Biosci. Biotechnol. Biochem., 72 (9), 2277–2284, 2008

Placental Transport during Pregnancy

Satoru YASUDA, Satoko HASUI, Chiaki YAMAMOTO, Chihiro YOSHIOKA, y Masaki KOBAYASHI, Shirou ITAGAKI, Takeshi HIRANO, and Ken ISEKI

Laboratory of Clinical Pharmaceutics and Therapeutics, Division of Pharmascience, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo, Nishi 6-chome, Kita-ku, Sapporo 060-0812, Japan

Received February 22, 2008; Accepted May 22, 2008; Online Publication, September 7, 2008 [doi:10.1271/bbb.80112]

The aim of this study was to elucidate the mechanism growth,5) and that deficiencies in impair fetal of folate transport in the placenta over the course of development.6) It has been reported that folate defi- pregnancy. We found that folate receptor (FR ) and ciency during pregnancy increases the risk of the reduced folate carrier (RFC) localized on the apical side development of neural-tube defects in the infant.7,8) of human placental villi. Since folate binding to Although folates are important vitamins, mammals placental brush-border membrane vesicles (BBMVs) cannot synthesize them by themselves. Hence, intake of was strongly inhibited by phosphatidylinositol-specific folates from dietary sources is essential, and systems phospholipase C (PI-PLC) treatment, it is possible that for the absorption of folates from the intestine play an FR , a glycosyl phosphatidylinositol linked glycopro- important role in this process. These systems have been tein, is a candidate for folate uptake from maternal described, and the mechanisms of carrier-mediated blood to the placenta. Moreover, additional inhibitory absorption of folates have been identified.9,10) Folate effects of thiamine pyrophosphate (TPP) and hemin on receptor (FR), a glycosylphosphatidylinositol linked folate uptake after PI-PLC treatment suggested that not glycoprotein, utilizes high-affinity binding of folates at only FR but also RFC and heme carrier 1 the membrane surface and mediates unidirectional flux (HCP1) are involved in the folate transport mechanism following internalization of the receptor-folate com- in the human placenta. It was also found that accumu- plex, in the same manner as that of many other ligand- lation of folate after intravenous injection increased receptor processes.11,12) Reduced folate carrier (RFC/ with the progress of gestation in the rat placenta and the SLC19A1) utilizes binding to a transporter at the fetus. Furthermore, increases in the expression levels of membrane surface to mediate internalization through mRNA of rFR , rRFC, and rHCP1 in the rat placenta membranes, and this process is capable of mediating during pregnancy were observed. These findings suggest bidirectional flux.13,14) It has been found that these that FR , RFC, and HCP1 are important carriers of carriers are pH-sensitive and are expressed not only in folate in the placenta during pregnancy. The results of the intestine but also in other organs, including the this study suggest that increases in the expression levels placenta.15–17) Furthermore, it is possible that intra- of FR , RFC, and HCP1 in the placenta play an cellular accumulation of folates reflects the operation important role in the response to increased need for of outward transport mediated by members of the folate for the placenta and fetus during development family of ATP-binding cassette transporters, such as with the progress of gestation. MRPs and breast cancer resistance protein (BCRP/ ABCG2).18) Key words: folate; placenta; folate receptor ; reduced It is known that the placenta is an organ that folate carrier; heme carrier protein 1 develops in a short period of time, and that its functions change with the progress of gestation. It has been The placenta is viewed as a protective barrier and a reported that expression levels of transporters that con- site for nutrient and waste exchange between mother and tribute to the transfer of nutrients and waste between fetus. It has been found that transporters play an the mother and the fetus change during pregnancy,19–21) important role in this nutrient and waste exchange in but the expression levels of carriers of folates in the the placenta.1–3) placenta over the course of pregnancy have not been Folates are essential nutrients required for the provi- investigated. sion of one-carbon moieties in biosynthetic processes.4) It is possible that heme carrier protein 1 (HCP1) is a It is known that they are essential for cell division and proton-coupled folate carrier in the intestine.22) Since

