Unidirectional efflux systems are required by cells for different physiological functions that include cellular detoxification(1, 2, 3, 4, 5, 6) , antigen presentation by the major histocompatibility complex system (7) , extracellular signaling(4, 8, 9) , and the regulation of ion channels across plasma membranes(10, 11) . A representative member of this group of transporters is P-glycoprotein, which mediates the ATP-dependent expulsion of various hydrophobic drugs(1, 2, 3) . The overproduction of P-glycoprotein is often associated with multidrug resistance, and the existence of different forms of P-glycoproteins suggests that families of structurally similar transport proteins exist with complementing substrate specificities. A multidrug resistance-associated protein also contains structural elements common to P-glycoproteins(12) , and recent findings suggest that multidrug resistance-associated protein is an efflux pump for glutathione conjugates(13) .

Efflux pumps for anions exhibit substrate and inhibitor specificities which are distinct from the P-glycoproteins overproduced in multidrug resistance(4, 5) . Anionic efflux systems have been described for glutathione S-conjugates(5, 14, 15, 16, 17, 18) , leukotrienes(4, 19, 20, 21) , cyclic AMP(8, 9) , methotrexate(22, 23, 24, 25, 26, 27, 28, 29, 30) , and cholate(9, 25, 30) . In L1210 mouse cells, the unidirectional efflux of methotrexate has been shown by inhibitor studies to consist of two components which mediate 70 and 30% of total methotrexate efflux, respectively. A variant L1210/C7 cell line has also been isolated which lacks the primary efflux route for methotrexate (system I) but exhibits elevated activity of the second route (system II)(27) . System II responds generally to the same compounds which inhibit system I, but differences exist in the concentrations required for half-maximal efflux inhibition, and system II does not appear to transport cholate(27) . A high sensitivity to inhibition by BPAA ()and indoprofen is a characteristic feature of system II.

PGA and other nucleophiles that include CDNB and ethacrynic acid are unusually potent inhibitors of efflux system I in L1210 cells(30) . The effectiveness of these inhibitors was attributed to the intracellular accumulation of the glutathione conjugates of these compounds and the subsequent inhibition of efflux by competition with methotrexate and cholate for binding sites on efflux proteins. The physiological function of efflux system I was suggested to involve the expulsion of a broad spectrum of compounds such as glutathione conjugates, large anions, and anion conjugates(30) . The latter conclusion was based on the ability of system I to transport anions of diverse structure (methotrexate and cholate), its inhibition by various large anions, and the apparent sensitivity to intracellular glutathione conjugates.

The present study was initiated to identify and characterize unidirectional and ATP-dependent efflux systems for DNP-SG in L1210 cells and to determine whether the specificities of these systems have common features with the efflux routes for methotrexate and cholate. The L1210/C7 cell line with a defect in the efflux of methotrexate and cholate was also examined for a corresponding defect in the efflux of DNP-SG. Results using inside-out vesicles established that L1210 cells contain two ATP-dependent efflux routes for DNP-SG and that these systems differ substantially in substrate affinity and sensitivity to inhibitors. Moreover, a high affinity system for DNP-SG in parental cells was found to be defective in the efflux-deficient L1210/C7 cell line.

MATERIALS AND METHODS

Chemicals

DNP-SG was synthesized chemically by a procedure based on the method of Vince et al.(32) . Glutathione (2.0 mmol) was dissolved in 2.0 ml of water and neutralized by the addition of 1.8 ml of 2 N NaOH (3.6 mmol). CDNB (2.4 mmol) dissolved in 15 ml of ethanol was then added dropwise to the glutathione solution, and the mixture was stirred for 16 h at 23 °C. The resulting precipitate was chilled on ice, washed with 25 ml each of ice-cold water and then ethanol, suspended in 15 ml of water, and dissolved by adjusting the pH to 7.0 with 2 N NaOH. Reprecipitation was achieved by adding 2 N HCl to pH 3.5 and chilling the sample on ice for 1 h. The final precipitate was washed with water and ethanol (as described above) and then with chloroform. DNP-SG was obtained as a dry powder in an amount corresponding to a theoretical yield of about 50% and was essentially free of CDNB as determined by HPLC (see below). Spectral analysis gave an absorbance maximum at 340 nm and a molar extinction coefficent of about 9,600.

