Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 263-8522, Japan
Submitted 30 September 2002 ; accepted in final form 20 March 2003
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ABSTRACT |
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MRP3; intestine; crypt
The intestinal epithelial cell (IEC)-6 is an immortalized epithelial cell line derived from neonatal rat ileum (32). IEC-6 cells have the characteristics of crypt-type intestinal cells but do not exhibit differentiated morphology or gene expression (32) by the lack of transcription factor Drosophila caudal (Cdx2) expression (42). It has been sometimes used as an in vitro intestinal model for the study of MTX transport, especially involving RFC-mediated uptake (33). RFC has been reported to be expressed in IEC-6 as well as small intestine (37), and optimum transport at low pH for MTX and folic acid, typical substrates for RFC, has also been observed both in vitro and in vivo (39). Although drug accumulation depends on the balance of cellular influx and efflux, little is known about the efflux of MTX in this cell line. Rajgopal et al. (33) found that uptake of MTX in RFC-transfected IEC-6 cells was enhanced when energy metabolism was blocked by azide and was reversed when glucose was present. They supposed that endogenous transporter(s), most likely the multidrug resistance-associated protein/ATP binding cassette C (MRP/ABCC) family, was involved in the efflux of MTX from IEC-6 cells, although there is still little evidence about which MRP member(s) is involved in the efflux. The MRP family has at least nine members in human, MRP1-9 (3, 15, 20, 24), all belonging to the ABC transporter superfamily. Over-expression of these transporters, especially MRP1-3 (ABCC1-3), often results in acquired resistance to cytotoxic drugs, such as the commonly used natural product antineoplastic agents, as well as MTX, by lowering the intracellular concentration of these compounds (4). MTX has actually been reported to be a typical nonconjugated substrate for MRP families including MRP1, MRP2, and MRP3 (21).
Fluorescein methotrexate (F-MTX), a fluorescent derivative of MTX, was
synthesized by coupling the carboxyl of the glutamate moiety of MTX through a
diaminopentane spacer to fluorescein isothiocyanate
(9). F-MTX has been used to
study the DHFR and membrane-associated MTX transport protein, by using the
fluorescence moiety in the molecule
(2,
6,
11). Fluorescence labeling of
the transporter in situ could be used for detecting and isolating cells by
flow cytometry and further monitoring localization and conformation of the
transporter within the membrane
(7). Recently, F-MTX transport
by RFC in human leukemia cell lines and leukemiablasts has also been
demonstrated by confocal laser scanning microscopy
(19). We
(30) have also suggested that
the transport of F-MTX in the small intestine is mediated partly by RFC by
using the in vitro everted intestine segments of rats. Recently, F-MTX has
also been suggested to be a fluorescent substrate for energy-driven efflux
transporter, possibly Mrp2, in fish
(10). Moreover, rabbit
Mrp2-mediated transport of
[3H]estradiol-17-D-glucuronide
(E217
G) was inhibited by F-MTX
(43). These reports imply the
interaction between F-MTX and Mrp families.
In the present study, to identify the efflux transporter(s) for MTX and its derivatives in IEC-6, we used F-MTX as a model compound. We were able to demonstrate that 1) F-MTX was a fluorescent substrate of rat Mrp3 and 2) F-MTX is preferentially transported in the apical-to-basolateral direction with the aid of Mrp3 on the basolateral membrane of IEC-6 cells.
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MATERIALS AND METHODS |
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Uptake study. IEC-6 cells were plated onto 24-well plates at a density of 2 x 105 cells/well. Uptake of F-MTX was examined in mature confluent IEC-6 monolayers (2-5 days postconfluence). The IEC-6 monolayers were incubated with 1 µM F-MTX in 37°C Krebs-Ringer buffer (in mM: 123 NaCl, 4.93 KCl, 1.23 MgSO4, 0.85 CaCl2, 5 glucose, 5 glutamine, 10 HEPES, and 10 MES, pH 5.5 at 37°C). Uptake was terminated by the addition of 1 ml ice-cold Krebs-Ringer buffer followed by immediate aspiration, washed three times with the same ice-cold buffer, and digested with 1% SDS. F-MTX was analyzed by HPLC as described previously (7). Briefly, the HPLC conditions were as follows: pump and detector, a 655 liquid chromatograph with a 650-10 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) (excitation and emission wavelengths: 497 and 518 nm, respectively); column, Inertsil ODS (GL Sciences, Tokyo, Japan); mobile phase, 0.1 M NH4OAc-CH3CN (7:3); flow rate, 1.0 ml/min. Protein content of cell digests was determined according to Lowry et al. (27), by using bovine serum albumin as the standard.
