminate between the effect of the difference in CMV preparation and such interindividual variability and, therefore, additional studies are needed to clarify the exact degree of biliary excretion of organic anions in humans.
It has been demonstrated that the clearance on drug metabolism in vivo can be extrapolated from in vitro data, i.e., the kinetic parameters for enzymatic reactions being estimated from in vitro studies using isolated hepatocytes or subcellar fractions such as microsomes and 9,000 g supernatants (20, 21). The parameters obtained can then be converted to values for the whole organ by taking into account the enzyme mass recovery for the preparation used (mg microsomal protein/g liver and/or nmol P-450/g liver; see Refs. 20 and 21). It is also desirable to be able to predict biliary excretion in vivo in humans from in vitro CMV uptake studies in a similar manner. In the case of biliary excretion, it may be possible to scale up CMV uptake data in biliary excretion activity in vivo by using the following equation
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(2) |
In the present study, we determined the enrichment for several marker
enzymes in human CMVs and found that enrichment differs for each marker
enzyme even in the same human CMV preparation (Table 2). If such
enzymes are exclusively located on the bile canalicular membrane, the
enrichment in the same membrane preparation should be the same for each
enzyme. The reason for this discrepancy in enrichment is still unknown,
although one possible reason may be that these marker enzymes are not
exclusively located on the bile canalicular membrane but are also
present in other organelles. In fact, there are some reports that LAP
and -GTP are also present in microsomal fractions (14, 24, 39).
Moreover, microsomal
-GTP activity is induced by ethanol (2). In the
light of such observations, it is difficult to determine the value for
enrichment (E). Because intracellular localization of the other two
marker enzymes is controversial, we assumed that E is equal to the
enrichment of ALP activity and calculated the
CLCMV for each ligand in both humans and rats where the average values of initial velocity
(Vinitial), R, E, and IO, shown in Table 2, were used. The
CLCMV for TCA was 8.7 times higher
in rats than in humans. The CLCMV
for glutathione conjugates (DNP-SG,
LTC4, and BSP-SG) and glucuronides
(E-glu, E2-17G, and GPFXG) was 12.2-39.5 and 1.9-4.1
times higher, respectively, in rats than in humans. Thus a species
difference seems to exist in the primary active transport, per gram
liver, of organic anions.
Further detailed in vivo studies are needed to confirm this hypothesis, but measurement of the biliary excretion in humans is not easy, and such information is quite limited. Therefore, another possible method for the prediction of biliary excretion in vivo is to use the information on a reference compound in which biliary excretion is already known. If the CLCMV and Vinitial for such a reference compound (CLCMV,reference and Vinitial,reference, respectively) have already been reported in humans, the corresponding values for test compounds (CLCMV,test and Vinitial,test, respectively) can be represented as
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(3) |
For certain types of drugs that are used to treat patients with renal failure, biliary excretion might be a more desirable elimination pathway because the pharmacokinetics of drugs mainly eliminated by urinary excretion usually exhibit a large interindividual variability in such patients. Actually, it has been reported that the plasma concentrations of temocaprilat, an angiotensin-converting enzyme inhibitor that is excreted in bile via cMOAT in rats (17), are not changed as much in patients with renal disease compared with other angiotensin-converting enzyme inhibitors, which are mainly eliminated via urine. Therefore, for the development of new drugs that will be used in patients with renal failure, uptake studies using human CMVs may offer a useful screening system to identify compounds that are recognized by transporters with preferentially high affinity and that are excreted in bile. On the other hand, it should also be remembered that efficient biliary excretion hinders the development of certain types of peptidic compounds, such as renin inhibitors (44, 55) and endothelin antagonists (35, 43). For example, BQ-123 is rapidly eliminated from the body in rats, with almost 90% of an intravenous dose being recovered in the bile (35). The biliary excretion of this compound is mainly mediated by cMOAT in rats (43). The present study indicated that seven out of eight CMV preparations exhibited significant ATP-dependent uptake of BQ-123 in rats, whereas only three out of six CMV preparations exhibited such ATP-dependent uptake in humans (Table 2). Thus it may be that the biliary excretion of such compounds is not as efficient but does exhibit a degree of interindividual variability in humans compared with that in rats. Such lower transport activity in humans was also observed in the case of pravastatin and MTX (Table 2). However, we cannot conclude from the present data alone that the contribution of the biliary excretion of these compounds to their overall elimination is lower in humans compared with rats, since the ratio of the amount excreted in bile to the injected dose is affected both by the biliary excretion activity and the total body clearance. Therefore, even if ATP-dependent uptake is very weak and below the detection limit in some human CMVs, the amount excreted in bile may still be high if the total body clearance is also low in such humans. As far as the development of new drugs is concerned, further studies are needed to support the validity of using human CMV uptake studies as a screening system to examine biliary excretion in humans.
