Reduced folate derivatives are endogenous substrates for cMOAT in rats

Hiroyuki Kusuhara1, Yong-Hae Han1, Minoru Shimoda2, Eiichi Kokue2, Hiroshi Suzuki1, and Yuichi Sugiyama1

1 Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113; and 2 Faculty of Agriculture, Tokyo College of Agriculture and Technology, Fuchu, Tokyo 183, Japan

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We examined the role of the canalicular multispecific organic anion transporter (cMOAT) in the biliary excretion of reduced folate derivatives in vivo and in vitro using normal [Sprague-Dawley rats (SDR)] and mutant [Eisai hyperbilirubinemic rats (EHBR)] rats whose cMOAT is hereditarily deficient. In vivo, the biliary excretion of endogenous tetrahydrofolate (H4PteGlu), 5-methyltetrahydrofolate (5-CH3-H4PteGlu), and 5,10-methylenetetrahydrofolate (5,10-CH2-H4PteGlu) in EHBR was reduced to 8.2%, 1.9%, and 5.5% of those in SDR, respectively, whereas that of 10-formyltetrahydrofolate (10-HCO-H4PteGlu) was detected only in SDR and not in EHBR. Bile drainage caused reduction of endogenous plasma folate concentrations in SDR but not in EHBR. In vitro, significant ATP-dependent uptake of 3H-labeled 5-CH3-H4PteGlu into canalicular membrane vesicles was observed only in SDR. This ATP-dependent uptake was saturable with a Michaelis constant (Km) value of 126 µM, which was comparable with its inhibitor constant (Ki) value of 121 µM for the ATP-dependent uptake of a typical cMOAT substrate, 2,4-dinitrophenyl-S-glutathione (DNP-SG). Vice versa, DNP-SG inhibited the uptake of 5-CH3-H4PteGlu with a Ki of 35 µM, which was similar to its Km value. In addition, H4PteGlu and 5,10-CH2-H4PteGlu also inhibited the ATP-dependent uptake of DNP-SG. These results indicate that 5-CH3-H4PteGlu and other derivatives are transported via cMOAT. Therefore, reduced folate derivatives are the first endogenous substrates for cMOAT that do not contain glutathione, glucuronide, or sulfate moieties.

enterohepatic circulation; organic anion; active transport; Eisai hyperbilirubinemic rats

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

REDUCED FOLATE IS REQUIRED to provide single-carbon units for the synthesis of purines and thymidine, for the interconversion of serine and glycine, for the degradation of histidine and glycine, and for the methylation of homocysteine to regenerate methionine (Fig. 1) (2, 3, 8, 28, 34). Figure 1 shows the structures and interconversions of bioactive reduced folate derivatives. Whereas 5-methyltetrahydrofolate (5-CH3-H4PteGlu) is the principal folate in plasma (8, 38), we found four reduced fo- late derivatives in rat bile, tetrahydrofolate (H4PteGlu), 5-CH3-H4PteGlu, 5,10-methylenetetrahydrofolate (5,10-CH2-H4PteGlu), and 10-formyltetrahydrofolate (10-HCO-H4PteGlu), as shown in Fig. 1 (29, 30, 31). Because bile drainage causes a rapid reduction in plasma folate concentration (30, 32), enterohepatic circulation plays an important role in folate homeostasis, i.e., maintenance of plasma folate concentrations. We (30) and Horne (11) proposed a physiological role for enterohepatic circulation of reduced folate derivatives as the source for the reutilization of folate, because 1) enteric infusion of 5-CH3-H4PteGlu and nonmethylated reduced folate derivatives maintained the plasma concentration of 5-CH3-H4PteGlu in rats with bile drainage (30), and 2) intravenous administration of folic acid (PteGlu), which has been postulated as a waste product (10, 32), elevated the biliary excretion of all reduced folate derivatives (30).


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Fig. 1.   Schematic diagram of folate biotransformations in the liver [information taken from Shane (28)]. Ser, serine; Gly, glycine; Cob, cobalamine; MCob, methylcobalamine; Hcy, homocysteine; Met, methionine. H4PteGlu, tetrahydrofolate; 5,10-CH2-H4PteGlu, 5,10-methylenetetrahydrofolate; 5-CH3-H4PteGlu, 5-methyltetrahydrofolate; 10-HCO-H4PteGlu, 10-formyltetrahydrofolate.

The hepatic elimination of compounds from the circulating blood consists of two steps: transport across both the basolateral and the bile canalicular membrane. The hepatic uptake of 5-CH3-H4PteGlu has been characterized in vitro using isolated hepatocytes (11) and basolateral membrane vesicles (12) in rats. Hepatic uptake of 5-CH3-H4PteGlu does not depend on the sodium concentration but depends on the proton concentration in the medium, since an inward proton gradient stimulated the uptake of 3H-labeled 5-CH3-H4PteGlu into isolated hepatocytes and basolateral membrane vesicles (11, 12), which is a common characteristic with the uptake of this substrate in the small intestine (21, 27). In spite of its structural similarity to 5-CH3-H4PteGlu, the hepatic uptake of methotrexate, an inhibitor for dihydrofolate reductase, showed different characteristics: 1) pH dependence was biphasic and showed maximal uptake at pH 7.0 (11), and 2) its uptake into isolated hepatocytes showed sodium dependency (11).

