ATP-dependent GSH and glutathione S-conjugate transport in skate liver: role of an Mrp functional homologue

James F. Rebbeor1,4, Gregory C. Connolly1,4, John H. Henson2,4, James L. Boyer3,4, and Nazzareno Ballatori1,4

1 Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, New York 14642; 2 Department of Biology, Dickinson College, Carlisle, Pennsylvania 17013; 3 Department of Medicine and Liver Center, Yale University School of Medicine, New Haven, Connecticut 06510; and 4 Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Multidrug resistance-associated proteins 1 and 2 (Mrp1 and Mrp2) are thought to mediate low-affinity ATP-dependent transport of reduced glutathione (GSH), but there is as yet no direct evidence for this hypothesis. The present study examined whether livers from the little skate (Raja erinacea) express an Mrp2 homologue and whether skate liver membrane vesicles exhibit ATP-dependent GSH transport activity. Antibodies directed against mammalian Mrp2-specific epitopes labeled a 180-kDa protein band in skate liver plasma membranes and stained canaliculi by immunofluorescence, indicating that skate livers express a homologous protein. Functional assays of Mrp transport activity were carried out using 3H-labeled S-dinitrophenyl-glutathione (DNP-SG). DNP-SG was accumulated in skate liver membrane vesicles by both ATP-dependent and ATP-independent mechanisms. ATP-dependent DNP-SG uptake was of relatively high affinity [Michaelis-Menten constant (Km) = 32 ± 9 µM] and was cis-inhibited by known substrates of Mrp2 and by GSH. Interestingly, ATP-dependent transport of 3H-labeled S-ethylglutathione and 3H-labeled GSH was also detected in the vesicles. ATP-dependent GSH transport was mediated by a low-affinity pathway (Km = 12 ± 2 mM) that was cis-inhibited by substrates of the Mrp2 transporter but was not affected by membrane potential or pH gradient uncouplers. These results provide the first direct evidence for ATP-dependent transport of GSH in liver membrane vesicles and support the hypothesis that GSH efflux from mammalian cells is mediated by members of the Mrp family of proteins.

glutathione; canalicular transport; bile secretion


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GSH IS SYNTHESIZED in the cell cytosol but is degraded exclusively in the extracellular space by the ectoproteins gamma -glutamyl transpeptidase and dipeptidase (4, 16, 17, 24). Transport of GSH across the cell membrane is therefore required for normal cellular turnover; however, the transport systems involved are not well defined. Recent studies indicate that GSH efflux from hepatocytes is mediated in part by Oatp1 (20), the hepatic basolateral organic solute transporter, by multidrug resistance-associated protein (Mrp)1 (12, 27), an ATP-dependent plasma membrane transport protein, and by Mrp2 (6, 11, 13, 26, 29), the liver canalicular ATP-dependent organic anion transporter. However, the evidence for the roles of Mrp1 and Mrp2 in GSH efflux is indirect (6). Studies that have attempted to demonstrate ATP-dependent GSH transport in isolated plasma membrane vesicles have provided negative results (6). A recent study of Mrp2-mediated GSH transport supports the hypothesis that GSH is a substrate for this transport protein but suggests that transport does not require ATP (39).

In contrast to these findings in mammalian systems, our studies in membrane vesicles isolated from the yeast Saccharomyces cerevisiae demonstrated the presence of a low-affinity ATP-dependent GSH transport process that was mediated by Ycf1p, the yeast orthologue of mammalian Mrp (28, 29). Ycf1p shows significant homology with rat Mrp1 (42.6%; Ref. 37) and Mrp2 (41.9%; Ref. 10), and these three proteins appear to transport similar compounds. In S. cerevisiae, Ycf1p is localized preferentially to the vacuolar membrane, where it functions to secrete organic anions such as glutathione S-conjugates into the vacuolar space (21, 22, 38, 40). Recent studies by Paulusma and co-workers (26) provide additional evidence for a role of Mrp1 and Mrp2 in GSH efflux. Using Madin-Darby canine kidney (MDCK)II cells stably expressing human MRP1 and MRP2, these investigators observed a direct correlation between MRP expression and GSH efflux from the cells; however, they were unable to detect ATP-dependent GSH transport in isolated membrane vesicles.

