©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Multiple Canalicular Transport Mechanisms for Glutathione S-Conjugates
TRANSPORT ON BOTH ATP- AND VOLTAGE-DEPENDENT CARRIERS (*)

(Received for publication, August 25, 1994; and in revised form, December 1, 1994)

Nazzareno Ballatori (§) Anh T. Truong

From the Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, New York 14642

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A large number of structurally distinct electrophiles are conjugated to glutathione within hepatocytes, and the resulting glutathione S-conjugates are selectively transported across the canalicular membrane into bile. To test the hypothesis that a single multi-specific, ATP-dependent carrier mediates biliary secretion of glutathione S-conjugates, the present study compared the driving forces and substrate specificity for canalicular transport of S-ethylglutathione (ethyl-SG), a low molecular weight and relatively hydrophilic thioether, and S-(2,4-dinitrophenyl)-glutathione (DNP-SG), a larger and more hydrophobic anion, using isolated rat liver canalicular membrane vesicles. In agreement with previous findings, DNP-SG transport was stimulated by ATP, although there was considerable transport in the absence of ATP. ATP-independent DNP-SG transport was unaffected by a Na gradient, was enhanced by a valinomycin-induced K diffusion potential, and was saturable, with both high affinity (K = 8 ± 2 µM) and low affinity (K = 0.5 ± 0.1 mM) components. High affinity ATP-independent DNP-SG uptake was cis-inhibited by GSH, GSH monoethyl ester, glutathione S-conjugates, other -glutamyl compounds, sulfobromophthalein, and 4,4`-diisothiocyanatostilbene-2,2`-disulfonic acid (DIDS). In contrast, ATP-dependent DNP-SG uptake was unaffected by GSH, GSH ester, S-methyl glutathione, or S-carbamidomethyl glutathione, but was strongly inhibited by sulfobromophthalein, DIDS, and by high molecular weight and relatively hydrophobic glutathione S-conjugates. Transport of the low molecular weight ethyl-SG conjugate was only minimally stimulated by ATP (10-20%). ATP-independent ethyl-SG uptake was electrogenic, saturable (K = 10 ± 1 µM) and was inhibited by GSH and all glutathione S-conjugates tested. These findings indicate the presence of multiple canalicular transport mechanisms for glutathione S-conjugates and demonstrate that the physicochemical properties of the S moiety are major determinants of transport. Relatively high molecular weight hydrophobic conjugates are substrates for both ATP-dependent and -independent mechanisms, whereas low molecular weight glutathione S-conjugates are transported largely by electrogenic carriers.


INTRODUCTION

Extrusion of cellular metabolites and xenobiotics from the hepatocyte across the canalicular plasma membrane into bile is mediated in part by at least three distinct ATP-dependent transport systems; the P-glycoproteins (multidrug resistance proteins), an export pump for monovalent conjugated bile acids, and a transporter for non-bile acid organic anions (reviewed by Arias et al., 1993; Meier and Steiger, 1993; Vore, 1993; Zimniak and Awasthi, 1993). An ATP-independent canalicular organic anion transporter has also been identified (Arias et al., 1993). These proteins display remarkably broad substrate specificities and together mediate the hepatocellular efflux of a variety of endogenous compounds and innumerable xenobiotics. Although the P-glycoproteins are well characterized at the biochemical and molecular level, the other transport mechanisms have only been examined in isolated membrane vesicle systems.

An important group of substrates for the ATP-dependent organic anion carrier is the glutathione S-conjugates, which are formed intracellularly by the reaction of GSH with both endogenous and exogenous electrophilic compounds (Boyland and Chasseaud, 1969; Chasseaud, 1979; Anderson and Meister, 1983). Studies with liver canalicular membrane vesicles (Ishikawa et al., 1989, 1990; Kobayashi et al., 1990, 1991; Akerboom et al., 1991; Nishida et al., 1992; Fernandez-Checa et al., 1992), as well as membrane preparations from red blood cells (Kondo et al., 1982; LaBelle et al., 1986a, 1986b), heart (Ishikawa, 1989; Ishikawa et al., 1989), and other tissues (Schaub et al., 1991; Ishikawa and Ali-Osman, 1993), have established that glutathione disulfide (GSSG), and glutathione S-conjugates of sulfobromophthalein (BSP(^1)-SG), 1-chloro-2,4-dinitrobenzene (DNP-SG), and a leukotriene (LTC(4)) are substrates for ATP-dependent carriers. Kinetic analysis of ATP-dependent DNP-SG transport in canalicular liver plasma membrane (cLPM) vesicles indicates a single high affinity component of transport, with a K ranging from 4 to 71 µM (Kobayashi et al., 1990; Akerboom et al., 1991), whereas erythrocytes have two ATP-dependent components with K values ranging from 1 to 8 µM and 100 to 900 µM, respectively (Bartosz et al., 1993; Kondo et al., 1982). Erythrocytes may also have separate ATP-dependent transporters for GSSG and DNP-SG (LaBelle et al., 1986b; Board et al., 1992), but this has not yet been firmly established. ATP-dependent organic anion transport has a broad substrate specificity, accepting a variety of anionic compounds that bear little structural similarity with each other or with the glutathione S-conjugate substrates (Kobayashi et al., 1990; Akerboom et al. 1991; Oude Elferink et al., 1991; Nishida et al., 1992a, 1992b; Bartosz et al., 1993). The mechanism by which these chemically diverse substrates are recognized by the transporter(s) is not known. The overall functional characteristics of the ATP-dependent organic anion transporters are comparable to those of the P-glycoproteins, suggesting similar structural motifs and a common evolutionary origin.

