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Am J Physiol Endocrinol Metab 291: E596-E603, 2006. First published April 18, 2006; doi:10.1152/ajpendo.00359.2005
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Cortisol stimulates system A amino acid transport and SNAT2 expression in a human placental cell line (BeWo)

Helen N. Jones,1 Cheryl J. Ashworth,2 Ken R. Page,3 and Harry J. McArdle1

1Maternal-Fetal Physiology, Rowett Research Institute, 2Animal Breeding and Development, Sustainable Livestock Systems Group, Scottish Agricultural College, Aberdeen, and 3School of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom

Submitted 3 August 2005 ; accepted in final form 15 February 2006


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Both placental system A activity and fetal plasma cortisol concentrations are associated with intrauterine growth retardation, but it is not known if these factors are mechanistically related. Previous functional studies using hepatoma cells and fibroblasts produced conflicting results regarding the regulation of system A by cortisol. Using the b30 BeWo choriocarcinoma cell line, we investigated the regulation of system A by cortisol. System A function was analyzed using methyl amino isobutyric acid (MeAIB) transcellular transport studies. Transporter expression [system A transporter (SNAT)1/2] was studied at the mRNA and protein levels using Northern and Western blotting, respectively. Localization was carried out using immunocytochemistry. The [14C]MeAIB transfer rate across BeWo monolayers after preincubation with cortisol for 24 h was significantly increased compared with control. This was associated with a relocalization of the SNAT2 transporter at lower cortisol levels and significant upregulation of mRNA and protein expression levels at cortisol levels >1 µM. This is the first study to show functional and molecular regulation of system A by cortisol in BeWo cells. It is also the first study to identify which system A isoform is regulated. These results suggest that cortisol may be involved in upregulation of system A in the placenta to ensure sufficient amino acid supply to the developing fetus.

transcellular transport; intrauterine growth retardation; placenta; system A transporter 2

TRANSPLACENTAL AMINO ACID TRANSPORT is vital for the development, growth, and function of both the placenta and fetus. Insufficiency in placental system A amino acid transport has been implicated in the development of intrauterine growth retardation (30). However, the cause of this insufficiency is unknown. It may be because of intrinsic failure of placental development/function or the actions of other factors on the amino acid transport systems. Changes in both fetal cortisol levels (1) and placental 11beta-hydroxysteroid dehydrogenase activity (20) are also associated with intrauterine growth retardation. Early in gestation, there is a positive correlation between fetal size and fetal cortisol plasma concentration (17, 22). This is reversed in later gestation, with smaller fetuses having higher plasma cortisol levels. Excess glucocorticoids in later gestation have been shown to be detrimental for both fetal growth and development, leading to fetal programming and possible consequences in later life (29) such as cardiovascular disease and obesity. Because there appear to be links between placental amino acid transport, fetal size, and cortisol levels, it is a necessity to identify any regulatory effect that cortisol might exert on the system A amino acid transport system in the placenta.

System A is a highly regulated sodium-dependent amino acid transport system capable of transporting small, nonbranched amino acids such as alanine and glycine. It is regulated by many effectors, including insulin (19), glucagon (37), cell volume (26), pH, and oxygen levels (21).

The regulation of amino acid transport system A by glucocorticoids has previously been shown to differ according to cell type. Gelehrter and McDonald (10) demonstrated that exposure of rat hepatoma tissue culture cells to dexamethasone or cortisol inhibited the transport of {alpha}-aminobutyric acid. However, in fibroblasts used by Russell et al. (27), exposure to increased cortisol levels resulted in an increase in methyl amino isobutyric acid (MeAIB) uptake and the system A proportion of proline, glycine, and alanine transport. Since publication of these data, several isoforms of system A have been identified and cloned in humans. System A transporter (SNAT)1, -2, and -4 encoded by the genes SLC38A1, -2, and -4 show differential tissue expression. In humans, SNAT1 is expressed predominantly in the brain and the placenta (35). SNAT2 shows ubiquitous tissue expression (11) and is associated with the adaptive regulation seen in system A transport under nutritional challenge (9). SNAT4 is predominantly expressed in liver and skeletal muscle (35), with some expression in the human placenta (5). The differences in isoform tissue expression may explain the differences seen in regulation of system A by cortisol in earlier studies.

