Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, 263-8675, Japan
Submitted 25 September 2003 ; accepted in final form 18 February 2004
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ABSTRACT |
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bile flow; multidrug resistance-associated protein; -lactam
Several -lactam antibiotics are known to induce choleresis. Verkade et al. (35) reported that intravenous administration of ampicillin (180 µmol/kg) to rats induced choleresis, but this was not observed in Mrp2-deficient TR rats. Gonzalez et al. (14) reported that 0.33 mmol/kg piperacillin also induced choleresis in rats, and this was mainly explained by the stimulation of BSIDF. Biliary excretion of
-lactam antibiotics also appears to be mediated by Mrp2, because excretion in bile was dramatically reduced in Mrp2-deficient rats (29, 35). These results suggest an interaction of
-lactams with Mrp2 acting as substrates and/or stimulators, although the precise mechanism remains to be elucidated.
Recently, MRP2 transport activity was shown to be stimulated by a number of compounds including probenecid, indomethacin, and bile acids (3, 17, 39). Of these, MRP2-mediated GSH export was reported to be stimulated in the presence of indomethacin, sulfinpyrazone, and vinblastine (11). Sulfinpyrazone is thought to be a substrate of MRP2 even in the absence of GSH, and it produced further stimulation in the presence of GSH via a cotransport mechanism (11). Vinblastine is cotransported with GSH, whereas indomethacin is a poor substrate of MRP2. Although no simple explanation is available, multiple sites are considered to be involved in the explanation of those results. Evers and colleagues (6, 11) proposed that MRP2, as well as perhaps MRP1, also has a G-site (GSH binding site) and a D-site (drug binding site). These two sites work by positive cooperation. These hypotheses need to be verified experimentally in the future. Importantly, there is no published report investigating whether such stimulatory compounds also exhibit a choleretic effect in vivo.
In this study, the physiological and pharmacological significance of these stimulatory effects on Mrp2 transport function was examined to help our understanding of the bile formation mechanism. In particular, the choleretic effect of benzylpenicillin (PCG) was studied in vivo and was also examined in vitro using Mrp2/MRP2 expression systems. As a result, we initially demonstrated that a compound that stimulates GSH efflux in vitro actually leads to in vivo choleresis via the stimulation of GSH excretion into bile.
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MATERIALS AND METHODS |
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[phenyl-4(n)-3H]PCG (1030 Ci/mmol) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). [3H]dinitrophenyl glutathione (DNP-SG; 50.0 Ci/mmol) was synthesized enzymatically using [glycine-2-3H]glutathione (New England Nuclear Life Science Products, Boston, MA) and 1-chloro-2,4-dinitrobenzene and glutathione S-transferase (Sigma, St. Louis, MO) as described previously (19). [3H]17-estradiol 17-(
-D-glucuronide) (E217
G; 55 Ci/mmol) was purchased from New England Nuclear Life Science Products. PCG potassium was purchased from Wako Pure Chemical Industries (Osaka, Japan).
Animals.
Male Sprague-Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBR) weighing 240300 g (78 wk old) were used throughout the experiments (Japan SLC, Shizuoka, Japan).
Blood and bile sampling.
SD rats and EHBR were maintained under ether anesthesia, and then the femoral artery and vein were cannulated with polyethylene tubing (PE-50) for PCG administration and blood sampling. The common bile duct was also cannulated with PE-10 tubing. [3H]PCG (180 µmol·ml1·kg1; 6.7 µCi/kg) in saline was administered intravenously through the femoral vein cannula. Blood samples were obtained at given times. Plasma and bile concentrations of [3H]PCG were determined by measuring the radioactivity in a liquid scintilation counter (model LSC5000; Aloka, Tokyo, Japan). Retrograde acivicin, an inhibitor of -glutamyl transpeptidase, was preinfused into the bile duct for the determination of GSH in the bile (18). Briefly, acivicin (Sigma) was dissolved in Krebs-Henseleit buffer (50 mM) and infused for 1 min via the bile cannula (100 µl/rat), which was occluded for 1 min and then released for 10 min to allow a constant bile flow rate to be reestablished. PCG or saline was administered after an additional 15 min. Rates of bile flow, bilirubin, and PCG excretion in the bile were unaffected by acivicin pretreatment. All procedures were approved by the animal care committee in our university.
Analysis of bilirubin and GSH in the bile.