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ACKNOWLEDGEMENTS |
---|
We acknowledge Sankyo, Eisai, Otsuka Pharmaceutical, and Banyu Pharmaceutical for providing pravastatin, E3040 and E-glu, GPFX and GPFXG, and BQ-123, respectively.
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FOOTNOTES |
---|
This work was supported in part by a Grant-in-Aid for Scientific Research provided by the Ministry of Education, Science, and Culture of Japan, in part by a grant for Cancer Research from the Ministry of Health and Welfare of Japan, and in part by CREST, Japan Science and Technology Corporation.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: Y. Sugiyama, Graduate School of Pharmaceutical Sciences, Univ. of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (E-mail: sugiyama{at}seizai.f.u-tokyo.ac.jp).
Received 3 April 1998; accepted in final form 6 January 1999.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anderson, M. P.,
R. J. Gregory,
S. Thompson,
D. W. Souza,
S. Paul,
R. C. Mulligan,
A. E. Smith,
and
M. J. Welsh.
Demonstration that CFTR is a chloride channel by alteration of its anion selectivity.
Science
253:
202-205,
1991[Medline].
2.
Barouki, R.,
M. Chobert,
J. Finidori,
M. Aggerbeck,
B. Nalpas,
and
J. Hanoune.
Ethanol effects in a rat hepatoma cell line: induction of -glutamyltransferase.
Hepatology
3:
323-329,
1983[Medline].
3.
Bissig, M.,
B. Hagenbuch,
B. Stieger,
T. Koller,
and
P. J. Meier.
Functional expression cloning of the canalicular sulfate transport system of rat hepatocytes.
J. Biol. Chem.
269:
3017-3021,
1994
4.
Bohme, M.,
M. Muller,
I. Leier,
G. Jedlitschky,
and
D. Keppler.
Cholestasis caused by inhibition of the adenosine triphosphate-dependent bile salt transport in rat liver.
Gastroenterology
107:
255-265,
1994[Medline].
5.
Bradford, M. M.
A rapid and sensitive method for the quantitation of microsomal quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:
248-254,
1976[Medline].
6.
Buchler, M.,
J. Konig,
M. Brom,
J. Kartenbeck,
H. Spring,
T. Horie,
and
D. Keppler.
cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMrp, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats.
J. Biol. Chem.
271:
15091-15098,
1996
7.
Che, M.,
D. F. Ortiz,
and
I. M. Arias.
Primary structure and functional expression of a cDNA encoding the bile canalicular, purine-specific Na+-nucreoside cotransporter.
J. Biol. Chem.
270:
13596-13599,
1995
8.
Chu, X.,
Y. Kato,
K. Niinuma,
K. Sudo,
H. Hakusui,
and
Y. Sugiyama.
Multispecific organic anion transporter is responsible for the biliary excretion of the camptothecin derivative irinotecan and its metabolites in rats.
J. Pharmacol. Exp. Ther.
281:
304-314,
1997
9.
Chu, X.,
Y. Kato,
and
Y. Sugiyama.
Multiplicity of biliary excretion mechanisms for irinotecan, CPT-11, and its metabolites in rats.
Cancer Res.
57:
1934-1938,
1997[Abstract].
10.
Chu, X.,
Y. Kato,
K. Ueda,
H. Suzuki,
K. Niinuma,
C. A. Tyson,
V. Weizer,
J. E. Dabbs,
R. Froehlich,
C. E. Green,
and
Y. Sugiyama.
Biliary excretion mechanism of CPT-11 and its metabolites in humans: involvement of primary active transporters.
Cancer Res.
58:
5137-5143,
1998[Abstract].
11.
Cole, S. P.,
G. Bhardwaj,
J. H. Gerlach,
J. E. Mackie,
C. E. Grant,
K. C. Almquist,
A. J. Stewart,
E. U. Kurz,
A. M. Duncan,
and
R. G. Deeley.
Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line.
Science
258:
1650-1654,
1992[Medline].
12.
Elferink, R. P. J. O.,
D. K. F. Meijer,
F. Kuipers,
P. L. M. Jansen,
A. K. Groen,
and
G. M. M. Groothuis.
Hepatobiliary secretion of organic compounds; molecular mechanism of membrane transport.