The mechanism for the transport of reduced folate derivatives across the bile canalicular membrane has not been clarified yet. Strum and Liem (35) reported that 5-CH3-H4PteGlu in bile was 14- to 16-fold larger than that in liver parenchyma, suggesting the presence of an active transporter on the bile canalicular membrane. Three kinds of primary active transporters for organic compounds have been identified on this membrane: a transporter for bile acids, P-glycoprotein, and canalicular multispecific organic anion transporter (cMOAT) (18, 20, 25, 42). cMOAT has been identified as a primary active transporter for organic anions, and its cDNA has been cloned recently (5, 14, 15, 17, 26). In the Eisai hyperbilirubinemic rats (EHBR) and TR- rats, which are derived from Sprague-Dawley rats (SDR) and Wistar rats, respectively, cMOAT is hereditarily deficient, while other primary active transporters are maintained. Using these animals, we and others (18, 20, 25, 42) have demonstrated that substrates for cMOAT include glutathione conjugates, glucuronides, sulfates of several bile acids, and some organic anions without conjugation. Because 1) we demonstrated previously (22) through examining the ATP-dependent uptake into canalicular membrane vesicles (CMV) prepared from SDR and EHBR that methotrexate is extruded via cMOAT, and 2) reduced folate derivatives have an anionic moiety, i.e., two carboxyl residues in their glutamic residue, which is a common characteristic among substrates for cMOAT (18, 20, 25, 42), we hypothesized that cMOAT may be responsible for the hepatobiliary transport of reduced folate derivatives and may play a role in folate homeostasis. In the present study, we examined this hypothesis in vivo and in vitro.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. PteGlu, (6R,S)-5-CH3-H4PteGlu calcium salt, (6R,S)-5,10-CH2-H4PteGlu magnesium salt, and (6R,S)-H4PteGlu trihydrochloride were purchased from Dr. B. Schircks Laboratories (Jona, Switzerland). Sodium ascorbate and paraformaldehyde were purchased from Wako Pure Chemicals (Osaka, Japan). All folate derivatives were dissolved in 0.2% sodium ascorbate solution except for 5,10-CH2-H4PteGlu, which was dissolved in 0.2% sodium ascorbate solution containing 3 × 10-3% paraformaldehyde. They were stored at -80°C. 3H-labeled 5-CH3-H4PteGlu was purchased from Moravek Biochemicals (Brea, CA). Unlabeled 2,4-dinitrophenyl-S-glutathione (DNP-SG) and [3H]DNP-SG (50.0 µCi/nmol) were synthesized enzymatically using [glycine-2-3H]glutathione (NEN, Boston, MA), 1-chloro-2,4-dinitrobenzene, and glutathione S-transferase (Sigma Chemical, St. Louis, MO) as described previously (19), and the purity was checked by TLC. ATP, creatine phosphate, and creatine phosphokinase were purchased from Sigma Chemical. All chemicals and reagents were of analytic grade.

Animals and sampling of biological fluids. SDR (male, 6 wk; Nihon Ikagaku, Tokyo, Japan) and EHBR (male, 6 wk; SLC Japan, Shizuoka, Japan) were used throughout the experiments. The animals were anesthetized with an intraperitoneal dose of urethan (1 g/kg body wt) throughout the experimental period. Blood was collected in test tubes containing sodium ascorbate (2 mg/ml plasma) as an antioxidant via a polyethylene catheter (PE-50) inserted into the femoral artery. After plasma separation, it was stored at -20°C until analysis. Bile specimens were collected into tubes placed on ice via a polyethylene catheter (PE-10) inserted into the bile duct. In the present study, sodium ascorbate (0.8%) was added to the bile specimens to prevent the oxidation of reduced folate derivatives (30). The volume of the sodium ascorbate was adjusted to provide a final concentration of 0.4%, taking into account the bile flow rate (~10 µl · min-1 · rat-1); e.g., for the collection at 30-min intervals, the bile specimens were collected in the tube containing 300 µl of the sodium citrate solution. After measuring the volume, we stored the bile samples at -20°C until analysis. Plasma and bile folate levels were determined using HPLC with electrochemical detection as reported previously (29, 31).

Intravenous administration of PteGlu. Rats were kept for 1 h under anesthesia with bile drainage before intravenous administration of oxidized folate (PteGlu) (1 mg/kg). Endogenous biliary excretion of reduced folate derivatives was determined before administration of PteGlu. Blood and bile specimens were collected as described previously for 4 h at 1-h and 30-min intervals, respectively.

Preparation of CMV. CMV were prepared from male SDR and EHBR using the slightly modified method of Meier and Boyer (23). After suspension of CMV in 10 mM Tris · HCl buffer (pH 7.4) containing 250 mM sucrose, CMV were frozen in liquid N2 and stored at -100°C until required. The transport activity of CMV used in this study was also checked by measuring the ATP-dependent uptake of [3H]DNP-SG (1 µM) for a 2-min incubation period at 37°C.