[H]DNP-SG was synthesized enzymatically by combining CDNB (2 µmol), glutathione (0.25 µmol), [H]glutathione (250 µC), and 2 units of glutathione S-transferase in 1.0 ml of 50 mM Tris-HCl, pH 7.4, and incubating the mixture for 2 h at 37 °C. The incubation mixture was then diluted with 1.0 ml of ice-cold distilled water and applied to a column of QAE-Sephadex (1.0-ml bed volume) equilibrated with 10 mM Tris-HCl, pH 7.4. After washing successively with 2 ml of 10 mM Tris-HCl, pH 7.4, and 3 ml of water to remove the CDNB and glutathione S-transferase, the bound [H]DNP-SG was eluted in 0.5-ml fractions with 0.7 N formic acid. The radioactive fractions were pooled, dried under reduced pressure, dissolved in about 1 ml of water, redried, and dissolved in 400 µl of water. Further purification by HPLC was achieved by applying the sample (in 200-µl portions) to an equilibrated 0.46 25-cm Altex C18 Ultrasphere-ODS column and eluting in 1.0-ml fractions with a gradient of solvent A (0.1% aqueous trifluoroacetic acid) and solvent B (0.1% trifluoroacetic acid, 10% water, and 90% acetonitrile) according to the following program: 0-5 min, 100% A, 5-30 min 100% A to 100% B. The sharp peak at approximately 19 min (detected at 278 nm) was collected, pooled with the duplicate run, dried under reduced pressure, and dissolved in 1.0 ml of water. Contaminating unreacted [H]glutathione and oxidized [H]glutathione disulfide eluted in a broad peak between 4 and 8 min under these conditions, and trace amounts of CDNB eluted at 24 min. The concentration of [H]DNP-SG was determined by comparison of HPLC profiles of unknown samples and standard solutions whose concentrations had been determined spectrally at 340 nm (molar extinction coefficient = 9, 600). Parallel samples subjected to scintillation counting gave specific activities in various preparations of 425,000 ± 50,000 counts/min/nmol. The recovery of tritium in the [H]DNP-SG usually exceeded 90%.

EA-SG was synthesized chemically by a procedure based on the method of Awasthi et al.(33) . Ethacrynic acid (0.10 mmol dissolved in 1.0 ml of ethanol) and glutathione (0.12 mmol dissolved in 1.0 ml of 10 mM potassium phosphate, pH 7) were combined, adjusted to pH 6.5 with 0.5 N KOH, and incubated for 24 h at 23 °C. After reducing the volume to about 0.4 ml (under reduced pressure), the EA-SG was purified by HPLC (in 200-µl portions) by the same procedure described above for [H]DNP-SG. EA-SG eluted at 18 min, whereas unreacted ethacrynic acid eluted at 23 min. After lyophilization, EA-SG was obtained as a dry white powder in a yield of about 75%. Spectral analysis revealed an absorbance maximum near 270 nm and an extinction coefficient (5,700) similar to that reported by Habig et al.(34) .

BSP-SG was synthesized enzymatically by a procedure based on the method of Garcia-Ruiz et al.(35) . BSP (0.10 mmol) and glutathione (0.20 mmol) were dissolved in 1 ml of 10 mM sodium-phosphate, pH 7, and adjusted to pH 7.5 with 0.5 N NaOH. Glutathione S-transferase (50 units) was then added, and the sample was incubated at 23 °C for 24 h. BSP-SG was separated from unreacted BSP and glutathione (in 200-µl aliquots) by the HPLC procedure described above for [H]DNP-SG, except that the gradient program was modified as follows: 0-5 min, 100% A; 5-40 min, 100% A to 30% B; 40-50 min, 30% B to 100% B. BSP-SG eluted at 37 min, whereas BSP eluted at 44 min. BSP-SG was obtained as a dry white powder in a yield of about 50%. BSP-SG and BSP exhibited the same spectral properties (above 450 nm) and appeared to have the same molar extinction coefficient (6,050) at 568 nm in 0.1 M Tris-HCl, pH 8.5.