Biochim. Biophys. Acta
1241:
215-268,
1995[Medline].
13.
Fernandez-Checa, J. C.,
H. Takikawa,
T. Horie,
M. Ookhtens,
and
N. Kaplowitz.
Canalicular transport of reduced glutathione in normal and mutant Eisai hyperbilirubinemic rats.
J. Biol. Chem.
267:
1667-1673,
1992
14.
Goldberg, D. M.
Structual, functional, and clinical aspects of -glutamyl transferase.
Crit. Rev. Clin. Lab. Sci.
12:
1-58,
1980.
15.
Hirohashi, T.,
H. Suzuki,
K. Ito,
K. Ogawa,
K. Kume,
T. Shimizu,
and
Y. Sugiyama.
Hepatic expression of multidrug resistance-associated protein (MRP)-like proteins maintained in Eisai hyperbilirubinemic rats (EHBR).
Mol. Pharmacol.
53:
1068-1075,
1998
16.
Ishikawa, T.,
M. Muller,
C. Klunemann,
T. Schaub,
and
D. Keppler.
ATP-dependent primary active transport of cysteinyl leukotrienes across liver canalicular membrane.
J. Biol. Chem.
265:
19279-19286,
1990
17.
Ishizuka, H.,
K. Konno,
H. Naganuma,
K. Sasahara,
Y. Kawahara,
K. Niinuma,
H. Suzuki,
and
Y. Sugiyama.
Temocaprilat, a novel angiotensin-converting enzyme inhibitor, is excreted in bile via an ATP-dependent active transporter (cMOAT) that is deficient in Eisai hyperbilirubinemic mutant rats (EHBR).
J. Pharmacol. Exp. Ther.
280:
1304-1311,
1997
18.
Ito, K.,
H. Suzuki,
T. Hirohashi,
K. Kume,
T. Shimizu,
and
Y. Sugiyama.
Molecular cloning of canalicular multispecific organic anion transporter defective in EHBR.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G16-G22,
1997
19.
Ito, K.,
H. Suzuki,
T. Hirohashi,
K. Kume,
T. Shimizu,
and
Y. Sugiyama.
Functional analysis of a canalicular multispecific organic anion transporter cloned from rat liver.
J. Biol. Chem.
273:
1684-1688,
1998
20.
Iwatsubo, T.,
N. Hirota,
T. Ooie,
H. Suzuki,
N. Shimada,
K. Chiba,
T. Ishizaki,
C. E. Green,
C. A. Tyson,
and
Y. Sugiyama.
Prediction of in vivo drug metabolism in the human liver from in vitro metabolism data.
Pharmacol. Ther.
73:
147-171,
1997[Medline].
21.
Iwatsubo, T.,
N. Hirota,
T. Ooie,
H. Suzuki,
and
Y. Sugiyama.
Prediction of in vivo drug disposition from in vitro data based on physiological pharmacokinetics.
Biopharm. Drug Dispos.
17:
273-310,
1996[Medline].
22.
Jansen, P. L. M.,
W. H. M. Peters,
and
W. H. Lamers.
Hereditary chronic conjugated hyperbilirubinemia in mutant rats caused by defective hepatic anion transport.
Hepatology
5:
573-579,
1985[Medline].
23.
Jedlitschky, G.,
I. Leier,
U. Buchholz,
M. Center,
and
D. Keppler.
ATP-dependent transport of glutathione S-conjugates by the multidrug resistance associated protein.
Cancer Res.
54:
4833-4836,
1994[Abstract].
24.
Kanno, T.,
M. Maekawa,
S. Kanda,
H. Kohno,
and
K. Sudo.
Evaluation of cytosolic aminopeptidase in human sera, evaluation in hepatic disorders.
Am. J. Clin. Pathol.
82:
700-705,
1984[Medline].
25.
Kobayashi, K.,
Y. Sogame,
H. Hara,
and
K. Hayashi.
Mechanism of glutathione S-conjugate transport in canalicular and basolateral rat liver plasma membranes.
J. Biol. Chem.
265:
7737-7741,
1990
26.
Kool, M.,
M. de Haas,
G. L. Scheffer,
R. J. Scheper,
M. J. van Eijk,
J. A. Juijn,
F. Baas,
and
P. Borst.
Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines.
Cancer Res.
57:
3537-3547,
1997[Abstract].
27.
Leier, I.,
G. Jedlitschky,
U. Buchholz,
M. Center,
S. P. Cole,
R. G. Deeley,
and
D. Keppler.
ATP-dependent glutathione disulphide transport mediated by the MRP gene-encoded conjugate export pump.
