Chelerythrine stimulates GSH transport by rat Mrp2 (Abcc2) expressed in canine kidney cells

Huan Lou, Murad Ookhtens, Andrew Stolz, and Neil Kaplowitz

Research Center for Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, California 90033

Submitted 25 June 2003 ; accepted in final form 29 July 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Rat multidrug resistant protein 2 (Mrp2; Abcc2), an ATP-driven pump located on the canalicular domain of hepatocytes, exports glutathione S-conjugates (GS-X) and GSH among its wide variety of substrates. Previous studies have shown that chelerythrine (CHEL), a quaternary benzophenanthridine cation, reacts with GSH to form a reversible adduct under physiological conditions. Here we report that CHEL can strongly stimulate GSH efflux by Mrp2, when it is constitutively expressed in polarized canine kidney cells, thereby leading to the depletion of cellular GSH. Transepithelial transport experiments indicate that Mrp2 transports GSH and CHEL with a 1:1 stoichiometry, which can be readily inhibited by GS-bimane, a GS-X substrate for Mrp2. Moreover, CHEL can block Mrp2-mediated leukotriene C4 uptake by membrane vesicles with an IC50 {approx} 100 µM in the presence of GSH, but not S-methyl GSH or ophthalmic acid. Thus the thiol group of GSH is required for inhibition of Mrp2 in the presence of CHEL. Our results suggest that CHEL stimulates GSH efflux by forming a reversible GS-CHEL adduct, which is transported by Mrp2 and dissociates extracellularly.

glutathione conjugates; glutathione-chelerythrine adduct; membrane transport; membrane vesicles; polarized cells


RAT MULTIDRUG RESISTANCE PROTEIN 2 (Mrp2), is a member of the ATP-binding cassette transporter family. Mrp2 is mainly expressed in the canalicular membrane domain of hepatocytes. It mediates transport into bile of a wide variety of organic compounds, including glutathione conjugates, glucuronides, sulfates, oxidized glutathione (GSSG), and GSH (3, 22). Although, a large body of evidence indicates that Mrp2 transports GSH conjugates efficiently as an ATP-driven export pump, the mechanism of its GSH export is not well understood. Mrp2-deficient GY/TR- mutant rats and Eisai hyperbilirubinemic rats exhibit defects in GSH export into bile, demonstrating that canalicular GSH transport in rat liver is Mrp2 dependent (10, 16, 29, 30). Consistent with this observation, the overexpression of human MRP2 in Madin-Darby canine kidney II (MDCKII) cells has been shown to lead to an increased export of GSH (30). GSH transport in intact MDCKII cells expressing MRP2 is sensitive to ATP depletion (30), although it has been difficult to demonstrate ATP-dependent GSH transport in membrane vesicles (14, 30, 31, 37), presumably because of the low affinity of the process for GSH. Indeed, in both perfused rat liver and MRP2-expressing cells, the rate of GSH transport is a linear function of intracellular GSH concentrations (18, 30). Aside from GSH alone being a substrate, it is also possible that Mrp2 transports GSH, in reversible complex with endogenous substance(s), as a GS-X substrate, with subsequent release of GSH under experimental conditions.

In this study, we show that CHEL can greatly increase GSH export in MDCKII cells overexpressing Mrp2. CHEL is a quaternary benzophenanthridine alkaloid present in many plant species (2, 23). It exists mainly as a quaternary cation or iminium ion (Fig. 1A, structure I) at neutral pH in aqueous solutions (pK = 8.8-9.0) and displays great susceptibility toward nucleophilic reagents, such as GSH (Fig. 1A, structure III), resulting in formation of an adduct, i.e., GS-CHEL (Fig. 1B, structure IV) (20, 36, 38, 39). The nucleophilic addition of the thiol group of GSH to the electrophilic iminium bond is reversible. The overall result of the reaction depends, therefore, on the position of an equilibrium. Under physiological conditions, the GS-CHEL adduct has an equilibrium constant of log K = 4.39, where K (M-1) = [adduct]/[GSH] · [CHEL] (38). Interestingly, the antimicrotubule activity of CHEL (39) and its inhibitory effect on rat liver alanine aminotransferase (38) are thought to result from the formation of the reversible adduct of CHEL with the thiol groups in tubulin and alanine aminotransferase. Our studies demonstrate a stimulatory action of CHEL on GSH efflux from MDCKII cells expressing Mrp2, most likely due to formation of the GS-CHEL adduct, which serves as a substrate for Mrp2 with high affinity compared with GSH alone.



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Fig. 1. Chemical structures of chelerythrine (CHEL) and GSH and formation of the glutathione S-CHEL (GS-CHEL) adduct. A: chemical structures of CHEL (structure I, quaternary cation or iminium ion; structure II, pseudobase) and GSH (structure III). B: GS-CHEL adduct (structure IV) formation. SG, glutathione.

 


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Materials. Acivicin, buthionine sulfoximine (BSO), GSH, and S-methyl GSH (S-MeGSH) were obtained from Sigma (St. Louis, MO). Chelerythrine chloride (CHEL) and verapamil (VRP) were from Calbiochem (San Diego, CA). Ophthalmic acid (L-{gamma}-glutamyl-L-{alpha}-aminobutyrylglycine) was from Bachem (Torrance, CA), and monochlorobimane (mBCl) was from Molecular Probes (Eugene, OR). [3H]VRP (60~85 Ci/mmol), [3H]leukotriene C4 (LTC4; 136.9 Ci/mmol), 3H2O (1.0 mCi/g), and [carboxyl-14C]inulin (1~3 mCi/g) were from NEN (Boston, MA).

