Mrp2 is involved in benzylpenicillin-induced choleresis

Kousei Ito, Tomokazu Koresawa, Koichi Nakano, and Toshiharu Horie

Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, 263-8675, Japan

Submitted 25 September 2003 ; accepted in final form 18 February 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Benzylpenicillin (PCG; 180 µmol/kg), a classic {beta}-lactam antibiotic, was intravenously given to Sprague-Dawley (SD) rats and multidrug resistance-associated protein 2 (Mrp2)-deficient Eisai hyperbilirubinemic rats (EHBR). A percentage of the [3H]PCG was excreted into the bile of the rats within 60 min (SD rats: 31.7% and EHBR: 4.3%). Remarkably, a transient increase in the bile flow (~2-fold) and a slight increase in the total biliary bilirubin excretion were observed in SD rats but not in the EHBR after PCG administration. This suggests that the biliary excretion of PCG and its choleretic effect are Mrp2-dependent. Positive correlations were observed between the biliary excretion rate of PCG and bile flow (r2 = 0.768) and more remarkably between the biliary excretion rate of GSH and bile flow (r2 = 0.968). No ATP-dependent uptake of [3H]PCG was observed in Mrp2-expressing Sf9 membrane vesicles, whereas other forms of Mrp2-substrate transport were stimulated in the presence of PCG. GSH efflux mediated by human MRP2 expressed in Madin-Darby canine kidney II cells was enhanced in the presence of PCG in a concentration-dependent manner. 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.

bile flow; multidrug resistance-associated protein; {beta}-lactam


THE MULTIDRUG RESISTANCE-ASSOCIATED protein (MRP) 2/ATP-binding cassette transporter family C2 (MRP2 for the human gene and Mrp2 for the rat gene are used throughout this manuscript) is highly expressed on the bile canalicular membrane of hepatocytes. It actively excretes structurally diverse organic anions, including GSH (25, 26), glutathione conjugates, bilirubin glucuronides, sulfated and glucuronidated bile salts, and nonconjugated organic anions that account for most of the bile salt independent flow (BSIDF). The other important carrier for the production of bile flow is the bile salt export pump (BSEP) that also actively excretes monovalent bile salts in an ATP-dependent manner to produce bile salt dependent flow (BSDF) (12). Alteration of the transport activity of these canalicular transporters will significantly affect bile flow. Internalization of these canalicular transporters will cause cholestasis (7, 24, 27), whereas reinsertion of these transporters will result in choleresis (20, 21, 30). Moreover, inhibition of the transport process also induces cholestasis (4, 5, 8). However, no direct evidence has been reported concerning choleresis due to stimulation of the intrinsic transport activity of these transporters.

Several {beta}-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 0.3–3 mmol/kg piperacillin also induced choleresis in rats, and this was mainly explained by the stimulation of BSIDF. Biliary excretion of {beta}-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 {beta}-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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.

[phenyl-4(n)-3H]PCG (10–30 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{beta}-estradiol 17-({beta}-D-glucuronide) (E217{beta}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 240–300 g (7–8 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·ml–1·kg–1; 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 {gamma}-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 1–2 x 108 Sf9 cells using the standard method described previously (16). The uptake of 1 µM [3H]PCG, 50 nM [3H]E217{beta}G, 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 (5–10 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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasma concentration profile and biliary excretion of PCG.

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 (18.7–23.1% 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.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1. Biliary excretion of [3H]benzylpenicillin ([3H]PCG). [3H]PCG (180 µmol/kg) was administered intravenously as a bolus to normal Sprague-Dawley (SD) rats ({bullet}) and Eisai hyperbilirubinemic rats (EHBR) ({circ}). A: plasma concentration time profile of [3H]PCG. B: biliary excretion rate of [3H]PCG. Inset: cumulative biliary excretion of [3H]PCG. Means ± SD of 3 studies were plotted.

 
Effect of PCG on the bile flow.

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.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2. PCG enhances biliary excretion of bilirubin, GSH, and exhibited choleresis in normal SD rats. PCG (180 µmol/kg) or saline was intravenously administered to normal SD rats ({bullet}, PCG; {blacktriangleup}, saline) and EHBR ({circ}, PCG; {triangleup}, saline) at time 0, and bile samples were analyzed to determine the bile flow rate (A), biliary excretion rate of total bilirubin (B), and GSH (C). GSH in the bile of EHBR was below the detection limit in both PCG- and saline-administered rats. Means ± SD of 3–6 rats were plotted. *P < 0.05 and **P < 0.01, significantly different from the points before PCG administration (–5 min) using ANOVA followed by Dunnett's test.