Uptake of 5-CH3-H4PteGlu into CMV. The transport study was performed using the rapid filtration technique described in a previous report (13). Fifteen microliters of transport medium (10 mM Tris, 250 mM sucrose, and 10 mM MgCl2, pH 7.4) containing radiolabeled compounds, ATP-regenerating system (10 mM creatine phosphate and 100 µg/ml creatine phosphokinase), 5 mM ATP (or 5 mM AMP), and 0.5% sodium ascorbate as an antioxidant with or without unlabeled substrate were preincubated at 37°C for 3 min and then rapidly mixed with 5 µl CMV suspension containing 10 µg protein. The transport reaction was stopped by the addition of 1 ml ice-cold buffer containing 250 mM sucrose, 100 mM NaCl, and 10 mM Tris · HCl (pH 7.4). The stopped reaction mixture was filtered through a 0.45-µm HAWP filter (Millipore, Bedford, MA) and washed twice with 5 ml of stop solution. Radioactivity retained on the filter was determined using a liquid scintillation counter (LSC-3500, Aloka, Tokyo, Japan). The uptake was normalized with respect to both the amount of CMV and the medium concentration of ligand. The saturation study for the uptake of 5-CH3-H4PteGlu was performed at designated concentrations of medium. ATP-dependent uptake of 5-CH3-H4PteGlu by isolated CMV was obtained by subtracting the uptake in the presence of AMP from that in the presence of ATP. The inhibitory effects of 5-CH3-H4PteGlu, H4PteGlu, and 5,10-CH2-H4PteGlu on the ATP-dependent uptake of [3H]DNP-SG and of DNP-SG on 3H-labeled 5-CH3-H4PteGlu uptake by CMV were measured at designated concentrations in the medium containing 0.5% sodium ascorbate and 3 × 10-3% paraformaldehyde (in the case of 5,10-CH2-H4PteGlu). The uptake of both [3H]DNP-SG and 3H-labeled 5-CH3-H4PteGlu was determined at 2 min after addition of CMV in the medium containing different concentrations of nonradiolabeled compounds.

Estimation of kinetic parameters. The kinetic parameters for the uptake of 5-CH3-H4PteGlu by CMV were estimated from the following equation
<IT>V</IT><SUB>0</SUB> = <IT>V</IT><SUB>max</SUB> × S/(<IT>K</IT><SUB>m</SUB> + S) + P<SUB>diffusion</SUB> × S (1)
where V0 is the initial uptake rate of the substrate (in pmol · min-1 · mg protein-1), S is the substrate concentration in the medium (in µM), Km is the Michaelis-Menten constant (in µM), Vmax is the maximum uptake rate (in pmol · min-1 · mg protein-1), and Pdiffusion is clearance for the nonspecific uptake (in µl · min-1 · mg protein-1). Equation 1 was fitted to the uptake data sets by an iterative nonlinear least-squares method using a MULTI program (41) to obtain estimates of the kinetic parameters. The input data were weighted as the reciprocal of the observed values, and the DampingGauss-Newton method algorithm was used for fitting. The fitted line was converted to the V0/S vs. V0 form (Eadie-Hofstee plot).

To analyze the results of the inhibition studies of the ATP-dependent uptake of DNP-SG and 5-CH3-H4PteGlu by reduced folate derivatives and DNP-SG, respectively, we used Eq. 2. The uptake clearance of substrate (V0/S) as a function of inhibitor concentrations (I) was fitted to Eq. 2 assuming a competitive inhibition to obtain the Ki for the inhibitor.
<IT>V</IT><SUB>0</SUB>/S = <IT>V</IT><SUB>max</SUB>/<IT>K</IT><SUB>m</SUB>(1 + I /<IT>K</IT><SUB>i</SUB>) + S (2)

    RESULTS
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Materials & Methods
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Endogenous levels of reduced folate derivatives in rat plasma and bile. Figure 2 shows the plasma concentrations and biliary excretion time profiles of reduced folate derivatives in SDR and EHBR with bile drainage. 5-CH3-H4PteGlu and H4PteGlu were detected in SDR and EHBR plasma (Fig. 2A). The endogenous plasma concentration of 5-CH3-H4PteGlu in EHBR was 58% of that in SDR, whereas the H4PteGlu concentration was similar for EHBR and SDR. Four hours after the initiation of bile drainage, plasma folate concentrations in SDR were reduced to 69% and 29% of the initial 5-CH3-H4PteGlu and H4PteGlu concentrations, respectively, whereas concentrations in EHBR were unaffected (Fig. 2A).


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Fig. 2.   Time profiles for plasma concentration and biliary excretion of folates in Sprague-Dawley rats (SDR) (A, B) and Eisai hyperbilirubinemic rats (EHBR) (A, C) with bile drainage. Plasma and bile in SDR (open symbols) and EHBR (solid symbols) were collected at the designated time via cannulas inserted into femoral artery and bile duct, respectively. Plasma and bile folates were determined as described in MATERIALS AND METHODS. Values are means ± SE (n = 5). Statistical importance was calculated using Student's t-test. * P < 0.05 vs. time 0. # P < 0.05 vs. SDR. Squares, H4PteGlu; circles, 5-CH3-H4PteGlu; triangles, 10-HCO-H4PteGlu; diamonds, 5,10-CH2-H4PteGlu.