y To whom correspondence should be addressed. Tel/Fax: +81-11-706-3770; E-mail: [email protected] 2278 S. YASUDA et al. folate uptake in placental cell lines has been found to be Preparation of BBMVs. BBMVs were prepared from pH-sensitive, it is conceivable that HCP1 also contrib- the human term placentas by the magnesium precipita- utes to folate transfer from mother to fetus. We have also tion method, with modifications.24) All steps were reported that HCP1 is a candidate folate carrier in the performed on ice or at 4 C. The placenta was washed placental cell line BeWo,23) but the function of HCP1 in with ice-cold saline. Then placental villi were cut as the human placenta has not determined. Thus, the tissue samples. The tissue samples (about 100 g wet mechanism by which folate is transported from mother weight) were stirred in 300 ml of ice-cold experimental to fetus, i.e., intake from the apical side of the placenta buffer (100 mMD-mannitol, 100 mM KCl, 20 mM villi, has not been clarified and investigation of this is HEPES/Tris, pH 7.4). After filtration with gauze, the necessary to identify the physiological system that samples were homogenized with a Waring blender at responds to folate demand in fetal development during 9,000 rpm for 4 min. MgCl2 solution (0.5 M) was added pregnancy. to a final concentration of 10 mM, and the homogenate The aim of this study was to determine the mecha- was allowed to stand for 20 min. The homogenate was nism of folate transport in the placenta over the course centrifuged at 4;300 g for 15 min, and then the super- of pregnancy. We investigated the folate transport natant was recentrifuged at 34;700 g for 30 min. The mechanism using human term placental brush-border resulting pellet was resuspended in 20 ml of experi- membrane vesicles (BBMVs). We also investigated mental buffer and homogenized in a glass/Teflon changes in folate accumulation in the placenta and fetus Dounce-type homogenizer at 10 strokes. After a final using pregnant rats at various days of gestation. centrifugation at 34;700 g for 30 min, the brush- border membranes were suspended in a buffer contain- Materials and Methods ing 100 mMD-mannitol, 100 mM KCl, and 20 mM MES/ Tris (pH 6.0), and homogenized with a Dounce-type Chemicals. [30,50,7,9-3H]Folic acid (23.0 Ci/mmol) homogenizer at 10 strokes. Then the samples were was purchased from Amersham Biosciences (Piscat- homogenized with a 25G syringe 10 times. When away, NJ). Folic acid (Folate) (pteroylglutamic acid, phosphatidylinositol-specific phospholipase C (PI-PLC) C19H19N7O6, Mw: 441.4) and folinic acid calcium salt treatment was needed, vesicles were incubated with (C20H21CaN7O7, Mw: 511.5) was purchased from Wako PI-PLC at 37 C for 30 min before the uptake experi- (Osaka, Japan) and Sigma-Aldrich (St. Louis, MO). All ment. The level of alkaline phosphatase (a marker other reagents were of the highest grade available and enzyme of the brush-border membrane) activity of the were used without further purification. brush-border membrane was more than 16-fold higher than that of the initial homogenate. Animals. Pregnant Wistar rats were obtained from Slc (Hamamatsu, Japan). The experimental protocols were Study of uptake by BBMVs. The uptake of [3H]-folic reviewed and approved by the Hokkaido University acid (0.5 mCi/ml) into brush-border membrane vesicles Animal Care Committee, in accordance with the ‘‘Guide was determined by the rapid filtration technique de- for the Care and Use of Laboratory Animals.’’ scribed previously.25) Twenty ml of membrane vesicle (8 mg protein/ml) suspension was added to 100 mlof Tissue collection. Human placental tissue (39–40 substrate mixture kept at 37 C. At the end of the incuba- weeks) was obtained, with informed consent and ethical tion period, uptake was stopped by diluting the incuba- committee approval, from patients admitted to Sapporo tion medium with 5 ml of ice-cold stop buffer (150 mM Maternity Women’s Hospital, Sapporo. Placentas were KCl, 20 mM HEPES/Tris, pH 7.5). The mixture was collected at term, following caesarean section, from immediately filtered through a Millipore filter (Millipore, women who had uncomplicated pregnancies. Billerica, MA; 0.45 mm in pore size, 2.5 cm in diameter; HAWP). The filter was rinsed with 5 ml of the same Immunohistochemistry. Tissue samples were obtained buffer. The radioactivity of the substrate trapped on from human placentas. They were snap-frozen in the filter was determined by scintillation spectrometry Optimal Cutting Temperature (OCT) compound and (1600TR, Packard Instruments, Meriden, CT). used for cryosections. Immunohistochemical staining was performed on paraformaldehyde-fixed and paraffin- Administration of folic acid and collection of placenta embedded sections. Tissue samples were incubated with and fetus. [3H]-Folic acid was administered in saline a monoclonal antibody of FR (Alexis, Lausen, Switzer- at a concentration of 282.5 ng/kg of body weight (13.5 land) or RFC (Alpha Diagnostic International, San Ci/ml) by intravenous injection from the jugular vein Antonio, TX) (dilution, 1:100). After washing with PBS, under ether anesthesia. The experimental rats were the samples were incubated with an FITC- or PE- beheaded 3 h after administration of folate, and samples conjugated secondary antibody at a dilution of 1:100. were excised at that time. Tissue samples were collected The nuclei were stained with DAPI. The samples were from dams on the 14th gestation day (gd), the 17th gd, then visualized using a confocal microscope (Zeiss and the 20th gd. About 10 placentas were obtained from LSM-510; Carl Zeiss, Thornwood, NY). each dam. Tissues were removed rapidly, washed, Folate Transport in Placenta 2279 weighed, and homogenized in 3 N NaOH using a Temecula, CA) (dilution, 1:500) for 1 h at room temper- polytoron homogenizer, and then shaken at 120 rpm ature, and then washed with PBS/T (3 10 min). They overnight. The tissue of the lysate was then sampled were subsequently incubated for 1 h at room temperature for scintillation spectrometry (1600TR Packard, Instru- with horseradish peroxidase-conjugated donkey anti- ments, Meriden, CT). goat secondary antibody or horseradish peroxidase- conjugated goat anti-mouse secondary antibody (Santa Quantitative real-time PCR. Total RNA was prepared Cruz Biotechnology, Santa Cruz, CA) at dilutions, of from rat placenta tissue using Isogen (Nippon , 1:1,000 and 1:4,000 respectively, and then washed with Tokyo). Single-strand cDNA was made from 2 mg of total PBS/T (3 10 min). The bands were visualized by RNA by reverse transcription (RT) using an Omniscript enhanced chemiluminescence according to the instruc- RT Kit (Qiagen, Tokyo). Quantitative real-time PCR was tions of the manufacturer (Amersham Biosciences). performed using an ABI PRISM 7700 Sequence Detector (Applied Biosystems, Foster City, CA) with 2SYBR Statistical analyses. Pairs of student’s t-test was used Green PCR Master Mix (Applied Biosystems), following to determine the significance of differences between the manufacturer’s protocol. PCR was performed using the two group means. Comparisons between more than rFR-, rRFC-, rHCP1-, and rGAPDH-specific primers two groups were made by the post-hoc Scheffe test. through 50 cycles of 94 C for 15 s, 50 C for 30 s, and Statistical significance was defined as P < 0:05. 