The reason for the inability to measure ATP-dependent GSH transport in mammalian membrane vesicles is not known but may be related to the combined effects of a low catalytic efficiency of GSH transport [i.e., a high Michaelis-Menten constant (Km), but only modest maximal velocity (Vmax)] and a comparatively high nonspecific permeability of inside-out mammalian plasma membrane vesicles, which tends to dissipate solute gradients generated by membrane pumps (23). Moreover, although vesicles that are in the inside-out configuration are required to demonstrate ATP-dependent transport, these vesicles may be particularly susceptible to a high nonspecific permeability because of the unnatural curvature of the membrane bilayer. Mammalian liver plasma membrane vesicles are able to maintain transmembrane ion or solute gradients for only relatively brief periods of time, on the order of minutes (1, 23, 25).

In contrast to mammalian liver vesicles, our recent studies with skate liver plasma membrane vesicles indicate that these vesicles can maintain comparable gradients for several hours (7), indicating a lower nonspecific permeability. The present study confirms this observation by showing that skate liver plasma membrane vesicles are also able to generate and maintain high transmembrane gradients for taurocholate and S-dinitrophenyl-glutathione (DNP-SG).

Like mammalian livers, skate livers secrete GSH, glutathione S-conjugates, and other organic anions into bile in relatively high concentrations (9, 34), indicating the presence of a functional homologue of the canalicular Mrp2 transporter. The present results support the hypothesis that an Mrp2-like transporter is present in skate liver and demonstrate the presence of ATP-dependent GSH and glutathione S-conjugate transport in skate liver membrane vesicles. These results also indicate that skate liver membrane vesicles may be a useful model system for studying this and other ATP-dependent hepatic transport processes.


    MATERIALS AND METHODS
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ABSTRACT
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Chemicals. N-7[(4-nitrobenzo-2-oxa-1,3-diazol)]-7beta -amino-3alpha ,12alpha -dihydroxy-5beta -cholan-24-oyl-2-aminoethanesulfonate (NBD-TC), a fluorescent bile salt derivative, was synthesized as described previously (31, 32) and was provided by David Miller, National Institute of Environmental Health Sciences (Research Triangle Park, NC). [glycine-2-3H]GSH (0.982 Ci/mmol) and [G-3H]taurocholate (2.1 Ci/mmol) were purchased from DuPont NEN. [glycine-2-3H]DNP-SG and 3H-labeled S-ethylglutathione (ethyl-SG) were synthesized as previously described (18). All other chemicals and reagents were purchased from Sigma, Aldrich, Amersham, J. T. Baker, or Fisher. The pH of incubation solutions containing high concentrations of GSH or other anions was routinely adjusted to 7.5 with Tris base immediately before use.

Animals. Male little skates (Raja erinacea; 0.7-1.2 kg body wt) were collected by net from Fisherman's Bay in Maine and maintained for up to 4 days in tanks equipped with flowing 15°C seawater at the Mount Desert Island Biological Laboratory, Salsbury Cove, ME. Male Sprague-Dawley rats (~250 g) were obtained from Charles River Laboratories (Kingston, NY) and fed Purina chow (formula 5001) ad libitum.

Membrane isolation. Plasma membranes from skate liver were isolated by a modification of methods developed by Sellinger et al. (33) and Song et al. (36). Three skate livers (30-40 g) were removed from skates anesthetized with pentobarbital sodium (5 mg/kg) administered via the caudal vein. The livers were perfused with 40 ml of ice-cold heparinized calcium- and magnesium-free elasmobranch Ringer solution (pH 7.4) and chilled on ice. Livers were minced with scissors in 100 ml of elasmobranch Ringer buffer diluted 1:1 with water and homogenized in a glass Dounce homogenizer (loose-fitting pestle type A) with 20 strokes. Portions of the homogenate were transferred to a 40-ml Dounce homogenizer and homogenized further with 10 strokes of a loose-fitting pestle. The homogenates were diluted to 690 ml with calcium- and magnesium-free 0.5× elasmobranch Ringer solution and centrifuged with a Sorvall GSA rotor at 4,000 g for 10 min. The supernatant was collected and centrifuged with a Sorvall SS-34 rotor for 20 min at 10,000 g. The soft outer white pellets were collected, leaving darker adherent pigment material, and combined in a prechilled 40-ml Dounce homogenizer and homogenized with 10 strokes of a type B pestle. Homogenates (3.5 ml) were layered over a discontinuous sucrose gradient made of 15 ml of 45%, 8 ml of 32%, and 8 ml of 16% sucrose. Gradients were then centrifuged for 60 min at 70,000 g in a Beckman SW-28 rotor. The banded material at the 16%/32% interface was collected and pooled, diluted with 150 ml of transport buffer containing (in mM) 10 HEPES, pH 7.4, 20 KCl, 0.1 CaCl2, and 250 sucrose, and centrifuged with a Sorvall SS-34 rotor for 10 min at 20,000 g. Supernatant was removed, and pelleted plasma membranes were resuspended with transport buffer (10 mM HEPES-Tris, pH 7.5, 250 mM sucrose, 20 mM KCl) to ~2.5 ml and passed through a 25-gauge needle 20 times. Samples were immediately placed in a -80°C freezer and were used within 6 mo of isolation.