All published studies describing cortisol and other hormone regulation of system A in the placenta (15, 13, 14) investigated the effect of cortisol on system A uptake, but not transcellular transfer of substrates or the regulation of the isoform expression at the mRNA or protein level. In this study, we chose to investigate the effect of cortisol on the transcellular transfer of MeAIB across a cell monolayer and determine a possible mechanism of regulation by studying isoform mRNA and protein expression.

To determine the effects of cortisol on amino acid transport system A in the placenta, we used the BeWo human choriocarcinoma cell line as a model for trophoblast. It is not possible to use isolated placental fragments or a primary trophoblast cell line for transcellular flux studies, since these cells spontaneously syncytialize, forming regions of fused cells separated by gaps. The BeWo cell line is frequently used as a trophoblast model because it maintains several characteristics of cytotrophoblasts, including human chorionic gonadotropin secretion, preservation of cytotrophoblast morphology (23), and cytokeratin-7 expression. BeWo cells grown on permeable filters also express functional polarity (3, 16). The BeWo cell line is recognized as a useful model for studying mechanisms and regulation of nutrient transport in the placenta. The system L amino acid transport system (25), the uptake of L-lysine (36), the transport of large neutral amino acids (6), and transepithelial glucose transport (34) have all been studied using the BeWo cell line. However, there are some limitations when using the BeWo cell line, for example, they do not express the SNAT4 isoform (5) so it was not possible to assess the SNAT4 response to cortisol in this study. We chose to use the b30 BeWo clone, since this has been shown previously both in our laboratory and others to form the confluent monolayers needed for transcellular transport studies (18, 33), and its cell lineage has been authenticated by European Cell and Culture Collection.

Given the importance that system A plays in placental amino acid transfer and its possible association with intrauterine growth retardation, this study was conducted to assess the role of cortisol in the regulation of system A amino acid transport and its isoform RNA and protein expression in the BeWo cell line.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. The b30 clone of the BeWo choriocarcinoma cell line was obtained from Dr A. L. Schwartz (St. Louis Children's Hospital, St. Louis, MO). The cells were routinely maintained in 80-cm2 flasks at 37°C under 5% CO2 and cultured in Dulbecco's modified Eagles medium + Glutamax 1 (1,500 mg/l glucose; Invitrogen, Paisley, UK) supplemented with 10% FCS and 2% penicillin/streptomycin. Medium was changed every 2 days, and cells were subcultured every 7 days. To study the effect of increased cortisol levels, cells were exposed to either control medium or control medium + a range of cortisol concentrations (5 nM-2.5 µM; see Refs. 12 and 25) for up to 24 h. For transport studies, cell monolayers were prepared by seeding 25-mm tissue culture inserts (Nunc, Hereford, UK) at a high density (5 x 105 cells). Cell monolayers were maintained at 37°C under 5% CO2. Confluence was estimated by determination of transepithelial resistance. Radiolabeled MeAIB (a nonmetabolizable system A substrate) and mannitol flux experiments were performed 6 days after seeding. Mannitol was included in the experiments to assess the passive component of MeAIB transepithelial transport. For immunocytochemistry, glass coverslips were seeded (5 x 104 cells) and grown for 2 days to allow cells to achieve a flattened morphology.

MeAIB transcellular transport. {alpha}-[1-14C]methylaminoisobutyric acid (sp act 50.5 mCi/mmol) and D-[1-3H(N)]mannitol (sp act 17 Ci/mmol) were obtained from PerkinElmer (Boston, MA). Cell monolayers were washed three times in balanced salt solution [BSS (in mM): 136 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, and 18 HEPES, pH 7.4 at 37°C] and placed in fresh six-well plates with 2 ml BSS. Radiolabeled MeAIB (0.2 µCi), with MeAIB added to give a final concentration of 10 µM, and mannitol (0.2 µCi) were added to the apical side in 1 ml serum-free medium. Samples (100 µl) were taken over a 20-min period. Basolateral radiolabel was determined by scintillation counting. Mannitol was included in the experiments to assess the passive component of MeAIB transepithelial transport. The ratio of apical [3H]mannitol to [14C]MeAIB was calculated. This ratio was applied to the basolateral [3H]mannitol counts, and the resulting figure (representing passive [14C]MeAIB transfer) was deducted from basolateral [14C]MeAIB counts. To calculate rates of transfer, we derived the slope of the line from the data at 1, 3, 5, 10, and 20 min. To demonstrate sodium-dependent MeAIB transfer, NaCl in the BSS used in the transport assay was replaced with LiCl on several cell monolayers.