The biliary excretion of reduced folate derivatives in SDR and EHBR was constant for at least up to 4 h (Fig. 2, B and C). The endogenous biliary excretion rate of H4PteGlu, 5-CH3-H4PteGlu, and 5,10-CH2-H4PteGlu in EHBR was 8.2%, 1.9%, and 5.5% of that in SDR (Table 1). 10-Formyltetrahydrofolate (10-HCO-H4PteGlu) was not detected in EHBR bile.

                              
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Table 1.   Endogenous biliary excretion rate in SDR and EHBR with bile drainage

Alteration of folate concentration in plasma and bile after exogenous administration of PteGlu. When PteGlu (1 mg/kg) was given intravenously, the maximal plasma concentration of 5-CH3-H4PteGlu and H4PteGlu increased 2- and 10-fold, respectively, relative to the initial value in SDR and EHBR (Fig. 3A). Increased plasma concentrations returned to endogenous levels at 4 h after administration in SDR. Although the maximal concentration was almost the same for SDR and EHBR, a prolonged elimination half-life was observed in EHBR.


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Fig. 3.   Time profiles for plasma concentration and biliary excretion of folates in SDR (A, B) and EHBR (A, C) after PteGlu (1 mg/kg) intravenous administration. Rats with constant bile drainage were kept for 1 h under anesthesia before intravenous administration of PteGlu (1 mg/kg). Plasma and bile in SDR (open symbols) and EHBR (solid symbols) were collected at the designated time after administration via cannulas inserted into femoral artery and bile duct, respectively. Plasma and bile folates were determined as described in MATERIALS AND METHODS. Values are means ± SE (n = 5). Statistical importance was calculated using Student's t-test. * P < 0.05 vs. time 0. # P < 0.05 vs. SDR. Squares, H4PteGlu; circles, 5-CH3-H4PteGlu; triangles, 10-HCO-H4PteGlu; diamonds, 5,10-CH2-H4PteGlu.

Intravenous administration of PteGlu increased the biliary excretion rate of all reduced folate derivatives four- to sixfold maximally, relative to the endogenous excretion rate in SDR (Fig. 3B). Although the increase in plasma concentration in EHBR was similar to that in SDR (Fig. 3A), a minimal increase in biliary excretion rate was observed in EHBR (Fig. 3C).

Uptake of 3H-labeled 5-CH3-H4PteGlu into CMV. Figure 4 shows the time profiles for the uptake of 3H-labeled 5-CH3-H4PteGlu (0.08 µM) into CMV prepared from SDR and EHBR in the presence of 5 mM ATP or AMP. ATP stimulated the uptake into CMV prepared from SDR but did not stimulate the uptake into CMV prepared from EHBR. The ATP-dependent uptake was linear up to 2 min and did not show any overshoot phenomenon even at 30 min (data not shown).


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Fig. 4.   Time profiles for uptake of 5-CH3-H4PteGlu into the canalicular membrane vesicles (CMV) prepared from SDR and EHBR. CMV (10 µg protein) prepared from SDR (open symbols) and EHBR (solid symbols) were incubated at 37°C in 20 µl medium (10 mM Tris · HCl, 250 mM sucrose, and 10 mM MgCl2, pH 7.4) containing 0.08 µM 3H-labeled 5-CH3-H4PteGlu in the presence of ATP (circles) or AMP (squares) and its regenerating system (5 mM ATP, 10 mM creatine phosphate and 100 µg/ml creatine phosphokinase). Values are means ± SE of triplicate experiments.

Saturation of ATP-dependent uptake of 3H-labeled 5-CH3-H4PteGlu into CMV. To obtain the kinetic parameters (Km, Vmax), we examined the concentration dependence of the ATP-dependent uptake of 3H-labeled 5-CH3-H4PteGlu into CMV, and the data are shown as an Eadie-Hofstee plot (Fig. 5). Nonlinear regression analysis revealed that the uptake can be described by a saturable and a nonsaturable component and yielded a Km of 125 ± 16 µM, Vmax of 272 ± 19 pmol · min-1 · mg protein-1, and a clearance for the nonsaturable component of 0.20 ± 0.02 µl · min-1 · mg protein-1 (Fig. 5).


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Fig. 5.   Eadie-Hofstee plot for the ATP-dependent uptake of 5-CH3-H4PteGlu into CMV. CMV (10 µg protein) prepared from SDR were incubated at 37°C with 1 µM 3H-labeled 5-CH3-H4PteGlu in 20 µl medium (10 mM Tris · HCl, 250 mM sucrose, and 10 mM MgCl2, pH 7.4) containing different concentrations of 5-CH3-H4PteGlu for 5 min, during which linearity is observed in the presence of ATP or AMP and its regenerating system (5 mM ATP, 10 mM creatine phosphate, and 100 µg/ml creatine phosphokinase). The ATP-dependent uptake was calculated by subtracting uptake in the presence of AMP from that of ATP. Values are means ± SE of triplicate experiments.