72 C for 30 s. Primers specific to rFR, rRFC, rHCP1, and rGAPDH were designed on the basis of sequences in Results the GenBankTM database (accession nos. NM 133527, U38180, NM 001013969, and AF106860 respectively). Expression of FR and RFC in human placental villi The sequences of the specific primers were as follows: In the first part of the study, we examined the sense sequence 50-CAA CAC AAG CCA GGA AGA expression of FR and RFC, strong candidates for CA-30 and antisense sequence 50-CAA GTT CGG placental folate carriers, in human term placentas by GGA ACA CTC AT-30 for rFR, sense sequence 50- immunohistochemistry (Fig. 1). Human placentas were CAT GCT AAG CGA ACT GGT GA-30 and antisense stained with each specific antibody for FR and RFC sequence 50-TTT TCC ACA GGA CAT GGA CA-30 for with DAPI for nuclear staining. Figure 1 shows ex- rRFC, sense sequence 50-CCT TCT GGG AGA TTT pression of FR on the apical side of the placenta villi CAA CG-30 and antisense sequence 50-CCA GAA AGG (green). Expression of RFC was also observed on the GTT GGC ATA AC-30 for rHCP1, sense sequence 50- apical side (red, closed arrow), but some of them ATG GGA AGC TGG TCA TCA AC-30 and antisense appeared to localize on the basal side (open arrow). sequence 50-GTG GTT CAC ACC CAT CAC AA-30 for rGAPDH. The expression level of each mRNA was Uptake of [3H]-folate into human placenta BBMVs normalized to the amplified the level of GAPDH mRNA, We investigated the folate transport mechanism using the internal reference gene. human placental BBMVs. Since it has been reported that folate transport increases under acidic conditions, we Western blot analysis. Total protein extracts were evaluated folate uptake under acidic conditions, (pH 6.0) prepared from placenta tissue homogenates. Tissue and neutral conditions, (pH 7.4). The amount of folate homogenates or cells were suspended in lysis buffer uptake into or binding to BBMVs was more than 10-fold containing 1.0% Triton X-100, 0.1% SDS, and 4.5 M greater at pH 6.0 than at pH 7.4 (data not shown) and urea. The suspension was left to stand for 5 min and hence we evaluated the folate transport mechanism using sonicated for 15 min at 4 C. Then it was centrifuged at a buffer at pH 6.0. Folate uptake into or binding to 12,000 rpm for 15 min at 4 C, and the protein concen- BBMVs was very rapid until 2 min. Moreover, the uptake tration in the clear supernatant was determined by the or binding of [3H]-folate equilibrated at 30 min (Fig. 2). method of Lowry et al.26) The samples were denatured at Hence we evaluated the folate transport mechanism at 85 C for 3 min in loading buffer containing 50 mM Tris– 1 min for uptake and for binding in further investigation. HCl, 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, We investigated the effects of structural analogs 0.002% BPB, and 3.6 M urea, and separated on 4.5% of folate on uptake into and on binding to BBMVs stacking and 10% SDS polyacrylamide gels. The (Table 1). Folate structural analogs inhibited about 90– were transferred electrophoretically onto nitro- 100% of folate uptake and binding at concentrations on cellulose membranes (Bio-Rad Hercules, CA) at 15 V the order of micro molars. These results suggest that for 90 min. The membranes were blocked with PBS uptake into and binding to BBMVs at 1 min is carrier- containing 0.05% Tween 20 (PBS/T) and 10% non-fat dependent. dry milk for 1 h at room temperature. After they were Next we investigated the contribution of FR to folate washed with PBS/T, the membranes were incubated binding to BBMVs. PI-PLC can inhibit the functions of with goat anti-FR monoclonal antibody (Santa Cruz glycosylphosphatidylinositol (GPI)-linked cell surface Biotechnology, Santa Cruz, CA) (dilution, 1:200) or proteins,27) such as FR. As Fig. 3 shows, PI-PLC mouse anti-actin monoclonal antibody (Chemicon, treatment decreased [3H]-folate binding to about 40% of 2280 S. YASUDA et al. A C E

