Comparison of intestinal folate carrier clone expressed in IEC-6 cells and in Xenopus oocytes

Chandira K. Kumar, Toai T. Nguyen, Francis B. Gonzales, and Hamid M. Said

Medical Research Service, Veterans Affairs Medical Center, Long Beach, 90822; and School of Medicine, University of California, Irvine, California 92697

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We recently identified a cDNA clone from mouse small intestine, which appears to be involved in folate transport when expressed in Xenopus oocytes. The open reading frame of this clone is identical to that of the reduced folate carrier (RFC) (K. H. Dixon, B. C. Lanpher, J. Chiu, K. Kelley, and K. H. Cowan. J. Biol. Chem. 269: 17-20, 1994). The characteristics of this cDNA clone [previously referred to as intestinal folate carrier 1 (IFC-1)] expressed in Xenopus oocytes, however, were found to be different from the characteristics of folate transport in native small intestinal epithelial cells. To further study these differences, we determined the characteristics of RFC when expressed in an intestinal epithelial cell line, IEC-6, and compared the findings to its characteristics when expressed in Xenopus oocytes. RFC was stably transfected into IEC-6 cells by electroporation; its cRNA was microinjected into Xenopus oocytes. Northern blot analysis of poly(A)+ RNA from IEC-6 cells stably transfected with RFC cDNA (IEC-6/RFC) showed a twofold increase in RFC mRNA levels over controls. Similarly, uptake of folic acid and 5-methyltetrahydrofolate (5-MTHF) by IEC-6/RFC was found to be fourfold higher than uptake in control sublines. This increase in folic acid and 5-MTHF uptake was inhibited by treating IEC-6/RFC cells with cholesterol-modified antisense DNA oligonucleotides. The increase in uptake was found to be mainly mediated through an increase in the maximal velocity (Vmax) of the uptake process [the apparent Michaelis-Menten constant (Km) also changed (range was 0.31 to 1.56 µM), but no specific trend was seen]. In both IEC-6/RFC and control sublines, the uptake of both folic acid and 5-MTHF displayed 1) pH dependency, with a higher uptake at acidic pH 5.5 compared with pH 7.5, and 2) inhibition to the same extent by both reduced and oxidized folate derivatives. These characteristics are very similar to those seen in native intestinal epithelial cells. In contrast, RFC expressed in Xenopus oocytes showed 1) higher uptake at neutral and alkaline pH 7.5 compared with acidic pH 5.5 and 2) higher sensitivity to reduced compared with oxidized folate derivatives. Results of these studies demonstrate that the characteristics of RFC vary depending on the cell system in which it is expressed. Furthermore, the results may suggest the involvement of cell- or tissue-specific posttranslational modification(s) and/or the existence of an auxiliary protein that may account for the differences in the characteristics of the intestinal RFC when expressed in Xenopus oocytes compared with when expressed in intestinal epithelial cells.