 
Effect of PCG on the biliary excretion of bilirubin and reduced glutathione.

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 min–1 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·min–1·kg–1 vs. 89.4 µl·min–1·kg–1 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).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3. A: relationship between bile flow and biliary [3H]PCG excretion in SD rats ({bullet}) and EHBR ({circ}) given PCG (180 µmol/kg) (y = 21.8x +54; r2 = 0.768). The point corresponding to 5 min after PCG administration in SD rats is indicated by the arrow. B: relationship between bile flow and biliary GSH excretion in SD rats given PCG (180 µmol/kg) (y = 169x +30; r2 = 0.968).

 
Vesicle uptake study of PCG in Mrp2-expressing membrane vesicles.

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 E217{beta}G had been previously demonstrated (16). The vesicles used were transport competent as confirmed by using [3H]E217{beta}G (55 nM) as a substrate; 6.75 pmol·mg–1·2 min–1 in the presence of 5 mM ATP that was ~10-fold higher than the uptake into control GFP-expressing vesicles (0.7 pmol·mg–1·2 min–1). 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{beta}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{beta}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).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4. Time profiles for the uptake of 1 µM [3H]PCG by membrane vesicles from rat multidrug resistance-associated protein 2 (Mrp2)-expressing Sf9 cells. Membrane vesicles (10 µg) from Mrp2-expressing Sf9 cells ({bullet}, {circ}) or green fluorescent protein (GFP)-expressing Sf9 cells ({blacktriangleup}, {triangleup}) were incubated with 5 mM AMP ({circ}, {triangleup}) or ATP ({bullet}, {blacktriangleup}). Means ± SD of 3 studies were plotted.

 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 5. Effects of PCG on the uptake of [3H]dinitrophenyl glutathione ([3H]DNP-SG) and [3H]E217{beta}G. Effects of PCG on the uptake of [3H]DNP-SG (50 nM; {bullet}, {circ}) and [3H]E217{beta}G (55 nM; {blacktriangleup}, {triangleup}) for initial 2 min. Open and closed symbols are the uptake in the presence of 5 mM AMP and ATP, respectively. Uptake into GFP-expressing vesicles was minor even in the presence of ATP and was unaffected by PCG (data omitted for clarity). Means ± SD of 3 studies were plotted. Where bars are not shown, the SD is contained within the limits of the symbols.

 
Effect of PCG on the efflux of GSH from MRP2-expressing cells.

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.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 6. Stimulation of GSH efflux by PCG in MRP2- Madin-Darby canine kidney II (MDCKII) cells. A: time profiles for the efflux of GSH from MDCKII ({triangleup}, {blacktriangleup}) and MRP2-MDCKII ({circ}, {bullet}) cells in the presence ({blacktriangleup}, {bullet}) or absence ({triangleup}, {circ}) of 1 mM PCG. B: concentration-dependent stimulation of the efflux of GSH from MDCKII (top graphs, n = 3) and MRP2-MDCKII cells (bottom graphs, n = 4). Cells were incubated for 2 h in the presence of the indicated concentration of PCG. GSH in the medium (open columns) and GSH remaining in the cell (filled columns) were analyzed by HPLC as described in MATERIALS AND METHODS. Total GSH (sum of the GSH exported and remaining in the cell at 2 h; {bullet}) was also plotted in the same figure. C: MRP2-dependent efflux of GSH. MRP2-dependent GSH efflux was calculated by subtracting the GSH efflux in MDCKII cells from that in MRP2-MDCKII cells as shown in B. Means ± SD were plotted. Where bars are not shown, the SD is contained within the limits of the symbols. *P < 0.05 and **P < 0.01, significantly different from those in the absence of PCG as determined by Student's t-test.

 
Transcellular transport of PCG in MRP2-expressing cells.

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.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 7. Time profiles for the transcellular transport of PCG across MDCKII monolayers. Transcellular transport of [3H]PCG (100 µM) across MDCKII monolayers were examined. Closed ({blacktriangleup}, {bullet}) and open ({triangleup}, {circ}) symbols represent the transcellular transport in the apical-to-basolateral and basolateral-to-apical directions, respectively. MDCKII (A) and MRP2-MDCKII (B) cells are shown. Means ± SD of 3 studies were plotted.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Transcellular transport clearance and accumulation of benzylpenicillin in the cells