Inhibitory effect of reduced folate derivatives on uptake of [3H]DNP-SG and vice versa. The inhibitory effect of reduced folate derivatives on the uptake of [3H]DNP-SG (1 µM) and that of DNP-SG on the uptake of 3H-labeled 5-CH3-H4PteGlu (1 µM) into CMV was examined (Fig. 6). As shown in Table 2, Ki values for 5-CH3-H4PteGlu, H4PteGlu, and 5,10-CH2-H4PteGlu for the uptake of DNP-SG by CMV were determined as 121, 359, and 269 µM, respectively. Ki (121 µM) for 5-CH3-H4PteGlu was comparable with its Km (126 µM). DNP-SG could inhibit the uptake of 3H-labeled 5-CH3-H4PteGlu with a Ki value of 34.5 µM, which is similar to its Km value (17.6 µM) (Fig. 6).


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Fig. 6.   Inhibition for the uptake of DNP-SG into CMV by reduced folate derivatives (A) and vice versa (B). A: CMV (10 µg protein) prepared from SDR were incubated at 37°C with 1 µM [3H]DNP-SG in 20 µl medium (10 mM Tris · HCl, 250 mM sucrose, 10 mM MgCl2, 5 mM ATP, and its regenerating system, pH 7.4) containing different concentrations of H4PteGlu (), 5-CH3-H4PteGlu (open circle ), and 5,10-CH2-H4PteGlu (star ) for 2 min, respectively. B: CMV (10 µg protein) prepared from SDR were incubated at 37°C with 1 µM 3H-labeled 5-CH3-H4PteGlu in 20 µl medium (10 mM Tris · HCl, 250 mM sucrose, 10 mM MgCl2, 5 mM ATP, and its regenerating system, pH 7.4) containing different concentrations of DNP-SG for 2 min, respectively. A and B: values are means ± SE of triplicate experiments.

                              
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Table 2.   Kinetic parameters for folate and DNP-SG uptake into CMV

    DISCUSSION
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In the present study, we examined the biliary excretion mechanism of reduced folate derivatives in vivo and in vitro using normal rats (SDR) and mutant rats (EHBR) whose cMOAT is hereditarily deficient.

Continuous bile drainage caused a reduction in the endogenous plasma concentration of 5-CH3-H4PteGlu and H4PteGlu in SDR (Fig. 2A), which is consistent with our previous findings (30). The biliary excretion was not affected as much by bile drainage, in spite of the reduction in plasma folate concentration (Fig. 2B). Because the major form (>90%) of folates in the bile is 5-CH3-H4PteGlu after its intravenous administration in normal rats (30), the source for endogenous reduced folates in the bile may not be 5-CH3-H4PteGlu in the blood. Therefore, another source(s) must be responsible for maintaining the folate levels in the bile; indeed, two possible sources are postulated. Because most of the folates in the liver exist in polyglutamate form (4, 6), their conversion to monoglutamate form may maintain their biliary excretion. The salvage pathway of PteGlu in the blood may be another source for the reduced folate derivatives in the bile (10, 32), since reduced folate concentrations in the plasma and bile were increased after PteGlu administration (Fig. 3, A and B). To elaborate, folates, including PteGlu, are released from dying cells such as senescent erythrocytes, and this process is known as the salvage pathway (7, 10, 16, 33).

In contrast to SDR, the biliary excretion of reduced folate derivatives in EHBR was only 3% of that in SDR (Table 1). Thus the minimal contribution of biliary excretion to the overall elimination of folates from the plasma in EHBR may explain why bile drainage had no effect on plasma concentration (Fig. 2A). In addition, intravenous administration of PteGlu failed to increase the biliary excretion of reduced folate derivatives in EHBR (Fig. 3C), although plasma concentration increased as observed in SDR (Fig. 3A). This holds for endogenous folates as well; i.e., the endogenous concentration of folate derivatives in the plasma was comparable between SDR and EHBR (Fig. 2A), though their levels in the bile were greatly reduced in EHBR. These observations suggest that plasma folate concentration in EHBR is maintained in a different manner from that of SDR. The prolonged elimination half-life in EHBR plasma after administration of PteGlu indicated the lower total body clearances of 5-CH3-H4PteGlu and H4PteGlu compared with those in SDR, which results from the diminishment of biliary excretion (Fig. 3C). Because enterohepatic circulation supplies 5-CH3-H4PteGlu to blood (30) in SDR as well as the salvation pathway described above, it is plausible that the folate supply into blood may be decreased in EHBR compared with SDR. Thus both the lower supply into blood and the lower plasma clearance of folates may compensate each other, which results in the minimum change in endogenous folate concentration in plasma.

The intestinal transport of folates has not been investigated in cMOAT-deficient rats. De Vries et al. (9) perfused the small intestine and found no difference in the transport of alpha -naphthyl beta -D-glucuronide across the brush-border and basolateral membranes between normal and cMOAT-deficient rats. Their results suggest that the contribution of cMOAT to the transport of its substrates in the small intestine is minimal, although a Northern blot indicated the expression of this transporter (15). The expression of transporters responsible for the intestinal uptake of folate has not been examined in cMOAT-deficient rats.