Because glutathione S-conjugates are good substrates for the ATP-dependent organic anion carrier, this transporter was named the GS-X pump by Ishikawa(1992). However, our recent finding that canalicular transport of a low molecular weight glutathione mercaptide (CH(3)Hg-SG) is independent of ATP indicates that not all glutathione S-conjugates are substrates for this GS-X pump and suggest that the physicochemical properties of the S moiety may determine whether the conjugate is transported on the ATP-dependent system (Dutczak and Ballatori, 1994). Furthermore, these data indicate the presence of at least two ATP-independent glutathione S-conjugate transport mechanisms. Studies of GSH transport in canalicular membrane vesicles also suggest the presence of a high affinity ATP-independent GSH transport system whose primary function may be to transport some glutathione S-conjugates, -glutamyl compounds, and other anions into bile (Ballatori and Dutczak, 1994). To test these hypotheses directly, the present study compared the driving forces and substrate specificity for canalicular transport of two glutathione S-conjugates that differ in their physicochemical properties, S-ethylglutathione (ethyl-SG) and S-(2,4-dinitrophenyl)glutathione (DNP-SG). Our findings identify and characterize both ATP-dependent and -independent glutathione S-conjugate transport mechanisms.


EXPERIMENTAL PROCEDURES

Materials

[Glycine-2-^3H]GSH (44.0 Ci/mmol) and [^3H(G)]taurocholate (2.1 Ci/mmol) were purchased from DuPont NEN. Ophthalmic acid (-Glu-AIB-Gly), -Glu-Gly-Gly, and Cys-Gly were obtained from Bachem Bioscience Inc. (Philadelphia, PA); and -Glu-Cys was a gift from the Kohjin Co. (Tokyo, Japan). Other chemicals and reagents were purchased from either Sigma, J. T. Baker (Phillipsburg, NJ), Fisher Scientific (Fair Lawn, NJ), or Aldrich.

DNP-SG (Hinchman et al., 1991; Saunders, 1934), BSP-SG (Whelan et al., 1970), and S-ethacrynic acid glutathione (Ploemen et al., 1990) were synthesized and purified as described previously. BSP-SG was further purified to remove small amounts of contaminating BSP, GSSG, and GSH by elution through a Sephadex G-50-fine column (1.5 times 30 cm). The Sephadex G-50 column was eluted with 100 mM Tris-HCl, pH 7.5, at a flow rate of 43 ml/h at room temperature. BSP-SG concentration was assessed by measuring absorbance at 580 nm in a solution containing 100 mM sodium pyrophosphate buffer, pH 8.4. Glutathione monoethyl ester was synthesized based on the method of Anderson et al.(1985) and characterized as described (Ballatori and Truong, 1992). S-Benzyl glutathione was synthesized from GSH and benzyl chloride by the method of Vince et al.(1971). The product was recrystallized in water.

S-Carboxymethyl glutathione was synthesized by dissolving equimolar amounts (1 mmol) of iodoacetate and GSH in water and immediately adding a solution of 2 M KOH containing 3.3 M KHCO(3) to raise the pH to 8.5. The reaction mixture was incubated in the dark at room temperature with continuous stirring for 15 min. The product was purified by ion exchange chromatography (Fig. 1B) and detected and quantitated by reaction with 0.2% ninhydrin in 99% ethanol, at a wavelength of 570 nm (Moore and Stein, 1948). DEAE-Sephadex A-25 column chromatography was carried out at a flow rate of 62 ml/h, using 50 mM potassium phosphate buffer, pH 7.5 (Fig. 1B). S-Carbamidomethyl glutathione was synthesized from GSH and iodoacetamide using a similar approach. Iodoacetamide and GSH (1 mmol each) were dissolved in of water, the pH was raised to 8.0 with saturated KHCO(3), and the solution incubated in the dark at room temperature, with continuous stirring for 1 h. The product was applied to a DEAE-Sephadex A-25 column and eluted with 10 mM potassium phosphate buffer, pH 7.5. S-Carbamidomethyl glutathione eluted in fractions 19-23 (using conditions as described in Fig. 1A) and was quantitated by reaction with ninhydrin. GSH and GSSG eluting from the column were detected by the method of Griffith(1980).