Cell lines. The MDCKII cell line was selected from subclones of the parental MDCK cell line (American Type Culture Collection, Manassas, VA) as described previously (15). The cells were maintained in flasks in DMEM supplemented with 10% FBS in 5% CO2 at 37°C. Stable clones of Mrp2-expressing cells (MDCKII-Mrp2) and control cells (MDCKII-V) were established by transfection of parental MDCKII cells with a rat Mrp2 expression vector (pCXN2/Mrp2) (21) and an empty vector (pCXN2), respectively. The vectors were kind gifts from Dr. Yuichi Sugiyama (University of Tokyo, Japan). Effectene transfection reagent (Qiagen, Valencia, CA) was used in the procedure according to the manufacturer's instructions, and G418 (0.8 mg/ml) was used for selection of stable clones. Finally, Mrp2 expression in MDCKII-Mrp2 but not in MDCKII-V cells was confirmed by Western blot analysis. For maintenance of these clones, G418 (0.2 mg/ml) was also present in the medium.

Western blot analysis. Crude cell membrane fractions from stable MDCKII-derived clones were prepared as described previously (1), separated on an 8% SDS-PAGE gel, and transferred to nitrocellulose membrane. Mrp2 bands were detected by enhanced chemiluminescence (Pierce, Rockford, IL) with M2III-6, a primary monoclonal antibody to human MRP2 (Alexis Biochemicals, San Diego, CA).

Confocal fluorescence microscopy. Polarized monolayers were processed as described previously (7) with some alterations. Briefly, cells were fixed, permeabilized (1% Triton X-100/PBS, 5 min), blocked (50% FBS/PBS, 60 min), and then incubated with M2III-6 (1:50 dilution, 30 min). Antibody binding was detected with FITC-conjugated secondary antibody (1:100 dilution). Microfilaments along the cell periphery were stained with rhodamine-phalloidin (1:100 dilution, 30 min). Cells were mounted and examined by using a Nikon PCM2000 confocal laser-scanning microscope, equipped with both argon blue laser and HeNe green laser and corresponding filters to give green and red signals, respectively. Images were compiled by using Simple PCI 3.6 software (C · IMAGING Systems, Cranberry Township, PA).

Transport assays in intact cells. All experiments were performed at 37°C with 5% CO2, essentially according to the procedure described previously (30). Briefly, cells were seeded at a density of 2.2 x 105 cells/cm2 on polyethylene terephthalate membranes (PTE; 23.1-mm diameter, 3-µm pore size; Becton Dickinson Labware, Franklin Lakes, NJ) in cell culture inserts with the growth medium (4 ml in both apical and basal compartments) and grown from day 1 to day 5. For MDCKII-Mrp2 cells, the medium was routinely changed 16 and 2 h before transport experiments to maintain intracellular GSH at a level of ~18 nmol/monolayer. For MDCKII-V cells, BSO (7 µM) in fresh medium was given 24 h before the experiments to inhibit intracellular GSH synthesis and reduce intracellular GSH to levels comparable to that of MDCKII-Mrp2 cells.

On the day of assay, cell monolayers (~3.7 x 106 cells/well) with transepithelial electrical resistance >=800 {Omega} · cm2 were washed and incubated in the preheated efflux medium (Hanks' balanced salt solution supplemented with 0.5 mM acivicin and 10 mM HEPES, pH 7.2; 2 ml in both apical and basal compartments). Acivicin was included in all experiments to inhibit GSH breakdown.

In experiments examining domain-specific GSH efflux, the drug or equivalent amount of the vehicle (as control) was present in chambers, as specified. At time points indicated, media (0.2 ml or as specified) were sampled from both chambers, and at the end of experiments, the cell extracts were prepared with 5% trichloroacetic acid (TCA). The total glutathione (GSH + GSSG) equivalents in the samples were measured by using the GSH-recycling assay. The GSH component in the samples was measured by the HPLC method of Fariss and Reed (13) with sample treatment including acidification and derivatization to form dinitrophenol derivatives.

For transepithelial transport experiments, drugs were present only in either the apical or the basal compartment (donor), as was [carboxyl-14C]inulin (25 nCi/ml). PSC833, a P-glycoprotein inhibitor, was present in both compartments at a concentration (0.1 µM) proven to inhibit the endogenous canine form (11). At time points indicated, media (2 ml) were collected from the opposite chambers (receiver), and equivalent volumes of fresh media were added back (for 30 min time point only). The samples were microcentrifuged (3,000 rpm, 5 min), and the supernatant was divided to three parts for measurement of CHEL by HPLC, [carboxyl-14C]inulin by liquid scintillation spectrometry, and GSH by the method mentioned above. The appearance of [carboxyl-14C]inulin in the apical medium was a measure of the paracellular flux of the cell monolayer, which was typically <0.5% of the dose per hour in our experiments. None of the treatments including CHEL and VRP affected the paracellular permeability.