BSP-SGSG (36) was synthesized enzymatically and purified by the same procedure as described above for BSP-SG, except that glutathione in the reaction mixture (0.50 mmol) was increased to a 5-fold molar excess over BSP (0.10 nmol), the pH was readjusted to 7.5 at 24-h intervals, and the incubation time was extended to 72 h. BSP-SGSG eluted during HPLC at 32 min and was obtained in a yield of about 15%. Concentration was determined from the extinction coefficient for BSP (at 568 nm and pH 8.5) of 6,050.

Cells

Preparation of Inside-out Vesicles

Transport Determinations in Inside-out Vesicles

RESULTS

General Characteristics of DNP-SG Transport into Inside-out Vesicles from L1210 Cells

Purified vesicles incubated in the complete assay mixture containing 1.5 µM [H]DNP-SG accumulated the labeled substrate by a process which was linear for 10 min at 37 °C, reached a maximum at 25 min, and then declined thereafter. Omission of the ATP-regenerating system or ATP (1.0 mM) led to a 3- and 20-fold reduction in the transport rate, respectively, and essentially no uptake occurred at 4 °C. A broad optimum was observed between pH 7.0 and 7.5. ATP-dependent uptake at 1.5 µM [H]DNP-SG after 25 min at 37 °C decreased by 1.6- and 2.4-fold when the sucrose concentration was increased to 0.5 and 1.0 M, respectively, indicating that the majority of uptake represented the accumulation of free substrate within the intravesicular space and not an ATP-dependent binding of substrate to the membrane. When the substrate concentration was increased to 100 µM, ATP-dependent uptake of [H]DNP-SG remained linear for 10 min at 37 °C, and reached a maximum after 20 min.

Uptake measurements at varying concentrations of [H]DNP-SG provided evidence for two ATP-dependent transport components. A double-reciprocal plot (Fig. 1) of transport at substrate concentrations ranging from 0.10 to 500 µM was biphasic and suggested the presence of a high and low affinity system with substantially different kinetic properties. Curve fitting of the data for multiple transport systems showed that the best fit was obtained for two independent transport components with K values for half-maximal transport of 0.63 µM (V = 6.6 pmol/min/mg protein) (Fig. 1) and 450 µM (V = 148 pmol/min/mg protein) (inset, Fig. 1). In a control experiment in which the transport interval was reduced from 10 to 5 min, no significant change was observed for the K of the high affinity system (0.60 µM) or the low affinity system (600 µM) for DNP-SG. Computer simulations which probed for a third transport component with a K between 2 and 20 µM [H]DNP-SG produced a poor fit for the data. Kinetic values for DNP-SG transport into L1210 cell vesicles are listed in Table 1.

Figure 1: Double-reciprocal plot of the substrate dependence for [H]DNP-SG transport by inside-out vesicles from L1210 cells. Data are shown for transport at [H]DNP-SG concentrations from 0.2 to 20 µM. Inset, double-reciprocal plot of transport (v) at [H]DNP-SG concentrations (s) from 5 to 500 µM.

DNP-SG Transport in Inside-out Vesicles from L1210/C7 Cells

Transport measurements at varying concentrations of [H]DNP-SG confirmed that different kinetics are expressed in vesicles from the variant cell line relative to the parent. A double-reciprocal plot of these data (Fig. 2) showed that two ATP-dependent components were present, but the high affinity system of parental cells was absent in the variant and had been replaced by a component with a similar V but a reduced affinity for [H]DNP-SG. Computer analysis indicated that the K for this intermediate affinity route was 4.5 µM (Table 1), a value 7-fold lower than the K of the high affinity route in parental cells. The low affinity system of variant cells (inset, Fig. 2) exhibited a K and V for DNP-SG transport which was comparable to parental cells (Table 1), suggesting that the same low affinity system was present in both cell lines.