folate uptake; reduced folate carrier; intestinal epithelial cells

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

FOLATE IS AN ESSENTIAL micronutrient that acts as a coenzyme in the synthesis of DNA and RNA and the interconversion and degradation of several amino acids (2-5). An adequate supply of folate is, therefore, necessary for normal cellular function, growth, and development. Folate deficiency has been suggested to be one of the most common vitamin deficiencies in the western hemisphere (8, 24). Humans and other mammals cannot synthesize folate and thus rely on exogenous sources to meet their metabolic requirements. Absorption of dietary folate has been shown to occur mainly in the proximal small intestine and involves a specialized, carrier-mediated system (12, 14a, 15, 17-19, 24). Despite the large volume of published work on folate absorption at the tissue, cellular, and subcellular levels, relatively little is known about the molecular identity of the systems involved. We recently isolated two cDNA clones that appear to be involved in folate transport, one from mouse small intestine and the other from human small intestine. (16, 13a) The mouse intestinal cDNA clone was found to have an open reading frame identical to the previously cloned reduced folate carrier (RFC) cDNA from mouse leukemia cell line L1210 (6). The human intestinal clone (hIFC-1) was found to have an open reading frame similar to previously identified cDNA clones of folate transporters from different human tissues (13, 14, 22, 23). Both clones, when expressed in Xenopus oocytes, caused significant and specific increase in the uptake of 5-methyltetrahydrofolate (5-MTHF); the expressed systems were saturable as a function of substrate concentration, with apparent Km similar to that observed with native intestinal preparations (12, 14a, 15, 17-19, 24). However, the pH dependence profile and sensitivity to inhibition by folate structural analogs were different. Although folate uptake in native intestinal preparations was markedly higher at acidic pH levels compared with neutral or alkaline pH levels, cRNA-induced folate uptake in Xenopus oocytes did not show such pH preference. Furthermore, although the folate transport system in native intestinal preparations handles both reduced (e.g., 5-MTHF) and oxidized (e.g., folic acid) folate derivatives with similar affinities, cRNA-induced folate uptake in Xenopus oocytes showed preferential inhibition by reduced over oxidized folate derivatives. To further study these differences, we determined the characteristics of RFC when expressed in an intestinal epithelial cell line (IEC-6 cells) and compared the findings with its characteristics when expressed in Xenopus oocytes.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials

[3,5,7,9-3H]folic acid (sp act 30 Ci/mmol; radiochemical purity > 97%) was purchased from American Radiolabeled Chemicals (St. Louis, MO). (6S)-5-[3',5,7,9-3H]MTHF ([3H]MTHF; sp act 27.1 Ci/mmol; radiochemical purity > 97%) was a generous gift from Dr. Barton Kamen (University of Texas Southwestern Medical Center, Dallas, TX). [3H]biotin (sp act 46.8 Ci/mmol; radiochemical purity > 98%) was obtained from DuPont NEN (Boston, MA). Fetal bovine serum (FBS) and G418-sulfate were obtained from GIBCO BRL (Grand Island, NY). 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), Dulbecco's modified Eagle's medium (DMEM), trypsin, and other cell culture ingredients were from Sigma Chemical (St. Louis, MO). Oocytes were obtained from healthy Xenopus laevis purchased from Xenopus I (Ann Arbor, MI). The plasmid containing the intestinal sodium-dependent glucose transporter-1 (SGLT-1) cDNA was a generous gift from Dr. M. A. Hediger (Harvard Medical School, Boston, MA). All other chemicals were of analytical grade and were purchased from commercial sources.

Antisense oligonucleotides. 5'-Cholesterol-modified DNA oligonucleotides for the study of folate uptake were purchased from Heligen Laboratories (Huntington Beach, CA). RFC-A (antisense), cholesterol-5'-CATGTTGCCCAGGTCCTCAT, is complementary to base pairs -17 to +3 relative to AUG of the mouse folate carrier, RFC. RFC-B (sense), cholesterol-5'-ATGAGGACCTGGGCAACATG, is base pairs -17 to +3 relative to AUG of RFC. RFC-C (scrambled), cholesterol-5'-CTTCGGGTCCTATACATCCG, has the same base composition as RFC-A, but the sequence is scrambled.

Methods

Cell culture. The rat-derived intestinal epithelial cells IEC-6 were obtained from the American Type Culture Collection (Rockville, MD) and were used between passages 18 and 25. IEC-6/WT (wild-type IEC-6; mock electroporated) cells and sublines were grown in 75-cm2 (Costar) plastic flasks in DMEM containing 5% FBS, 100 U/ml penicillin, 50 U/ml streptomycin, and 0.25 µg/ml amphotericin B and were maintained in a 5% CO2-95% air atmosphere at 37°C. As applicable, Geneticin (G418 sulfate) was added to medium at a concentration of 0.7 mg/ml (by activity). Culture medium was changed every 3 days. Cells were subcultured by trypsinization with 0.05% trypsin and 0.6 mM EDTA in Ca2+-free and Mg2+-free phosphate-buffered saline solution and plated onto 12-well plates at a cell density of 5 × 105 cells/well. Uptake of folic acid was studied 2-5 days following confluence. Cell growth was observed by periodic monitoring with an inverted microscope. Cell viability was tested by the trypan blue dye exclusion method and found to be >95%.