Kinetic studies in MDCKII-Mrp2 monolayers were performed at 37°C over a 15-min interval. The apical medium was reduced to 1 ml to accommodate shortened incubation time, and CHEL (120 nmol · 2 ml-1 · well-1) was present only in the basal compartment; otherwise, the conditions were identical to the transepithelial transport experiments. GSH accumulation in the apical medium over 15 min was measured to approximate the initial rate of efflux. (Note: in all our transport experiments, the rate of apical GSH accumulation did not decrease in 60 min.) The corresponding intracellular GSH content was measured at 15 min. Monolayers with reduced intracellular GSH levels at time 0 were obtained by pretreatment with various doses of BSO (0, 5, 7, and 50 µM) for 24 h before the experiments. The data for transport rates were expressed in nmol · min-1 · 108 cells-1 (3.7 x 106 cells/well) for MDCKII-Mrp2. The intracellular glutathione concentrations (mM) were calculated by using assayed glutathione contents and the intracellular water space. The intracellular water space was determined by using both 3H2O and [carboxyl-14C]inulin. By subtracting the extracellular trapped water volume from the total water space in the cell pellet, intracellular volume was found to be 21.1 ± 0.6 µl/107 cells for both MDCKII-Mrp2 and MDCKII-V.

GS-bimane (GS-B) efflux experiments were performed, as described in kinetic studies, with mBCl present in the apical compartment at time 0. At indicated time points, media (0.2 ml) were collected from both chambers, and cells were lysed at the end of experiments with 0.4 ml of 5% TCA. GS-B in the media and in the cell extracts was measured by its fluorescence at an excitation wavelength of 386 nm and an emission wavelength of 476 nm and was quantified by a standard curve established with known standards (21). In the inhibition experiments, CHEL (120 nmol · 2 ml-1 · well-1) was present in the basal compartment, and GSH accumulations in the media and remaining in the cells were also determined.

Quantification of CHEL. CHEL in the samples was analyzed by an ion-pairing HPLC method as described previously (33). The column used was an Apex methyl column (5 mm, 25 x 4.6 mm inner diameter; Jones Chromatography, Lakewood, CO). The mobile phase (pH 2.7) was prepared with ion-pairing reagents hexanesulfonic acid (2.75 mM) and hexyltriethylammonium phosphate (2.25 mM) from 0.5 M stocks (Regis Chemical, Morton Grove, IL) in HPLC-grade water and was then mixed with acetonitrile (60:40, vol/vol). The flow rate was 3 ml/min. CHEL was eluted at a 9.5-min retention time and was measured at 268 nm, or at 316 nm for samples containing VRP. Samples from the efflux medium were diluted (1:4 vol/vol) with 0.5% HCl-methanol (vol/vol), and 100 µl were injected on the column. The concentration of CHEL in the samples was calculated using CHEL standards.

Preparation of membrane vesicles and vesicle transport studies. Plasma membrane vesicles were prepared from MDCKII-Mrp2 and MDCKII-V cells at 4°C as described (19, 30) and stored at -80°C. The protein concentration of the vesicles was determined by the method of Lowry and was typically 6~8 mg/ml.

Transport of [H3]LTC4 by membrane vesicles was measured as previously described (5, 19, 32). Briefly, membrane vesicles were thawed, diluted (3 mg/ml) with acivicin (0.5 mM) in incubation buffer B (0.25 M sucrose, 20 mM HEPESTris, pH 7.0), vesiculated, and kept on ice for 1 h. Transport buffer was prepared with incubation buffer B to contain final concentrations of [3H]LTC4 (6.8 nM, 40 nCi), ATP or AMP (4 mM), MgCl2 (10 mM), and acivicin (0.5 mM), after being mixed (4:1) with the diluted vesicles. For the experiments using the ATP-regeneration system, creatine phosphate (10 mM) and creatine kinase (100 µg/ml) were included (32). The assay was carried out at 37°C for 5 min unless otherwise specified. The reaction was initiated by the addition of transport buffer (40 µl) to membrane vesicles (30 µg protein/10 µl), both of which were preheated to 37°C for 5 min, and was stopped by dilution and rapid filtration (5). All data were corrected for nonspecific binding measured as the amount of radioactivity bound to vesicles and filter at 4°C and zero time as described (4). The net ATP-dependent LTC4 uptake was determined by subtracting the value obtained in the presence of AMP from that obtained in the presence of ATP.

GSH (2 mM) was included in all the vesicle studies except as otherwise noted. The presence of GSH strongly reduced ATP-independent [3H]LTC4 association with membrane vesicles (data not shown), most likely due to binding of membrane-bound glutathione S-transferase (GST) as mentioned by others (19). To minimize GSH oxidation, incubation buffer B was gassed with nitrogen for 15 min to remove oxygen before use, and GSH stock solutions were freshly prepared. In addition, transport experiments were carried out under nitrogen.