Figure 2: Double-reciprocal plot of the substrate dependence for [H]DNP-SG transport by inside-out vesicles from L1210/C7 cells. Data are shown for transport at [H]DNP-SG concentrations from 0.8 to 50 µM. Inset, double-reciprocal plot of transport (v) at [H]DNP-SG concentrations (s) from 10 to 500 µM.

Inhibitor Specificity of the High Affinity Transport System

Figure 3: Semi-log plot of the concentration dependence for inhibition of [H]DNP-SG transport in vesicles from L1210 cells by various compounds. Measurements were performed at 1.5 µM [H]DNP-SG. MTX, methotrexate.

Large anionic compounds which are not glutathione conjugates were also examined for the ability to inhibit the high affinity transport system for DNP-SG (Table 2). The most potent of these compounds was the taurate diconjugate of bilirubin, whose K of 0.10 µM was comparable to the most effective glutathione conjugates. QUIN-2 (a tetraanionic quinoline derivative), bilirubin, BSP, and indomethacin inhibited with an intermediate affinity, whereas other aromatic anions (e.g. BPAA and indoprofen) were poor inhibitors of this system. An unusual response was observed for cholate, which produced an increase in transport to 125% of the control at 50 µM, a subsequent decline to control levels at 150 µM, and inhibition by 50% at 350 µM (data not shown). Methotrexate also inhibited the high affinity system for DNP-SG (Fig. 3) but had a relatively low affinity (K = 300 µM).

BPAA (Fig. 4A), flurbiprofen, and indoprofen inhibited [H]DNP-SG transport (at 1.5 µM) in parental vesicles, but the extent of inhibition reached only 10% at concentrations below 20 µM and then remained relatively constant thereafter. This latter result suggested that these compounds had inhibited the portion of total uptake representing the low affinity system, but had little or no effect on the high affinity system.

Figure 4: [H]DNP-SG transport in vesicles from L1210 cells at varying concentrations of BPAA in the absence (A) and presence of EA-SG (B). A, response of transport at 1.5 µM [H]DNP-SG to increasing concentrations of BPAA. B, response of transport at 5.0 µM [H]DNP-SG to increasing concentrations of BPAA in the presence of a fixed concentration (40 or 100 µM) of EA-SG. Transport in the controls without inhibitor were 6.1 and 8.6 pmol/min/mg protein at 1.5 and 5.0 µM [H]DNP-SG, respectively.

Separation of the High Affinity and Low Affinity Transport Systems in L1210 Cells

Isolation of the low affinity system was a more challenging problem since this route contributed a majority of total transport only at relatively high substrate concentrations. To improve the relative contribution from this route, the substrate concentration was raised to 5.0 µM, and EA-SG was added (at 40 µM) to inhibit the high affinity system. The resulting ATP-dependent transport activity for [H]DNP-SG was then evaluated for a BPAA-sensitive transport component (Fig. 4B). Under these conditions, maximal inhibition by BPAA reached 50%, and the concentration for half-maximal inhibition was 6 µM (K = 5.9 µM). At 100 µM EA-SG, the BPAA-sensitive portion of [H]DNP-SG transport increased to 75%, and the IC for BPAA remained at 6 µM (Fig. 4B). Using the latter conditions, K values for the low affinity system were also obtained for indoprofen (K = 3.0 µM), indomethacin (K = 10 µM), and bilirubin dituarate (K = 1.8 µM) (Table 2). Hence, EA-SG could be shown to be a potent and relatively specific inhibitor of the high affinity transport system, but conditions were not achieved for a complete separation of the two transport activities. Even at 100 µM EA-SG, interference from the high affinity system was approximately 25%.