Plasmid and sublines. Plasmid pIFC1 (the cDNA insert of this plasmid is actually what we refer to as RFC in this paper) is the product of in vivo excision of the IFC-1 lambda ZAP clone (16); in effect it is derived from plasmid pBK-CMV (Stratagene, La Jolla, CA). Plasmid pIFC1b is pIFC1 from which the 358-base pair Nhe I-Nde I fragment containing the bacterial lac promoter and operator has been deleted. Plasmid pTN223 is pIFC1b from which the 2.3-kilobase EcoR I-Xho I fragment containing the RFC gene has been deleted. IEC-6/WT cells are mock-electroporated IEC-6 cells; IEC-6/RFC cells are IEC-6 cells stably transfected with plasmid pIFC1b; IEC-6/PTN cells are IEC-6 cells stably transfected with plasmid pTN223.

Electroporation. Electroporation was done using Gene Pulser transfection apparatus as per the instruction manual supplied by Bio-Rad Laboratories (Hercules, CA), with minor modifications. IEC-6 cells in log phase (50-75% confluence) were trypsinized and suspended in DMEM with 5% FBS (no antibiotics). The cells were pelleted and washed twice with Hanks' balanced salt solution without Ca2+ or Mg2+ (HBSS) to remove all FBS and antibiotics. Approximately 4-5 × 106 cells were resuspended in FBS-free DMEM without antibiotics and with 10 µg of linearized plasmid DNA in a final volume of 400 µl. Electroporation was done at room temperature (500 µF, 250 V, 0.4-cm cuvette). After electroporation, cells were incubated at room temperature for 10 min, diluted in DMEM with 10% FBS and without antibiotics, and then plated onto a 6- or 12-well plate. The medium was replaced with DMEM containing 10% FBS and antibiotics after cells had adhered to the plate. Afterwards, every 3 days, the medium was replaced with DMEM containing 10% FBS, antibiotics and G418, until the cells reached confluence.

Northern blot analysis. Cell samples were lysed in guanidium thiocyanate solution (5.5 M guanidium thiocyanate, 0.025 M sodium citrate, 0.5% sarkosyl, and 0.5% 2-mercaptoethanol; pH 7) and centrifuged at 3.5 × 103 g for 15 min. Total RNA was isolated by ultracentrifugation of clarified guanidium thiocyanate homogenates through cesium trifluoroacetate or CsCl (7). Poly(A)+ RNA was isolated by oligo(dT) cellulose chromatography and quantitated by spectrophotometry. Two micrograms of poly(A)+ RNA were applied to each lane, electrophoresed in 1% agarose-formaldehyde RNA-denaturing gels, pressure blotted onto Magna 0.22-µm nylon membrane (Micron Separation, West Port, MA), and ultraviolet cross-linked at 120 mJ/cm2 (Stratagene, La Jolla, CA). The RNA blot was then hybridized with radiolabeled probe in 1× prehybridization/hybridization solution (Life Technologies, Grand Island, NY) and 50% deionized formamide at 42°C, washed at high stringency [0.2× saline sodium citrate and 0.1% sodium dodecyl sulfate (SDS) for 30 min at 55°C], and exposed to Kodak phosphor screens at room temperature for 1-3 days. The presence of intact RNA in all the lanes was confirmed by stripping the blots (0.1% SDS, 95°C, 1 min), and rehybridizing them with a rat cyclophilin probe (10). The hybridization signals were quantitated with a phosphorimager (ImageQuant, Storm 860, Molecular Dynamics, Sunnyvale, CA).