Direct and indirect bilirubin concentrations of bile were determined by using a kit (bilirubin BII-Test Wako; Wako Pure Chemical Industries). Reduced GSH in bile was determined by a postlabel HPLC method as described previously (18). Briefly, 10-µl bile specimens were directly mixed with ice-cold 990 µl 25% metaphosphoric acid-0.1% EDTA and frozen at 20°C until analysis. This initial treatment minimized the degradation of GSH even in the presence of the bile component. Samples were centrifuged at 20,000 g for 5 min at 4°C, and the resulting supernatants were mixed with 3-fluorotyrosine as an internal standard followed by filtration through a 0.45-µm syringe filter (Millex-LH; Millipore, Bedford, MA). HPLC was performed on an Inertsil Octa Decyl Silyl column (4.6 mm inner diameter x 250 mm; GL Sciences, Tokyo, Japan). Elution was performed by using a mobile phase (0.1% trifluoroacetic acid/methanol = 18:1) at a flow rate of 1.0 ml/min. The eluate from the column was mixed with solution containing 18.6 mM O-phthalaldehyde and 17.1 mM 2-mercaptoethanol in 100 mM carbonate buffer (pH 10.5) that was delivered at a rate of 0.2 ml/min. The mixture was then passed through a stainless steel coil at 70°C to facilitate derivatization. A fluorescence detector was used and operated at an excitation wavelength of 355 nm and an emission wavelength of 425 nm. The concentration of GSH was calculated from the ratio of the height of the standard GSH sample.
Vesicle isolation and transport study.
Recombinant baculovirus was prepared as described (16). Sf9 cells were infected with appropriate amounts of the respective virus and cultured for 60 h in the presence of 5% FBS. Membrane vesicles were isolated from 12 x 108 Sf9 cells using the standard method described previously (16). The uptake of 1 µM [3H]PCG, 50 nM [3H]E217G, and 55 nM [3H]DNP-SG into the vesicle was measured as described previously (16). Briefly, 16 µl transport medium (in mM: 10 Tris, 250 sucrose, 10 MgCl2, 5 ATP or AMP, pH 7.4) was preincubated at 37°C for 3 min and then rapidly mixed with 4 µl membrane vesicle suspension (510 µg) and subsequently incubated for the indicated period. The transport reaction was stopped by the addition of 1 ml ice-cold buffer containing 250 mM sucrose, 100 mM NaCl, and 10 mM Tris·HCl (pH 7.4). The stopped reaction mixture was passed through a 0.45-µm HAWP filter (Millipore) and then washed twice with 5 ml stop solution. Radioactivity retained on the filter was determined by using a liquid scintillation counter (model LSC5000; Aloka). Mrp2-dependent uptake was calculated by subtracting the uptake into green fluorescent protein (GFP)-expressing vesicles (negative control) from uptake into Mrp2-expressing vesicles in the presence of 5 mM ATP (16).
Cell culture and transport assay.
Parental Madin-Darby canine kidney (MDCK) II cells and MDCK II cells expressing human MRP2 (MRP2-MDCK II) were kindly supplied by Dr. Piet Borst (The Netherlands Cancer Institutes, Amsterdam, The Netherlands) (11). Cells were cultured in MEM (cat. no. M4655; Sigma) supplemented with 10% FBS at 37°C in the presence of 5% CO2. MRP2-MDCKII cells were maintained in the presence of 0.4 mg/ml geneticin (Sigma). Cells were seeded on plates with 24 wells at a density of 1.68 x 105 cells per well. The cells were then cultured for 3 days to allow them to form polarized monolayers. GSH efflux assay was performed in HBSS (Life Technologies, Gaithersburg, MD) containing 0.5 mM acivicin (Sigma) at 37°C in the presence of 5% CO2 (250 µl/well). Samples (200 µl) were taken and immediately mixed with ice-cold 25% metaphosphoric acid-0.1% EDTA (400 µl) and then vortexed and stored at 20°C until analysis. At the end of the experiment, cells were disrupted with 1 ml 25% metaphosphoric acid-0.1% EDTA for 30 min at 37°C and then vortexed and stored at 20°C until analysis. HPLC analysis was performed as described above. For [3H]PCG transport assay, cells were seeded on polycarbonate transwell filter (6.5-mm diameter, 3-µm pore size, model 3415; Corning Costar, Corning, NY) at a density of 1.3 x 105 cells per well and cultured for 3 days. Cells were washed twice with HBSS and preincubated at 37°C for 20 min. The experiments were initiated by replacing the buffer at either the apical or basal side of the cell layer with HBSS containing 100 µM [3H]PCG (5 µCi/ml). The cells were incubated at 37°C, and aliquots of buffer were taken from each compartment at several time points. Radioactivity in 50 µl of medium was measured in a liquid scintillation counter (model LSC5000; Aloka). At the end of the experiments, the cells were washed three times with ice-cold HBSS and solubilized in 200 µl of 0.1% SDS. A portion of cell lysate (100 µl) was transferred to a scintillation vial, and the rest was used to determine the protein concentration.
