Primary active transport of organic anions on bile canalicular membrane in humans

Kayoko Niinuma1, Yukio Kato1, Hiroshi Suzuki1, Charles A. Tyson2, Valorie Weizer2, Jack E. Dabbs2, Ritchie Froehlich2, Carol E. Green2, and Yuichi Sugiyama1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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), beta -estradiol 17-(beta -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


    INTRODUCTION
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INTRODUCTION
METHODS
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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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Materials. [3H]taurocholate ([3H]TCA, 3.47 µCi/nmol), beta -[3H]estradiol 17-(beta -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 gamma -glutamyltranspeptidase (gamma -GTP) were determined by, respectively, the method of Yachi et al. (51) and assay kits for LAP and gamma -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 gamma -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
<IT>V</IT><SUB>o</SUB>/S = <IT>V</IT><SUB>max</SUB>/(<IT>K</IT><SUB>m</SUB> + S) + <IT>P</IT><SUB>dif</SUB> (1)
where Vo is the initial uptake rate of ligand by CMV (pmol · min-1 · mg protein-1), S is the ligand concentration in the medium (µM), Km is the Michaelis constant (µM), Vmax is the maximum uptake rate (pmol · min-1 · mg protein-1), and Pdif is the nonspecific uptake clearance (µl · min-1 · mg protein-1). The above equation was fitted to the uptake data by an iterative nonlinear least-squares method using a MULTI program (52) to obtain estimates of the kinetic parameters. The input data were weighted as the reciprocal of the square of the observed values, and the algorithm used for the fitting was the Damping Gauss Newton method (52).


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Effect of acivicin on gamma -GTP activity in CMVs. In the present study, we pretreated human CMVs with acivicin to avoid the degradation of glutathione conjugates by gamma -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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Fig. 1.   Stability of 2,4-dinitrophenyl-S-glutathione (DNP-SG) during 2 min incubation with human canalicular membrane vesicles (CMV) preincubated with acivicin for various time periods. After preincubation of human CMV (H1) with the indicated concentration of acivicin at 25°C for the periods shown, DNP-SG was then added and incubated for 2 min at 37°C. The ratio of the intact form was determined by TLC (see METHODS). Each value represents the mean (n = 2 determinations).


                              
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Table 1.   Stability of DNP-SG and BSP-SG during various incubation periods with CMV in the presence or absence of acivicin

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 gamma -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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Table 2.   Characterization of human and rat CMV

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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Fig. 2.   Time profiles for the uptake of [3H]taurocholic acid ([3H]TCA; A and B), [3H]leukotriene C4 ([3H]LTC4; C and D), [14C]E3040 glucuronide ([14C]E-glu; E and F), and beta -[3H]estradiol 17-(beta -D-glucuronide) ([3H]E2-17G; G and H) by CMV prepared from human (H1; A, C, E, and G) and rat (R17; B, D, F, and H). After 3 min of preincubation, the reaction was started by adding CMV (10 µg of protein). Reaction mixtures were incubated at 37°C, with or without ATP (5 mM) and the ATP-regenerating system in the medium. , Total uptake (uptake with ATP and the ATP-regenerating system); open circle , ATP-independent uptake (uptake without ATP, AMP, or ATP-regenerating system). Concentration of [3H]TCA, [3H]LTC4, [14C]E-glu, and [3H]E2-17G was 1.0, 0.1, 20, and 0.05 µM, respectively. For [3H]LTC4 uptake, CMV were preincubated at 25°C for 30 min with 1 mM acivicin. Uptake amount (dpm/mg protein) was normalized by the ligand concentration (dpm/µl) in the medium. Each point represents the mean of 2 determinations.

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 · min-1 · 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 ~<FR><NU>1</NU><DE>3</DE></FR> 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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Fig. 3.   Comparison of the initial uptake for ATP-dependent uptake of several compounds by CMV prepared from humans and rats. Uptake rate (dpm · min-1 · mg protein-1) was normalized by the ligand concentration (dpm/µl) in the medium. Average values for ATP-dependent uptake in human and rat CMV were cited from Table 2. Data for methotrexate were not used, since only one CMV preparation from human showed ATP-dependent uptake (see Table 2). Broken line indicates 1-to-1 correlation. BSP-SG, sulfobromophthalein glutathione; GPFXG, grepafloxacin glucuronide.