Inhibitor Specificity of the Transport Systems for DNP-SG in L1210/C7 Cells

Figure 5: [H]DNP-SG transport at increasing concentrations of EA-SG (A) or BPAA (B) in vesicles from L1210/C7 cells. [H]DNP-SG concentrations, 5.0 µM; transport in the control without inhibitor, 4.0 pmol/min/mg protein.

DNP-SG transport in L1210/C7 vesicles in the presence of combinations of EA-SG and BPAA is shown in Fig. 6. Transport at a fixed concentration of EA-SG (40 µM) and varying levels of BPAA is shown in Fig. 6A. The resulting inhibition by BPAA appeared monophasic and extrapolated to greater than 90% at high levels of BPAA. Similarly, transport at a fixed concentration of BPAA (100 µM) and varying levels of EA-SG produced a similar monophasic profile and a maximum inhibition that also exceeded 90% (Fig. 6B). Hence, EA-SG and BPAA can be employed for a nearly quantitative separation of the intermediate and low affinity transport systems in L1210/C7 vesicles.

Figure 6: Inhibition of [H]DNP-SG transport by combinations of EA-SG and BPAA in vesicles from L1210/C7 cells. Transport at 5.0 µM [H]DNP-SG was measured in the presence of 40 µM EA-SG (+EA-SG) (A) and varying concentrations of BPAA or 100 µM BPAA (+BPAA) (B) and varying concentrations of EA-SG.

The inhibitor specificity of the transport systems for DNP-SG in L1210/C7 vesicles was determined by measuring inhibitor responses in the presence of 100 µM BPAA or 40 µM EA-SG. [H]DNP-SG transport in the presence of a constant level of EA-SG or BPAA and increasing concentrations of three selected inhibitors is shown in Fig. 7. Flurbiprofen (Fig. 7A) mediated a potent inhibition of DNP-SG transport in the presence of EA-SG, but not BPAA, indicating that only the low affinity system was sensitive to this inhibitor. Conversely, BSP-SG (Fig. 7B) was a potent inhibitor of [H]DNP-SG transport in the presence of BPAA, but was much less effective with added EA-SG, indicating that the intermediate affinity system was more sensitive to this inhibitor. Bilirubin ditaurate (Fig. 7C) inhibited transport in the presence of either BPAA or EA-SG.

Figure 7: Response of the intermediate and low affinity transport systems of L1210/C7 cells to (A) flurbiprofen (FB), (B) BSP-SG, and (C) bilirubin ditaurate (BDT). Transport by the intermediate affinity system was determined at 5.0 µM [H]DNP-SG in the presence of 100 µM BPAA (+BPAA), whereas the low affinity system was determined in assay mixtures containing 40 µM EA-SG (+EA-SG).

Various compounds and their K values for inhibition of the intermediate and low affinity transport systems for DNP-SG in L1210/C7 vesicles are shown in Table 3. These two transport systems responded to different types of compounds, although an overlap in specificity was evident with some inhibitors. The intermediate affinity system of L1210/C7 cells responded generally to the same compounds which inhibited the high affinity system of parental cells. The most effective compounds were glutathione conjugates, bilirubin ditaurate, QUIN-2, bilirubin, and BSP, whereas GS-SG and various monovalent aromatic anions were poor inhibitors of this system. K values for unlabeled DNP-SG coincided with K values for [H]DNP-SG transport. Inhibition was also obtained with methotrexate (K = 220 µM), whereas cholate produced a stimulation similar to that observed for the high affinity system. The extent of this stimulation was somewhat higher in L1210/C7 vesicles (50% at 50 µM cholate), although the general pattern was the same; stimulation at low levels of cholate, a maximum at 50 µM, and inhibition at higher concentrations. A double-reciprocal plot of transport at varying concentrations of [H]DNP-SG showed that 50 µM cholate increased the V for the intermediate affinity system by 50% but had little effect on the K (4.0 µM) for DNP-SG (data not shown).

