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REPORT
MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Division of Human Development, St. Mary's Hospital, The Medical School, University of Manchester, Manchester, United Kingdom
Submitted 2 June 2005 ; accepted in final form 1 September 2005
ABSTRACT
The system A amino acid transporter is encoded by three members of the Slc38 gene family, giving rise to three subtypes: Na+-coupled neutral amino acid transporter (SNAT)1, SNAT2, and SNAT4. SNAT2 is expressed ubiquitously in mammalian tissues; SNAT1 is predominantly expressed in heart, brain, and placenta; and SNAT4 is reported to be expressed solely by the liver. In the placenta, system A has an essential role in the supply of neutral amino acids needed for fetal growth. In the present study, we examined expression and localization of SNAT1, SNAT2, and SNAT4 in human placenta during gestation. Real-time quantitative PCR was used to examine steady-state levels of system A subtype mRNA in early (6–10 wk) and late (10–13 wk) first-trimester and full-term (38–40 wk) placentas. We detected mRNA for all three isoforms from early gestation onward. There were no differences in SNAT1 and SNAT2 mRNA expression with gestation. However, SNAT4 mRNA expression was significantly higher early in the first trimester compared with the full-term placenta (P < 0.01). We next investigated SNAT4 protein expression in human placenta. In contrast to the observation for gene expression, Western blot analysis revealed that SNAT4 protein expression was significantly higher at term compared with the first trimester (P < 0.05). Immunohistochemistry and Western blot analysis showed that SNAT4 is localized to the microvillous and basal plasma membranes of the syncytiotrophoblast, suggesting a role for this isoform of system A in amino acid transport across the placenta. This study therefore provides the first evidence of SNAT4 mRNA and protein expression in the human placenta, both at the first trimester and at full term.
SNAT1; SNAT2; gestational expression; syncytiotrophoblast
-(methylamino)isobutyric acid (MeAIB) (7). This nonmetabolized amino acid analog has been used extensively to study system A in the placenta (21, 27, 37). Recent data show that system A is encoded by three different members of the SLC38 gene family (Slc38a1, Slc38a2, and Slc38a4), giving rise to the three subtypes of this Na+-coupled neutral amino acid transporter (SNAT): SNAT1, SNAT2, and SNAT4 (previously referred to as ATA1, ATA2, and ATA3, respectively) (31). SNAT1 was the first isoform to be cloned from rat brain and initially was designated GlnT because of its preference for glutamine as a substrate (49). Cloning of the human homolog as well as SNAT2 and SNAT4 followed (18, 19, 44, 45, 50, 51). These previous studies showed the three isoforms to be highly homologous: amino acid sequences for SNAT1 and SNAT2 demonstrated 52% homology, and SNAT4 was found to be 48% homologous to SNAT1 and 57% homologous to SNAT2. Functionally, SNAT1 and SNAT2 operate via similar mechanisms (19, 50, 51). SNAT4 has a lower substrate affinity for neutral amino acids than SNAT1 and SNAT2 (18, 45) and also interacts with cationic amino acids in a Na+-independent manner such that it resembles system y+L (18). Gene expression for the three system A subtypes varies between tissues. Northern blot analysis has shown that only Slc38a2 (SNAT2) is expressed ubiquitously in mammalian tissues; therefore, this subtype is likely to represent the classic system A amino acid transporter (19, 44). Slc38a1 (SNAT1) is expressed predominantly in the human heart, brain, and placenta and may represent an important subtype at these sites (50). Slc38a4 (SNAT4) has previously been described to be uniquely expressed in the liver (18, 45).
The syncytiotrophoblast is the transporting epithelium of the placenta. Maternofetal exchange of amino acids is mediated largely by transporter systems in its microvillous plasma membrane (MVM; maternal facing) and basal plasma membrane (BM; fetal facing). Reduced system A activity was found in MVM vesicles isolated from placentas in which the fetus had shown intrauterine growth restriction (IUGR) (15, 23, 33). Whether the altered activity of this transporter represents a primary event in cases of IUGR or is secondary to the growth restriction remains to be established. However, inhibition of this transporter in animal models leads to fetal growth restriction (10), suggesting that the reduced activity of system A in placentas from IUGR pregnancies could be a cause rather than an effect of this pathological condition.