Fitting of kinetic data. The SAAM II program (35) was used to conduct the fits and obtain the estimates of kinetic parameters as described before (28).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Depletion of cellular GSH by CHEL. To characterize GSH transport by rat Mrp2 in detail, we have generated stable clones of Mrp2-expressing (MDCKII-Mrp2) and control (MDCKII-V) cells (see MATERIALS AND METHODS). Rat Mrp2 is a membrane transporter that is routed to the apical domain of polarized MDCKII monolayer after transfection. We confirmed membrane expression and apical domain localization of Mrp2 in MDCKII-Mrp2 cells, but not in MDCKII-V cells, using Western blot and confocal immunofluorescence microscopy (Fig. 2). The GSH transport activity of Mrp2 was verified by comparing basal levels of GSH efflux in polarized monolayers of MDCKII-Mrp2 and MDCKII-V cells with comparable GSH levels at time 0 (see MATERIALS AND METHODS). Under basal conditions, apical GSH efflux was much greater in MDCKII-Mrp2 than in MDCKII-V cells (1.01 ± 0.16 vs. 0.03 ± 0.02 nmol · 60 min-1 · well-1). In contrast, basolateral GSH efflux was the same (0.32 ± 0.14 vs. 0.37 ± 0.11 nmol · 60 min-1 · well-1). Taken together, these data indicate that Mrp2 mediates apical GSH transport from MDCKII-Mrp2 cell monolayers.



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Fig. 2. Expression and localization of Mrp2 in MDCKII-derived clones. MDCKII-derived clones stably transfected by rat Mrp2 expression vector (MDCKII-Mrp2) and by vector alone (MDCKII-V) were analyzed for expression of Mrp2. Top: Western blot analysis of crude membrane fractions from these clones. Mrp2 (arrow) was detected by M2III-6, a monoclonal antibody for human MRP2. Bottom: immunolocalization of Mrp2 in polarized cell monolayers (MDCKII-V and MDCKII-Mrp2) observed by confocal microscopy. Rhodamine-phalloidin stain (red) shows cell boundary; M2III-6 stain (green) shows Mrp2. Blue lines in XY views indicate positions selected for XZ views. AP, apical domain; BL, basolateral domain.

 

We investigated the effect of CHEL on the GSH transport activity of Mrp2 at first with CHEL present in both the apical and basal efflux media. Addition of CHEL to MDCKII-derived monolayers resulted in a dose-dependent increase in GSH efflux with a matching loss in intracellular GSH (Fig. 3); the total (intracellular + extracellular) GSH remained unchanged. The largest increments in apical GSH accumulation in MDCKII-Mrp2 cells were observed at lower doses (40-80 nmol CHEL/well), with lesser additional increments at higher doses (>80 nmol CHEL/well), indicating that CHEL-stimulated export by Mrp2 is apparently saturable. The molecular form (i.e., oxidized vs. reduced) of effluxed glutathione was determined by HPLC and was found to be 97 ± 0.4% in the reduced form.



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Fig. 3. GSH transport by Mrp2 and the effect of CHEL. Monolayers of MDCKII-Mrp2 and MDVKII-V cells grown on membrane filters (see MATERIALS AND METHODS) were incubated at 37°C and 5% CO2 with the efflux medium (2 ml) in both apical and basal compartments. CHEL at indicated doses was present in both compartments. Domain-specific GSH efflux, determined at t = 30 and 60 min as the amount accumulated in the corresponding apical or basal medium, is shown in A. Corresponding intracellular GSH of monolayers (hatched bars) are shown in B. The total GSH per well (open bars, B) is the sum of GSH effluxed and remaining in the cells. Data are means ± SD of a typical experiment in triplicate. The molecular form of glutathione in the media was confirmed to be mainly GSH (97 ± 0.4%).

 

Mrp2 transports CHEL concurrently with GSH. We hypothesized that the observed increase in apical GSH accumulation in the presence of CHEL is due to transported GS-CHEL, which undergoes postefflux dissociation in the highly diluted extracellular milieu. We first tested whether Mrp2 transports both CHEL and GSH. Transepithelial transport experiments followed by HPLC analysis were used to assess the apical CHEL export from monolayers of MDCKII-MRP2 and MDCKII-V cells. To minimize the possible transport of CHEL by P-glycoprotein, these experiments were conducted in the presence of PSC833 (0.1 µM), which inhibits the endogenous P-glycoprotein activity, so that we could directly address the role of Mrp2 in CHEL transport. There was a striking increase in both apical directional CHEL flux and apical GSH export in MDCKII-Mrp2 compared with MDCKII-V cells (Fig. 4, A and B). Thus Mrp2 mediates both CHEL and GSH transport. Moreover, CHEL stimulated GSH transport by Mrp2 to a similar extent regardless of whether added to the basal or apical compartment (Fig. 4B), or both (Fig. 3A). It appears that CHEL enters cells without domain preference, most likely by passive diffusion in its hydrophobic pseudobase form (Fig. 1A, structure II). Additionally, CHEL flux in basal to apical direction in MDCKII-Mrp2 was at least 30-fold greater than that in the opposite direction (0-30 min), indicating that CHEL export was predominantly mediated by Mrp2. Therefore, we conclude that CHEL enters cells by diffusion but is restricted to be exported out of the cell with GSH predominantly by Mrp2 in our cell model. This conclusion strengthens the interpretation that it is the GS-CHEL that serves as a substrate for Mrp2.



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Fig. 4. Time- and dose-dependent transepithelial transport of CHEL by Mrp2 and concurrent GSH transport. Experiments were performed as described in Fig. 3 legend, with the addition of 0.1 µM PSC833 in the efflux medium to inhibit the endogenous P-glycoprotein. CHEL at indicated doses was present only in either the apical or basolateral compartment (donor). CHEL appearing in the opposite compartment medium (A) was determined by HPLC. GSH accumulation in both compartments was determined simultaneously (B), along with intracellular GSH at t =0 and 60 min (not shown). Each data point represents the mean ± SD of experiments in triplicate.