Treatment with antisense oligonucleotides. Treatment with antisense oligonucleotides was done as described by Bishop et al. (1). Essentially, cells were plated in DMEM containing 5% FBS with G418 (IEC-6/RFC, IEC-6/PTN) and without G418 (IEC-6/WT) at a cell density of 1-5 × 105 cells/well in 12-well plates. When the cells were 80% confluent (i.e., day 5), medium was replaced with DMEM. After 24 h, cholesterol-modified oligonucleotides (5 µM) in DMEM were added to the cells. Exposure to oligonucleotides continued for a total of 48 h, with a fresh medium change with oligonucleotides every 24 h. An uptake study (with 5-MTHF and folic acid) was done on day 8 (i.e., 2 days postconfluence).

Transport studies in IEC-6 cell sublines. Uptake experiments were performed at 37°C. The incubation buffer was Krebs-Ringer buffer containing (in mM) 123 NaCl, 4.93 KCl, 1.23 MgSO4, 0.85 CaCl2, 5 glucose, 5 glutamine, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 10 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.5 (unless otherwise stated). [3H]folic acid or [3H]MTHF was added to the incubation buffer at the beginning of the experiment, and uptake was terminated after 3 min of incubation (unless otherwise specified) by the addition of 1 ml of ice-cold Krebs-Ringer buffer followed by immediate removal by aspiration. The monolayers were rinsed twice with ice-cold Krebs-Ringer buffer and digested with 1 ml of 1 N NaOH, neutralized by HCl, and then counted for radioactivity in a liquid scintillation counter. Protein contents of cell digests were estimated on parallel wells or on the same wells using a protein assay kit from Bio-Rad based on the method of Lowry et al. (11), with bovine serum albumin as the standard. Data are means ± SE of multiple separate monolayers and are expressed in picomoles or femtomoles per milligram protein per unit time. P values were calculated using the Student's t-test. Kinetic parameters of the carrier-mediated (i.e., DIDS-sensitive) folic acid and 5-MTHF uptake, Vmax and apparent Km, were calculated using a computerized model of the Michaelis-Menten equation as described by Wilkinson (21). Inhibition constants (Ki) for the different folate structural analogs on the uptake of [3H]folic acid and [3H]MTHF were calculated by the method of Dixon et al. (6).

Expression of RFC in Xenopus oocytes and uptake studies. Microinjection of RFC cRNA and uptake experiments were carried out as described earlier (16). Briefly, oocytes (stage V-VI) were defolliculated and microinjected with in vitro transcribed cRNA from RFC cDNA or from SGLT-1 cDNA. Water-injected oocytes were used as a control. Oocytes were incubated for 3-5 days and then used for the uptake study. Uptake experiments were performed at room temperature by incubating 6-8 oocytes for 1 h in 200 µl of Barth's buffer containing (in mM) 88 NaCl, 1 KCl, 0.325 Ca(NO3)2, 0.4 CaCl2, 0.825 MgSO4, 2.4 NaHCO3, 5 MES, and 5 HEPES. [3H]MTHF was added to the incubation buffer at the beginning of the experiments, and uptake was terminated after 1 h of incubation by the addition of 5 ml of ice-cold Barth buffer followed by four successive washes with the same buffer. Cells were transferred individually to scintillation vials and dissolved in 250 µl of 10% SDS, scintillation fluid was added, and radioactivity was counted. Data for oocyte uptake are presented as means ± SE of six to eight oocytes and are expressed as femtomoles per oocyte per hour. P values were calculated using the Student's t-test. Ki values for the different folate analogs and DIDS were determined as described earlier (6).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Characteristics of RFC Expressed in IEC-6 Cells

mRNA expression. Northern blot analysis of poly(A)+ RNAs from IEC-6/WT, IEC-6/PTN, and IEC-6/RFC was done to determine the level of RFC mRNA present in each of the sublines used (Fig. 1). The study was repeated three times using different poly(A)+ RNA preparations. The cyclophilin-normalized IEC-6/WT-to-IEC-6/PTN-to-IEC-6/RFC ratio of RFC hybridizing poly(A)+ RNA is 1:1.06 ± 0.05:1.95 ± 0.14 (means ± SE). This shows that there was an approximately twofold increase in RFC mRNA levels in IEC-6/RFC cells compared with IEC-6/PTN cells and IEC-6/WT cells (P < 0.001).