Rat liver canalicular membranes were isolated according to methods previously performed in this laboratory (5, 8, 23). They were stored in the same transport buffer (in mM: 10 HEPES-Tris, pH 7.5, 250 sucrose, 20 KCl) at -80°C.

Isolation of skate hepatocyte clusters. Skate hepatocytes were isolated as described by Smith et al. (35), using a two-step collagenase perfusion protocol. Cells were washed and resuspended in elasmobranch Ringer solution (in mM: 270 NaCl, 4 KCl, 2.5 CaCl2, 3 MgCl2, 0.5 Na2SO4, 1 KH2PO4, 8 NaHCO3, 350 urea, 5 D-glucose, and 5 HEPES-Tris, pH 7.5) at a concentration of 30-50 mg wet wt/ml (~3-5 ×106 cells/ml). Cells were maintained at 15°C under room air.

Electrophoresis and immunoblotting. Skate liver plasma membranes were added to an equal volume of sample loading buffer (50 mM Tris·HCl, pH 6.8, 2% SDS, 0.1 mM dithiothreitol, 10% glycerol) and subjected to SDS-PAGE on 6-20% (wt/vol) gradient gels. The separated polypeptides were electrotransferred to a 0.45-µm nitrocellulose membrane for 2 h at 130 mA using a semidry transfer apparatus. The filters were blocked and then incubated for 2 h at room temperature with a 1:5,000 dilution of EAG-15 rabbit antisera raised against the rat liver Mrp2 antigen (kindly provided by D. Keppler; Heidelberg, Germany). Immunoreactive bands were detected by incubating washed membranes with a 1:3,000 dilution of anti-rabbit IgG conjugated with horseradish peroxidase enzyme (Sigma) and then visualized by enhanced chemiluminescence according to the manufacturer's instructions (Amersham). Nitrocellulose filters were also incubated with a 1:500 dilution of MIII-5 mouse monoclonal antibody (kindly provided by R. P. J. Oude Elferink, Amsterdam, The Netherlands), and immunoreactive bands were detected by incubating washed membranes with a 1:2,000 dilution of anti-mouse IgG conjugated with horseradish peroxidase enzyme (Sigma) and visualized as described above.

Immunocytochemical localization. Skate hepatocyte clusters were first allowed to adhere onto glass coverslips coated with 1 mg/ml high-molecular-weight poly-L-lysine. The cells were fixed for 30 min in -20°C acetone, rehydrated in phosphate-buffered saline, and then blocked in phosphate-buffered saline plus 1% bovine serum albumin and 0.2% Triton X-100. Cells were then incubated in 1:200 dilution of the EAG-15 anti-Mrp2 primary antibody followed by a 1:400 dilution of goat anti-rabbit IgG conjugated with Alexa 488 fluorophore (Molecular Probes, Eugene, OR). Stained cells were viewed on an Olympus Fluoview laser scanning confocal microscope using a ×40 (1.15 NA) water-immersion objective lens.

Vesicle transport. Transport was measured as uptake of radiolabeled substrate into vesicles collected by rapid filtration on a Millipore 0.45-µm filter under vacuum, essentially as described previously (5, 23). Skate liver plasma membrane vesicles were thawed by immersion in a 20°C water bath and diluted in transport buffer (10 mM HEPES, pH 7.5, 250 mM sucrose, 20 mM KCl, with an ATP regenerating system consisting of 10 mM phosphocreatine, 10 mM MgCl2, 100 µg/ml creatine phosphokinase, and either 5 mM Na2ATP or 10 mM NaCl). Diluted vesicles were treated with 0.5 mM acivicin to inhibit resident gamma -glutamyltranspeptidase enzyme, passed repeatedly through a 26-gauge needle (×10), and incubated at 20°C for 15 min before the transport reaction was started. Freshly prepared dithiothreitol was added to 5 mM in all transport solutions used to measure GSH uptake to maintain glutathione in its reduced form. Transport was started by adding 20 µl of diluted plasma membrane vesicles to 80 µl of incubation transport buffer (with substrate) at 4°C or 20°C for timed intervals. Transport was quenched by adding 1 ml of ice-cold stop buffer (in mM: 300 sucrose, 10 HEPES-Tris, pH 7.5, and 20 KCl), and vesicles were collected by applying 1 ml of quenched reaction solution to the prewetted filter under vacuum and washing the filter with an additional 4 ml of ice-cold stop buffer. Filters were collected and dissolved in 5 ml of Opti-Fluor (Packard Instruments, Meriden, CT), and radiolabeled drug uptake was quantitated by liquid scintillation measurements. Controls for nonspecific binding of drug to filters and vesicles were performed by measuring retention of radiolabeled substrate on filters in the absence of vesicles or on secretory vesicles incubated in transport buffer at 4°C for each time point.