 

CHEL transport by Mrp2 is dependent on the cellular GSH content. We further characterized the effects of cellular GSH content on CHEL transport by Mrp2. Accordingly, we lowered cellular GSH content by treatment of MDCKII-Mrp2 monolayers with a lower amount of fresh medium or with BSO (25 or 50 µM) 24 h before the experiments (see Table 1). In each group, half of the monolayers were used in transepithelial transport experiments and the other half for measuring initial intracellular GSH contents. Apical accumulations of CHEL and GSH vs. initial intracellular GSH are shown in Fig. 5. Mrp2-mediated export of CHEL was lower in monolayers with lower cellular GSH, as was the apical GSH export. Thus CHEL appears to require GSH for transport by Mrp2.


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Table 1. Effect of media and BSO on cellular GSH in MDCKII-Mrp2 monolayers

 


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Fig. 5. Dependence of CHEL transport by Mrp2 on intracellular GSH content. Monolayers of MDCKII-Mrp2 with normal and decreasing initial cellular GSH were obtained by pretreatments (see Table 1). In each group, half of the monolayers were used for measuring initial intracellular GSH content, and the other half assayed as described in Fig. 4 legend with CHEL (120 nmol/well) present only in the basal compartment. Each bar represents the mean ± SD of experiments in triplicate. Data for the initial cellular GSH content of ~17.8 nmol/well are from Fig. 4.

 

Effect of CHEL on kinetics of Mrp2-mediated GSH transport. Rates of apical GSH transport by Mrp2 over a range of intracellular GSH concentrations were determined at 37°C for 15 min in the presence and absence of CHEL (see MATERIALS AND METHODS and Table 1). CHEL was present in the basolateral compartment of MDCKII-Mrp2 monolayers at 120 nmol · 2 ml-1 · well-1, at which a steady rate (linear accumulation) of apical export of CHEL and GSH was observed in previous experiments (Fig. 4).

As shown in Fig. 6, the rate of apical GSH efflux in the absence of CHEL (control) appeared to have a linear relationship to intracellular GSH concentration, indicative of a low-affinity system (Km >> 2 mM), in agreement with earlier reports (30). In contrast, in the presence of CHEL, the rate was substantially higher than the control and was saturable. Fitting these data with the Michaelis-Menten model revealed the estimates of kinetic parameters, with apparent Km = 0.28 ± 0.04 mM and apparent Vmax = 3.3 ± 0.2 nmol · min-1 · 108 cells-1. We conclude that the increase in GSH transport by Mrp2 in the presence of CHEL is the result of a high-affinity transport process.



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Fig. 6. Kinetics of Mrp2-mediated GSH transport and effect of CHEL. Rates of apical GSH efflux from MDCKII-Mrp2 monolayers as a function of intracellular GSH concentrations in the presence ({bullet}) and absence ({circ}) of CHEL (120 nmol · 2 ml-1 · well-1, in basal compartment only) are shown. Lines indicate fits obtained by using linear regression in the absence of CHEL or the Michaelis-Menten equation in the presence of CHEL. Experiments were carried out at 37°C for 15 min on monolayers with normal and decreasing cellular GSH (see MATERIALS AND METHODS and Table 1). Each data point represents the mean ± SD of experiments in triplicate.

 

Inhibition of CHEL-stimulated GSH efflux by GS-bimane. It is possible that the substrate-binding site in the presence of CHEL is the same as GS-X substrates for Mrp2, such as GS-B. If so, GS-B would be expected to inhibit CHEL-stimulated GSH efflux by Mrp2 in a dose-dependent manner. mBCl diffuses freely into cells and is then conjugated with GSH by GST to form GS-B, which leads to decreased cellular GSH concentration. Thus we conducted the inhibitory experiments under conditions identical to those for the earlier kinetic studies (Fig. 6) for easy comparison, with mBCl present in the apical medium at concentrations that resulted in steady apical GS-B export (not shown). Treatment with mBCl decreased the rate of CHEL-stimulated apical GSH efflux in a dose-dependent manner (Fig. 7A), accompanied by declining intracellular GSH. To distinguish inhibition of Mrp2 from substrate (GSH) depletion, we compared GSH transport in the presence of both mBCl and CHEL to that with CHEL alone at comparable cellular GSH levels derived from the concentration-dependent kinetic curve (Fig. 7B). With cellular GSH in the 5 nmol/well (monolayer) range, CHEL-stimulated GSH transport was inhibited by ~50% in the presence of mBCl.



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Fig. 7. Inhibition of CHEL-stimulated apical GSH efflux by GS-bimane (GS-B). Experiments were performed on MDCKII-Mrp2 monolayers grown under normal conditions (as described in Fig. 6 legend), with CHEL present only in the basal compartment and monochlorobimane (mBCl) in the apical compartment in the amount (per well) indicated. The apical GSH efflux rates measured over 15 min are shown (A). Each bar represents the mean ± SD of a typical experiment in triplicate. *P < 0.001, compared with monolayers in the absence of both CHEL and mBCl (ANOVA analysis). {dagger}P < 0.001, compared with monolayers treated with CHEL only (ANOVA analysis). To rule out the contribution from the declining cellular GSH due to formation of GS-B, the rate in the presence of both mBCl and CHEL as a function of cellular GSH concentration is shown (B) compared with the kinetic curve established in the presence of CHEL alone (dashed line represents fit to kinetics data in Fig. 6). The intracellular GS-B concentrations were determined to be 35 ± 2 and 64 ± 2 µM for mBCl doses of 15 and 25 nmol/well, respectively.