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Fig. 1.   Northern blot analysis of poly(A)+ RNA isolated from mock- electroporated IEC-6 cells (IEC-6/WT) and from IEC-6 cells stably transfected with plasmids pTN223 (IEC-6/PTN) or pIFC1b (IEC-6/RFC). Poly(A)+ RNA (2 µg) was applied to a denaturing formaldehyde agarose gel as follows: lane 1, IEC-6/WT; lane 2, IEC-6/PTN; lane 3, IEC-6/RFC. Top: blot was hybridized with randomly labeled 2.3-kilobase EcoR I-Xho I RFC fragment (16), washed at high stringency (0.2× saline sodium citrate and 0.1% SDS at 55°C, 15 min), and exposed to a Kodak phosphor screen film at room temperature for 2 days. Hybridization signals were quantitated by Storm 860 system (Molecular Dynamics). Bottom: blot was stripped (0.1% SDS, 95°C, 1 min), hybridized with 0.9-kilobase BamH I rat cyclophilin (CYC) fragment (10), washed at high stringency, exposed for 1 day, and quantitated as above. Arrows, RFC- and cyclophilin-hybridizing bands. Experiment was performed on 3 separate occasions, using different poly(A)+ RNA preparations; shown is a representative scan of phosphor screen of 1 such experiment.

Uptake of folic acid and 5-MTHF by different cell sublines. Uptake of [3H]folic acid (5.4 nM) by IEC-6/WT, IEC-6/PTN, and IEC-6/RFC cell sublines was examined at pH 5.5. Uptake was found to be fourfold higher in IEC-6/RFC cells compared with uptake by IEC-6/PTN cells, which was similar to uptake in the wild-type IEC-6/WT cells (140 ± 1, 36 ± 2, and 34 ± 1 fmol · mg protein-1 · 3 min-1, respectively). Similarly, uptake of the reduced folate derivative [3H]MTHF (6.7 nM) was found to be higher in IEC-6/RFC cells compared with IEC-6/PTN, which was similar to that of IEC-6/WT cells (161 ± 5, 34 ± 2, and 33 ± 3 fmol · mg protein-1 · 3 min-1, respectively). Compared with uptake of folate compounds, uptake of the unrelated [3H]biotin (4.6 nM) was found to be similar in all of the cell sublines tested (13 ± 1, 12 ± 0.1, and 11 ± 0.3 fmol · mg protein-1 · 3 min-1 for IEC-6/WT, IEC-6/PTN, and IEC-6/RFC, respectively).

Effects of RFC antisense oligonucleotides. In this study, we examined the effects of exposing the IEC-6 cells expressing RFC (i.e., IEC-6/RFC cells) to cholesterol-modified antisense, sense, and scrambled RFC DNA oligonucleotides on the uptake of [3H]folic acid (5.4 nM) and [3H]MTHF (6.7 nM). The results showed that exposure of the cells to antisense oligonucleotides caused a significant decrease in folic acid and 5-MTHF uptake, whereas exposing them to sense and scrambled oligonucleotides (i.e., controls) had no significant effects on the induced folate uptake (Table 1).

                              
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Table 1.   Uptake of [3H]folic acid and [3H]MTHF by IEC-6/WT, IEC-6/PTN, and IEC-6/RFC cells treated with oligonucleotides