To demonstrate directly the contribution of cMOAT to the biliary excretion of reduced folate derivatives, we examined the uptake of 3H-labeled 5-CH3-H4PteGlu into CMV prepared from SDR and EHBR (Fig. 4). As reported previously from our laboratory (36), the CMV prepared from SDR exhibited ATP-dependent uptake for DNP-SG and TCA, while ATP-dependent uptake of DNP-SG by CMV prepared from EHBR was diminished to approximately one-thirtieth relative to that by CMV from SDR. ATP stimulated the uptake of 3H-labeled 5-CH3-H4PteGlu into the CMV from SDR, while this ATP-dependent uptake was diminished in EHBR (Fig. 4). Overshoot phenomena, however, was not observed in ATP-dependent uptake of 3H-labeled 5-CH3-H4PteGlu into CMV from SDR. Because overshoot phenomena are caused by ATP consumption and the subsequent release of substrates from inside the vesicles (40), it may be possible that low membrane permeability due to high hydrophilicity of 5-CH3-H4PteGlu may prevent its detectable release from the vesicles, even after the consumption of ATP.

Kinetic analyses revealed that Km and Ki values for 5-CH3-H4PteGlu were comparable (Table 2). The Ki value for DNP-SG was also comparable with a Km value reported from this laboratory (18 µM) (24) (Table 2). These results suggest that DNP-SG and 5-CH3-H4PteGlu share an efflux transporter on the bile canalicular membrane. We conclude that 5-CH3-H4PteGlu is a substrate for cMOAT, because, along with in vivo results, 1) DNP-SG is extruded via cMOAT on the bile canalicular membrane (24, 36, 37) and 2) ATP-dependent uptake of 5-CH3-H4PteGlu was diminished in the CMV prepared from EHBR (Fig. 4). Organic anions with a hydrophobic group and two carboxylate groups are liable to higher affinity substrates for cMOAT than compounds without such characteristics. Also, the glutathione moiety is important for maximum affinity for cMOAT. The Km values for the cMOAT of leukotriene C4 (LTC4), glutathione disulfide, beta -estradiol 17-(beta -D-glucuronide), methotrexate, and 5-CH3-H4PteGlu were 0.25, 400, 75, 295, and 126 µM, respectively (1, 13, 22, 39). The Ki values for cMOAT of 5,10-CH2-H4PteGlu and H4PteGlu are 269 and 358 µM. Although both LTC4 and glutathione disulfide have a glutathione moiety, there was a 1,600-fold difference in their Km values. This seems to be due to the fact that LTC4 has a hydrophobic domain in its structure, while glutathione disulfide does not. Reduced folate derivatives are endogenous substrates for cMOAT with comparable Km values with beta -estradiol 17-(beta -D-glucuronide) and methotrexate. However, their affinity for cMOAT is 500- to 1,400-fold lower than that of LTC4, which has a glutathione moiety in its structure.

Cumulative in vivo and in vitro studies revealed the endogenous substrates for cMOAT, which include glucuronides [such as beta -estradiol 17-(beta -D-glucuronide), bilirubin diglucuronide, and the glucuronides of several bile acids], glutathione conjugates (such as oxidized glutathione and LTC4), and sulfates of several bile acids (18, 20, 25, 42). The excretion into bile mediated by cMOAT is one of the inactivation/detoxification pathways for these endogenous substrates. Because reduced folate derivatives are substrates for cMOAT without requiring conjugation, this is the first report of an endogenous substrate for cMOAT without a glutathione, glucuronide, or sulfate moiety in its structure.

In conclusion, cMOAT is responsible for the biliary excretion of reduced folate derivatives. It is possible that this biliary excretion provides an efficient reutilization system of folate together with the uptake by small intestine and may supply reduced folate constantly to intestinal mucosa in which rapid cell division occurs and requires reduced folates as a cofactor for biosynthesis of DNA and RNA.

    ACKNOWLEDGEMENTS

This study was supported in part by grants from the Ministry of Education, Science, Sports, and Culture of Japan, Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation, and Tokyo Biochemical Research Foundation.

    FOOTNOTES

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.

Address for reprint requests: Y. Sugiyama, Graduate School of Pharmaceutical Sciences, Univ. of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.

Received 15 January 1998; accepted in final form 8 June 1998.

    REFERENCES
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Discussion
References

1.   Akerboom, T., M. Inoue, H. Sies, R. Kinne, and I. M. Arias. Biliary transport of glutathione disulfide studied with isolated rat-liver canalicular membrane vesicles. Eur. J. Biochem. 141: 211-215, 1984[Abstract].

2.   Appling, D. R. Compartmentation folate-mediated one-carbon metabolism in eukaryotes. FASEB J. 5: 2645-2651, 1991[Abstract/Free Full Text].

3.   Benkovic, S. J. On the mechanism of action of folate- and biopterin-requiring enzymes. Annu. Rev. Biochem. 49: 227-251, 1980[Medline].