The low affinity system for DNP-SG in vesicles from L1210 cells exhibited a high affinity for several large anions (Table 3). The most effective compound was bilirubin ditaurate (K = 1.5 µM), although strong inhibition (K = 3.0-6.0 µM) was also observed with monovalent anions (indoprofen, flurbiprofen, and BPAA) which were not inhibitors of the high or intermediate affinity systems (cf. Table 2and Table 3). Bilirubin (K = 40 µM) and methotrexate (K = 150 µM) were moderate inhibitors of this transport system. The most pronounced inhibition by a glutathione conjugate was observed with BSP-SG, but the K for BSP-SG (60 µM) was indistinguishable from the K for BSP alone (50 µM). Inhibition by unlabeled DNP-SG (at concentrations up to 1.0 mM) produced a K (450 µM) which was consistent with the observed K (500 µM) for [H]DNP-SG. Analogs of BPAA with decreasing molecular size (naphthalene acetic acid and phenyl acetic acid) were progressively weaker inhibitors of the low affinity system.

Michaelis Constants for ATP

DISCUSSION

L1210 mouse cells contain two ATP-dependent transport systems which mediate the efflux of DNP-SG. These systems were detected and characterized using inside out vesicles which had been purified from isolated plasma membranes. The two systems can be readily distinguished by their substantially different K values for half-maximal transport of [H]DNP-SG (0.63 and 450 µM, respectively) and by differences in relative contribution at different substrate concentrations. Transport at DNP-SG concentrations below 1 µM proceeds almost exclusively by the high affinity system, whereas these two systems contribute approximately equally to total transport when the concentration of DNP-SG is raised to 30 µM. Numerical values for K that had been determined for each route by influx analysis (Table 1) were confirmed by inhibitor analysis with unlabeled DNP-SG ( Table 2and Table 3). The present finding of multiple transport routes for DNP-SG in L1210 cells is consistent with prior studies in which human erythrocytes were also shown to contain two energy-dependent efflux routes with substantially different K values for DNP-SG. K values of 1.4 and 700 µM were detected in intact cells(42) , whereas slightly higher K values of 3.9 and 1,600 µM were observed in inside-out vesicles(43) . High affinity, ATP-dependent efflux systems for DNP-SG with a K of 4 and 20 µM, respectively, have also been demonstrated in rat liver canalicular membrane vesicles (15) and in vesicles from rat heart sarcolemma(14) .

The inhibitor specificity of the high affinity transport system in L1210 vesicles was determined with minimal interference from the low affinity route by employing a low concentration of [H]DNP-SG. The most effective inhibitor of this system was BSP-SG, whose affinity (K = 0.09 µM) was 7-fold higher than DNP-SG (K = 0.63 µM). Other large anionic conjugates (BSP-SGSG, LTC, EA-SG, and bilirubin ditaurate) were also bound with a higher affinity than DNP-SG, whereas a lower affinity was observed for S-hexyl glutathione and GS-SG. Glutathione conjugates with aromatic (BSP-SG) or aliphatic (LTC) substituents were accommodated with a comparably high affinity, and conjugation with glutathione was not required for binding since a high affinity (K = 0.10 µM) was also observed for the taurate diconjugate of bilirubin. The same high affinity observed in the present study for LTC (K = 0.20 µM) had also been observed for LTC transport via a glutathione conjugate pump in membrane vesicles from rat heart sarcolemma (K = 0.15 µM) and rat liver (K = 0.25 µM)(20) . Our competition studies support the model of Awasthi (5, 44) in which the substrate binding site on the high affinity glutathione conjugate pump is suggested to have a relatively broad specificity. Using the designations of Ishikawa(4, 45) , the G-domain for binding of the glutathione portion of conjugates appears to accept both the zwitterionic glutathione structure as well as the anionic composition of taurate diconjugates, and the C-domain for the adduct portion of conjugates accommodates uncharged compounds with an aromatic dinitrophenyl or aliphatic S-hexyl group, as well as anionic compounds with large aliphatic (LTC) or aromatic (BSP) constituents. A high affinity for the glutathione diconjugate of BSP is a further indication of substantial flexibility at the substrate binding site.