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bile flow involves BSDF and BSIDF. These are produced by the active efflux of bile salts and reduced glutathione by BSEP and Mrp2, respectively. Cholestatic compounds such as cyclosporin A and PSC833 are potent inhibitors of these carriers as assessed by the membrane vesicle study and are the direct cause of cholestasis observed in in vivo situations (4, 5). Some toxic compounds as well as hyperosmotic treatment are reported to affect canalicular localization of Mrp2 and/or BSEP by reducing the total bile flow (7, 10, 20, 21, 27). However, to date, there is no example of a direct correlation between the stimulation of transport activity assessed in an in vitro membrane vesicle study and the in vivo choleretic effect. The compound itself may become a driving force if its concentration in the bile is high enough after the use of active efflux carriers. In the case of PCG, the highest biliary concentration is 20 mM, whereas it is 0.2 mM in the plasma at 10 min after PCG administration (Fig. 1). Such a steep gradient likely causes the osmotic traction of water from the blood to the bile via the tight junctions of hepatocytes and cholangiocytes. If PCG excretion is directly related to choleresis, the choleretic index can be calculated as 21.8 µl/µmol PCG excreted into bile, and this is comparable with the other choleretics reported previously (13, 14, 36). However, the biliary excretion of PCG itself was not exactly correlated with the bile flow rate (r2 = 0.768), suggesting that other transported ion(s) may be the additional driving force of the choleretic phenomenon. BSIDF is mainly produced by the excretion of GSH into the bile and is readily affected by some forms of drug treatment (2). As shown in Fig. 3A, the biliary excretion of GSH correlated fairly well with the change in bile flow (r2 = 0.968). This indicates that the biliary excretion rate of GSH as well as PCG itself may be the driving force of bile flow after PCG administration. A similar choleretic effect mediated by the stimulation of GSH has been reported previously. Nifedipine (30 µM), a Ca2+ channel blocker used clinically, increased bile flow by 1.6-fold in the isolated perfused rat liver and is accompanied by enhanced biliary excretion of GSH (~5-fold) (38). Our in vivo correlation analysis and in vitro GSH export experiment suggested that the stimulation of GSH efflux as well as PCG excretion itself are involved in PCG-induced choleresis.

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 {beta}-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 {beta}-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, {alpha}-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{beta}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{beta}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{beta}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 2–3 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.


    FOOTNOTES
 

Address for reprint requests and other correspondence: T. Horie, Lab. of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba Univ., 1-8-1 Inohana, Chuo-ku, Chiba 263-8675, Japan (E-mail: horieto{at}p.chiba-u.ac.jp).