Statistical analysis.
Statistical analysis was performed by Student's t-test or ANOVA followed by a post hoc test (Dunnett or Tukey) as described in the figure legends. Differences were considered to be statistically significant when P < 0.05.
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RESULTS |
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Elimination of [3H]PCG (180 µmol/kg) from plasma followed typical two-exponential kinetics and was delayed in EHBR compared with SD rats (Fig. 1A). In all, 31.7% of the radioisotope was recovered in the bile of SD rats until 60 min (Fig. 1B). In a separate experiment, HPLC analysis revealed that only about half of the radioisotope recovered in the bile of SD rats was in the intact form (unpublished data), although we have not identified the metabolite(s) at present. As a result, 15% of the dose was recovered as intact PCG in the bile during 60 min, which was comparable with a previous report (18.723.1% of the dose was recovered in intact form) (35). On the other hand, only 4.3% of the radioisotope was recovered in the bile of EHBR over a 60-min period after [3H]PCG administration (Fig. 1B). These results indicate the role of Mrp2 in the biliary excretion of PCG and its metabolite(s) as well as their systemic elimination from the body.
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The bile flow was transiently increased by 100% after PCG administration (180 µmol/kg) in SD rats, whereas this was not observed in EHBR or saline-injected SD rats (Fig. 2A). The peak value of the bile flow was obtained at 10 min after the administration. This choleretic effect was similar to that in a previous report in which a 70% increase in bile flow was observed after intravenous administration of ampicillin (180 µmol/kg) to Wistar rats, whereas no such effect was seen in Mrp2-deficient TR rats (35). These results indicate that Mrp2 is necessary for PCG-induced choleresis.
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The biliary excretion of total bilirubin including conjugated (direct) and unconjugated (indirect) forms was transiently increased by 34% at 5 min after PCG administration in SD rats, and this higher level was maintained thereafter (Fig. 2B). In EHBR, the biliary excretion rate was about half that of SD rats before PCG administration and gradually decreased after PCG administration (Fig. 2B). The biliary excretion rate of GSH, which has recently been shown to be a low-affinity substrate of Mrp2, was dramatically increased by 210% after PCG administration, and its concentration in bile was increased despite the increase in bile volume (Fig. 2C). That was not due to the altered degradation rate of GSH in the presence of millimolar concentration of PCG in the bile. Degradation rate constant of GSH in bile was not affected in the presence of 20 mM PCG at 37°C as determined by an in vitro experiment (0.298 ± 0.11 and 0.326 ± 0.05 min1 in the absence and presence of 20 mM PCG, respectively). GSH in the bile from saline-treated SD rats was not altered during the experiment (Fig. 2C). No GSH was detected in the bile of EHBR even after the PCG administration (data not shown). The correlation between the biliary excretion rate of PCG or GSH and the bile flow rate are shown in Fig. 3. The biliary excretion rate of PCG was correlated with the bile flow rate (r2 = 0.768; Fig. 3A), although not all the points were on the linear line (0.72 µmol·min1·kg1 vs. 89.4 µl·min1·kg1 for the biliary excretion rate of PCG and bile flow at 5 min, respectively, indicated by the arrow in Fig. 3A). On the other hand, the GSH excretion rate correlated fairly well with the bile flow rate as shown in Fig. 3B (r2 = 0.968).