ATP analysis. Skate liver plasma membrane vesicles and rat liver canalicular plasma membrane vesicles (100 µg protein/100 µl incubation solution) were incubated in transport buffer (in mM: 10 HEPES, pH 7.5, 250 sucrose, and 20 KCl) containing an ATP regenerating system (10 mM phosphocreatine, 10 mM MgCl2, 100 µg/µl creatine phosphokinase, and 5-6 mM Na2ATP). Rat canalicular membrane vesicles and controls with no membranes added were incubated at 37°C. Skate liver plasma membrane vesicles were incubated at 20°C. At specific time points, 20 µl was removed from the incubation vial and diluted in 2 ml of ice-cold 2% perchloric acid. After a 10-min incubation on ice, samples were filtered using a 0.45-µm syringe filter, and ATP content was measured using the HPLC method previously described (15).

Statistical analysis. Kinetic data from experiments measuring uptake of radiolabeled substrate in plasma membrane vesicles were fit to the Michaelis-Menten equation by nonlinear least-squares regression analysis using SigmaPlot 4.16. Vmax and Km values with standard errors were derived from these curves. Comparison of data measuring initial rates of uptake of [3H]GSH in the presence and absence of inhibitors was performed by Student's t-test and correlated to P < 0.05.


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RESULTS
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Skate liver plasma membrane vesicles can accumulate and maintain high transmembrane gradients of organic anions. The uptake of 1 µM [3H]taurocholate (Fig. 1) into skate liver plasma membrane vesicles was measured at 20°C for timed intervals with 5 mM MgATP and without ATP. The difference represents the time course for ATP-dependent uptake of 1 µM [3H]taurocholate (Fig. 1). ATP-dependent accumulation was seen during the first 2 h and then remained stable for up to 10 h. The kinetics and substrate specificity of taurocholate transport in skate liver vesicles have been characterized in a separate study (7). These previous data indicate that the apparent Km for ATP-dependent taurocholate uptake is 40 µM, and the substrate specificity is comparable to that of the mammalian Bsep/Spgp transporter (14).


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Fig. 1.   Time course of uptake of 1 µM [3H]taurocholate in skate liver plasma membrane vesicles. Skate liver plasma membranes (100 µg) were incubated in transport buffer (10 mM HEPES, pH 7.5, 250 mM sucrose, and 20 mM KCl, with an ATP regenerating system consisting of 10 mM phosphocreatine, 10 mM MgCl2, and 100 µg/ml creatine phosphokinase) with 1 µM [3H]taurocholate in the presence () or absence (open circle ) of 5 mM MgATP at 20°C for varied amounts of time. The difference in the amount of uptake with MgATP compared with that in the absence of MgATP was plotted as the ATP-dependent uptake (down-triangle). Data points represent the means ± SE of triplicate measurements in a representative experiment. Bars indicating SE are smaller than the symbols

The intravesicular volume of skate liver plasma membrane vesicles was estimated to be ~1 µl/mg protein according to the equilibrium values for L-alanine uptake measured previously (33). Accordingly, the intravesicular concentration of [3H]taurocholate was calculated to be 250 µM, or 250-fold higher than that of the incubation medium at the 10 h time point. Thus skate liver membrane vesicles are able to actively accumulate this organic anion and maintain a large outwardly directed gradient for at least 10 h, indicating that the vesicles are highly transport competent and are most likely tightly sealed.