Figure 1: Separation of glutathione and glutathione S-conjugates by ion exchange chromatography. Approximately 5 µmol of the indicated compounds were applied to a DEAE-Sephadex A-25 column (1.5 times 30 cm), and eluted at room temperature with either 10 mM (A) or 50 mM (B) potassium phosphate buffer, pH 7.5, at a flow rate of 62 ml/h. Fractions of 3 ml were collected and analyzed by reaction with ninhydrin (as illustrated), but also by enzymatic analysis for GSH and GSSG (Griffith, 1980) and direct absorbance at 365 nm for DNP-SG.



Synthesis of [^3H]Ethyl-SG and [^3H]DNP-SG

[^3H]Ethyl-SG was synthesized enzymatically from [^3H]GSH and iodoethane using glutathione S-transferase from equine liver (Sigma). Dithiothreitol was removed from the [^3H]GSH solution by extraction with a 10-fold excess of ethyl acetate, in samples acidified to pH 2 with 0.5 M HCl (Butler et al., 1976). Extracted [^3H]GSH (6 nmol) was reacted with excess iodoethane (10 µmol) in 50 mM potassium phosphate buffer, pH 7.5, at 37 °C in the presence of 60 µg/ml of glutathione S-transferase for 60 min. [^3H]Ethyl-SG was purified from residual [^3H]GSH and [^3H]GSSG on a DEAE-Sephadex A-25 column (Fig. 1A). Fractions containing [^3H]ethyl-SG were combined and concentrated on a Speed-Vac evaporator (Jouan Inc., Winchester, VA). Purity of [^3H]ethyl-SG was confirmed using the high performance liquid chromatography method of Fariss and Reed(1987).

[^3H]DNP-SG was synthesized using a similar approach. [^3H]GSH (6 nmol) was reacted with 1-chloro-2,4-dinitrobenzene (25 nmol) at 37 °C for 20 min. 1-Chloro-2,4-dinitrobenzene was recrystallized from ethanol and water (3:2 v/v) prior to use. [^3H]DNP-SG was purified from residual [^3H]GSH and [^3H]GSSG as illustrated in Fig. 1B. Radiolabeled compounds were added to the membrane incubation solutions at a concentration of 2.5 µCi/ml, such that the final concentration of potassium phosphate was less than 5 mM.

Preparation of Rat Canalicular Liver Plasma Membrane (cLPM) Vesicles

Male Sprague-Dawley rats (250 g) were obtained from Charles River Laboratories (Kingston, NY) and fed Purina rodent chow (formula 5001) ad libitum. The methods for isolating cLPM subfractions are as described by Meier et al. (1984a) and previously performed in this laboratory (Ballatori et al., 1986; Ballatori and Dutczak, 1994; Dutczak and Ballatori, 1994; Simmons et al., 1990, 1992). The degree of purification of the cLPM vesicles has been extensively analyzed previously by intracellular and plasma membrane marker enzyme activities (Meier et al., 1984a). In the present study, the purity of each membrane preparation was analyzed by measurement of Na K-ATPase and Mg-ATPase activities (Scharschmidt et al., 1979) and -glutamyltransferase activity (Orlowski and Meister 1963). The relative enrichments for these enzymes, defined as the ratio of specific activities in the cLPM subfraction to those of liver homogenate, were 65 ± 18 for -glutamyltransferase, 94 ± 19 for Mg-ATPase, and 2 ± 3 for Na K-ATPase. These values are comparable to those reported by Meier et al. (1984a) and indicate a highly enriched plasma membrane subfraction and virtually complete separation of cLPM from basolateral membranes as reflected by minimal Na K-ATPase activity. Protein was determined by the method of Lowry et al.(1951) using bovine serum albumin as the standard.

Transport Measurements

Frozen membrane suspensions were quickly thawed by immersion in a 37 °C water bath, diluted to the desired protein concentration, and passed repeatedly (10 times) through a 25-gauge needle. Acivicin (250 µM), an irreversible inhibitor of -glutamyltransferase, was added to all vesicle preparations to inhibit the catabolism of glutathione-containing compounds (Ballatori and Dutczak, 1994), and vesicles were preincubated at 25 °C for 15 min.

In some experiments, the membrane potential was clamped using the potassium ionophore valinomycin (10 µg/mg protein) in the presence of 20 mM KCl in the intra- and extravesicular spaces. Valinomycin was dissolved in dimethyl sulfoxide: the final concentration of dimethyl sulfoxide was less than 0.1%, a concentration that had no effect on transport (data not shown). Some incubation solutions were supplemented with dithiothreitol (5 mM) to ensure that sulfhydryl-containing compounds remained in their reduced forms. The corresponding controls also received 5 mM dithiothreitol. The pH of incubation solutions containing high concentrations of glutathione S-conjugates, GSH, or other anions was routinely adjusted to 7.5 immediately before use with either 1 M KOH or Tris base. Specific details regarding the composition of the vesicle incubation media are provided in the individual figure and table legends.