Biochem. J.
314:
433-437,
1996[Medline].
28.
Lokiec, F.,
P. Canal,
C. Gay,
E. Chatelut,
J. P. Armand,
H. Roche,
R. Bugat,
E. Goncalves,
and
B. A. Mathincu.
Pharmacokinetics of irinotecan and its metabolites in human blood, bile, and urine.
Cancer Chemother. Pharmacol.
36:
79-82,
1995[Medline].
29.
Madon, J.,
U. Eckhardt,
T. Gerloff,
B. Stieger,
and
P. J. Meier.
Functional expression of the rat liver canalicular isoform of the multidrug resistance-associated protein.
FEBS Lett.
406:
75-78,
1997[Medline].
30.
Mayer, R.,
J. Kartenbeck,
M. Buchler,
G. Jedlitschky,
I. Leier,
and
D. Keppler.
Expression of the MRP gene-encoded conjugate export pump in liver and its selective absence from the canalicular membrane in transport-deficient mutant hepatocytes.
J. Cell Biol.
131:
137-150,
1995[Abstract].
31.
Mikami, T.,
T. Nozaki,
O. Tagaya,
S. Hosokawa,
T. Nakura,
H. Mori,
and
S. Kondou.
The characters of a new mutant in rats with hyperbilirubinuria syndrome.
Cong. Anom.
26:
250-251,
1986.
32.
Moseley, R. H.,
L. J. Zugger,
and
R. W. V. Dyke.
The neurotoxin 1-methyl-4-phenylpyridinium is a substrate for the canalicular organic cation/H+ exchanger.
J. Pharmacol. Exp. Ther.
281:
34-40,
1997
33.
Muller, M.,
C. Meijer,
G. J. Zaman,
P. Borst,
R. J. Scheper,
N. H. Mulder,
E. G. de Vries,
and
P. L. Jansen.
Overexpression of the gene encoding the multidrug resistance-associated protein results in increased ATP-dependent glutathione S-conjugate transport.
Proc. Natl. Acad. Sci. USA
91:
13033-13037,
1994
34.
Muller, M.,
H. Roelofsen,
and
P. L. Jansen.
Secretion of organic anions by hepatocytes: involvement of homologues of the multidrug resistance protein.
Semin. Liver Dis.
16:
211-219,
1996[Medline].
35.
Nakamura, T.,
A. Hisaka,
Y. Sasaki,
Y. Suzuki,
K. Ishikawa,
M. Yano,
and
Y. Sugiyama.
Carrier mediated active transport of BQ-123 a peptidic endothelin antagonist, into rat hepatocytes.
J. Pharmacol. Exp. Ther.
278:
564-572,
1996[Abstract].
36.
Niinuma, K.,
O. Takenaka,
T. Horie,
K. Kobayashi,
Y. Kato,
H. Suzuki,
and
Y. Sugiyama.
Kinetic analysis of the primary active transport of conjugated metabolites across the bile canalicular membrane: comparative study of S-(2,4-dinitrophenyl)-glutathione and 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl)benzothiazole glucuronide.
J. Pharmacol. Exp. Ther.
282:
866-872,
1997
37.
Nishida, T.,
C. Hardenbrook,
Z. Gatmaitan,
and
I. M. Arias.
ATP-dependent organic anion transport system in normal and TR rat liver canalicular membranes.
Am. J. Physiol.
262 (Gastrointest. Liver Physiol. 25):
G629-G635,
1992
38.
Oksenberg, D.,
S. A. Marsters,
B. F. O'Dowd,
H. Jin,
S. Havlik,
S. J. Peroutka,
and
A. Ashkenazi.
A single amino-acid difference confers major pharmacological variation between human and rodent 5-HT1B receptors.
Nature
360:
161-163,
1992[Medline].
39.
Patterson, E. K.,
S. Hsiao,
and
A. Keppel.
Studies on dipeptidases and aminopeptidases. I. Distinction between leucine aminopeptidase and enzymes that hydrolyze L-leucyl--naphthylamide.
J. Biol. Chem.
238:
3611-3620,
1963
40.
Paulusma, C. C.,
P. J. Bosma,
G. J. R. Zaman,
C. T. M. Bakker,
M. Otter,
G. L. Scheffer,
R. S. J. Scheper,
P. Borst,
and
P. J. Oude Elferink.
Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene.
Science
271:
1126-1128,
1996[Abstract].
41.