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bakos E, Evers R, Sinko E, Varadi A, Borst P, and Sarkadi B. Interactions of the human multidrug resistance proteins MRP1 and MRP2 with organic anions. Mol Pharmacol 57: 760–768, 2000.[Abstract/Free Full Text]
  2. Ballatori N and Truong AT. Relation between biliary glutathione excretion and bile acid-independent bile flow. Am J Physiol Gastrointest Liver Physiol 256: G22–G30, 1989.[Abstract/Free Full Text]
  3. Bodo A, Bakos E, Szeri F, Varadi A, and Sarkadi B. Differential modulation of the human liver conjugate transporters MRP2 and MRP3 by bile acids and organic anions. J Biol Chem 278: 23529–23537, 2003.[Abstract/Free Full Text]
  4. Böhme M, Büchler M, Müller M, and Keppler D. Differential inhibition by cyclosporins of primary-active ATP-dependent transporters in the hepatocyte canalicular membrane. FEBS Lett 333: 193–196, 1993.[CrossRef][ISI][Medline]
  5. Böhme M, Müller M, Leier I, Jedlitschky G, and Keppler D. Cholestasis caused by inhibition of the adenosine triphosphate-dependent bile salt transport in rat liver. Gastroenterology 107: 255–265, 1994.[ISI][Medline]
  6. Borst P, Evers R, Kool M, and Wijnholds J. A family of drug transporters: the multidrug resistance-associated proteins. J Natl Cancer Inst 92: 1295–1302, 2000.[Abstract/Free Full Text]
  7. Crocenzi FA, Mottino AD, Cao J, Veggi LM, Sanchez Pozzi EJ, Vore M, Coleman R, and Roma MG. Estradiol-17{beta}-D-glucuronide induces endocytic internalization of Bsep in the rat. Am J Physiol Gastrointest Liver Physiol 285: G449–G459, 2003.[Abstract/Free Full Text]
  8. Deters M, Nolte K, Kirchner G, Resch K, and Kaever V. Comparative study analyzing effects of sirolimus-cyclosporin and sirolimus-tacrolimus combinations on bile flow in the rat. Dig Dis Sci 46: 2120–2126, 2001.[CrossRef][ISI][Medline]
  9. Dietrich CG, Ottenhoff R, de Waart DR, and Oude Elferink RP. Role of MRP2 and GSH in intrahepatic cycling of toxins. Toxicology 167: 73–81, 2001.[CrossRef][ISI][Medline]
  10. Dombrowski F, Kubitz R, Chittattu A, Wettstein M, Saha N, and Häussinger D. Electron-microscopic demonstration of multidrug resistance protein 2 (Mrp2) retrieval from the canalicular membrane in response to hyperosmolarity and lipopolysaccharide. Biochem J 348: 183–188, 2000.[CrossRef][ISI][Medline]
  11. Evers R, de Haas M, Sparidans R, Beijnen J, Wielinga PR, Lankelma J, and Borst P. Vinblastine and sulfinpyrazone export by the multidrug resistance protein MRP2 is associated with glutathione export. Br J Cancer 83: 375–383, 2000.[CrossRef][ISI][Medline]
  12. Gerloff T, Stieger B, Hagenbuch B, Madon J, Landmann L, Roth J, Hofmann AF, and Meier PJ. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver. J Biol Chem 273: 10046–10050, 1998.[Abstract/Free Full Text]
  13. Gonzalez J, Fernandez C, Marino E, Morales A, and Jimenez R. Biliary excretion and choleretic effect of cefmetazole in rats. Antimicrob Agents Chemother 33: 1970–1974, 1989.[ISI][Medline]
  14. Gonzalez P, Mauriz JL, Jimenez R, Gonzalez-Gallego J, and Tunon MJ. Choleresis and inhibition of biliary lipid secretion induced by piperacillin in the rat. Clin Exp Pharmacol Physiol 29: 880–884, 2002.[CrossRef][ISI][Medline]
  15. Huang L and Vore M. Multidrug resistance p-glycoprotein 2 is essential for the biliary excretion of indocyanine green. Drug Metab Dispos 29: 634–637, 2001.[Abstract/Free Full Text]
  16. Ito K, Suzuki H, and Sugiyama Y. Charged amino acids in the transmembrane domains are involved in the determination of the substrate specificity of rat Mrp2. Mol Pharmacol 59: 1077–1085, 2001.[Abstract/Free Full Text]
  17. Ito K, Suzuki H, and Sugiyama Y. Single amino acid substitution of rat MRP2 results in acquired transport activity for taurocholate. Am J Physiol Gastrointest Liver Physiol 281: G1034–G1043, 2001.[Abstract/Free Full Text]
  18. Ji B, Ito K, Suzuki H, Sugiyama Y, and Horie T. Multidrug resistance-associated protein2 (MRP2) plays an important role in the biliary excretion of glutathione conjugates of 4-hydroxynonenal. Free Radic Biol Med 33: 370–378, 2002.[CrossRef][ISI][Medline]
  19. Kobayashi K, Sogame Y, Hara H, and Hayashi K. Mechanism of glutathione S-conjugate transport in canalicular and basolateral rat liver plasma membranes. J Biol Chem 265: 7737–7741, 1990.[Abstract/Free Full Text]
  20. Kubitz R, D'Urso D, Keppler D, and Häussinger D. Osmodependent dynamic localization of the multidrug resistance protein 2 in the rat hepatocyte canalicular membrane. Gastroenterology 113: 1438–1442, 1997.[ISI][Medline]
  21. Kubitz R, Warskulat U, Schmitt M, and Häussinger D. Dexamethasone- and osmolarity-dependent expression of the multidrug-resistance protein 2 in cultured rat hepatocytes. Biochem J 340: 585–591, 1999.[CrossRef][ISI][Medline]