 

Effect of CHEL on apical efflux of GS-B. To address whether GSH/CHEL inhibits transport of GS-conjugates by Mrp2, apical and basal GS-B efflux and intracellular GS-B were determined in the presence and absence of CHEL; the conditions matched those in Fig. 7 (see MATERIALS AND METHODS). The rate of apical GS-B export decreased in the presence of CHEL (120 nmol · 2 ml-1 · well-1) (Fig. 8A). This decrease corresponded to an increase in intracellular GS-B accumulation (Fig. 8B). Thus CHEL/GSH inhibits GS-B efflux by Mrp2.



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Fig. 8. Apical GS-B efflux by Mrp2 and the effect of CHEL. GS-B efflux experiments were carried out in MDCKII-Mrp2 monolayers under conditions matching those in Fig. 7. MBCl was present at indicated doses in the apical compartment alone or in combination with CHEL (120 nmol · 2 ml-1 · well-1, in basal compartment). Apical and basolateral GS-B efflux and intracellular GS-B were determined at t = 15 min by measuring the fluorescence of GS-B. The apical GS-B efflux (A) and cellular GS-B (B) are shown. Each bar represents the mean ± SD of an experiment in triplicate. {dagger}P < 0.01; {ddagger}P < 0.001, compared with the corresponding monolayers in the absence of CHEL (ANOVA analysis).

 

CHEL requires GSH and thiol group of GSH to inhibit transport of GS-conjugates by Mrp2. To further define the substrate specificity of the Mrp2 system, we examined inhibition of Mrp2-mediated [3H]LTC4 uptake into vesicles from MDCKII-Mrp2 cells. This assay was chosen because radiolabeled CHEL was not available, and it has been difficult to detect ATP-dependent GSH transport in vesicle studies. Membrane vesicles prepared from MDCKII-Mrp2 cells exhibited an ATP-dependent uptake of [3H]LTC4 (6.8 nM) that was time dependent (up to 5 min) and osmotically sensitive (data not shown). The rate of uptake was 13-fold higher than that in control vesicles from MDCKII-V cells: 147 ± 9.1 vs. 11 ± 1.3 fmol · mg-1 · 5 min-1. This result agrees well with that from intact cells. Given that GSH (2 mM) was used in the transport medium to reduce ATP-independent uptake in our assay, we repeated these experiments by replacing GSH with two other molecules: S-MeGSH, a GSH thioether derivative, and ophthalmic acid (OPTA), a GSH analog with a methyl group in place of the thiol group. A comparable rate for the ATP-dependent LTC4 uptake was observed in both cases (Fig. 9A). In the presence of GSH (2 mM), CHEL inhibited the ATP-dependent uptake of [3H]LTC4 (6.8 nM) with an IC50 {approx} 100 µM (Fig. 9B). HPLC analysis confirmed that, under our experimental conditions, formation of GSSG was not increased by the addition of CHEL (100 µM).



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Fig. 9. CHEL requires the thiol group of GSH to inhibit Mrp2-mediated [3H]leukotriene C4 (LTC4) uptake by membrane vesicles. A: GSH is not essential for ATP-dependent [3H]LTC4 uptake. [3H]LTC4 uptake by membrane vesicles from MDCKII-Mrp2 (30 µg) was measured at 37°C for 5 min with [3H]LTC4 (6.8 nM) and 2 mM GSH, S-methyl GSH (S-MeGSH), or ophthalmic acid (OPTA) in the presence of ATP (4 mM) or AMP (4 mM), as described in MATERIALS AND METHODS. Each bar represents the mean ± SD of experiments in triplicate. B: CHEL inhibits ATP-dependent [3H]LTC4 uptake in the presence of GSH. Rates of [3H]LTC4 uptake in the presence of 2 mM GSH and different concentrations of CHEL (0, 10, 30, 100, 500 µM) were measured as in A. Data are presented as percentages of the uptake in the presence of GSH alone (control). Each point represents the mean ± SD of a typical experiment in triplicate. C: the thiol group of GSH is essential for CHEL to inhibit [3H]LTC4 uptake by Mrp2. Rates of [3H]LTC4 uptake were measured as in A with 2 mM GSH, S-MeGSH, or OPTA alone or in combination with 100 µM CHEL. Data are presented as percentages of uptake in the presence of GSH alone (control). Each bar represents the mean ± SD of experiments in triplicate. *P < 0.001, compared with the control in the presence of GSH only (ANOVA analysis).

 

We used S-MeGSH or OPTA to assess whether the thiol group of GSH is required for the inhibitory effect of CHEL/GSH on the ATP-dependent [3H]LTC4 uptake by Mrp2 (Fig. 9C). Unlike GSH, S-MeGSH or OPTA failed to inhibit [3H]LTC4 uptake with CHEL. Because the thiol group of GSH was required for CHEL to inhibit transport of LTC4 by Mrp2, it is reasonable to expect that the GS-CHEL adduct is a substrate for Mrp2 under physiological conditions. However, because the adduct is labile under various assay conditions and dilutions, it has not been possible to directly demonstrate that the adduct is released from the cells.