In normal pregnancy, fetal body weight increases at a faster rate than placental weight during the second half of pregnancy (40, 47). An appropriate increase in the transport capacity of the placenta is therefore required to support the rising nutrient demands of the growing fetus. Indeed, there is a fourfold increase in the activity of system A in MVM vesicles isolated from term placentas compared with those isolated from first-trimester placentas (32), although such a change was not observed in whole placental villous fragments (11), which comprise a heterogeneous mix of various cell types.
Since the identification of system A at the molecular level, no investigation has addressed whether the expression of system A is gestationally regulated in the human placenta. In this study, we used the sensitive method of real-time quantitative PCR (QPCR) to detect and measure mRNA expression for Slc38a1, Slc38a2, and Slc38a4 in first-trimester and term placentas. Interestingly, we discovered that the placenta does express Slc38a4, the liver-specific isoform of system A, and that gene expression for this subtype was regulated gestationally. Therefore, we determined protein expression for SNAT4 in first-trimester and term placentas using Western blot analysis, and we investigated SNAT4 protein localization at these stages of gestation using immunohistochemistry. Distribution of SNAT4 to both the MVM and BM in term syncytiotrophoblasts was confirmed using Western blot analysis.
MATERIALS AND METHODS
Tissue Acquisition
All tissue was obtained with written informed consent as approved by the Local Research Ethics Committee. Term (38–40 wk) placentas were collected after cesarean section or vaginal delivery from uncomplicated singleton pregnancies. First-trimester (6–13 wk) placentas were obtained after elective medical or surgical termination of pregnancy. Gestational age was estimated from the date of last menstrual period and confirmed using ultrasound dating.
mRNA Expression
Preparation of tissue for QPCR. Total RNA was extracted from placental tissue, cytotrophoblast cells, and BeWo choriocarcinoma cells and quantified as described previously (29). Cytotrophoblast and BeWo cells were cultured and processed in our laboratory as described previously (2, 8). All RNA samples (100 ng) were reverse transcribed simultaneously to generate a cDNA template, ensuring comparable reverse transcription efficiency.
Primers and probe design. Gene sequences for Slc38a1, Slc38a2, and Slc38a4 were derived from GenBank. Primers and probes (Table 1) were designed for each of these sequences using Beacon Designer software (Premier Biosoft International, Palo Alto, CA), and specificity was confirmed by performing Basic Local Alignment Search Tool assessment. Primers and probes were synthesized by MWG-Biotech (Ebersberg, Germany).
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Statistical Analysis
QPCR data are presented as median values of percentage expression relative to a 40-wk placental sample, designated the calibrator, which was included in each QPCR run to serve as an internal standard as described previously (29). First-trimester data were split into two groups, <10 wk (n = 23) and 10–13 wk (n = 12), to compare gene expression before and after the onset of maternal blood flow to the placenta at 
10 wk (5). Expression in these groups was compared with expression in the term group (n = 21). For the first-trimester groups, no distinctions were made between the two modes of delivery (medical terminations <10 wk, n = 18; medical terminations 10–13 wk, n = 5; surgical terminations <10 wk, n = 6; surgical terminations 10–13 wk, n = 6), because we have previously shown that different modes of delivery do not affect mRNA expression for a range of genes examined (29). The data were analyzed using a nonparametric Kruskal-Wallis test, followed by Dunn's post hoc test, with P < 0.05 considered significant using GraphPad software (San Diego, CA).