Transcellular transport study. IEC-6 cells were seeded in Transwell membrane inserts (24-well format, 3-µm pore size, Becton Dickinson Labware) at a density of 5 x 104 cells/well and cultured for 2 to 5 days postconfluence. Cells were washed with Krebs-Ringer buffer and incubated at 37°C with 1 µM F-MTX in the apical or basolateral compartment with or without 10 µM MK-571. The transported F-MTX in the opposite compartment was measured by HPLC as described above.
Northern blot hybridization. Specific probes for Mrp1, Mrp2, Mrp3, and GAPDH for Northern blot analysis were prepared from the sequence between bases 328 and 740 (413 bp) of rat Mrp1 partial cDNA (GenBank accession no. X96394 [GenBank] ), full-length (4880 bp) rat Mrp2 (16), full-length (5174 bp) rat Mrp3 (12), and bases 523-1003 (481 bp) of rat GAPDH, respectively. Northern blot hybridization was performed according to the method described previously (16). Poly(A)+RNA was separated on 0.7% agarose gel containing 3.7% formaldehyde and transferred to a nylon membrane (Biodyne A; Pall Gelman Laboratory, Ann Arbor, MI) before fixation by baking for 2 h at 80°C. Membranes were prehybridized in buffer containing 4x SSC (1x SSC consists of 150 mM NaCl and 150 mM sodium citrate, pH 7.0), 5x Denhardt's solution (Wako Pure Chemical Industries), 0.2% SDS, 0.1 mg/ml sonicated salmon sperm DNA, and 50% formamide for 2 h at 42°C. Hybridization was performed overnight in the same buffer with 32P-labeled cDNA probes prepared by a random-primed labeling method (Rediprime; Amersham Biosciences, Piscataway, NJ) according to the manufacturer's instructions. The hybridized membrane was washed in 2x SSC and 0.1% SDS at room temperature for 20 min, followed by washing in 2x SSC and 0.1% SDS at 55°C for 20 min and then in 0.1x SSC and 0.1% SDS at 55°C for 20 min. Filters were exposed to Fuji imaging plates (Fuji Photo Film, Kanagawa, Japan) and analyzed by a BAS 2000 imaging analyzer (Fuji Photo Film).
Semiquantitative RT-PCR analysis of Mrp expression in IEC-6 cells. Single-stranded cDNA was synthesized from 1 µg mRNA by using 50 pmol random 9-mer and 50 units RT XL (Takara Bio, Shiga, Japan) in a volume of 20 µl. The tube was incubated in a PC-960G Microplate gradient thermal cycler (Corbett Research) at 30°C for 10 min, 42°C for 30 min, 50°C for 30 min, 60°C for 30 min, and 99°C for 5 min. The resulting cDNA product (1 µl) was subjected to a step-down PCR reaction with rTaq (Takara Bio) and respective primer sets. The tubes (10 µl each) were incubated at 95°C for 1 min to denature the cDNA and primers. The subsequent step-down cycling program was 95°C for 30 s, 74°C for 15 s, 72°C for 45 s involving 3 cycles, 95°C for 30 s, 70°C for 15 s, 72°C for 45 s involving 3 cycles, 95°C for 30 s, 66°C for 15 s, 72°C for 45 s involving 3 cycles, 95°C for 30 s, 74°C for 15 s, 62°C for 45 s involving 3 cycles, 95°C for 30 s, 58°C for 15 s, 72°C for 45 s involving 3 cycles, 95°C for 30 s, 54°C for 15 s, 72°C for 45 s involving 11-30 (26-45 in total) cycles. Primers specific for rat Mrp1 (sense: 5'-CTGATCTCTGGCATATCTGATG-3'; antisense: 5'-GACTGTAGGAGACAGAAGA-3'), rat Mrp2 (sense: 5'-CAGGACTGTTCTGAAAGGTTACAAGCATCG-3'; antisense: 5'-ACATTAGGATTGCACAGATATAGCCAAACC-3'), and rat Mrp3 (sense: 5'-GCACGCTGCGCATGAACCTCG-3'; antisense: 5'-GGCGAGTCCTGCATCTTTGG-3') were used resulting in amplified products of 413, 463, and 449 bp, respectively. Each PCR product was separated on an 8% polyacrylamide gel and stained with ethidium bromide.