Uptake of radiolabeled substrates into cLPM vesicles was measured by a rapid Millipore filtration technique (Ballatori et al., 1986; Meier et al., 1984b). Membrane suspensions (80-130 µg of protein in 20 µl) were preincubated at 25 or 37 °C for 15 min. Uptake studies were initiated by the addition of 80 µl of incubation medium, also prewarmed, containing various concentrations of the radiolabeled substrates. Transport was terminated by the addition of 1 ml of ice-cold stop solution and immediate rapid filtration through a 0.45-µm filter (Millipore, HAWP) that had been premoistened with ice-cold stop solution. Vesicles were then washed with an additional 4 ml of stop solution. Unless otherwise indicated the stop solution consisted of 300 mM sucrose, 10 mM HEPES-Tris, pH 7.5, 0.2 mM CaCl(2), and 20 mM KCl. Filters were dissolved in 5 ml of Opti-Fluor (Packard Instrument Co., Downers Grove, IL) and counted in a Packard Tri Carb Scintillation Counter (model 4530). Nonspecific binding of isotope to filters and vesicles was determined in each experiment by the rapid addition of ice-cold incubation solution, followed by cold stop solution, to 20 µl of membrane vesicle suspension also kept at 0-4 °C. This blank was subtracted from all determinations. All incubations were performed in triplicate and all observations confirmed with three or more separate membrane preparations. Kinetic parameters were calculated from the Eadie-Hofstee transformation of the data by the method of residuals (Gibaldi and Perrier, 1975).

The effect of ATP on [^3H]ethyl-SG, [^3H]DNP-SG, and [^3H]taurocholate uptake by cLPM vesicles was measured at 37 °C in an incubation solution containing 290 mM sucrose, 10 mM HEPES-Tris, pH 7.5, 20 mM KCl, 0.2 mM CaCl(2), 10 mM MgCl(2), 10 mM phosphocreatine, 100 µg/ml of creatine phosphokinase, and either 1 mM ATP (disodium salt) or 2 mM NaCl.


RESULTS

ATP-Dependence of Ethyl-SG, DNP-SG, and Taurocholate Uptake in cLPM Vesicles

Uptake of DNP-SG and taurocholate into cLPM vesicles was markedly stimulated by ATP (Fig. 2), confirming previous findings (Kobayashi et al., 1990; Akerboom et al., 1991; Muller et al., 1991; Nishida et al., 1991). In contrast to DNP-SG, ethyl-SG uptake was only stimulated 10-20% by ATP in the same membrane preparations (Fig. 2). However, for both DNP-SG and ethyl-SG there was considerable uptake in the absence of ATP, indicating the presence of ATP-independent transport mechanism(s).


Figure 2: Effect of ATP on ethyl-SG, DNP-SG, and taurocholate uptake in cLPM vesicles. Vesicles containing 250 mM sucrose, 10 mM HEPES-Tris, pH 7.5, 20 mM KCl, and 0.2 mM CaCl(2) were pretreated with 0.25 mM acivicin. Uptake of 10 µM [^3H]ethyl-SG, 10 µM [^3H]DNP-SG, and of 1 µM [^3H]taurocholate was measured at 37 °C in an incubation solution containing 290 mM sucrose, 10 mM HEPES-Tris, pH 7.5, 20 mM KCl, 0.2 mM CaCl(2), 10 mM MgCl(2), 10 mM phosphocreatine, 100 µg/ml creatine phosphokinase, and either 1 mM ATP (disodium salt) or 2 mM NaCl. The stop solution contained 300 mM sucrose, 10 mM HEPES-Tris, pH 7.5, 20 mM KCl, and 0.2 mM CaCl(2). Data are means ± S.E. of three experiments, each performed in triplicate.



ATP-independent ethyl-SG and DNP-SG uptake occurred into an osmotically sensitive space (Fig. 3). Uptake of both solutes was linearly decreased as vesicle size diminished at higher osmolarities. The data for DNP-SG are comparable to those presented by Inoue et al.(1984), whereas those for ethyl-SG are similar to those for high affinity GSH uptake by the cLPM vesicles (Ballatori and Dutczak, 1994) and indicate a relatively small degree of binding to the vesicles. The apparent intravesicular volumes calculated from these uptake values are approximately 2.5-3.5 µl/mg of protein, providing further evidence for low nonspecific binding to the canalicular membranes.