An additional factor that may contribute to the prolonged ATP-dependent accumulation of [3H]taurocholate in skate liver membrane vesicles is a prolonged maintenance of the ATP gradient with these vesicles, compared with rat liver membrane vesicles. To test this possibility, we measured ATP levels in incubation solutions containing either skate liver membrane vesicles or rat liver canalicular membrane vesicles (Fig. 2). Skate membrane vesicles were incubated at 20°C and rat liver membrane vesicles at 37°C, but the composition of the medium was identical. With the rat liver membranes, ATP declined slowly during the first 10-15 min of incubation and was essentially completely hydrolyzed by 30 min (Fig. 2). In contrast, ATP content was unchanged during the first 3 h in incubation solutions containing skate membrane vesicles and was decreased only slightly at the 4 h time point (Fig. 2). These results indicate that skate membranes have lower ATP hydrolytic capacity and that the ATP regenerating system can maintain ATP concentrations relatively constant for extended periods of time under these conditions.


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Fig. 2.   Prolonged maintenance of ATP concentrations in incubation solutions containing skate liver plasma membrane vesicles but not in those containing rat liver canalicular membrane vesicles. Rat liver canalicular membrane vesicles (100 µg/100 µl incubation solution; black-triangle) and controls with no membranes added () were incubated at 37°C in transport buffer containing ~5-6 mM ATP and an ATP regenerating system. Skate plasma membrane vesicles (100 µg/100 µl incubation solution; ) were incubated at 20°C in the same buffer. ATP content of the buffer was measured at specific time points for up to 4 h. Data points represent the means ± SE of 3 experiments with different plasma membrane preparations.

Detection of an Mrp2-like protein in skate liver plasma membranes. Skate liver plasma membranes were subjected to SDS-PAGE electrophoresis, and the separated proteins were immunoblotted with two different antibodies raised against rat liver canalicular Mrp2 (EAG-15 and MIII-5). Rat liver canalicular plasma membranes containing the Mrp2 antigen, seen as an immunoreactive band at ~190 kDa, were included as a control. A protein band with a molecular mass of between 170 and 180 kDa was also detected with both the EAG-15 and MIII-5 antibodies in skate liver plasma membranes (Fig. 3), indicating the presence of an Mrp2-like protein in skate liver membranes. Each of these two antibodies also reacted weakly with lower-molecular-weight bands, but these bands were distinct for the two Mrp2 antibodies. No signal was detected in membranes isolated from skate muscle or heart (data not shown).


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Fig. 3.   Immunodetection of Mrp2-like antigen in skate liver plasma membranes. Plasma membranes isolated from the skate liver were subjected to SDS-PAGE, and separated proteins were immunoblotted with the EAG-15 and MIII-5 polyclonal antibodies against rat liver Mrp2. Rat liver canalicular plasma membranes (0.5 µg) were also run as a control. The antibodies react with a 190-kDa antigen in the rat liver membranes and also cross-react with an antigen of slightly smaller molecular mass in the skate liver membranes.

Immunolocalization of Mrp2-like protein in skate hepatocyte clusters. Staining of isolated skate hepatocyte clusters with anti-Mrp2 revealed an apical/pericanalicular staining pattern (Fig. 4). Interestingly, this pattern was often discontinuous, suggesting an apparent differential accumulation of the transporter in different domains of the apical membrane.


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Fig. 4.   Immunolocalization of Mrp2 to the apical region of skate hepatocytes. A: an XY confocal longitudinal slice of an anti-Mrp2-labeled skate hepatocyte cluster. B: an XZ confocal cross-sectional slice of the same cluster at the position of the arrows in A. Note the intense and focal distribution of the anti-Mrp2 labeling in the apical/pericanalicular regions of the cells. The cross section in B demonstrates how the Mrp2 staining surrounds the lumen of the bile canaliculus. Bar, 10 µM.

ATP-dependent transport of [3H]DNP-SG, [3H]ethyl-SG, and [3H]GSH in skate liver plasma membrane vesicles. Additional studies tested for the presence of ATP-dependent DNP-SG transport in skate liver membrane vesicles. DNP-SG is secreted into skate bile in high concentrations (34), indicating the presence of an active canalicular transport mechanism for this organic anion.

Uptake of 10 µM [3H]DNP-SG in skate liver plasma membrane vesicles was measured at 20°C for timed intervals up to 4 h, with 5 mM MgATP and without ATP (Fig. 5A). Uptake was mediated by both ATP-dependent and ATP-independent mechanisms in this mixed population of skate liver plasma membranes (i.e., canalicular and sinusoidal membrane domains). The difference represents the time course for ATP-dependent uptake. ATP-dependent uptake was linear for up to 45 min and continued throughout the duration of these experiments (4 h).