cDNA probes and antibodies. A human specific SNAT1 probe, 2,964 bp, was prepared from the full-length I.M.A.G.E clone ID 3871101. The I.M.A.G.E clone was obtained from the Human Genome Mapping Project resource center (Cambridge, UK). A 328-bp human specific SNAT2 probe was prepared by RT-PCR from RNA using standard protocols and primers designed from human sequences. The sense and antisense primers correspond to 2438–2457 and 2746–2766 of human SNAT2 (Genbank Accession no. AF259799). A probe against 18S RNA was used to normalize the mRNA expression data. Antibodies prepared against peptide sequences specific to human SNAT2 were obtained (ESNLGKKKYETEFHPG; PRIMM, Milan, Italy). Mouse anti-human beta-actin (Sigma, Gillingham, UK) antibody was used to normalize protein expression data. Antibody specificity was verified using peptide block methods in immunohistochemistry (see below).

Western blotting. BeWo cells were washed three times in ice-cold BSS, pH 7.4. Cells were homogenized and sonicated. Total protein (30 µg) was separated on a Tris-glycine gel and transferred to a nitrocellulose membrane (Amersham International) by electrophoresis. Membranes were incubated with the appropriate primary antibody (1:500) overnight at 4°C and an horseradish peroxidase secondary antibody (1:5,000, Sigma) for 2 h at room temperature. Protein bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Tattenhall, UK). Protein expression was quantified using densitometry and normalized for loading against beta-actin.

Immunocytochemistry. Immunofluorescence staining was performed on BeWo cells on glass coverslips. Cells were fixed in frozen methanol for 5 min. To quench autofluorescence, cells were incubated in 50 mM NH4Cl for 10 min at room temperature. To prevent nonspecific binding, cells were incubated with 0.2% fish skin gelatin (FSG)-PBS for 5 min at room temperature. Primary antibody (1:50) was applied in 0.2% FSG-PBS and allowed to bind overnight at 4°C in a humidified atmosphere. After washes in 0.2% FSG/PBS the anti-rabbit fluoroscein conjugated antibody (1:100; Vector Laboratories) was applied for 1 h at room temperature in a humidified atmosphere. The cells were rinsed in PBS and deionized water, and coverslips were mounted in Vectashield mounting medium. Cells were observed, and images were captured using an Axioscope microscope (x100), Axiocam, and AxioVision software (Carl Zeiss Vision).

Antibody specificity was verified by preincubating antibody (1:50 dilution) with peptide (100 µg/ml) for 2 h at room temperature before adding to the coverslips.

Northern analysis. BeWo RNA was isolated using TRI reagent (Helena Biosciences, Sunderland, UK). Total RNA (10 µg) was separated on a 1% formaldehyde agarose gel, transferred to a nylon membrane (Amersham International, Little Chalfont, UK) by electrophoresis at 100 mA and 4°C overnight, and cross-linked using an ultraviolet cross-linker (Ultra-Violet Products, Upland, CA). The cDNA probe was labeled with [{alpha}-32P]dCTP Ready-To-Go labeling beads (Amersham International). Hybridizations were performed overnight at 42°C. mRNA was quantified by measuring the amount of radioactivity hybridizing to the bands on the Northern blot using a wire proportional counter (Packard instant imager; Packard Bioscience, Pangbourne, Reading, UK).

Statistical analysis. Results were assessed for statistical significance using the Student's unpaired t-test and one-way ANOVA with Tukey's multiple comparison tests. A P value <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Transepithelial electrical resistance and passive flux. Incubation of BeWo monolayers with an increasing concentration of cortisol for 24 h did not alter the transepithelial electrical resistance compared with control monolayers (Table 1). The passive component of transfer was assessed by measuring [3H]mannitol in the basal chamber. Incubation of monolayers with cortisol did not alter the transfer rate of mannitol (Table 1).