B D F

20 µm

Fig. 1. Expression of FR and RFC in Human Placenta Villi. Expression of FR (A) and RFC (B) was determined using antibodies against FR (A, E, green) and RFC (B, F, red) respectively. Nuclei were stained with DAPI (C, D). (E) and (F) show merged images of (A) and (C) and of (B) and (D) respectively. Data shown are typical results of three independent experiments.

12 10

10 8 8 6 6 4 4

Uptake (pmol/mg protein) 2 2 Uptake (pmol/mg protein)

0 10 20 30 0 0.05 0.10 0.15 0.20 Time (min) PI-PLC concn. (units/ml) Fig. 2. Time Course of Folate Uptake into Human Placental BBMVs. [3H]-folate (22 nM) uptake was measured in an experimental Fig. 3. Effect of PI-PLC Treatment on Folate Binding. buffer with a pH of 6.0. Each point represents the mean S.D. of BBMVs were treated with PI-PLC at concentrations of 0– 3 four determinations. 0.2 units/ml, as described in ‘‘Materials and Methods.’’ [ H]-folate (22 nM) uptake was measured in an experimental buffer with a pH of 6.0 for 30 min. Each point represents the mean S.D. of four determinations.

Table 1. Effects of Structural Analogs on Folate Uptake the level without PI-PLC treatment at 30 min, suggesting Inhibitor Concn. (mM) Percent of control that about 60% of [3H]-folate bound to FR. We also No addition 100 8:9 investigated the amount of folate binding to BBMVs by Pteroic acid 5 12:7 0:4 shrinking the vesicles under a high osmolarity buffer Folinic acid 50 9:6 1:1 condition, and the results suggested that about 70% of 50 1:6 2:0 [3H]-folate bound to BBMVs (data not shown). These Effects of structural analogs on folate uptake. [3H]-folate (22 nM) uptake was findings suggest that PI-PLC inhibited almost all of the measured with and without an inhibitor in a buffer at pH 6.0 for 1 min. Each value is the mean S.D. for three different samples. , significantly binding of folate to BBMVs. different from no addition at p < 0:05, by Student’s unpaired t-test. We also investigated the inhibitory effect of PI-PLC Folate Transport in Placenta 2281