Protein Expression
Western blot analysis. Protein was extracted from first-trimester (7–13 wk; n = 8) and term placental villous tissue homogenates (38–40 wk; n = 8) as described previously (6). Total liver homogenate (Sigma Aldrich, Dorset, UK) was used as the positive control tissue for SNAT4, and BeWo choriocarcinoma cell homogenate was used as the negative control because these cells do not express Slc38a4 (25, 36). To compare expression in the MVM and BM of the syncytiotrophoblast, paired samples from term placentas were prepared as described previously (14, 16). Purity of MVM and BM isolates had been determined previously by standard markers and polarized expression of p-glycoprotein and plasma membrane Ca2+-ATPase to MVM and BM, respectively (42). For all samples, including the positive and negative controls, 30 µg of protein were mixed at a ratio of 1:1 with loading buffer (22% glycerol, 139 mM Tris·HCl, pH 6.8, 154 mM SDS, 4.4 M urea, 0.002% bromophenol blue, and 10% vol/vol 2-mercaptoethanol) and heat reduced for 5 min at 95°C. PAGE was performed using 3% stacking gels and 7% resolving gels. The proteins were then electrotransferred onto nitrocellulose membrane, and antibody probing was performed as described previously (6). A rabbit anti-SNAT4 affinity-purified polyclonal antibody was used (1:100 dilution; 2 µg/ml). This antibody had been raised to the amino acid sequence YGEVEDELLHAYSKV of human SNAT4 (Eurogentec, Seraing, Belgium). For a negative control, the purified antigenic peptide was used in 15x excess to preabsorb antibody overnight at 4°C. Primary and horseradish peroxidase-conjugated secondary antibody incubations were performed for 1 h at room temperature. Positive signals were detected using ECL, and the density of immunoreactive species was assessed using a GS 700 Imaging Densitometer (Bio-Rad Laboratories, Hemel Hempstead, UK) with Molecular Analyst software.
Statistical analysis. To correct for any differences between the two blots used to compare gestations, SNAT4 expression in each sample was normalized to expression in a term calibrator sample that had been included on both blots. The data were analyzed using a nonpaired, two-tailed t-test and GraphPad software, and P < 0.05 was considered significant.
Immunohistochemistry. Specimens of first-trimester (8–12 wk, n = 4) and term placentas (38–40 wk, n = 3) were immersion fixed in ice-cold Zn2+ fix (4) within 30 min of delivery, prepared for immunohistochemistry, and treated as described previously (6). The localization of SNAT4 was examined using the rabbit anti-SNAT4 antibody used for Western blot analysis (1:20 dilution; 10 µg/ml). For the negative controls, the primary antibody was preabsorbed with antigenic peptide as described for Western blot analysis. Slides were viewed under a Leitz Dialux 22 microscope using a x40 magnification lens objective.
RESULTS
Slc38a1, Slc38a2, and Slc38a4 mRNA Expression Over Gestation
The amplification product generated for each of the genes examined was visualized as a single band of appropriate size after agarose gel electrophoresis, confirming primer specificity (Fig. 1). This finding was further confirmed by generation of a single peak on the associated dissociation curve after QPCR (data not shown). The shape of this curve was determined by the size and nucleotide content of the amplicon, and therefore a single peak represents a single product and confirms the specificity of the primers. Figure 1 demonstrates that cytotrophoblast cells, first-trimester placental samples, and term placental samples express Slc38a1, Slc38a2, and Slc38a4 mRNA, whereas BeWo cells express only Slc38a1 and Slc38a2 mRNA. Figure 2, A and B, shows that there were no significant differences in mRNA expression of Slc38a1 and Slc38a2 from 6 to 9 wk and from 10 to 13 wk gestation or between these gestation periods and full term. In contrast, Slc38a4 mRNA expression was significantly higher (P < 0.01) at 6–9 wk compared with term (Fig. 2C).
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Figure 3A shows that an immunoreactive signal was observed at 60 kDa in all placental samples. This immunoreactive signal comigrated with the signal observed in the liver, which was included as a positive control. These data accord well with the predicted size of 60 kDa for SNAT4. Preabsorption of the antibody with 15x peptide abolished this signal (Fig. 3B), confirming antibody specificity for SNAT4. Figure 3C shows that no immunoreactive signal was detectable in the BeWo sample, which was used as a negative control, further confirming the specificity of this antibody. Densitometric analysis of the blots shown in Fig. 3A revealed that SNAT4 protein expression was significantly higher (P < 0.05) at term than during the first trimester (Fig. 3D), a finding that is also visually apparent. To ensure that this finding was not attributable to unequal protein loading, the same samples were probed for 
-actin (Fig. 4A). Densitometric analysis revealed that the expression of this housekeeping protein in first-trimester and term samples was not significantly different (Fig. 4B), confirming equal protein loading.