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  		Table 1. Effect of pretreatment of BeWo cells with increasing cortisol concentrations on the transepithelial electrical resistance and passive permeability of the monolayer

 
Transcellular MeAIB transfer. The rate of flux across the cell layer was measured by counting [14C]MeAIB in the basal chamber. Transcellular transfer can be thought of as occurring in the following three stages: uptake, transfer across the cell, and efflux in the basal chamber. It took ~1 min before we could measure significant counts in the basal chamber. This time presumably represents the uptake and intracellular transfer. After this period, levels in the basal chamber rose linearly with time. Therefore, we measured from 1 to 20 min as the transcellular transfer rate. Passive flux of [14C]MeAIB only represented ~10% total flux. The concentration of MeAIB on the apical membrane was 10 µM. On the basolateral side, it never exceeded 1 pM, indicating that the flux can be considered as essentially unidirectional.

Sixty percent of [14C]MeAIB transfer was inhibited by replacing Na+ with Li+ in the transport buffer, indicating that 60% of transport was sodium dependent, corresponding to system A transport (Fig. 1).


Figure 1
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  		Fig. 1. Effect of replacing NaCl (136 mM) with LiCl (136 mM) in the transport medium on transcellular MeAIB transfer. Histogram shows mean of 6 determinations for each condition + SE. **P < 0.005.

 
Over 60% of transcellular [14C]MeAIB transfer was inhibited by addition of 10 mM alanine or unlabeled MeAIB, whereas addition of 10 mM lysine had no effect (Fig. 2). Similar results were obtained in cells exposed to 1 µM cortisol for 24 h. These data demonstrate the specificity of transport and show that the majority is through system A.


Figure 2
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  		Fig. 2. Inhibition of [14C]MeAIB transfer by 10 mM unlabeled amino acids. Histogram shows mean of 6 determinations for each condition. Statistical analysis was carried out using 1-way ANOVA (P < 0.001) with Tukey's multiple comparison: control vs. Lys not significant (NS); control vs. Ala, P > 0.01; and control vs. MeAIB, P < 0.001.

 
Incubation of BeWo cells with 1,000 nM cortisol caused a significant increase in the amount of MeAIB transferred from the apical to basolateral chambers over the 20-min time period compared with control cells (Fig. 3). Incubation of BeWo cells in cortisol for 24 h caused a dose-dependent increase in the transcellular transfer rate of [14C]MeAIB compared with control levels (Fig. 4).


Figure 3
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  		Fig. 3. Transcellular transfer rate of [14C]MeAIB across BeWo monolayers pretreated with either control medium or control medium + 1,000 nM cortisol. Mean of 6 determinations ± SE for each condition is shown.

 

Figure 4
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  		Fig. 4. Percent stimulation of transcellular [14C]MeAIB transport by increasing concentrations of cortisol (64.77 nM) produces 50% maximal stimulation.

 
Effect of cortisol on system A mRNA levels. SNAT1 mRNA levels showed no significant differences from control mRNA levels after exposure of the BeWo cells to a range of cortisol concentrations (0–2.5 µM; Fig. 5).


Figure 5
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  		Fig. 5. Representative Northern blot for system A transporter 1 (SNAT1) in control BeWo (lanes 1 and 6), BeWo exposed to 1,000 nM cortisol for 24 h (lanes 2, 4, and 7), and BeWo exposed to 2,500 nM cortisol for 24 h (lanes 3, 5, and 8). RNA expression of 18S was used to normalize data. Graph shows the effect of a range of cortisol concentrations on SNAT1 mRNA expression (mean of 6 determinations ± SE for each condition). No significant differences were seen using 1-way ANOVA and Tukey's multiple comparison test.