4.   Brody, T., J. E. Watson, and E. L. Stokstad. Folate pentaglutamate and folate hexaglutamate mediated one-carbon metabolism. Biochem. J. 21: 276-282, 1982.

5.   Buchler, M., J. Konig, M. Brom, J. Kartenbeck, H. Spring, T. Horie, and D. Keppler. cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMrp, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats. J. Biol. Chem. 271: 15091-15098, 1996[Abstract/Free Full Text].

6.   Connor, M. J., and J. A. Blair. The identification of the folate conjugates found in rat liver 48 h after the administration of radioactively labeled folate tracers. Biochem. J. 186: 235-242, 1980[Medline].

7.   Cossins, E. A. Folates in biological materials. In: Folate and Pterins, edited by R. L. Blakely, and S. J. Bencovic. New York: Wiley Intersciences, 1984, vol. 1, p. 1-59.

8.   Davis, R. E., and D. J. Nicol. Folic acid. Int. J. Biochem. 20: 133-139, 1988[Medline].

9.   De Vries, M. H., F. A. Redegeld, A. S. Koster, J. Noordhoek, J. G. de Haan, R. P. J. Oude Elferink, and P. L. M. Jansen. Hepatic, intestinal and renal transport of 1-naphthol-beta -D-glucuronide in mutant rats with hereditary-conjugated hyperbilirubinemia. Naunyn Schmiedebergs Arch. Pharmacol. 340: 588-592, 1989[Medline].

10.   Hillman, R. S., C. M. Campbell, and S. E. Steinberg. Role of the enterohepatic cycle in the recovery of folate from senescent red cells. Trans. Assoc. Am. Physicians 95: 237-243, 1982[Medline].

11.   Horne, D. W. Studies on the mechanism of folate transport of 5-methyltetrahydrofolic acid and methotrexate transport in freshly isolated hepatocytes. Biochim. Biophys. Acta 1023: 47-55, 1990[Medline].

12.   Horne, D. W., K. A. Reed, and H. M. Said. Transport of 5-methyltetrahydrofolate in basolateral membrane vesicles of rat liver. Arch. Biochem. Biophys. 298: 121-128, 1992[Medline].

13.   Ishikawa, T., M. Mueller, C. Klunemann, T. Schaub, and D. Keppler. ATP-dependent primary active transport of cysteinyl leukotrienes across the liver canalicular membrane. Role of the ATP-dependent transport system. J. Biol. Chem. 265: 19279-19286, 1990[Abstract/Free Full Text].

14.   Ito, K., H. Suzuki, T. Hirohashi, K. Kume, T. Shimizu, and Y. Sugiyama. Expression of the putative ATP-binding cassette region homologous to that in multidrug resistance associated protein (MRP), is hereditarily defective in Eisai hyperbilirubinemic rats (EHBR). Int. Hepatol. Commun. 4: 292-299, 1996.

15.   Ito, K., H. Suzuki, T. Hirohashi, K. Kume, T. Shimizu, and Y. Sugiyama. Molecular cloning of canalicular multispecific organic anion transporter defective in EHBR. Am. J. Physiol. 272 (Gastrointest. Liver Physiol. 35): G16-G22, 1997[Abstract/Free Full Text].

16.   Iwai, K., P. M. Luttner, and G. Toennies. Blood folic acid studies. VII. Purification and properties of the folic acid precursors of human erythrocytes. J. Biol. Chem. 239: 2365-2369, 1964[Free Full Text].

17.   Kartenbeck, J., U. Leuschner, R. Mayer, and D. Keppler. Absence of the canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin-Johnson syndrome. Hepatology 23: 1061-1066, 1996[Medline].

18.   Keppler, D., and I. M. Arias. Hepatic canalicular membrane. Introduction: transport across the hepatocyte canalicular membrane. FASEB J. 11: 15-18, 1997[Free Full Text].

19.   Kobayashi, L., Y. Sogame, H. Hara, and K. Hayashi. Mechanism of glutathione-S-conjugate transport in canalicular and basolateral rat liver plasma membranes. J. Biol. Chem. 265: 7737-7741, 1990[Abstract/Free Full Text].

20.  Kusuhara, H., H. Suzuki, and Y. Sugiyama. The role of P-glycoprotein and canalicular multispecific organic anion transporter (cMOAT) in the hepatobiliary excretion of drugs. J. Pharm. Sci. In press.

21.   Mason, J. B., R. Shoda, M. Haskell, J. Selhub, and I. H. Rosenberg. Carrier affinity as a mechanism for the pH-dependent of folate transport in the small intestine. Biochim. Biophys. Acta 1024: 331-335, 1990[Medline].

22.   Masuda, M., Y. Iizuka, M. Yamazaki, R. Nishigaki, Y. Kato, K. Niinuma, H. Suzuki, and Y. Sugiyama. Methotrexate is excreted into the bile by canalicular multispecific organic anion transporter in rats. Cancer Res. 57: 3506-3510, 1997[Abstract].

23.   Meier, P. J., and J. L. Boyer. Preparation of basolateral (sinusoidal) and canalicular plasma membrane vesicles for the study of hepatic transport processes. Methods Enzymol. 192: 334-345, 1990.