Notably, in the presence of the ATP-regeneration system, we obtained similar results, except that the ATP-dependent [3H]LTC4 uptake was increased up to 30 min.

VRP enhances Mrp2-mediated transport of GSH and CHEL. VRP is a multidrug resistance (MDR) reversing agent and a calcium channel blocker. Previous studies have shown that VRP can enhance MRP1-mediated ATP-dependent GSH uptake in the absence of other substrates in vesicle studies and that it is not actively transported by MRP1 (27). Therefore, we examined the effects of VRP on Mrp2-mediated GSH transport. The apical export of [3H]VRP was comparable in MDCKIIMrp2 and MDCKII-V cells in transepithelial transport experiments (37 ± 3.7 vs. 37 ± 1.5 and 105 ± 6.0 vs. 102 ± 7.8 nmol · 30 min-1 · well-1 at VRP doses of 200 and 500 nmol/well, respectively). Thus Mrp2 is un-likely to transport VRP. In these experiments, PSC833 and acivicin were not present in the efflux medium. In the presence of PSC833 (0.1 µM), VRP alone in the basal compartment (600 nmol · 2 ml-1 · well-1) caused a slight but not significant increase in apical GSH efflux from MDCKII-Mrp2 monolayers (1.24 ± 0.01 vs. 1.01 ± 0.16 nmol · 60 min-1 · well-1, P > 0.05). However, with the addition of CHEL in basal compartment (80 and 120 nmol · 2 ml-1 · well-1), VRP synergistically elevated apical efflux of both GSH (Fig. 10A) and CHEL (Fig. 10B). In these experiments, VRP or CHEL alone, or in combination, stimulated the endogenous basolateral GSH transporter to a comparable level (Fig. 10A). It appears that the response of this putative transporter to the cotreatment of VRP + CHEL is not synergistic.



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Fig. 10. Verapamil (VRP) effect on Mrp2-mediated GSH and CHEL transport. Monolayers of MDCKII-Mrp2 cells were assayed as described in Fig. 4 legend, with VRP (600 nmol · 2 ml-1 · well-1) present only in the basal compartment. CHEL was added to the basal compartment either alone or in combination with VRP, at the doses indicated. The control was in the absence of both CHEL and VRP. Shown are the amounts of GSH accumulated in apical and basal media (A) and the amount of CHEL accumulated in the apical medium (B). Each data point represents the mean ± SD of experiments in triplicate.

 

Mrp2 mediates transport of CHEL and GSH with a 1:1 molar ratio. CHEL and GSH efflux by Mrp2 has been observed in three sets of experiments with varying CHEL doses (Fig. 4) or initial intracellular GSH contents (Fig. 5) and with coincubation with VRP (Fig. 10). Data pairs from these experiments show an excellent correlation (two-tailed P < 0.0001), regardless of whether they were analyzed separately or in combination (Fig. 11).



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Fig. 11. Linear correlation of CHEL and GSH transport by Mrp2. To analyze the relationship of Mrp2-mediated GSH and CHEL transport, values of apical GSH efflux from previous experiments (Figs. 4, 5, and 10) were compiled and plotted against their corresponding apical CHEL accumulations. The linear regression analysis (straight line) resulted in a slope = 0.91 ± 0.05 and a y-intercept = 1.0 ± 0.3, with a correlation coefficient r = 0.984 (r2 = 0.968, P < 0.0001). Symbols represent data from different CHEL doses (squares), initial intracellular GSH contents (triangles), and coincubation with VRP (hexagons).

 

To determine the stoichiometry of CHEL and GSH transport by Mrp2, values of apical accumulation of GSH from all previous data were compiled and plotted against the corresponding apical accumulation of CHEL (Fig. 11). Regression analysis revealed a linear relationship with a slope = 0.91 ± 0.05, and a y-intercept = 1.0 ± 0.3. This outcome indicates that CHEL transport and CHEL-dependent GSH transport by Mrp2 have a 1:1 ratio (slope ~1) and that a fraction of the total GSH accumulated in apical medium is independent of CHEL transport (the y-intercept).

Because CHEL can enter the MDCKII cells from either pole, recycling of CHEL leading to excess GSH vs. CHEL needs to be considered. In transepithelial transport experiments, CHEL was added to the basolateral compartment at 80 and 120 nmol/well. When the apical medium was reduced to 0.5 ml from 4 ml (see MATERIALS AND METHODS), apical GSH accumulation at 60 min was sustained, but there was a 38 and 30% drop in apical CHEL accumulation, respectively, suggesting that the decline in the accumulation was due to recycling of CHEL rather than its export.


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
The putative role of CHEL as a PKC inhibitor should be noted. Although originally thought to be a PKC inhibitor, recent evidence has revealed that CHEL does not inhibit PKC (8, 24, 40). We originally performed our experiments to assess the effects of various PKC inhibitors on Mrp2 function in our cell model. Among a wide variety of inhibitors, only minor effects on GSH efflux in Mrp2-expressing cells were seen (unpublished observations) except for CHEL. This led us to seek an alternative explanation for the apparent stimulation of GSH efflux in Mrp2-expressing cells. Our present work is the first description of the effect of CHEL on the transport of cellular GSH by Mrp2. In our cell model, rat Mrp2 is overexpressed in the apical domain of MDCKII-Mrp2 monolayers, as reported for human MRP2 by others (6, 12, 30), and plays a predominant role in the apical GSH efflux. We demonstrated that CHEL could deplete intracellular GSH apparently through a dramatic stimulation of GSH efflux by Mrp2. Although the endogenous apical transporter in MDCKII-V cells (a stable clone transfected with an empty vector) responded similarly to CHEL, the magnitude of apical GSH transport was very low.