Transport systems involved in cellular detoxification would be expected to possess a general ability to translocate various competitive inhibitors which exhibit structural similarities to known substrates (e.g. DNP-SG), and in some cases, this possibility has been confirmed by direct measurements. Transport of LTC and DNP-SG has been determined in rat liver plasma membranes(20) , and each compound was deduced from kinetic studies to be a substrate of the same transport system. The high affinity system for DNP-SG in L1210 vesicles is inhibited by various structurally similar glutathione conjugates and by other compounds (e.g. bilirubin ditaurate) which may also be transported by this system, although this has not been verified by direct measurements. Compounds with a relatively low affinity for this system may also be transport substrates. Regardless of affinity, efficient transport of inhibitors could occur as long as the V increases proportionally with decreasing affinity.

Prior kinetic studies have suggested that methotrexate may exit L1210 cells via systems which also function in the extrusion of glutathione conjugates(30) . This latter conclusion was based, in part, on the potent inhibition of methotrexate efflux by compounds, such as prostaglandin A, ethacrynic acid, and CDNB, which can be readily converted to intracellular glutathione conjugates. The present study shows that methotrexate is an inhibitor of the high affinity carrier for DNP-SG and hence supports the notion that methotrexate may utilize the latter system to exit L1210 cells. The relatively low affinity of the conjugate pump for methotrexate (K = 300 µM) does not detract from this model since a low affinity for methotrexate had been suggested from prior studies. Methotrexate efflux showed no evidence of saturation in intact L1210 cells which had been loaded with methotrexate to internal concentrations up to 30 µM (120 pmol/mg protein). ()The possibility that methotrexate efflux occurs via a glutathione conjugate pump is also supported by measurements in inverted human erythrocyte ghosts(46) . Methotrexate transport in this system was inhibited by several compounds which also inhibit glutathione conjugate transport. Moreover, the reported K for methotrexate transport (470 µM) was similar to the K observed in the present study (300 µM) for inhibition of the high affinity system by methotrexate.

The proposal that methotrexate efflux by system I in intact cells occurs via the high affinity efflux system for DNP-SG is further supported by transport studies using vesicles from L1210/C7 cells. The loss in ability to efflux methotrexate by L1210/C7 cells (27) is accompanied by a corresponding loss in the ability of L1210/C7 vesicles to transport DNP-SG (cf.Fig. 1and Fig. 2), and the defect was traced to the high affinity transport system for DNP-SG.

Vesicles from L1210/C7 cells contain an intermediate affinity transport route for DNP-SG which could not be detected in the parental cells. This additional system may be a mutated form of the high affinity route of parental cells since these systems exhibit a comparable V for DNP-SG transport (Table 1), and both have a similar binding preference for glutathione conjugates (cf. Table 2and Table 3). Alternatively, the intermediate affinity system may represent a separate route which had been induced or stimulated from low levels in the variant, or had been obscured in the parent by interference from the high affinity system.

The low affinity transport system for DNP-SG was isolated by performing transport measurements in the presence of a specific inhibitor of the higher affinity route. EA-SG (at concentrations of 40-100 µM) substantially reduced transport via the high affinity system in parental vesicles (Fig. 4) and essentially eliminated interference from the intermediate affinity system in L1210/C7 vesicles ( Fig. 5and Fig. 6). Parental and variant cells appear to express the same low affinity transport system for DNP-SG since no differences were observed in the K or V for DNP-SG transport (Table 1), and these two routes exhibited the same response to inhibition by BPAA, indoprofen, bilirubin ditaurate, and indomethacin (cf.Table 2and Table 3).