Figure 3: Transport of ethyl-SG and DNP-SG into an osmotically active intravesicular space. Canalicular vesicles were resuspended in media containing 10 mM HEPES-Tris, pH 7.5, 250 mM sucrose, and 0.25 mM acivicin. They were then incubated at 25 °C for 30 min in media containing 10 mM HEPES-Tris, pH 7.5, 10 µM of either [^3H]ethyl-SG (circles) or [^3H]DNP-SG (triangles), and from 0.3-0.7 M sucrose. Membrane blanks were performed at each sucrose concentration. Data are means ± S.E. (n = 3).



Driving Forces for ATP-independent Canalicular Transport of Ethyl-SG and DNP-SG

The effects of an inwardly directed 100 mM Na gradient on ethyl-SG and DNP-SG uptake are illustrated in Fig. 4. Uptake of these glutathione S-conjugates was unaffected when Na was replaced with either K or Li. In contrast, transport of ethyl-SG and DNP-SG was stimulated by a valinomycin-induced K diffusion potential (Table 1). Uptake of these conjugates into cLPM vesicles was increased when an inside-positive potential was generated with an inwardly directed K gradient and valinomycin, indicating that canalicular transport is electrogenic and that the membrane potential may serve as the driving force for their biliary secretion.


Figure 4: Time course of ethyl-SG and DNP-SG uptake into cLPM vesicles in the presence of various inwardly directed cation gradients. Canalicular membrane vesicles were resuspended in 250 mM sucrose, 10 mM HEPES-Tris, pH 7.5, and 0.25 mM acivicin. Uptake of 10 µM [^3H]ethyl-SG or 10 µM [^3H]DNP-SG was measured at 25 °C in an incubation solution containing 170 mM sucrose, 10 mM HEPES-Tris, pH 7.5, 10 µM [^3H]ethyl-SG or 10 µM [^3H]DNP-SG, and 100 mM of either NaCl, KCl, or LiCl. Insets illustrate uptake at short time intervals. Values are means ± S.E. of three experiments, each performed in triplicate.





Concentration Dependence of DNP-SG and Ethyl-SG Uptake into cLPM Vesicles in the Absence of ATP

Initial rates of [^3H]DNP-SG and [^3H]ethyl-SG uptake into cLPM vesicles were measured under ion-equilibrated conditions and in the presence of valinomycin (10 µg/mg protein) and KCl (20 mM) in the intra- and extravesicular spaces to prevent the establishment of a membrane potential. To detect the presence of both high and low affinity transport components, initial rates of uptake were measured over a large concentration range, from 1 µM to 6 mM for DNP-SG (Fig. 5) and from 1 µM to 10 mM for ethyl-SG (Fig. 6). Uptake was measured at 25 °C. The nonsaturable component of uptake (dashed lines in Fig. 5and Fig. 6) was assessed by least-square linear regression analysis of the data at high extravesicular substrate concentrations (geq2 mM). The slopes of the dashed lines in Fig. 5, A and B, are similar, as are those in Fig. 6, A and B.


Figure 5: Concentration dependence of initial rates of DNP-SG uptake in cLPM vesicles in the absence of ATP. Vesicles containing 250 mM sucrose, 10 mM HEPES-Tris, pH 7.5, 20 mM KCl, and 0.2 mM CaCl(2) were pretreated with 0.25 mM acivicin and 10 µg valinomycin/mg protein. They were then incubated at 25 °C in the same media supplemented with [^3H]DNP-SG (1 µM to 6 mM), and uptake was measured after 10 s. The dashed lines represents the nonsaturable component of uptake, obtained by least-square linear regression analysis of the data at high extravesicular substrate concentrations (geq2 mM). The slopes of the dashed lines in panels A and B are similar. Inset shows the Eadie-Hofstee plot of these data over the entire concentration range. Values are means ± S.E. of three experiments, each performed in triplicate.




Figure 6: Concentration dependence of initial rates of ethyl-SG uptake in cLPM vesicles. Explanatory information is similar to that of Fig. 5legend. Uptake of [^3H]ethyl-SG (1 µM to 10 mM) was measured after 10 s. Values are means ± S.E. of three experiments, each performed in triplicate.



Initial rates of DNP-SG uptake were a nonlinear function of concentration (Fig. 5). When corrected for the nonsaturable component (dashed lines) data were obtained that follow Michaelis-Menten kinetics. An Eadie-Hofstee plot of all of the data (Fig. 5, inset) revealed the presence of at least two saturable transport components: a high affinity system with an apparent K(m) of 8 ± 2 µM and a V(max) of 33 ± 6 pmolbulletmgbullet10 s, and a low affinity system with a K(m) of 0.5 ± 0.1 mM and a V(max) of 526 ± 75 pmolbulletmgbullet10 s.