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The above in vivo experiments suggested that PCG is a substrate of Mrp2 as well as its stimulator. To determine whether PCG is a substrate of Mrp2, an uptake experiment was performed by using membrane vesicles isolated from rat Mrp2-expressing Sf9 cells in which the ATP-dependent uptake of E217G had been previously demonstrated (16). The vesicles used were transport competent as confirmed by using [3H]E217
G (55 nM) as a substrate; 6.75 pmol·mg1·2 min1 in the presence of 5 mM ATP that was
10-fold higher than the uptake into control GFP-expressing vesicles (0.7 pmol·mg1·2 min1). However, neither Mrp2-dependent nor ATP-dependent uptake of [3H]PCG was observed (Fig. 4). Although several compounds are known to become substrates of MRP1 and MRP2 only in the presence of GSH, that was not the case for PCG, because no uptake was observed even in the presence of 5 mM GSH (data not shown). Mrp2-dependent [3H]GSH uptake was not observed both in the absence or presence of 1 mM PCG as well (data not shown). These results indicate that PCG is not a substrate of Mrp2 or is a rather low-affinity substrate as reported for GSH (25). We then examined the effect of PCG on the Mrp2 transport activity using a typical glutathione conjugate ([3H]DNP-SG, 50 nM, 2 min) and glucuronide conjugate ([3H]E217
G, 55 nM, 2 min) as substrates. Uptake into Mrp2-expressing vesicles was stimulated in the presence of ATP, whereas it was unaffected in the presence of AMP. Uptake into GFP-expressing vesicles was minor even in the presence of ATP and was unaffected by PCG (data omitted for clarity). ATP-dependent uptake of these substrates was significantly enhanced in the presence of PCG with maximum transport activity at 5 mM and 1 mM PCG for DNP-SG (492 ± 51%) and E217
G (189 ± 7.4%), respectively (Fig. 5). An inhibitory effect was observed in the presence of a high concentration of PCG (Fig. 5). A similar effect was observed by using canalicular membrane vesicles (CMVs) isolated from SD rat liver (data not shown).
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Transport of GSH by Mrp2 is hardly detectable in a standard membrane vesicle study, but its export has been observed in an intact cell system (11, 25). To examine whether the GSH efflux mediated by Mrp2/MRP2 is stimulated by PCG, human MRP2-expressing MDCKII cells (MRP2-MDCKII) were used. In these cells, GSH export in the apical direction is enhanced and results in a lower intracellular GSH concentration as reported previously (11, 25). In our experiment, the time-dependent GSH efflux over 2 h was also high compared with the parent MDCKII cells and was stimulated by 1 mM PCG (Fig. 6A). This stimulatory effect was concentration dependent up to 10 mM (Fig. 6, B and C). Accordingly, the intracellular GSH concentration was reduced in PCG-treated MRP2-MDCKII cells (Fig. 6B). The total GSH (cell + medium) recovered during the experiment was not affected (Fig. 6B), indicating that the increase in the GSH efflux was achieved by enhanced efflux rather than accelerated synthesis of GSH. Such an effect was not observed, or was only minor, in parental cells.
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The possibility of the MRP2-mediated transport of PCG was further examined in an intact cell system at a concentration of 100 µM. In a parental MDCKII cells, transport of [3H]PCG in the basolateral-to-apical direction was somewhat higher than in the opposite direction, indicating an endogenous efflux carrier on the apical membrane or an uptake carrier on the basolateral membrane (Fig. 7A). If PCG is a substrate of MRP2, vectorial transport in the basolateral-to-apical direction should be observed, because MRP2 localizes on the apical membrane. However, such preferential transport was not observed (Fig. 7B). Transcellular transport clearance in the basolateral-to-apical direction, calculated from the initial 0.5 to 1 h, was rather lower in MRP2-MDCKII cells compared with that in parental MDCKII cells (Table 1). PCG remaining in the cells after 2-h exposure from the apical compartment were lower compared with those from the basolateral compartment (Table 1). This also implies the presence of endogenous transporter(s) as described above.