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Figure 5 shows representative images of immunohistochemical staining for SNAT4 in first-trimester (Fig. 5A) and term placental tissue (Fig. 5C). SNAT4 is localized to the syncytiotrophoblast, with positive staining on MVM, BM, and the stroma at both stages of gestation. There is also strong staining in the fetal blood vessels that is more apparent in term tissue, probably because vascularization of the tissue is much more advanced at this stage of gestation. Negative control sections of both first-trimester (Fig. 5B) and term tissue (Fig. 5D) lacked detectable staining. SNAT4 expression in term MVM and BM was confirmed using Western blot analysis (Fig. 6A). A single 60-kDa immunoreactive species was detected in both MVM and BM, which was abolished by preabsorption of antibody with excess antigenic peptide (Fig. 6B). Densitometric analysis of immunoreactive signal revealed no significant difference between SNAT4 expression in MVM and BM (data not shown).
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In the present study, we have demonstrated for the first time mRNA and protein expression for the SNAT4 isoform of the system A amino acid transporter in the human placenta. SNAT4 previously was considered a liver-specific subtype of system A because Northern blot analysis failed to detect mRNA for this isoform in any other tissue, with the exception of rat skeletal muscle (18, 45). The method of QPCR used in the current study is, of course, more sensitive than Northern blot analysis (13). Furthermore, we have shown that mRNA expression for this subtype of system A is significantly higher in early first-trimester villous tissue compared to term. This observation might underlie the lack of positive signal observed previously in term placenta (18). Expression of Slc38a4 RNA also has been confirmed in rat placenta (35) and early gestation mouse placenta (41) using Northern blot analysis, and it has been reported in other mouse tissues analyzed using RT-PCR (34). Using RT-PCR, the researchers who originally reported a novel human amino acid transporter, hNAT3, assigned to the system N family demonstrated high levels of RNA expression in liver; lower levels in muscle, kidney, and pancreas; and trace levels in heart and placenta (17). This transporter can now be reclassified as SNAT4 on the basis of further characterization of this isoform of system A. These observations demonstrate that although expression of Slc38a4 mRNA is relatively high in the liver, it is not uniquely expressed by this tissue and should no longer be regarded as liver specific.
The mRNA data reveal considerable variability in the levels of Slc38a1, Slc38a2, and Slc38a4 gene expression between placentas within the same gestational groups. This phenomenon has been reported previously and is thought to be due partly to individual variability (genetic component) and partly to the heterogeneous cellular composition of the placenta (39). Despite this variability, statistical analysis has revealed that Slc38a4 mRNA expression is significantly higher in placentas from the 6- to 10-wk gestation first-trimester group compared with the term group. There is no difference, however, between expression in the 6- to 10-wk gestation group and the 10- to 13-wk gestation first-trimester group, indicating that the decrease in Slc38a4 mRNA expression may take place after this time.
The gestational regulation of Slc38a4 mRNA expression in placenta prompted us to compare SNAT4 protein expression in first-trimester and term tissue. The specificity of the SNAT4 antibody was confirmed by the absence of an immunoreactive product in the BeWo choriocarcinoma cell line, which does not express Slc38a4 mRNA (25, 36). These cells do express Slc38a1 and Slc38a2 mRNA (25, 26, 36, 46), however, with translation into protein confirmed for both SNAT1 and SNAT2 (26, 36). In contrast to the observation of a decrease in mRNA expression for this subtype of system A with advancing gestation, Western blot analysis revealed that protein expression was significantly higher at term compared with the first trimester. These data add to the increasing body of evidence for complex gestational changes in placental transporter expression and activity (1). A change in mRNA expression is not necessarily paralleled by a similar change in protein expression, perhaps because of differences between regulation of transcription, mRNA degradation, translation into protein, and posttranscriptional processes. Each of these events and the rate at which they occur may differ between cell types. In the placenta, the relative proportion of syncytiotrophoblasts, cytotrophoblast cells, and endothelial cells changes quite markedly during gestation. If variations in transcription and translation of SNAT4 in these different cell types exist, they could account for the observations reported herein. For example, immunohistochemical detection of SNAT4 shows strong staining in fetal blood vessels, which are widespread at term but poorly developed during the first trimester.