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Fig. 5.   Time course of uptake of 10 µM 3H-labeled S-dinitrophenyl-glutathione (DNP-SG), 0.5 mM 3H-labeled S-ethylglutathione (ethyl-SG), and 1 mM 3H-labeled GSH in skate liver plasma membrane vesicles. Skate liver plasma membranes (100 µg) were incubated in transport buffer (10 mM HEPES, pH 7.5, 250 mM sucrose, 20 mM KCl, with an ATP regenerating system consisting of 10 mM phosphocreatine, 10 mM MgCl2, 100 µg/ml creatine phosphokinase) with 10 µM [3H]DNP-SG (A), 0.5 mM [3H]ethyl-SG (B), or 1 mM [3H]GSH (C) in the presence () or absence () of 5 mM MgATP at 200C for varied amounts of time (inset). Dithiothreitol (5 mM) was also present in the GSH uptake experiments to prevent the oxidation of GSH to GSSG. The difference in the amount of uptake with MgATP compared with that in the absence of MgATP for each compound was plotted as the ATP-dependent uptake. Data points represent the means ± SE of at least 3 experiments with different plasma membrane preparations.

Of significance, uptake of 0.5 mM [3H]ethyl-SG and 1 mM [3H]GSH (Fig. 5, B and C) in skate liver plasma membrane vesicles was also mediated by both ATP-dependent and ATP-independent mechanisms. The time course for ethyl-SG and GSH uptake was similar to that of DNP-SG.

ATP concentration dependence of 10 µM [3H]DNP-SG and 5 mM [3H]GSH uptake in skate liver plasma membrane vesicles. The initial rate of ATP-dependent uptake of 10 µM [3H]DNP-SG (Fig. 6A) or 5 mM [3H]GSH (Fig. 6B) was measured after 45 min of incubation with varying concentrations of MgATP to correlate the kinetics of substrate transport and ATP availability. Apparent Km values for MgATP of 0.4 ± 0.2 and 0.5 ± 0.2 mM were measured for DNP-SG and GSH, respectively (Fig. 6). These values are similar to those measured in rat liver canalicular membranes (a Km for ATP of 0.26 mM; Ref. 18).


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Fig. 6.   ATP-concentration dependence of 10 µM [3H]DNP-SG and 5 mM [3H]GSH uptake in skate liver plasma membrane vesicles. Skate liver plasma membranes (100 µg) were incubated in transport buffer (10 mM HEPES, pH 7.5, 250 mM sucrose, 20 mM KCl, and 10 mM MgCl2) either with no ATP or with increasing concentrations of ATP in the presence or absence of 10 µM [3H]DNP-SG (A) or 5 mM [3H]GSH (B) at 20°C for 45 min. Dithiothreitol (5 mM) was also present in the GSH uptake experiments. The difference in the amount of uptake with each concentration of ATP compared with that in the absence of ATP is plotted as the ATP-dependent uptake. Data points represent the means ± SE of at least 3 experiments. Data were fit with a nonlinear least-squares regression curve following the Michaelis-Menten equation.

Concentration dependence of ATP-driven transport of [3H]DNP-SG and [3H]GSH in skate liver plasma membrane vesicles. ATP-dependent uptake of [3H]DNP-SG and [3H]GSH was measured for a range of concentrations in the presence and absence of 5 mM MgATP to determine the kinetic parameters of ATP-dependent transport for each. The data for ATP-dependent uptake were plotted against the concentration of either [3H]DNP-SG (Fig. 7A) or [3H]GSH (Fig. 7B) and fit by nonlinear least-squares regression curves after the Michaelis-Menten equation. The apparent Km values for DNP-SG and GSH were 32 ± 9 µM and 12 ± 2 mM, and the Vmax values were 262 ± 27 pmol · mg-1 · 45 min-1 and 24 ± 5 nmol · mg-1 · 45 min-1, respectively. These kinetic data demonstrate that skate liver plasma membrane vesicles possess a high-affinity transporter for DNP-SG and a relatively low-affinity ATP-dependent transporter for GSH.


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Fig. 7.   Concentration-dependent uptake of [3H]DNP-SG and [3H]GSH in skate liver plasma membrane vesicles. Skate liver plasma membranes (100 µg) were incubated in transport buffer (10 mM HEPES, pH 7.5, 250 mM sucrose, and 20 mM KCl, with an ATP regenerating system consisting of 10 mM phosphocreatine, 10 mM MgCl2, and 100 µg/ml creatine phosphokinase) with increasing concentrations of either [3H]DNP-SG (A) or [3H]GSH (B) in the presence () or absence () of 5 mM MgATP at 20°C for 45 min. Dithiothreitol (5 mM) was also present in the GSH uptake experiments. The difference in the amount of uptake with MgATP compared with that in the absence of MgATP at each concentration is plotted as the ATP-dependent uptake (black-triangle). Data points represent the means ± SE of at least 3 experiments with several different plasma membrane preparations. ATP-dependent data were fit with a nonlinear least-squares regression curve following the Michaelis-Menten equation.