The inhibitor specificity of the low affinity transport system for DNP-SG suggests that zwitterionic glutathione conjugates are not the primary efflux substrates for this system. Affinity measurements (Table 3) indicate that this system is an organic anion pump since it has a relatively high affinity for large monovalent anions (BPAA or indoprofen), anionic conjugates (bilirubin ditaurate), and large divalent anions (bilirubin and BSP). A preference for conjugated divalent anions is indicated since this system exhibits a 40-fold higher affinity for bilirubin ditaurate relative to bilirubin. Since BSP and BSP-SG interact with about the same affinity, the glutathione constituent of BSP-SG does not appear to enhance or hinder the binding of BSP. The minimum size requirement for anion binding is about 200 Daltons, as determined from the increase in affinity with size in a series of structurally related monovalent anions: phenylacetic acid (K >200 µM); naphthalene acetic acid (K = 70 µM); and biphenylacetic acid (K = 5.5 µM). Candidates for physiological substrates of this anion efflux system include various mono- and diglucuronide conjugates and anionic mercapturates derived from the metabolism of zwitterionic glutathione conjugates.

The low affinity efflux system for DNP-SG may also be the same system which mediates the efflux of methotrexate by system II in intact L1210 cells(26, 27) . Methotrexate structurally fits the general specificity of this system for large organic anions and was shown to bind with a moderate affinity (K = 150 µM). The primary evidence linking these two transport systems is the close correlation in the ability of BPAA, indoprofen, and flurbiprofen to inhibit both the low affinity transport system for DNP-SG in vesicles (see Table 2and Table 3) and efflux system II for methotrexate in intact cells(26, 27) .

Electrophiles are usually detoxified in mammalian cells via a glutathione S-transferase dependent conjugation with glutathione and subsequent elimination of the hydrophilic products via efflux pumps. Endogenous compounds may also require conjugation without the involvement of glutathione and excretion of the anionic products. Bilirubin ditaurate is representative of the latter group of compounds. Since xenobiotics, electrophiles, and endogenous compounds programmed for catabolism may exhibit a variety of possible structures, detoxifying systems must be able to accommodate a range of possible anions and anion conjugates to protect cell integrity. Glutathione S-transferases contribute to this diversity by comprising a family of enzymes with differing specificities(47) . Similarly, the extrusion of unwanted anions and zwitterions of various sizes and structures may be achieved most efficiently by a combination of efflux pumps of varying specificities. The notion of multiple efflux routes for glutathione conjugates is supported in the present study with L1210 cells and previously in human erythrocytes. The latter contain high and low affinity efflux systems for DNP-SG(42, 43) , and a separate activity for the efflux of oxidized glutathione(14) . Multiple anion efflux systems have also been demonstrated in intact L1210 cells by the presence of two unidirectional efflux routes for methotrexate(26, 27) . An inhibitor analysis of the high and low affinity transport systems for DNP-SG in L1210 cells shows that these two effux systems, in combination, accommodate and presumably extrude a broad range of structurally diverse compounds with a molecular requirement which includes only a moderate-to-large size and a net negative charge.

FOOTNOTES

*

This work was supported by Research Grant CA23970 from the National Cancer Institute, Department of Health and Human Services. This is manuscript number 8776-MEM from The Scripps Research Institute.

§

To whom correspondence should be addressed: Dept. of Molecular and Experimental Medicine (NX-6), The Scripps Research Institute, 10666 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-554-8091; Fax: 619-554-6223.

()

The abbreviations used are: CDNB, 1-chloro-2,4-dinitrobenzene; DNP-SG, 2,4-dinitrophenyl-S-glutathione; BPAA, 4-biphenylacetic acid; BSP, bromosulfophthalein; BSP-SG, glutathione conjugate of BSP; BSP-SGSG, glutathione diconjugate of BSP; EA-SG, glutathione conjugate of ethacrynic acid; hexyl-SG, S-hexyl glutathione; LTC, leukotriene C; QUIN-2, 2-[(2-bis-[carboxymethyl]amino-5-methylphenoxy)-methyl]-6-methoxy-8-bis(carboxymethyl)aminoquinoline; GS-SG, oxidized glutathione; DTE, dithioerythreitol; HPLC, high performance liquid chromatography.

()

G. B. Henderson, unpublished results.

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