The identification of SNAT4 in human placenta and its localization to the transporting epithelium of this tissue, the syncytiotrophoblast, raises the question of its functional importance in amino acid transport from mother to fetus. The broader substrate specificity of SNAT4 suggests that it could mediate the Na+-independent transport of cationic amino acids such as arginine (18) as well as the Na+-dependent transport of neutral amino acids typical of system A. This broad specificity complicates studies to identify definitively the contribution of SNAT4 to system A activity. Furthermore, this subtype has a low affinity for MeAIB, the system A-specific substrate (18). Comprehensive vesicle studies in which many substrates are used may provide insight into the role of SNAT4 but could prove difficult to interpret, considering the overlap between the different transporters and the substrates with which they interact (22, 31). The lack of gestational change in system A activity (measured as Na+-dependent 14C-MeAIB uptake) in placental villous fragments from first-trimester and term human tissue (11) is surprising in view of the gestational increase previously reported in MVM vesicles (32) and considering the gestational changes in placental SNAT4 protein expression demonstrated herein. Perhaps this phenomenon can be explained by the heterogeneous nature of villous fragments, the variable distribution of the SNAT isoforms, and the relatively low substrate specificity of SNAT4 for MeAIB. These issues require further investigation.
It is likely that transport of amino acids by system A is acutely regulated in vivo by various hormones and growth factors as well as by substrate availability evidenced by in vitro studies (21, 28, 43). Long-term regulation of system A by these factors is mediated through changes in transcription and translation of the genes coding for system A. Research into this area has already begun with respect to substrate regulation of SNAT2 (12, 30), but much more work remains to be done. Knocking out placenta-specific IGF-II (Igf2) in mice causes placental growth restriction from embryonic day 12 (E12) and fetal growth restriction from E19 (9). There is increased maternofetal 14C-MeAIB transport per gram of placenta weight in these mice at E16, consistent with maintained fetal growth despite reduced placental size at this stage of gestation. This observation is of interest because there is increased expression of Slc38a4 mRNA in these placentas at E16, whereas expression of Slc38a1 and Slc38a2 remains unaltered (Constância M, unpublished observations). The relationship between Slc38 mRNA, SNAT protein expression, and system A activity in the placentas of these mice remains to be elucidated. However, the selective increase in Slc38a4 gene expression in parallel with the increased MeAIB transport suggests that this isoform contributes to system A activity in the placenta.
In view of the current observations and other recent studies, SNAT4 is probably expressed functionally in tissues other than the liver and placenta, including muscle, kidney, pancreas, and heart. SNAT4 in the liver is thought to provide the major route for uptake of cationic amino acids such as arginine because this tissue does not express any of the other high-affinity cationic amino acid transporters (38). To date, evidence exists for only relatively low levels of CAT2 mRNA expression in liver, an isoform of the low-affinity cationic amino acid transporter y+ (20). Lack of mRNA expression for the high-affinity cationic amino acid transporter y+L in human muscle and heart (48) also suggests an inverse relationship between SNAT4 and y+L expression in these tissues, raising the possibility that SNAT4 may provide a route for cationic amino acid transport. There is evidence for y+L expression and activity in the human placenta (3, 48), and therefore the importance of SNAT4 in this tissue in terms of cationic amino acid transport may not be as great as in these other tissues. The present study makes it important to determine the function of SNAT4 in the placenta and to address its contribution to amino acid transport and fetal growth.
GRANTS
This work was funded by the Wellcome Trust. M. Desforges has a Wellcome Trust Prize studentship.
ACKNOWLEDGMENTS
We thank the midwives and nursing staff of St. Mary's Hospital for assistance in obtaining placentas. We are grateful to Dr. Carolyn Jones for advice and assistance with immunohistochemistry and also to Dr. Melissa Westwood for comments on the manuscript. We also thank Dr. Diane Atkinson for donating the BeWo samples used in this study.
FOOTNOTES
Address for reprint requests and other correspondence: J. D. Glazier, Academic Unit of Child Health, Univ. of Manchester, St. Mary's Hospital, Hathersage Road, Manchester M13 0JH, United Kingdom (e-mail: j.glazier{at}manchester.ac.uk)
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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