Sasabe, H.,
A. Tsuji,
and
Y. Sugiyama.
Carrier-mediated mechanism for the biliary excretion of a quinolone antibiotic, grepafloxacin and its glucuronide in rats.
J. Pharmacol. Exp. Ther.
284:
1033-1039,
1998
42.
Saxena, M.,
and
G. B. Henderson.
ATP-dependent efflux of 2,4-dinitrophenyl-S-glutathione.
J. Biol. Chem.
270:
5312-5319,
1995
43.
Shin, H.,
Y. Kato,
T. Yamada,
K. Niinuma,
A. Hisaka,
and
Y. Sugiyama.
Hepatobiliary transport mechanism for the cyclopentapeptide endothelin antagonist BQ-123.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G979-G986,
1997
44.
Takahashi, H.,
R. B. Kim,
P. R. Perry,
and
G. R. Wilkinson.
Characterization of the hepatic canalicular membrane transport of a model oligopeptide: ditekiren.
J. Pharmacol. Exp. Ther.
281:
297-303,
1997
45.
Takenaka, O.,
T. Horie,
H. Suzuki,
K. Kobayashi,
and
Y. Sugiyama.
Kinetic analysis of hepatobiliary transport for conjugative metabolites in the perfused liver of mutant rats (EHBR) with hereditary conjugative hyperbilirubinemia.
Pharm. Res.
12:
1746-1755,
1995[Medline].
46.
Takikawa, H.,
N. Sano,
T. Narita,
Y. Uchida,
M. Yamanaka,
T. Horie,
T. Mikami,
and
O. Tagaya.
Biliary excretion of bile acid conjugates in a hyperbilirubinemic mutant Sprague-Dawley rat.
Hepatology
14:
352-360,
1991[Medline].
47.
Takikawa, H.,
R. Yamazaki,
N. Sano,
and
M. Yamanaka.
Biliary excretion of estradiol-17-glucuronide in the rat.
Hepatology
23:
607-613,
1996[Medline].
48.
Taniguchi, K.,
M. Wada,
K. Kohno,
T. Nakamura,
T. Kawabe,
M. Kawakami,
K. Kagotani,
K. Okumura,
S. Akiyama,
and
M. Kuwano.
A human canalicular multispecific organic anion transporter (cMOAT) gene is overexpressed in cisplatin-resistant human cancer cell lines with decreased drug accumulation.
Cancer Res.
56:
4124-4129,
1996[Abstract].
49.
Wolters, H.,
F. Kuipers,
M. J. H. Slooff,
and
R. J. Vonk.
Adenosine triphosphate-dependent taurocholate transport in human liver plasma membranes.
J. Clin. Invest.
90:
2321-2326,
1992[Medline].
50.
Wolters, H.,
M. Spiering,
A. Gerding,
M. J. H. Slooff,
F. Kuipers,
M. J. Hardonk,
and
R. J. Vonk.
Isolation and characterization of canalicular and basolateral plasma membrane fractions from human liver.
Biochim. Biophys. Acta
1069:
61-69,
1991[Medline].
51.
Yachi, K.,
Y. Sugiyama,
Y. Sawada,
T. Iga,
Y. Ikeda,
G. Toda,
and
M. Hanano.
Characterization of rose bengal binding to sinusoidal and canalicular plasma membrane from rat liver.
Biochim. Biophys. Acta
978:
1-7,
1989[Medline].
52.
Yamaoka, K.,
Y. Tanigawara,
Y. Nakagawa,
and
T. Uno.
A pharmacokinetic analysis program (MULTI) for microcomputer.
J. Pharmacobio-Dyn.
4:
879-885,
1981[Medline].
53.
Yamazaki, M.,
K. Kobayashi,
and
Y. Sugiyama.
Primary active transport of pravastatin across the liver canalicular membrane in normal and mutant Eisai hyperbilirubinemic rats.
Biopharm. Drug Dispos.
17:
607-621,
1996[Medline].
54.
Yamazaki, M.,
H. Suzuki,
and
Y. Sugiyama.
Recent advances in carrier-mediated hepatic uptake and biliary excretion of xenobiotics.
Pharm. Res.
13:
497-513,
1996[Medline].
55.
Ziegler, K.,
C. Kolac,
and
W. Ising.
ATP-dependent transport of the linear renin-inhibiting peptide EMD-51921 by canalicular plasma membrane vesicles of rat liver: evidence of drug-stimulatable ATP-hydrolysis.
Biochim. Biophys. Acta
1196:
209-217,
1994[Medline].