24.   Niinuma, K., O. Takenaka, T. Horie, K. Kobayashi, Y. Kato, H. Suzuki, and Y. Sugiyama. Kinetic analysis of the primary active transport of conjugated metabolites across the bile canalicular membrane: comparative study of (2,4 dinitrophenyl) glutathione and 4 hydroxy 5,7 dimethyl 2 methylamino 4 (3 pyridylmethyl) benzothiazole glucuronide. J. Pharmacol. Exp. Ther. 282: 866-872, 1997[Abstract/Free Full Text].

25.   Oude-Elferink, R. P. J., D. K. F. Meijer, F. Kuipers, P. L. M. Jansen, A. K. Groen, and G. M. M. Groothuis. Hepatobiliary secretion of organic compounds, molecular mechanisms of membrane transport. Biochim. Biophys. Acta 1241: 215-268, 1995[Medline].

26.   Paulusma, C. C., P. J. Bosma, G. J. Zaman, C. T. Bakker, M. Otter, G. L. Scheffer, R. J. Scheper, P. Borst, and R. P. J. Oude Elferink. Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene. Sci. Am. 271: 1126-1128, 1996.

27.   Selhum, J., G. M. Powell, and I. H. Rosenberg. Intestinal transport of 5-methyltetrahydrofolate. Am. J. Physiol. 246 (Gastrointest. Liver Physiol. 9): G515-G520, 1984[Abstract/Free Full Text].

28.   Shane, B. Folylpolyglutamate synthesis and role in the regulation of one-carbon metabolism. Vitam. Horm. 45: 263-335, 1989[Medline].

29.   Shin, H. C., M. Shimoda, and E. Kokue. Identification of 5,10-methylenetetrahydrofolate in rat bile. J. Chromatogr. Biomed. Appl. 661: 237-244, 1994.

30.   Shin, H. C., M. Shimoda, and E. Kokue. Enterohepatic circulation kinetics of bile-active folate derivatives and folate homeostasis in rats. Am. J. Physiol. 269 (Regulatory Integrative Comp. Physiol. 38): R421-R425, 1995[Abstract/Free Full Text].

31.   Shin, H. C., M. Shimoda, E. Kokue, and Y. Takahashi. Identification of 10-formyltetrahydrofolate, tetrahydrofolate and 5-methyltetrahydrofolate as major reduced folate derivatives in rat bile. J. Chromatogr. Sci. 620: 39-46, 1993.

32.   Steinberg, S. E. Mechanisms of folate homeostasis. Am. J. Physiol. 246 (Gastrointest. Liver Physiol. 9): G319-G324, 1984[Abstract/Free Full Text].

33.   Steinberg, S. E., C. L. Campbell, and R. S. Hillman. The role of the enterohepatic cycle in folate supply to tumor in rats. Br. J. Haematol. 50: 309-316, 1982[Medline].

34.   Stockstad, E. L. R., and J. Koch. Folic acid metabolism. Physiol. Rev. 47: 85-115, 1967.

35.   Strum, W. B., and H. H. Liem. Hepatic uptake, intracellular protein binding, and biliary secretion of 5-methyltetrahydrofolate. Res. Commun. Chem. Pathol. Pharmacol. 30: 493-507, 1980[Medline].

36.   Takenaka, O., T. Horie, K. Kobayashi, H. Suzuki, and Y. Sugiyama. Kinetic analysis of hepatobiliary transport for conjugated metabolites in the perfused liver of mutant rats (EHBR) with hereditary conjugated hyperbilirubinemia. Pharm. Res. 12: 1746-1755, 1995[Medline].

37.   Takenaka, O., T. Horie, H. Suzuki, and Y. Sugiyama. Different biliary excretion systems for glucuronide and sulfate of a model compound; study using Eisai hyperbilirubinemic rats. J. Pharmacol. Exp. Ther. 274: 1362-1369, 1995[Abstract].

38.   Tani, M., and K. Iwai. High-performance liquid chromatographic separation of physiological folate monoglutamate compounds. J. Chromatogr. Sci. 267: 175-181, 1983.

39.   Vore, M., T. Hoffman, and M. Gosland. ATP-dependent transport of beta -estradiol 17-(beta -D-glucuronide) in rat canalicular membrane vesicles. Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G791-G798, 1996[Abstract/Free Full Text].

40.   Watanabe, T., H. Suzuki, Y. Sawada, M. Naito, T. Tsuruo, M. Inaba, M. Hanano, and Y. Sugiyama. Induction of hepatic P-glycoprotein enhances biliary excretion of vincristine in rats. J. Hepatol. 23: 440-448, 1995[Medline].

41.   Yamaoka, K., T. Tanigawara, T. Nakagawa, and T. Uno. A pharmacokinetic analysis program (MULTI) for microcomputer. J. Pharmacobio-Dyn. 4: 879-885, 1981[Medline].

42.   Yamazaki, M., H. Suzuki, and Y. Sugiyama. Recent advances in carrier-mediated hepatic uptake and biliary excretion of xenobiotics. Pharm. Res. 13: 497-513, 1993.


Am J Physiol Gastroint Liver Physiol 275(4):G789-G796
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