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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Fig. 4.   Eadie-Hofstee plots of [3H]TCA (A and B), [3H]DNP-SG (C and D), and [14C]E-glu (E and F) uptake by CMV prepared from humans (H2; A, C, and E) and rats (R12; B, D, and F). CMV were incubated at 37°C for 1 min (TCA and E-glu) or 2 min (DNP-SG) with or without ATP and the ATP-regenerating system in the medium (open circle ). ATP-dependent uptake () was obtained by subtracting the value in the absence of ATP from that in its presence. For [3H]DNP-SG uptake, each human CMV preparation was preincubated at 25°C for 30 min with 1 mM acivicin. Each point and vertical bar represents the mean ± SE of 3 determinations. Solid lines are the fitted lines based on Eq. 1. Kinetic parameters obtained are listed in Table 3. Vo, initial uptake rate; S, ligand concentration in medium.


                              
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Table 3.   Kinetic parameters for ATP-dependent uptake of TCA, DNP-SG, and E-glu by CMV from human and rat

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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Table 4.   Characterization of CMV prepared from fresh and frozen human liver or that prepared after 9 mo

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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Table 5.   Correlation of transport activity in human CMV


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
CL<SUB>CMV</SUB> = (<IT>V</IT><SUB>initial</SUB> × R)/(E × IO) (2)
where CLCMV and Vinitial represent the biliary excretion clearance defined as the biliary excretion rate divided by the unbound hepatic concentration in vivo (µl · min-1 · g liver-1) and the initial ATP-dependent uptake velocity in CMVs normalized both by medium concentration and membranous protein (µl · min-1 · mg protein-1). The terms R and E are the recovery of liver homogenate protein (mg homogenate protein/g weight of original liver sample) and enrichment of the CMV fraction. In this extrapolation, we should note the artificial nature of CMVs, which may lead to the observation that in vitro data from rat CMVs do not always correlate well with data from hepatocytes or in vivo biliary excretion data in rats. For example, contamination of sinusoidal membranes and other organelles in the CMV preparation and/or the nonspecific adsorption of substrates, especially those that are highly hydrophobic, on CMVs cannot be completely excluded in in vitro transport studies. To verify the extrapolation method based on Eq. 2, systematic analysis of the biliary excretion of a series of compounds, both in vivo and in vitro, has to be performed in experimental animals. Such analysis requires the direct determination of CLCMV in vivo, by analyzing the biliary excretion rate, hepatic concentration, and unbound fraction inside the liver. Because the measurement of the hepatic concentration in vivo in humans is very limited, this approach should first be used in experimental animals. It is also important to evaluate the in vivo biliary clearance, defined as the ratio of the biliary excretion rate divided by the plasma concentration. Therefore, the relationship between CLCMV and such biliary clearance should also be clarified using experimental animals.

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 gamma -GTP are also present in microsomal fractions (14, 24, 39). Moreover, microsomal gamma -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
CL<SUB>CMV,test</SUB>/CL<SUB>CMV,reference</SUB> = <IT>V</IT><SUB>initial,test</SUB>/<IT>V</IT><SUB>initial,reference</SUB> (3)
Therefore, when Vinitial,test is determined by a CMV uptake study, we can estimate CLCMV,test without considering E and IO. In this extrapolation method, reference compounds should be structurally similar or at least be likely to share the same transport system in the CMVs. Several reports have been published involving measurement of the biliary excretion of the anti-cancer agent CPT-11 and its metabolites in patients with hepatic metastasis after cannulation of their bile duct (28). Because the primary active transport system (cMOAT) is involved in the biliary excretion of CPT-11 and its metabolites (9, 10), these compounds might be useful as reference compounds to calculate the biliary excretion clearance of other compounds excreted in bile mainly by cMOAT.

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.


    ACKNOWLEDGEMENTS

We acknowledge Sankyo, Eisai, Otsuka Pharmaceutical, and Banyu Pharmaceutical for providing pravastatin, E3040 and E-glu, GPFX and GPFXG, and BQ-123, respectively.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 276(5):G1153-G1164
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