Immunofluorescence microscopy. IEC-6 cells were seeded on a cover glass (15 mm in diameter, uncoated) in a 12-well plate at a density of 5 x 105 cells/well and cultured for 2 to 5 days postconfluence. For confocal laser scanning microscopy, cells were washed in PBS and fixed for 10 min in 4% (vol/vol) formaldehyde in PBS at room temperature, followed by permeabilization for 5 min in 1% (vol/vol) Triton X-100 in PBS at room temperature. Cells were incubated with anti-rat Mrp3 rabbit antiserum (1:100) (14) for 1 h at room temperature. Antibody binding was detected with fluorescein-labeled anti-rabbit IgG (1:250) (Vector Laboratories, Burlingame, CA). Nucleic acids were stained by using SYTO61 (1:1,000) (Molecular Probes). Cells were examined by LSM510 confocal laser scanning microscopy (Carl Zeiss, Jena, Germany). The 488-nm wavelength of an argon laser and the 633-nm wavelength of a Helium-Neon laser were used with appropriate combinations for excitation and emission filters.
Production of recombinant baculovirus and viral infection. Recombinant baculovirus was prepared as described previously (17). Sf9 cell suspension was poured into a 150-mm culture dish and allowed to attach for 1 h at 27°C. The medium was changed to fresh medium supplemented with 5% FBS and an appropriate amount of virus carrying rat Mrp3 (1) or green fluorescent protein (GFP; negative control) cDNA and cultured for 60 h. Cells were harvested, and membrane vesicles were subsequently isolated.
Preparation of membrane vesicles and transport study. IEC-6 cells were seeded in 100-mm dishes at a density of 1 x 107 cells/dish and cultured for 2 to 5 days postconfluence. Membrane vesicles were prepared from IEC-6 monolayers, and recombinant Sf9 cells were prepared as reported previously (17). Cell pellets were diluted 40-fold with a hypotonic buffer (1 mM Tris · HCl, 0.1 mM EDTA, 2 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 µg/ml pepstatin, pH 7.4 at 4°C) and stirred gently for 1 h on ice. The cell lysate was centrifuged at 100,000 g for 30 min at 4°C. The resulting pellet was suspended in 10 ml isotonic TS buffer (10 mM Tris · HCl and 250 mM sucrose, pH 7.4 at 4°C) and homogenized with a Dounce B homogenizer (glass/glass, tight pestle, 30 strokes). The crude membrane fraction was layered on the top of a 38% (wt/vol) sucrose solution in 5 mM Tris · HEPES (pH 7.4 at 4°C) and centrifuged at 280,000 g for 45 min. The turbid layer at the interface was collected, diluted to 20 ml with TS buffer, and centrifuged at 100,000 g for 30 min at 4°C. The resulting pellet was suspended in TS buffer. Vesicles were formed by passing the suspension 30 times through a 25-gauge needle by using a 1-ml syringe. Membrane vesicles were finally frozen in liquid nitrogen and stored at -80°C until use. Protein content was measured by the Lowry method (27). The transport study was performed by using the rapid filtration technique described previously (16). Briefly, 16 µl transport buffer (in mM: 10 Tris · HCl, 250 sucrose, 10 MgCl2, 5 ATP or AMP) and an ATP-regenerating system (10 mM creatine phosphate, 100 µg/ml creatine phosphokinase, pH 7.4 at 37°C) containing F-MTX were preincubated at 37°C for 3 min and then rapidly mixed with 4 µl membrane vesicle suspension (10 µg protein). The transport reaction was stopped by the addition of 1 ml ice-cold buffer containing 250 mM sucrose, 0.1 mM NaCl, and 10 mM Tris · HCl (pH 7.4 at 4°C). The stopped reaction mixture was filtered through a 0.45-µm cellulose acetate filter (model HAWP02500; Millipore, Bradford, MA) and then washed twice with 5 ml stop solution. F-MTX was extracted with 0.5 ml of 0.1 M NH4OH and analyzed by HPLC as described in Uptake study. Membrane vesicles from rat red blood cells (RBCs) were prepared as described previously (41).