7 * 3 6 5 * 2 * 4 † 3 1 2 1 Uptake (pmol/mg protein) 0 Uptake (pmol/mg protein) 0 PI-PLC treatment - ++ PI-PLC treatment + ++ TPP (5 mM) --+ Hematoporphyrin- + - Hemin -+- Fig. 4. Inhibitory Effects of TPP on Folate Uptake. BBMVs were treated with PI-PLC at a concentration of 0.2 Fig. 5. Inhibitory Effects of Hematoporphyrin and Hemin on Folate units/ml, as described in ‘‘Materials and Methods.’’ [3H]-folate Uptake. (22 nM) uptake was measured in an experimental buffer with a pH of BBMVs were treated with PI-PLC at a concentration of 0.2 6.0 for 1 min. Each column represents the mean with S.D. for three units/ml, as described in ‘‘Materials and Methods.’’ [3H]-folate determinations. , p < 0:05; y, p < 0:05. (22 nM) uptake was measured in an experimental buffer with a pH of 6.0 for 1 min. Each column represents the mean with S.D. for three determinations. , significantly different from PI-PLC treatment on folate binding to BBMVs at 1 min. It was found that alone at p < 0:05. the amount of [3H]-folate counted decreased to about 40% of the level of total [3H]-folate binding to and uptake into non-treated BBMVs. Hence it is possible AB that about 40% of [3H]-folate was ingested by BBMVs 20 70 and 60% of [3H]-folate bound to FR at that time. Placenta Placenta * 60 * Furthermore, we investigated the effect of thiamine Fetus Fetus 15 50 pyrophosphate (TPP), a substrate of RFC,28) on [3H]- 40 folate uptake into PI-PLC-treated BBMVs. A further 10 3 * * 30 decrease in [ H]-folate uptake was observed under the * Weight (g) 20 addition of TPP (about 80% of the level of PI-PLC Percent of dose 5 * treatment alone) (Fig. 4). 10 * * Recently, it has been reported that HCP1 is a novel 0 0 candidate folate carrier.22) Hence we investigated the 14 17 20 14 17 20 Gestation day Gestation day effects of hematoporphyrin and hemin, weak inhibitors 3 of HCP1, on [ H]-folate uptake into PI-PLC-treated Fig. 6. Accumulations of Folate in the Placenta and Fetus in the BBMVs (Fig. 5). Further decreases were observed with Pregnant Rat. both the addition of hematoporphyrin and the addition of Administration of [3H]-folate and sample collection were per- hemin, suggesting that HCP1 is also involved in the formed as described in ‘‘Materials and Methods.’’ Accumulation of folate transport mechanism in the human full-term folate in the placenta and fetus were normalized to the amount of [3H]-folate administered to each rat (A). B shows the wet weight of placenta. total placenta and fetus obtained from each dam. Each column represents the mean with S.D. for 3–4 determinations. , signifi- Accumulation of folate in the rat placenta cantly different from the 14th gd at p < 0:05. We investigated the transfer of folate in pregnant rats. We administered [3H]-folate solution to pregnant rats. Three hs after administration, we confirmed that more in the placenta, we investigated the mRNA expression than 80% of the [3H]-folate administered had disap- levels of rFR, rRFC, and rHCP1 using pregnant rats peared from whole blood (data not shown), and we (Fig. 7). The mRNA expression levels of rFR and investigated the accumulation of folate in the placenta rRFC were higher on the 17th gd than on the 14th gd, and the fetus at that time. It was found that the weights and the expression level of rFR mRNA was increased of the placenta and the fetus increased with the progress by about 10-fold on the 20th gd. Moreover, we of gestation, but the accumulation of [3H]-folate in both determined the expression level of rFR in the rat the placenta and the fetus also increased (Fig. 6). placenta by Western blot analysis. It was found that that level also increased with the progress of gestation Changes in expression levels of folate carriers in rat (Fig. 8). The expression level of rHCP1 mRNA did not placenta with the progress of gestation change between the 14th gd and the 17th gd, but it was To clarify the contribution of carriers to folate transfer increased by 6-fold on the 20th gd. 2282 S. YASUDA et al.

rFR α Since FR might be localized on the apical side of the 10 * 13) rRFC * human placenta, localization of RFC has been inves- rHCP1 * tigated only using placental cell lines, and not using human placenta. Our study showed that both FR and 5 RFC were expressed on the apical (maternal) side of the * placenta villi (Fig. 1). It has been reported that RFC

