1 Graduate School of
Pharmaceutical Sciences, 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
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).
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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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.
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MATERIALS AND METHODS |
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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 × 103% 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 × 103% 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
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(1) |
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(2) |
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RESULTS |
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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).
|
|
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.
|
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).
|
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 · min1 · mg
protein
1, and a clearance
for the nonsaturable component of 0.20 ± 0.02 µl · min
1 · mg
protein
1 (Fig. 5).
|
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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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 -naphthyl
-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,
-estradiol
17-(
-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
-estradiol
17-(
-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 -estradiol
17-(
-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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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