There are two possible molecular mechanisms as to how CHEL may be transported with and influence the transport of GSH from cells expressing Mrp2. One is cotransport, with the drug and GSH binding to the transport site(s), as well as undergoing transport as separate molecules. Alternatively, a reversible adduct of the drug and GSH could be bound, as well as transported as a single molecule, and could subsequently dissociate extracellularly, releasing GSH and the drug. A cotransport mechanism, for example, has been proposed in MRP1-mediated transport of Vinca alkaloid vincristine (VCR). The evidence in support of such a mechanism is as follows: 1) Mrp1 transports VCR with GSH (25, 26, 34); 2) the thiol group of GSH is not required for the transport, given that S-MeGSH is able to replace GSH in supporting transport of VCR; and 3) there is a lack of evidence in vivo or in vitro that VCR forms an adduct with GSH. However, thus far, the stoichiometry of the proposed cotransport has not been determined because of the hydrophobicity of VCR. Similarly, cotransport mechanism has been proposed for Mrp2-mediated transport of Vinca alkaloid vinblastine (VBL) and GSH (11, 37). In vesicle studies, transport of VBL did not require the thiol group of GSH, and it had a ratio of VBL to GSH of 2-3:1 in transepithelial transport experiments. It should be noted that, unlike CHEL, VBL is hydrophobic and easily diffuses into and out of cells, which provides a molecular basis for its behavior in transepithelial transport experiments. On the other hand, GS-adduct transport has been suggested in Mrp2-mediated transport of arsenite (17) and {alpha}-naphthylisothiocyanate (ANIT) (9). These compounds form reversible adducts with GSH in vitro and stimulate GSH export into bile in intact liver or from MRP2-expressing cells. The stoichiometry in both cases has been difficult to assess because of recycling of arsenite and ANIT across the cell membrane.

CHEL is known to form a reversible adduct with GSH under physiological conditions. In our present studies, we have shown that apical CHEL export by Mrp2 occurs concurrently with GSH with a 1:1 stoichiometry and that VRP synergistically increases export of both GSH and CHEL. The kinetics of Mrp2-mediated apical GSH efflux were markedly different in the presence of CHEL compared with its absence (apparent Km = 0.28 mM vs. >>2 mM). This low Km value is in the order of the reported Km (70 ± 12 µM) for ATP-dependent GS-dinitrophenyl (GS-DNP) uptake by membrane vesicles prepared from MRP2-expressing MDCKII cells (30). Additionally, GS-B, a model substrate for Mrp2, blocked the CHEL-stimulated GSH transport by Mrp2 and vice versa, suggesting that they share a common substrate site. Finally, we were able to show that CHEL inhibited (IC50 {approx} 100 µM) Mrp2-mediated ATP-dependent LTC4 uptake by vesicles in the presence of GSH (2 mM), whereas neither S-MeGSH nor OPTA, substituted for GSH, were able to support the inhibition. Thus not only GSH but also its thiol group is required.

Because the intracellular GSH concentration in MDCKII-Mrp2 cells (~2 mM) is at least an order of magnitude greater than the maximum CHEL concentration (unpublished observations), nearly all the CHEL molecules in the cells available for transport should be in the adduct form. Upon release from the cells, however, a new equilibrium is reached, in which most of the adduct dissociates. This occurs because GSH is no longer in excess and both GSH and CHEL concentrations are low. Unfortunately, because of the CHEL and GSH assay conditions, it is not possible to directly measure the adduct.

In summary, CHEL stimulates GSH efflux by Mrp2 through an apparent high-affinity process that transports the two molecules with a stoichiometry of 1:1. This mode of action appears to be consistent with and can best be interpreted with the reversible adduct of GS-CHEL being the actual substrate for Mrp2. The transport of reversible adducts of GSH by Mrp2 has a number of implications. The high rate of GSH secretion into bile mediated by Mrp2 may be partly due to the transport of reversible adducts with unidentified endogenous substances. The transport of reactive drugs or metabolites into bile as reversible GSH adducts can be considered as either a protective function for removing certain toxic drugs from hepatocytes or a toxic mechanism for exposing cholangiocytes to these reactive substances after dissociation of the adducts in bile, leading to cholestatic injury.


    DISCLOSURES
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 ABSTRACT
 MATERIALS AND METHODS
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This work was supported by National Institutes of Health (NIH) Grants R01-DK-30312 and R01-AG-7467. The resources and services of the Cell Biology (Confocal Microscopy) and Mathematical Analysis and Modeling cores of the University of Southern California Research Center for Liver Diseases, supported by NIH Grant P30-DK-48522, were also utilized.


    FOOTNOTES
 

Address for reprint requests and other correspondence: N. Kaplowitz, Keck School of Medicine, Univ. of Southern California, 2011 Zonal Ave., HMR101, Los Angeles, CA 90033 (E-mail: kaplowit{at}usc.edu).

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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 MATERIALS AND METHODS
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