Uptake of folic acid and 5-MTHF as a function of substrate concentration in the different IEC-6 cell sublines. Uptake of folic acid and 5-MTHF by the different IEC-6 sublines was examined as a function of increasing the substrate concentration in the incubation medium (from 0.0054 to 4 µM). The study was done both in the absence and presence of the organic anion transport inhibitor DIDS (1 mM). The results showed that, in the absence of DIDS, folic acid and 5-MTHF uptake includes a saturable component, whereas the uptake of both substrates was lower and linear in the presence of DIDS. Uptake of folic acid and 5-MTHF by the DIDS-sensitive component was determined by subtracting the uptake in the presence of DIDS from the uptake in its absence. Kinetic parameters of the saturable uptake processes were then determined as described in Methods. As can be seen in Table 2, the Vmax of the uptake process of both substrates was significantly (P < 0.01 for both) higher in IEC-6/RFC cells than in IEC-6/PTN cells and IEC-6/WT cells. In the case of 5-MTHF, there was also a small yet significant (P < 0.01) increase in Vmax in IEC-6/PTN compared with IEC-6/WT (Table 2). With regard to the apparent Km, on the other hand, no clear trend was observed. In the case of folic acid uptake, the apparent Km in IEC-6/PTN and IEC-6/RFC cells were lower than that of IEC-6/WT cells, being significantly lower (P < 0.01) in the case of IEC-6/PTN cells. In the case of 5-MTHF, the apparent Km in both IEC-6/PTN and IEC-6/RFC cells were significantly (P < 0.01) higher than that of IEC-6/WT cells.

                              
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Table 2.   Kinetic parameters of folic acid and 5-MTHF uptake in IEC-6/WT, IEC-6/PTN, and IEC-6/RFC cells

Effects of membrane transport inhibitors. In this experiment, we examined the effects of the anion transport inhibitors DIDS and SITS (both at 1 mM) on the uptake of [3H]MTHF (6.7 nM) by the different cell sublines. The results showed significant inhibition (P < 0.01) of 5-MTHF uptake by both compounds (Table 3). Ki values for DIDS and SITS were then calculated using the Dixon method. At buffer pH 5.5, the Ki values were 0.44, 0.51, and 0.37 mM (for DIDS) and 0.32, 0.87, and 0.54 (for SITS) for IEC-6/WT, IEC-6/PTN, and IEC-6/RFC, respectively. However, at buffer pH 7.5, very little to no inhibition was observed in all the cell sublines tested (data not shown).

                              
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Table 3.   Effect of anion transport inhibitors DIDS and SITS on uptake of [3H]MTHF by IEC-6/WT, IEC-6/PTN, and IEC-6/RFC cells

Effects of incubation buffer pH. In this study, we examined the effect of incubation buffer pH on the uptake of [3H]folic acid (5.4 nM) and [3H]MTHF (6.7 nM) by the different cell sublines. The results showed that uptake of folic acid was significantly higher (P < 0.01) at pH 5.5 than at pH 7.5 (Fig. 2A). Similarly, uptake of 5-MTHF was significantly higher (P < 0.01) at pH 5.5 compared with pH 7.5 (Fig. 2B)


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Fig. 2.   Effect of incubation buffer pH on uptake of folic acid (A) and 5-methyltetrahydrofolate (5-MTHF; B) by IEC-6/WT (square ), IEC-6/PTN (star ), and IEC-6/RFC (open circle ) cells. Cells were incubated for 3 min at 37°C in Krebs-Ringer buffer containing 10 mM MES and 10 mM HEPES of varying pH. [3H]folic acid (5.4 nM; A) or [3H]MTHF (6.7 nM; B) was added to incubation buffer at start of uptake. Data are means ± SE of 6 separate uptake determinations.

Effects of folate structural analogs on the uptake of [3H]folic acid and [3H]MTHF. The effects of different concentrations of folate structural analogs (5-formyltetrahydrofolate, 5-MTHF, and folic acid) on the uptake of [3H]folic acid (5.4 nM) and [3H]MTHF (6.7 nM) by IEC-6/RFC cells were examined in this study. The results showed that there was a concentration-dependent inhibition in the uptake of [3H]folic acid by 5-MTHF and 5-formyltetrahydrofolate with Ki values of 0.75 and 0.33 µM, respectively. Similarly, uptake of [3H]MTHF was inhibited by folic acid and 5-formyltetrahydrofolate with Ki values of 0.55 and 0.41 µM, respectively.