Fold * localizes in the basolateral membrane in placental cell 32) 2 lines BeWo, and our study also showed that some of RFC localize on basal side of the placental villi. We have reported that RFC does not contribute to folate 1 uptake from the apical membrane of BeWo cells.23) However, as shown in Fig. 1, it was found a large 14 17 20 proportion of RFC localizes on the apical side of the Gestation day villi, suggesting that both FR and RFC are involved in folate transport from maternal blood to villi in the Fig. 7. Expression Levels of mRNA of Folate Carriers in the Rat Placenta. human placenta. 3 The expression of each mRNA was determined by real-time PCR. [ H]-folate uptake and binding was strongly inhibited Each column represents the mean with S.D. for three determinations. by structural analogs of folate (Table 1), suggesting that Data show the ratio of the expression level of mRNA to that of each uptake into and binding to BBMVs was not nonspecific mRNA in the 14th gd. , significantly different from the 14th gd at uptake or binding, and that some carriers for folate are p < 0:05. involved in this mechanism. PI-PLC is known to have an inhibitory effect on GPI-linked cell-surface proteins.27) It was found that PI-PLC reduced folate binding in a α 64 kDa rFR concentration-dependent manner (Fig. 3), and that not all of the folate uptake or binding was inhibited by 49 kDa PI-PLC even when BBMVs were treated with a high Actin 37 kDa concentration of PI-PLC. These facts suggest that FR 14 17 20 contributes greatly to folate transport in the placenta but Gestation day that other carriers are also involved in folate transport in the human placenta. Fig. 8. Expression Level of rFR in the Rat Placenta. Hence it was hypothesized that RFC, the expression of The expression levels of rFR were determined by Western blot which was observed by immunohistochemistry, is also analysis. The data shown are typical results from three independent involved in the mechanism of folate transport into the experiments. placenta. [3H]-folate binding to FR was inhibited by treatment with PI-PLC (0.2 units/ml), and the addition Discussion of TPP inhibited [3H]-folate uptake more strongly than did treatment with PI-PLC alone (Fig. 4). These results The blood-placental barrier plays an important role in suggest that RFC was involved in folate transport in the the transfer of various compounds between mother and placental brush-border membranes, in addition to FR. fetus. It is known that various kinds of transporters are However, since TPP did not inhibit all of the [3H]-folate expressed in the placenta, and are involved in the uptake in PI-PLC-treated BBMVs even at a high constitution of the blood-placental barrier.29) It has also concentration of 5 mM, not all of folate transport is been found that the expression levels in the placenta of mediated by FR and RFC. On the other hand, the certain transporters that transport nutrients to the fetus, concentration of TPP was high (5 mM). We suggest that such as the amino acid transporter and the glucose RFC is involved in folate transport in placental brush- transporter, change over the course of gestation.19,20) border membranes, and that TPP competes with folate These findings suggest that the function of the placenta transport under physiological conditions. varies depending on the demands of the developing fetus. It has been reported that HCP1 is a novel candidate Folates are essential nutrients for the fetus protecting folate carrier. It has been characterized as a proton- it from various risks. It is possible that FR, which coupled, pH-sensitive folate carrier, and that it is also transports folates by endocytosis, and RFC, which expressed in the placenta.22) Thus it is possible that mediates bidirectional folate transport, are involved in HCP1 also mediates folate uptake in human placental the folate transport mechanisms in the placenta,30,31) but BBMVs. The inhibitory effects of hematoporphyrin the contributions of these carriers to folate transport and hemin on [3H]-folate uptake into PI-PLC-treated between mother and fetus are not known. Furthermore, BBMVs were weak, but they reduced [3H]-folate uptake changes in the expression levels of folate carriers in the to 70–75% of the control level (PI-PLC treatment placenta with the progress of gestation have not yet been alone), suggesting that HCP1 is also involved in the investigated in detail. folate transport mechanism in the human term placenta. Folate Transport in Placenta 2283 Accumulations of [3H]-folate in the placenta and fetus References increased with the progress of gestation (Fig. 6). Since it has been found that some folate changes to the reduced 1) Baumann, M. U., Deborde, S., and Illsley, N. P., form after administration to rats, folate carriers might Placental glucose transfer and fetal growth. Endocrine, transport it.33) Hence we could not identify the form of 19, 13–22 (2002). folate that accumulated in the placenta, and it is possible 2) Cetin, I., Placental transport of amino acids in normal that the total amount of chemiluminescense from and growth-restricted pregnancies. Eur. J. Obstet. accumulated tritium reflects the folate transport ability Gynecol. Reprod. Biol., 110, S50–54 (2003). 