A similar kinetic analysis for ethyl-SG (Fig. 6) revealed the presence of a single high affinity saturable component, with an apparent K(m) of 10 ± 1 µM and a V(max) of 6 ± 2 pmolbulletmgbullet10 s. At extravesicular ethyl-SG concentrations up to 10 mM, there was no evidence for a second low affinity system (Fig. 6B).

Substrate Specificity of Glutathione S-conjugate Transport in cLPM Vesicles

To compare the substrate specificity of ATP-dependent and -independent glutathione S-conjugate transport, initial rates of DNP-SG and ethyl-SG (5 µM) uptake were measured in the presence of a number of potential transport inhibitors, under voltage-clamped and ion equilibrated conditions (Table 2). Most of the inhibitors were added at a concentration of 1 mM. ATP-dependent DNP-SG uptake was calculated as the difference in uptake observed with and without 1 mM ATP at 37 °C, whereas ATP-independent uptake of DNP-SG and ethyl-SG was calculated as the difference in uptake at 25 and 4 °C in the absence of extravesicular ATP.



ATP-dependent DNP-SG transport was unaffected by GSH, GSH monoethyl ester, or by low molecular weight and relatively hydrophilic glutathione S-conjugates, such as S-methyl glutathione, S-carbamidomethyl glutathione, or glutathionesulfonic acid (Table 2). In contrast, larger and more hydrophobic glutathione S-conjugates were powerful inhibitors. ATP-dependent transport was essentially completely inhibited by 1 mM DNP-SG, BSP-SG, GSSG, S-octyl, S-(p-nitrobenzyl), S-(p-chlorophenacyl), and S-ethacrynic acid glutathione. For the S-alkyl conjugates, the extent of inhibition increased as the chain length increased from methyl to octyl (Table 2), confirming previous findings (Kobayashi et al., 1990; Ishikawa et al., 1990), and indicating that hydrophobicity is an important determinant of ATP-dependent glutathione S-conjugate transport.

A comparison of the effects of S-carboxymethyl glutathione (GS-CH(2)-COOH) to those of S-carbamidomethyl glutathione (GS-CH(2)-CO-NH(2)) indicates that charge may also be an important determinant of transport on the ATP-dependent system. The dianionic S-carboxymethyl conjugate decreased uptake to 69% of control, whereas S-carbamidomethyl glutathione had no effect (Table 2), even though these compounds have similar molecular weights and hydrophobicity.

ATP-dependent DNP-SG uptake (5 µM) was also nearly completely inhibited by BSP (100 µM) and DIDS (100 µM) and significantly decreased by taurocholate (250 µM), but was unaffected by other anionic compounds including the GSH analogs -Glu-AIB-Gly and -Glu-Gly-Gly, the dipeptides -Glu-Cys and Cys-Gly, and the amino acids glutamate and cysteine (Table 2). The inhibition by BSP, DIDS, and taurocholate supports previous studies indicating that the ATP-dependent carrier can transport a variety of organic anions, in addition to glutathione S-conjugates (Ishikawa et al., 1989, 1990; Kobayashi et al., 1990, 1991; Akerboom et al., 1991; Oude Elferink et al., 1991; Nishida et al., 1992; Fernandez-Checa et al., 1992; Bartosz et al., 1993).

In contrast to the ATP-dependent mechanism, ATP-independent uptake of both DNP-SG and ethyl-SG was inhibited by GSH, GSH monoethyl ester, and by low molecular weight glutathione S-conjugates (Table 2). For example, GSH and S-methyl glutathione decreased ATP-independent uptake of [^3H]DNP-SG and [^3H]ethyl-SG to 53 and 33%, and 67 and 53% of control, respectively. Similarly, the GSH analog -Glu-AIB-Gly (ophthalmic acid) had no effect on ATP-dependent transport but inhibited ATP-independent uptake by 30% (Table 2). Another difference between the ATP-dependent and -independent mechanisms was observed with 100 µM DIDS as the inhibitor. DIDS nearly completely blocked ATP-dependent transport but only decreased ATP-independent transport to 63-73% of control (Table 2).

Similar to the ATP-dependent mechanism, ATP-independent uptake of DNP-SG and ethyl-SG was strongly inhibited by BSP and relatively large or hydrophobic glutathione S-conjugates but was unaffected by glutamate, cysteine, or dipeptides. -Glu-Cys produced a small inhibition of ethyl-SG uptake (Table 2). In general, the various inhibitors produced similar effects on ATP-independent ethyl-SG and DNP-SG uptake, although the extent of inhibition was larger for ethyl-SG (Table 2).

Fig. 7illustrates the relation between the inhibitory effects of glutathione S-conjugates and their molecular weight, using data from Table 2. Note that the overall pattern of inhibition is similar for both ATP-dependent and -independent transport, but there is also a clear divergence of effects at low molecular weights. For the ATP-dependent system, there is an abrupt increase in inhibition as the molecular weight reaches 370 and essentially complete inhibition at molecular weights greater than 420 (Fig. 7).