Western blot analysis. Membrane vesicles from IEC-6 cells and Sf9 cells or RBCs were size fractionated in an 8.5% polyacrylamide slab gel containing 0.1% SDS. Fractionated proteins were transferred to Immobilon Transfer Membrane (Millipore) by electroblotting. Blotted membranes were blocked with Tris-buffered saline containing 0.05% Tween 20 and 3% BSA for 1 h at room temperature and probed for 1 h at room temperature with anti-rat Mrp3 rabbit antiserum (1:1,000) or anti-MRP1 monoclonal antibody (MRPr1, 1:200; Kamiya Biomed, Seattle, WA) diluted with Tris-buffered saline containing 0.05% Tween 20 and 0.1% BSA. The membranes were then allowed to bind donkey anti-mouse (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-rabbit IgG conjugated with horseradish peroxidase (Amersham Biosciences, Piscataway, NJ) (1:3,000) at room temperature for 1 h and analyzed by using an enhanced chemiluminescence kit (Amersham Biosciences).
Data analysis. Uptake rates were fitted to the Michaelis-Menten equation by using a nonlinear least-squares program, MULTI (44), to calculate the kinetic parameters.
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RESULTS |
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Transcellular transport of F-MTX by IEC-6 monolayers. To obtain
insight into the functional localization of Mrp-type transporter(s) in IEC-6
cells, the transcellular transport experiment was performed. IEC-6 cells were
grown on transwell membrane inserts and incubated with F-MTX at a
concentration of 1 µM in the apical or basolateral compartments with or
without 10 µM MK-571. As shown, transport of F-MTX in the
apical-to-basolateral direction was significantly higher than that in the
basolateral-to-apical direction (Fig.
2). The apical-to-basolateral transport of F-MTX was significantly
reduced in the presence of 10 µM MK-571, whereas the basolateral-to-apical
transport of F-MTX was slightly but significantly enhanced in the presence of
10 µM MK-571. These results indicate that the transporter, likely a member
of the Mrp family, localized on the basolateral membrane of IEC-6 monolayers
is responsible for F-MTX efflux and is blocked by MK-571. Transport of F-MTX
from the apical to the basolateral compartment is still twice as high as
transport from basolateral to the apical compartment in the presence of MK-571
(Fig. 2). One of the possible
reasons is the ability of inefficient concentration of MK-571 (10 µM) to
completely block the Mrp3-mediated transport (see
Table 1). However, MK-571 is
usually used at 10 µM because of its cytotoxicity at higher
concentration.
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Expression of Mrp1, Mrp2, and Mrp3 in IEC-6 cells. Until recently, three members of the Mrp family, including Mrp1, Mrp2, and Mrp3, were reported likely to accept MTX as a substrate (21). Among them, Mrp2 was also reported to accept F-MTX as a substrate (10, 43). To examine the expression of Mrp1, Mrp2, and Mrp3 in IEC-6 cells, Northern blot analysis was performed. As shown in Fig. 3A, an Mrp1 and Mrp3 transcript was detectable in IEC-6 cells. In contrast, no Mrp2-specific bands were obtained in IEC-6 cells. Furthermore, Mrp3 expression seems to be dominant in IEC-6 cells as assessed by semiquantitative RT-PCR (Fig. 3B). The expression of Mrp3 was readily detectable after 26 cycles, whereas Mrp1 was detected to an equal extent only after 42 cycles. Mrp2 was not detected even after 40 cycles (data not shown), which was consistent with the data obtained by Northern blot analysis.