3) Young, A. M., Allen, C. E., and Audus, K. L., Efflux of the placenta. Although as Fig. 6 shows, that both the transporters of the human placenta. Adv. Drug Deliv. placenta and fetus changed in size during pregnancy, Rev., 55, 125–132 (2003). these findings suggest that the increase in capacity for 4) Shane, B., and Stokstad, E. L. R., Vitamin B12-folate placental folate transport is involved in the increase in interrelationships. Annu. Rev. Nutr., 5, 115–141 (1985). folate accumulation in them with the progress of 5) Blount, B. C., Mack, M. M., Wehr, C. M., MacGregor, gestation. Since folates are essential nutrients required J. T., Hiatt, R. A., Wang, G., Wickramasinghe, S. N., for the provision of one-carbon moieties in biosynthetic Everson, R. B., and Ames, B. N., Folate deficiency processes, it is thought that the transport of folate in the causes uracil misincorporation into human DNA and placenta increases in response to increased need for breakage: implications for cancer and folates in the process of fetal development. neuronal damage. Proc. Natl. Acad. Sci. USA, 94, The transport mechanisms of folates in the placenta 3290–3295 (1997). 6) Thenen, S. W., Gestational and neonatal folate defi- have been investigated previously.34,35) Furthermore, ciency in rats. Nutr. Res., 11, 105–116 (1991). expression of rFR, rRFC, and rHCP1 in the rat has 7) Hibbard, B. M., The role of Folic acid in pregnancy; with 36–38) also been reported. But their expression levels and particular reference to anaemia, abruption and absorp- changes in their expression levels in the placenta over the tion. J. Obstet. Gynaecol. Br. Commonw., 71, 529–542 course of gestation have not been detrmined. As shown in (1964). Fig. 7, we found that the mRNA expression levels of all 8) Smithells, R. W., Sheppard, S., and Schorah, C. J., of the folate carriers increased with the progress of Vitamin deficiencies and neural tube defects. Arch. Dis. gestation. Moreover, it was found that the protein Child., 51, 944–950 (1976). expression level of rFR also increased in the placenta 9) Sirotnak, F. M., and Tolner, B., Carrier-mediated (Fig. 8). We have reported that the expression level of membrane transport of folates in mammalian cells. ABCG2 in the rat placenta decreased from the mid stage Annu. Rev. Nutr., 19, 91–122 (1999). 10) Said, H. M., Recent advances in carrier-mediated to the end of gestation.39) Assaraf reported that ABCG2 intestinal absorption of water-soluble vitamins. Annu. plays an important role in the maintenance of folate Rev. Physiol., 66, 419–446 (2004). 18) homeostasis. These findings suggest that the expres- 11) Sabharanjak, S., and Mayor, S., Folate receptor endocy- sion levels of the carriers involved in placental folate tosis and trafficking. Adv. Drug Deliv. Rev., 56, 1099– transport are regulated to supply folate to the placenta 1109 (2004). and fetus, and that it increases with their development 12) Antony, A. C., Kane, M. A., Portillo, R. M., Elwood, during pregnancy. The expression levels of other nutrient P. C., and Kolhouse, J. 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F., Wiley-Liss, New York, increases with the progress of gestation. The results of pp. 91–120 (1992). this study also suggest that increases in the expression 15) Damaraju, V. L., Hamilton, K. F., Seth-Smith, M. L., levels of FR, RFC, and HCP1 in the placenta play an Cass, C. E., and Sawyer, M. B., Characterization of important role in the response to increased needs for binding of folates and antifolates to brush-border folate on the part of the placenta and fetus during membrane vesicles isolated from human kidney. Mol. development. Pharmacol., 67, 453–459 (2005). 16) Rajgopal, A., Sierra, E. E., Zhao, R., and Goldman, I. D., Expression of the reduced folate carrier SLC19A1 in Acknowledgment IEC-6 cells results in two distinct transport activities. Am. J. Physiol. Cell Physiol., 281, C1579–1586 (2001). This work was supported by a Grant-in-Aid for 17) Prasad, P. D., Ramamoorthy, S., Moe, A. J., Smith, Scientific Research from the Ministry of Education, C. H., Leibach, F. 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