Figure 7: Relation between the molecular weight of transport inhibitors (glutathione S-conjugates and other -glutamyl compounds) and ATP-dependent and -independent glutathione S-conjugate uptake into cLPM vesicles. Data are from Table 2. The compounds shown are, in increasing molecular weights, 1) -Glu-Cys, 2) -Glu-p-nitroanilide, 3) -Glu-Gly-Gly, 4) -Glu-Glu, 5) -Glu-AIB-Gly, 6) GSH, 7) S-methyl glutathione, 8) GSH monoethyl ester, 9) S-ethyl glutathione, 10) glutathionesulfonic acid, 11) S-butyl glutathione, 12) S-carbamidomethyl glutathione, 13) S-carboxymethyl glutathione, 14) S-lactoyl glutathione, 15) S-benzyl glutathione, 16) S-octyl glutathione, 17) S-(p-nitrobenzyl)-glutathione, 18) S-(p-chlorophenacyl)-glutathione, 19) DNP-SG, 20) S-ethacrynic acid glutathione, 21) GSSG, and 22) BSP-SG.




DISCUSSION

Conjugation of electrophilic compounds with GSH functions as a key cellular defense mechanism against endogenous reactive metabolites and xenobiotics and may also be involved in inflammation, signal transmission, and modulation of cell proliferation (Boyland and Chasseaud, 1969; Chasseaud, 1979; Anderson and Meister, 1983; Ishikawa, 1992). Glutathione S-conjugates formed intracellularly must be released from the cell before further processing can occur in extracellular spaces (Hinchman and Ballatori, 1994). Current evidence indicates that the principal mechanism by which glutathione S-conjugates are transported across the cell membrane is via an ATP-dependent organic anion transporter (Kobayashi et al., 1990; Akerboom et al., 1991; Ishikawa, 1992).

The present study demonstrates that in addition to the ATP-dependent system glutathione S-conjugates are substrates for at least two ATP-independent mechanisms. [^3H]DNP-SG uptake by isolated cLPM vesicles was mediated by high affinity (K(m) 8 µM) and low affinity (K(m) 0.5 mM) ATP-independent transport components, as well as an ATP-stimulated mechanism. Substrate specificity studies indicate that these systems transport a wide range of glutathione S-conjugates and other anionic compounds. Although there is considerable overlap in substrate selectivity between the ATP-dependent and -independent systems, there are also distinct substrate requirements (Table 2).

Glutathione S-conjugates vary considerably in molecular size, charge, and water solubility (Boyland and Chasseaud, 1969; Chasseaud, 1979). The present findings demonstrate that the physicochemical properties of the S moiety are key determinants of transport across the canalicular membrane. Relatively low molecular weight hydrophilic conjugates are substrates for electrogenic carriers, but not for the ATP-dependent mechanism, whereas larger and more hydrophobic conjugates are substrates for both. Charge also appears to be important: S-carboxymethyl glutathione (GS-CH(2)-COOH) decreased ATP-dependent uptake to 69% of control, whereas S-carbamidomethyl glutathione (GS-CH(2)-CO-NH(2)) had no effect (Table 2). Both of these glutathione S-conjugates inhibited ATP-independent transport of DNP-SG and ethyl-SG to a similar extent, although the dianionic compound was a somewhat more effective inhibitor.

DNP-SG appears to be an excellent substrate for both the ATP-dependent and -independent systems. A comparison of the kinetic parameters for ATP-dependent and high affinity ATP-independent DNP-SG transport indicates that the apparent K(m) values are comparable (4 µM (Kobayashi et al., 1990) versus 8 µM (Fig. 5), respectively), as are the V(max) values (30 versus 33 pmolbulletmgbullet10 s), indicating that DNP-SG should be transported at similar rates on these competing pathways. This conclusion is supported by studies in isolated rat hepatocytes showing that approximately 50% of DNP-SG efflux in this experimental system is independent of ATP (Oude Elferink et al., 1990). These investigators reported that ATP depletion to 16% of control levels with fructose decreased DNP-SG efflux to 55% of control. However, in contrast with the present findings, Oude Elferink et al.(1990) noted that membrane depolarization in high K media had no significant effect on DNP-SG efflux from intact hepatocytes, suggesting that membrane potential is not a driving force. However, because only a fraction of the total DNP-SG efflux measured in intact hepatocytes is expected to be sensitive to changes in membrane potential, the potential-sensitive component may not have been readily detected in this experimental system.