Characteristics of RFC Expressed in Xenopus Oocytes

Characteristics of RFC expressed in Xenopus oocytes have been determined in a previous study (16), and only relevant experiments were repeated here for the purpose of comparison. As a control for oocyte quality, in vitro transcribed SGLT-1 cRNA was microinjected into oocytes and assayed for the uptake of [14C]methyl-alpha -D-glucopyranoside (0.1 µM). Uptake was performed as described in Methods. There was usually a 100- to 200-fold increase in the substrate uptake compared with water-injected control oocytes (e.g., 2.69 ± 1.7 and 336.7 ± 29.0 fmol · oocyte-1 · h-1 for the water- and SGLT-1 cRNA-injected oocytes, respectively). Oocytes from donors demonstrating a good expression of SGLT-1 were used for the studies on the expression of RFC.

Effects of incubation buffer pH. In this study, we examined the effect of incubation buffer pH on the uptake of [3H]MTHF (33.5 nM) by the oocytes expressing RFC. The results showed significantly (P < 0.01) higher uptake at neutral and alkaline pH (pH 7.5) compared with pH 5.5 (19.1 ± 1.0, 17.9 ± 0.9, and 9.7 ± 0.9 fmol · oocyte-1 · h-1 at pH 7.5, 6.5, and 5.5, respectively).

Effects of folate structural analogs and the anion transporter inhibitor DIDS on the uptake of [3H]MTHF. The effect of different concentrations of folate structural analogs (5-formyltetrahydrofolate and folic acid) and that of DIDS on the uptake of [3H]MTHF (33.5 nM) by oocytes expressing RFC were examined in this study. As seen before (16), there was a concentration-dependent inhibition in the uptake of [3H]MTHF by 5-formyltetrahydrofolate and folic acid with Ki values of 2.3 and 40.7 µM, respectively. Similarly, the uptake of [3H]MTHF was inhibited by DIDS with a Ki value of 59 µM.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Our laboratory has recently cloned two folate transporter cDNA from mammalian small intestine: RFC from mouse small intestine (16) and hIFC1 from human small intestine (13a). The open reading frames of the mouse and human intestinal cDNA clones were found to be identical or similar to the folate carrier clones previously isolated by others (6, 13, 14, 22, 23). Both clones induced significant 5-MTHF uptake in Xenopus oocytes compared with water-injected controls. In both cases, the induced uptake was also found to be saturable, with apparent Km values that were similar to those of their corresponding native tissue preparations (1.99 ± 0.32 and 0.71 ± 0.06 µM for RFC and hIFC-1, respectively) (12, 14a, 15, 17-19, 24). However, the pH profile and sensitivity of the induced folate transporter to the inhibitory effects of folate analogs were found to be different from those of the native intestinal tissue preparation. Although uptake of folate in intestinal preparations is pH dependent and is markedly higher at acidic pH (e.g., pH 5.5) compared with neutral or alkaline pH (e.g., pH 7.5), uptake of folate by the induced carrier in Xenopus oocytes following injection of cRNA of RFC and hIFC-1 was not so. Furthermore, although oxidized and reduced folate derivatives cause a similar degree of inhibition in folate uptake by intestinal tissue, the induced folate uptake in Xenopus oocytes was found to be more sensitive to the inhibitory effect of reduced compared with oxidized folate derivatives. Our aim in the present study was to further understand these differences. To do so, we stably transfected an intestinal epithelial cell line (IEC-6) with RFC clone by electroporation and determined the characteristics of the induced folate uptake process and compared our findings with the characteristics of the induced folate uptake following microinjection of RFC cRNA into Xenopus oocytes.

First, we determined the level of expression of RFC mRNA in the different cell sublines by Northern blot analysis and found a twofold increase in RFC mRNA levels in IEC-6/RFC cells compared with IEC-6/PTN and IEC-6/WT cells. We then compared the degree of folic acid uptake by the different cell sublines and found a fourfold increase in folate uptake in IEC-6/RFC compared with IEC-6/PTN and IEC-6/WT cells. Similar results were obtained when 5-MTHF was used as the folate substrate. When the uptake of the unrelated biotin was examined, on the other hand, similar uptake was found in all three cell sublines. This demonstrates the specificity of induction of folate uptake in the IEC-6/RFC cells.