Of significance, DNP-SG and GSH were mutually competitive inhibitors of ATP-dependent uptake in skate liver plasma membrane vesicles (Fig. 8). The apparent inhibitory constant (Ki) values calculated from these data are 14 mM for GSH (Fig. 8A) and 33 µM for DNP-SG (Fig. 8B), consistent with the respective Km values for these substrates. These results indicate that DNP-SG and GSH are transported by similar ATP-dependent transport systems.


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Fig. 8.   GSH and DNP-SG are mutual competitive inhibitors of ATP-dependent transport in skate liver plasma membrane vesicles. A: ATP-dependent [3H]DNP-SG uptake was measured in vesicles incubated in transport buffer containing 5 mM ATP and an ATP regenerating system with no added GSH (black-triangle) or with 20 mM GSH (). Data were plotted as function of velocity (pmol · mg protein-1 · 45 min-1) vs. velocity/substrate concentration (pmol · mg protein-1 · 45 min-1/µM) (inset). Data points represent the means ± SE of at least 3 experiments with different plasma membrane preparations. B: ATP-dependent [3H]GSH uptake was measured in vesicles incubated in transport buffer containing an ATP regenerating system with no added DNP-SG (black-triangle) or with 70 µM DNP-SG (). Data points represent the means ± SE of at least 3 experiments with different plasma membrane preparations.

Substrate specificity of ATP-dependent DNP-SG and GSH transport in skate liver plasma membrane vesicles. The nonhydrolyzable analog of ATP, AMP-PNP, could not stimulate uptake of either 10 µM [3H]DNP-SG or 1 mM [3H]GSH, and 100 µM vanadate, a putative competitive analog of ATP, inhibited transport by nearly 70%, providing further evidence that ATP hydrolysis is necessary for uptake (Table 1).

                              
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Table 1.   Effects of substrates and inhibitors of ABC transporters on the ATP-dependent uptake of [3H]DNP-SG and [3H]GSH in skate liver plasma membrane vesicles

Several potential substrates and inhibitors of ATP binding cassette transporters were tested for their ability to inhibit ATP-dependent DNP-SG and GSH transport (Table 1). The overall pattern of inhibition was remarkably similar between DNP-SG and GSH, consistent with transport of these anions by a common mechanism. Uptake of both compounds was inhibited by vanadate, DIDS, leukotriene C4, and sulfobromophthalein, whereas p-aminohippuric acid, verapamil, vincristine, and digoxin had only minimal effects (Table 1). The latter observations also indicate that multidrug resistance (MDR1)-like transporters are not involved in the transport of these anions. Taurocholate (0.1 mM) had no effect on DNP-SG uptake and slightly decreased GSH uptake (Table 1). GSSG was an effective inhibitor of DNP-SG transport but was not tested with [3H]GSH because 5 mM dithiothreitol was added to these solutions to keep [3H]GSH in the reduced form.


    DISCUSSION
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ABSTRACT
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RESULTS
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The present study demonstrates the presence of ATP-dependent GSH, ethyl-SG, and DNP-SG transport in skate liver plasma membrane vesicles and suggests that transport of these anions is mediated by a functional analog of mammalian Mrp. Thus this study supports the hypothesis that some Mrp proteins mediate GSH efflux from cells and provides the first direct evidence of ATP-dependent GSH transport.

Western blotting with either EAG-15 or MIII-5 anti-Mrp2 antibodies indicates that skate liver plasma membrane vesicles contain protein(s) that may be homologous to Mrp2. A protein band of ~180 kDa cross-reacted with both of these antibodies, indicating the presence of a similar protein in these fish liver plasma membranes. Furthermore, immunofluorescence labeling detected intense areas of fluorescence at the apical canalicular membranes and in the pericanalicular region. This apical distribution is reminiscent of that seen in isolated rat hepatocyte couplets, in which Mrp2 is localized in both the apical membrane and in vesicles adjacent to the apical plasma membrane (30).