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DISCUSSION |
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We were unable to detect any ATP-dependent transport in the recombinant Mrp2 expression system. We were also unable to detect any ATP-dependence or difference in the transport activity into CMVs isolated from SD rat and EHBR liver (data not shown) as reported previously (32). Similarly, no ATP-dependent transport of other -lactam antibiotics including ampicillin and cefodizim was demonstrated, whereas the biliary excretion was significantly impaired in Mrp2-deficient rats (29, 35). One of the possible reasons for this discrepancy is that these
-lactams are very poor substrates of Mrp2, hardly detectable in the membrane vesicle study. GSH is a typical example of this type of "poor" substrate. It has been reported that no ATP-dependent transport of GSH was detected in CMVs by using a standard protocol although its transport was readily detectable in an intact cell system (25) or under particular vesicle study conditions in which a reducing agent was omitted and an inert gas was added instead (26). The other possibility is the lack of cofactor(s) for the recognition of PCG as a substrate. Some compounds, including chelerythrine,
-naphtylisothiocyanate, and arsenite are transported by Mrp2/MRP2 in a reversible conjugation form with GSH (9, 22). Such a cotransport mechanism with GSH or labile formation of GSH conjugate is, however, unlikely for PCG because: 1) fivefold excess PCG [including its metabolite(s)] over GSH was excreted in the bile of SD rats (Figs. 1B and 2C), 2) 5 mM GSH did not stimulate [3H]PCG transport in Mrp2-expressing vesicles (data not shown), and 3) apical preferential transport was not observed in MRP2-MDCKII cells in which sufficient amounts of intracellular GSH and exogenous MRP2 are equipped (Fig. 7, Table 1). Similarly, Zelcer et al. (39) reported that PCG transport was not observed in MRP2-expressing Sf9 cell membrane vesicles and MRP2-MDCKII cells. Still, we could not deny the presence of other unidentified endogenous cofactor(s) necessary for Mrp2/MRP2-mediated transport. The other possibility is that PCG is not a substrate of Mrp2/MRP2 and its absence in bile of EHBR is a secondary cause of Mrp2-deficiency. There are some compounds that are not substrates of Mrp2 but exhibit impaired biliary excretion in vivo. The biliary excretion of indocyanine green (ICG), a relatively hydrophobic anion, is significantly reduced in EHBR, whereas Mrp2 is not a major carrier of ICG. Inhibition of intracellular trafficking by endogenous bilirubin accumulated in hepatocytes may be the primary cause of the impaired biliary excretion in EHBR (29). Supporting this hypothesis, biliary excretion of ICG was also diminished in Mdr2 knockout mice (15) whose plasma bilirubin concentration is elevated and Mrp2 expression is maintained (31). In our case, however, bilirubin is not a major endogenous inhibitor of the biliary excretion pathway of PCG in EHBR liver; there was no biliary excretion of PCG in the EHBR liver perfusion study even after hepatic bilirubin was unloaded by preperfusion with 3% BSA for 60 min (unpublished observation). In any case, future studies need to be carried out to determine whether PCG is a substrate or merely a stimulator of Mrp2.
The stimulatory effect of PCG on the transport activity of the MRP family has been reported (1, 39) using membrane vesicles expressing MRP2. The transport of the glutathione conjugate of N-ethylmaleimide into MRP2-expressing membrane vesicles from Sf9 cells was stimulated by PCG in a concentration-dependent manner (maximum 300% of the control in the presence of 5 mM PCG) (1). The transport of [3H]E217
G by MRP2 was also stimulated by PCG in a concentration-dependent manner (maximum
300% of the control in the presence of 1 mM PCG) (39). In our [3H]E217
G uptake experiment, we used concentrations of 0.2, 1, 5, and 10 mM PCG and obtained maximum stimulation at 1 mM. An inhibitory effect was observed in our experiment above 10 mM for [3H]DNP-SG transport and 5 mM for [3H]E217
G. One of the possible reasons for these biphasic patterns may be the existence of a low-affinity interacting (of the order of mM) site for PCG. If PCG is a substrate of Mrp2, this low-affinity site could be a candidate transport site for PCG. Such a low-affinity site (Ki = 20 mM) has been reported for GSH (25). The stimulatory effect of PCG on the efflux of GSH from MRP2-MDCKII strongly suggests that PCG interacts with MRP2 to stimulate its GSH transport activity. In this experimental cell system, PCG is not actively taken up without known specific carriers. The distribution volume of [3H]PCG in MDCKII and MRP2-MDCKII at 2 h was as low as 23 µl/mg (calculated from Table 1), which is similar to the distribution volume of water in the cell (23). This results in an intracellular concentration of
1 mM in the presence of 1 mM PCG in the medium for 2 h. Because PCG is a substrate of organic anion transporter-C/SLC21A6 (33), double-transfected cells expressing the uptake carrier on the basolateral membrane and MRP2 on the apical membrane will be a valuable tool for detecting the transcellular transport of such hydrophilic compounds (28). In contrast, specific carriers are reported to be involved in the hepatic uptake of PCG from blood (33, 34, 37) to achieve a higher concentration in hepatocytes. As a result, Mrp2-mediated GSH efflux could be stimulated by PCG to a substantial degree in vivo.
In conclusion, the choleretic effect of PCG is caused by the stimulation of biliary GSH efflux as well as the concentrative biliary excretion of PCG itself, both of which were Mrp2 dependent.
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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