Antisense oligonucleotides can selectively inhibit gene expression in a sequence-specific manner and therefore have the potential to be used as sensitive and specific probes for the induced biological function of proteins (20). Delivering a sufficient intracellular concentration of antisense oligonucleotides can be facilitated by conjugating a cholesterol group to the 5' end of the oligonucleotide (9). When cells were preincubated with cholesterol-modified antisense DNA oligonucleotides, there was a decrease in the uptake of folic acid and 5-MTHF in the case of IEC-6/RFC cells. Sense and scrambled oligonucleotides did not cause any significant decrease in IEC-6/RFC cells, indicating that the increased folate uptake in IEC-6/RFC was specific to RFC sequences.

The increase in folic acid and 5-MTHF uptake observed in IEC-6/RFC cells compared with IEC-6/PTN and IEC-6/WT appeared to be mainly mediated through an increase in the Vmax of folate uptake in the former cell subline. The apparent Km of folic acid and 5-MTHF uptake also changed in the different cell sublines (range was between 0.31 and 1.56 µM); however, no specific trend was seen. These observations, together with the above finding of increased levels of RFC mRNA in IEC-6/RFC cells compared with IEC-6/PTN and IEC-6/WT, suggest that the increase in folate uptake in IEC-6/RFC compared with other cell sublines involves an increase in the number of the folate carriers in IEC-6/RFC cells.

A known characteristic of folate transport in native intestinal preparation and in intestinal cell lines such as IEC-6 is the sensitivity of the folate carrier to the inhibitory effects of the anion transport inhibitors DIDS and SITS (Refs. 15, 19; see also RESULTS). In the present study, the increased folate uptake in IEC-6/RFC was also found to be inhibited by DIDS and SITS. This inhibition was observed at pH 5.5 but was minimal to nonexistent at buffer pH 7.5. The effect of incubation buffer pH on the uptake of folic acid and 5-MTHF by the different cell sublines was also examined. The results showed the uptake of folic acid and 5-MTHF to be significantly higher at acidic pH 5.5 compared with alkaline pH 7.5. This characteristic of folate uptake is similar to that of the native small intestinal epithelial cells (15, 19). It is, however, different from the characteristic of cRNA-induced folate uptake in Xenopus oocytes, where the uptake was higher at neutral and alkaline pH compared with acidic pH (see RESULTS). Furthermore, similar to effects in native intestine, reduced and oxidized folate derivatives caused a similar degree of inhibition in [3H]folic acid and [3H]MTHF uptake by RFC-expressing IEC-6 cells, i.e., IEC-6/RFC. In contrast, the cRNA-induced folate uptake in Xenopus oocytes was more sensitive to the inhibitory effect of reduced compared with oxidized folate derivatives (see RESULTS). These findings demonstrate that the characteristics of RFC vary depending on the cellular system in which it is expressed. Furthermore, the results may suggest the involvement of a cell- or tissue-specific posttranslational modification mechanism(s) that could account for the differences in the characteristics of the expressed RFC in these two different cellular systems. Of course, the possibility that the differences in folate uptake characteristics between intestinal cells expressing RFC and Xenopus oocytes expressing the same carrier is due to existence of an auxiliary protein(s), species differences, and/or a difference in membrane composition cannot be excluded at present.

    ACKNOWLEDGEMENTS

We thank Alvaro Ortiz for excellent technical assistance.

    FOOTNOTES

This study was supported by grants from the Department of Veterans Affairs and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-47203-01.

Address for reprint requests: H. M. Said, UCI and Long Beach VA Medical Program, VA Medical Center-151, Long Beach, CA  90822.

Received 10 January 1997; accepted in final form 24 July 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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