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Immunolocalization of Mrp3 protein in IEC-6 cells. The localization of Mrp3 protein was determined in IEC-6 monolayers with anti-Mrp3 antiserum by using confocal laser scanning microscopy. Mrp3 was predominantly found to be localized on the basolateral membrane domain of IEC-6 monolayers (Fig. 4B). No fluorescence was observed in IEC-6 monolayers when primary antibody was omitted (Fig. 4A). Basolateral localization of Mrp3 was consistent with that found in vitro by using the canine kidney epithelial cell line Madin-Darby canine kidney transfected with human MRP3 (23) as well as under in vivo conditions present in the rat intestinal epithelium (36). Mrp1 was not detected under the same culture condition in IEC-6 cells.
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Expression of Mrp3 protein in IEC-6 cells. The expression of Mrp3 protein in IEC-6 cell membrane vesicles was also confirmed by Western blot analysis. A 190-kDa band was detected with anti-Mrp3 antiserum and was consistent with the size reported previously in liver and intestine (36). As positive and negative controls, membrane vesicles from Mrp3-expressing (Mrp3-Sf9) and GFP-expressing (GFP-Sf9) Sf9 cells were loaded (6 µg/lane), respectively. A 175-kDa band was detected in Mrp3-Sf9 but not in GFP-SF9 (Fig. 5A) as reported previously (1). The difference in the molecular weight between IEC-6 cells and Sf9 cells may be accounted for by the lower degree of sugar modification in insect cells as reported for rabbit Mrp2 expressed in Sf9 cells (43). Densitometric analysis revealed a 55-fold higher expression of Mrp3 protein in Mrp3-Sf9 compared with membrane vesicles from IEC-6 cells. Expression of Mrp1 protein (190 kDa) in IEC-6 cells was only a tracer level compared with that in RBCs (Fig. 5B). The intense bands detected around 70 kDa in IEC-6 cells (Fig. 5B) are unknown proteins cross-reactive to the secondary antibody because they were still detected in the absence of primary antibody (data not shown).
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Transport of F-MTX by Mrp3-Sf9 and membrane vesicles from IEC-6
cells. To further characterize the F-MTX efflux directly, membrane
vesicles were isolated from IEC-6 monolayers. The time-dependent uptake of
F-MTX into membrane vesicles was significantly stimulated fourfold in the
presence of ATP compared with that in the presence of AMP
(Fig. 6A). In
addition, there was clear Mrp3 and ATP dependence in the uptake of F-MTX into
Mrp3-Sf9, indicating that F-MTX is a good substrate for rat Mrp3
(Fig. 6B). No
endogenous ATP-dependent uptake was observed in control GFP-Sf9
(Fig. 6B). To compare
the transport characteristics observed in IEC-6 cells and Mrp3 expressed in
Sf9 cells, the concentration dependence and inhibitor sensitivity were
examined. As shown in Fig. 7,
the ATP-dependent transport was saturable with similar Km
values of 11.0 ± 1.8 and 4.5 ± 1.1 µM for IEC-6 and Mrp3-Sf9,
respectively, whereas the ATP-independent transport was not saturated 40
µM. The maximum uptake rate (Vmax) values obtained were 96.6
± 9.7 pmol · mg-1 · 15 min-1 and
1,226 ± 155 pmol · mg-1 · 15 min-1
for IEC-6 and Mrp3-Sf9, respectively. The large difference in the
Vmax values may be due to the protein expression level and other
factors as described in DISCUSSION. The LTD4-receptor
antagonist MK-571, a potent inhibitor of human MRP1 (Ki
0.6 µM) (25) and rat
Mrp2 (Ki
2.6 µM)
(16), E217
G
(Km
67 µM)
(13,
14), and TC
(Km
3.0 µM)
(13,
14), both typical substrates
of rat Mrp3, all inhibited the ATP-dependent uptake of F-MTX in IEC-6 membrane
vesicles and Mrp3-Sf9 with comparable sensitivity
(Table 1).