To determine whether there was Mrp-like transport activity in the skate liver, we initially tested for the presence of ATP-dependent uptake of 10 µM [3H]DNP-SG in isolated plasma membrane vesicles (Fig. 5). DNP-SG is a high-affinity substrate of human MRP2 (13) and rat Mrp2 (2, 8, 18). DNP-SG was transported by both ATP-dependent and -independent mechanisms in a mixed preparation of sinusoidal and canalicular membrane vesicles (Fig. 5). The ATP-stimulated uptake was clearly more robust in the first hour and then steadily decreased over the duration of these experiments. Uptake was not stimulated by AMP-PNP, indicating that ATP hydrolysis is required. The initial rates of 10 µM [3H]DNP-SG uptake exhibited saturation kinetics with respect to MgATP, with an apparent Km for MgATP of 0.4 ± 0.2 mM. This Km value is similar to that measured in rat liver canalicular membranes (a Km for ATP of 0.26 mM; Ref. 18). The Km for DNP-SG itself (32 µM) was also comparable to that measured for Mrp2-mediated transport in the rat (18). Together, these data suggest that the ATP-dependent DNP-SG transport observed in skate liver plasma membrane vesicles is mediated by a homologue of Mrp2.

The major finding from the present study is the demonstration of an ATP-dependent GSH transport system in skate liver and the indication that GSH is a substrate for a skate equivalent of an Mrp transporter. ATP-dependent DNP-SG and GSH transport in skate membranes had a similar affinity for MgATP, neither was stimulated by a nonhydrolyzable analog of ATP, and both were cis-inhibited by the same compounds. However, ATP-dependent GSH transport was of relatively low affinity, with an apparent Km of 12 ± 2 mM.

Our ability to detect ATP-dependent GSH transport in skate vesicles, as opposed to studies in rat liver membrane vesicles, is most likely related to the relatively low nonspecific permeability of the skate liver membrane vesicles. This low permeability most likely allowed the vesicles to accumulate GSH in an ATP-dependent fashion, despite the low catalytic efficiency of the transport process. Another important factor is the relatively low ATP hydrolytic capacity of skate membranes, which allowed ATP levels to be maintained in the vesicle incubation solutions for extended periods of time (Fig. 2). The Km for GSH transport in skate liver plasma membranes was found to be 12 mM, a value comparable to that measured for GSH transport by yeast Ycf1p (28, 29) and to that estimated for GSH efflux from MDCKII cells (26). This high Km, coupled with only a modest Vmax (Fig. 7), would normally preclude detection of transport activity in membrane vesicle systems, unless the efflux of the accumulated GSH were somehow restricted by the overall permeability properties of the vesicles. Indeed, the present results support the previous suggestion that skate liver plasma membranes have a relatively low nonspecific permeability (7). The data in Fig. 1 indicate that skate membrane vesicles are able to concentrate the bile acid [3H]taurocholate by 1-2 orders of magnitude over an extended period of time, at least 10 h. By comparison, rat liver membrane vesicles generally reach maximum uptake values within several minutes and then gradually decline thereafter (1, 23, 25). These differences may be due in part to differences in plasma membrane lipid and protein composition between these cold-water fish and rat livers and to differences in the temperatures at which the experiments were performed (20°C in skate vs. 37°C for mammalian membrane vesicles).

The overall kinetic parameters and substrate specificity for ATP-dependent GSH transport in the skate membrane vesicles are also comparable to those measured for Ycf1p-mediated GSH transport (28, 29) and are consistent with the Ki (20 mM) measured for competitive inhibition of ATP-dependent [3H]DNP-SG transport by GSH in membrane vesicles isolated from MDCKII cells expressing MRP2 (26). Together, these results in three different experimental models provide further evidence that GSH is a low-affinity substrate for the Mrp proteins.


    ACKNOWLEDGEMENTS

We thank Drs. D. Keppler and R. P. J. Oude Elferink for kindly providing antibodies to Mrp2.


    FOOTNOTES

This work was supported in part by National Institutes of Health Grants DK-48823, DK-25636, and ES-06484, National Institute of Environmental Health Sciences Center Grants ES-03828 and ES-01247, and Environmental Toxicology Training Grant ES-07026.

Address for reprint requests and other correspondence: N. Ballatori, Dept. of Environmental Medicine, Box EHSC, Univ. of Rochester School of Medicine, 575 Elmwood Ave., Rochester, NY 14642 (E-mail: Ned_Ballatori{at}urmc.rochester.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. §1734 solely to indicate this fact.

Received 8 November 1999; accepted in final form 29 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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