  22. Lou H, Ookhtens M, Stolz A, and Kaplowitz N. Chelerythrine stimulates GSH transport by rat Mrp2 (Abcc2) expressed in canine kidney cells. Am J Physiol Gastrointest Liver Physiol 285: G1335–G1344, 2003.[Abstract/Free Full Text]
  23. Mills JW and Lubin M. Effect of adenosine 3',5'-cyclic monophosphate on volume and cytoskeleton of MDCK cells. Am J Physiol Cell Physiol 250: C319–C324, 1986.[Abstract/Free Full Text]
  24. Mottino AD, Cao J, Veggi LM, Crocenzi F, Roma MG, and Vore M. Altered localization of canalicular Mrp2 in estradiol-17{beta}-D-glucuronide-induced cholestasis. Hepatology 35: 1409–1419, 2002.[ISI][Medline]
  25. Paulusma CC, van Geer MA, Evers R, Heijn M, Ottenhoff R, Borst P, and Oude Elferink RP. Canalicular multispecific organic anion transporter/multidrug resistance protein 2 mediates low-affinity transport of reduced glutathione. Biochem J 338: 393–401, 1999.[CrossRef][ISI][Medline]
  26. Rebbeor JF, Connolly GC, and Ballatori N. Inhibition of Mrp2- and Ycf1p-mediated transport by reducing agents: evidence for GSH transport on rat Mrp2. Biochim Biophys Acta 1559: 171–178, 2002.[ISI][Medline]
  27. Rost D, Kartenbeck J, and Keppler D. Changes in the localization of the rat canalicular conjugate export pump Mrp2 in phalloidin-induced cholestasis. Hepatology 29: 814–821, 1999.[ISI][Medline]
  28. Sasaki M, Suzuki H, Ito K, Abe T, and Sugiyama Y. Transcellular transport of organic anions across a double-transfected Madin-Darby canine kidney II cell monolayer expressing both human organic anion-transporting polypeptide (OATP2/SLC21A6) and multidrug resistance-associated protein 2 (MRP2/ABCC2). J Biol Chem 277: 6497–6503, 2002.[Abstract/Free Full Text]
  29. Sathirakul K, Suzuki H, Yasuda K, Hanano M, Tagaya O, Horie T, and Sugiyama Y. Kinetic analysis of hepatobiliary transport of organic anions in Eisai hyperbilirubinemic mutant rats. J Pharmacol Exp Ther 265: 1301–1312, 1993.[Abstract]
  30. Schmitt M, Kubitz R, Lizun S, Wettstein M, and Haussinger D. Regulation of the dynamic localization of the rat Bsep gene-encoded bile salt export pump by anisoosmolarity. Hepatology 33: 509–518, 2001.[CrossRef][ISI][Medline]
  31. Smit JJ, Schinkel AH, Oude Elferink RP, Groen AK, Wagenaar E, van Deemter L, Mol CA, Ottenhoff R, van der Lugt NM, van Roon MA, van der Valk MA, Offerhaus GJA, Berns AJM, and Borst P. Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell 75: 451–462, 1993.[ISI][Medline]
  32. Tamai I, Maekawa T, and Tsuji A. Membrane potential-dependent and carrier-mediated transport of cefpiramide, a cephalosporin antibiotic, in canalicular rat liver plasma membrane vesicles. J Pharmacol Exp Ther 253: 537–544, 1990.[Abstract]
  33. Tamai I, Nezu J, Uchino H, Sai Y, Oku A, Shimane M, and Tsuji A. Molecular identification and characterization of novel members of the human organic anion transporter (OATP) family. Biochem Biophys Res Commun 273: 251–260, 2000.[CrossRef][ISI][Medline]
  34. Tsuji A, Terasaki T, Takanosu K, Tamai I, and Nakashima E. Uptake of benzylpenicillin, cefpiramide and cefazolin by freshly prepared rat hepatocytes. Evidence for a carrier-mediated transport system. Biochem Pharmacol 35: 151–158, 1986.[CrossRef][ISI][Medline]
  35. Verkade HJ, Wolbers MJ, Havinga R, Uges DR, Vonk RJ, and Kuipers F. The uncoupling of biliary lipid from bile acid secretion by organic anions in the rat. Gastroenterology 99: 1485–1492, 1990.[ISI][Medline]
  36. Xia Y, Lambert KJ, Schteingart CD, Gu JJ, and Hofmann AF. Concentrative biliary secretion of ceftriaxone. Inhibition of lipid secretion and precipitation of calcium ceftriaxone in bile. Gastroenterology 99: 454–465, 1990.[ISI][Medline]
  37. Yabuuchi H, Tamai I, Morita K, Kouda T, Miyamoto K, Takeda E, and Tsuji A. Hepatic sinusoidal membrane transport of anionic drugs mediated by anion transporter Npt1. J Pharmacol Exp Ther 286: 1391–1396, 1998.[Abstract/Free Full Text]
  38. Yang B and Hill CE. Nifedipine modulation of biliary GSH and GSSG/conjugate efflux in normal and regenerating rat liver. Am J Physiol Gastrointest Liver Physiol 281: G85–G94, 2001.[Abstract/Free Full Text]
  39. Zelcer N, Huisman MT, Reid G, Wielinga P, Breedveld P, Kuil A, Knipscheer P, Schellens JH, Schinkel AH, and Borst P. Evidence for two interacting ligand binding sites in human multidrug resistance protein 2 (ATP binding cassette C2). J Biol Chem 278: 23538–23544, 2003.[Abstract/Free Full Text]




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Google Scholar
Articles by Ito, K.
Articles by Horie, T.
Articles citing this Article
PubMed
PubMed Citation
Articles by Ito, K.
Articles by Horie, T.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.