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DISCUSSION |
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The functional characteristics of F-MTX transport into IEC-6 membrane
vesicles were also similar to those into Mrp3-Sf9 membrane vesicles, implying
the involvement of Mrp3 in the transport of F-MTX in IEC-6 cells. The
Km values (11 vs. 4.5 µM) were similar in both membrane
vesicles. Hirohashi et al.
(13) have reported that the
IC50 value of MTX on the ATP-dependent uptake of
E217G (100 nM) into rat Mrp3-expressing vesicles is between
50 and 100 µM, which is nearly equal to the Ki value
under tracer experiment conditions. Because MTX is also a transport substrate
of Mrp3 (1,
13), the
Km value is also supposed to be between 50 and 100 µM.
The affinity of MTX for human MRP3 was as low as 776 µM
(45). Thus our data clearly
demonstrate that F-MTX is a substrate with much higher affinity for Mrp3
compared with MTX itself (13,
45). Although information
about structure-recognition relationships is limited, the addition of the FITC
moiety with a monocarboxylate and a degree of hydrophobicity may be favorable
as far as rat Mrp3 recognition is concerned. A similar phenomenon has been
observed in rabbit Mrp2 (43).
The ATP-dependent uptake of [3H]E217
G by rabbit
Mrp2 was reduced to 94 and 55% in the presence of fluorescein (1 mM) and MTX
(1 mM), respectively, whereas it was reduced to 22% in the presence of as
little as 100 µM F-MTX
(43).
Inhibitor sensitivity of F-MTX was similar but not completely identical for
IEC-6 and Mrp3-Sf9. For example, 75 µM E217G and 200 µM
TC inhibited the ATP-dependent uptake of F-MTX into Mrp3-Sf9 vesicles by 80%
but only inhibited it into IEC-6 vesicles by 60%. These differences may be
derived from a contribution by other ATP-dependent transporter(s) in IEC-6
cells whose inhibitor sensitivity is lower than that of Mrp3. As far as human
MRP1 is concerned, the Km value for E217
G
is reported to be 2.5 µM
(26), a rather higher affinity
than that of rat Mrp3 (67 µM)
(13). Our data showing that 75
µM E217
G was not enough to inhibit the ATP-dependent
uptake of F-MTX into IEC-6 vesicles (Table
1) supports the hypothesis that Mrp1 is a minor and Mrp3 a major
efflux pump for F-MTX in IEC-6 cells given that rat Mrp1 has a similar
affinity to human MRP1 for E217
G. Although the expression of
Mrp3 protein in Mrp3-Sf9 was 55-fold higher than that in IEC-6 vesicles, the
Vmax value of the transport in Mrp3-Sf9 was only 12-fold higher
than that in IEC-6 vesicles. Such a difference may be due to the following:
1) the contribution of other transport-er(s) may not be negligible,
2) the transport activity is reduced by immature sugar modification
(Fig. 5A) or the
different membrane environment in insect cells
(28), and 3) the
difference in the content of inside-out vesicles between Mrp3-Sf9 and IEC-6
vesicles may affect the net transport activity. Because ATP is membrane
impermeable and the ATP-hydrolysis domain is located in the cytosolic region
of the Mrp molecule, only inside-out vesicles but not right-side-out vesicles
are involved in the ATP-dependent transport. Although we do not know which is
the case, the above factors may account for such a discrepancy.
In conclusion, F-MTX is also a good substrate for rat Mrp3 and will be of great use for the study of Mrp transporters. The vectorial transport of F-MTX from the apical to the basolateral compartment in IEC-6 is mainly mediated by Mrp3 expressed on the basolateral membrane.
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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