In contrast to DNP-SG, ethyl-SG is a poor substrate for the ATP-dependent system and is transported almost exclusively by a high affinity electrogenic carrier. Uptake of ethyl-SG into cLPM vesicles was only minimally stimulated by ATP (Fig. 2), and ethyl-SG was only a weak inhibitor of ATP-dependent DNP-SG transport (Table 2). The absence of a low affinity ATP-independent transport component for ethyl-SG (Fig. 6) was surprising since both DPN-SG (Fig. 5) and CH(3)Hg-SG (Dutczak and Ballatori, 1994) exhibit high and low affinity ATP-independent systems. The reason for this apparent discrepancy is not known but may be due to a low V(max) for ethyl-SG transport which would preclude detection in the vesicle system.

Although ATP-independent transport of glutathione S-conjugates was observed in all previous studies of GSSG, BSP-SG, DNP-SG, and LTC(4) transport, this component was considered insignificant as it usually constituted less than 50% of the total uptake or was attributed to transport by contaminating membrane fractions. Kobayashi et al.(1990) observed that ATP-independent DNP-SG uptake was higher in basolateral liver membrane vesicles than in canalicular vesicles and proposed that this electrogenic transport mechanism was localized exclusively to the basolateral domain. Their data demonstrate that uptake of radioisotope (from [^3H]DNP-SG) is higher in basolateral vesicles incubated in media containing either 100 mM KCl or NaCl, as compared to sucrose, in the absence of valinomycin and ATP. However, although the mechanism by which Na and K stimulate uptake is still not clear, it cannot be explained by membrane potential, particularly since chloride, a highly permeant anion, was present at a concentration of 120.4 mM in these studies. It is important to note that the experiments of Kobayashi et al.(1990) were performed in the absence of a -glutamyltransferase inhibitor. In our experience, there is considerable degradation of GSH or glutathione S-conjugates by canalicular vesicles, which are highly enriched in -glutamyltransferase activity, unless this enzyme is inhibited. This could contribute to the observed difference in radioisotope uptake between canalicular and basolateral vesicles.

Early studies of GSSG and DNP-SG transport in canalicular membrane vesicles identified low affinity ATP-independent systems (K(m) values of 0.4 and 1.0 mM, respectively; Akerboom et al., 1984; Inoue et al., 1984), but these findings were subsequently discounted when comparatively high affinity ATP-dependent systems were described. In contrast, the present findings indicate that both ATP-dependent and high and low affinity ATP-independent systems are present on the canalicular membrane and most likely play a functionally important role in biliary secretion of specific glutathione S-conjugates. The reason Akerboom et al.(1984) and Inoue et al.(1984) failed to detect high affinity, ATP-independent transport is explained by the relatively high substrate concentrations used in their kinetic analyses (above 50 µM) which prevented detection of the high affinity component (i.e.K(m) values of 8-10 µM; Fig. 5and Fig. 6).

Studies of BSP and bilirubin glucuronide transport in cLPM vesicles by Nishida et al. (1992a, 1992b) also indicate the presence of both ATP-dependent and membrane potential-dependent organic anion transport mechanisms. Mutant Wistar rats (TR rats) that exhibit conjugated hyperbilirubinemia and reduced biliary excretion of certain organic anions lack ATP-dependent BSP and bilirubin glucuronide transport, but retain voltage-dependent canalicular transport of these anions (Nishida et al., 1992a, 1992b). ATP-independent bilirubin glucuronide transport has a lower V(max) than the ATP-dependent system (1.8 versus 17 nmolbulletmgbullet20 s), but a higher affinity (K(m) value of 26 and 71 µM), indicating that the ATP-independent system may play an important role under physiological conditions. Because BSP, bilirubin glucuronide, and DNP-SG are mutually competitive inhibitors of transport, they probably share at least one transport mechanism (Nishida et al., 1992a, 1992b). The present findings support this hypothesis but also indicate that there may be more than one ATP-independent organic anion transport mechanism. ATP-independent transport of DNP-SG (Fig. 5), CH(3)Hg-SG (Dutczak and Ballatori, 1994), and GSH (Ballatori and Dutczak, 1994), is mediated by both high and low affinity components.

Although these data indicate the presence of multiple transport mechanisms, the actual number and identity of the transport proteins remains unknown. The broad and overlapping substrate specificities of the multiple transport systems, their complex kinetic behavior, and the absence of selective transport inhibitors, make it difficult to distinguish and characterize these transporters in isolated membrane vesicle systems. A complete characterization of the transporters will require their physical separation or the functional expression of the cloned cDNA.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant ES-06484 and National Institute of Environmental Health Sciences Center Grant ES-01247. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Environmental Medicine, University of Rochester School of Medicine, Rochester, NY 14642. Tel.: 716-275-0262; Fax: 716-256-2591.

(^1)
The abbreviations used are: BSP, sulfobromophthalein; cLPM, canalicular liver plasma membrane; DNP-SG, S-(2,4-dinitrophenyl)glutathione; AIB, aminoisobutyric acid; DIDS, 4,4`-diisothiocyanatostilbene-2,2`-disulfonic acid.


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