 
SNAT2 mRNA expression remained at control levels when the BeWo cells were exposed to lower levels of cortisol (20 to 50 nM) for 24 h (Fig. 6). However, after 24 h incubation with 1 µM cortisol, SNAT2 mRNA levels were significantly (P < 0.05, n = 6) increased by 21% compared with control (Fig. 6). Exposure of cells to 2.5 µM cortisol for 24 h produced a more significant (P < 0.01) 30% increase in mRNA levels compared with control. One-way ANOVA showed a significant (P = 0.0059) increase between control and with 1 and 2.5 µM cortisol.


Figure 6
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  		Fig. 6. Representative Northern blot for SNAT2 in control BeWo (lanes 1 and 6), BeWo exposed to 1,000 nM cortisol for 24 h (lanes 2, 4, and 7), and BeWo exposed to 2,500 nM cortisol for 24 h (lanes 3, 5, and 8). RNA expression of 18S was used to normalize data. Graph shows the effect of a range of cortisol concentrations on SNAT2 mRNA expression ± SE. No change was seen in SNAT2 mRNA expression after exposure to 20 or 30 nM cortisol. However, significant upregulation of SNAT2 RNA expression was observed after addition of 1 and 2.5 µM cortisol. *P < 0.05, n = 6, control vs. 1 µM cortisol by Tukey's multiple comparison test. **P < 0.01, n = 6, control vs. 2.5 µM cortisol by Tukey's multiple comparison test.

 
Effect of cortisol on system A protein expression and localization. We could not measure SNAT1 protein levels; however, in light of the RNA expression results, it is unlikely that they would change with increasing cortisol levels.

SNAT2 protein levels remained at control levels when BeWo cells were treated with low concentrations of cortisol (20 to 50 nM) for 24 h. However, in 50 nM cortisol SNAT2 relocalized to the plasma membrane (Fig. 7B) within 2 h of incubation compared with control (Fig. 7A). This relocalization persisted throughout a 24-h exposure (8, 12, 18, and 24 h; Fig. 7, C–F).


Figure 7
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  		Fig. 7. Cellular localization of SNAT2 in BeWo cells exposed to control medium (A) or control medium + 50 nM cortisol for 2 (B), 8 (C), 12 (D),18 (E), and 24 (F) h. Scale bar = 10 µm.

 
Control cells (Fig. 8A) expressed SNAT2 at the cell membrane and in intracellular compartments. The fluorescent staining of both compartments was markedly increased (Fig. 8B) after incubation of the cells in 1 µM cortisol for 24 h. SNAT2 antibody specificity was shown by preincubation of anti-SNAT2 with its antigenic peptide as described in EXPERIMENTAL PROCEDURES (Fig. 8C).


Figure 8
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  		Fig. 8. Cellular localization of SNAT2 in BeWo cells (A), BeWo cells exposed to 1 µM cortisol for 24 h (B), and a negative control (C). Scale bar = 10 µm.

 
The increase in intracellular SNAT2 expression seen in Fig. 8b was confirmed by Western blotting. After exposure of the cells to 1 µM cortisol for 24 h, an 11% (P = 0.018, n = 8) increase in SNAT2 protein expression over control was seen (Fig. 9).


Figure 9
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  		Fig. 9. Graphs show SNAT2 protein expression. No change after exposure to 20 or 50 nM cortisol compared with control. However, SNAT2 protein expression is upregulated by exposure to 1,000 nM cortisol compared with control. *P < 0.05, n = 8, 1,000 nM cortisol (Student's unpaired t-test). Representative Western blot for SNAT2 in control BeWo (lanes 3, 4, 5, and 7) vs. BeWo exposed to 1,000 nM cortisol (lanes 1, 2, 6, and 8). beta-Actin protein expression was used to normalize data.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This is the first study to show that cortisol stimulates the transcellular transfer of a system A substrate and the first to link it with the regulation of expression and localization of the system A isoforms SNAT1 and -2. There are, currently, three isoforms of system A transporters (SNAT1, -2, and -4). In this study, we have concentrated primarily on SNAT1 and -2, since SNAT4 has been shown not to be expressed in BeWo cells (5). We show that cortisol can alter transfer in many ways, by alteration of the localization of SNAT2 and, at higher levels, by changing protein and mRNA expression. The effect is specific, since passive permeability properties are not changed, and is also different for the two isoforms.

Cortisol is vital for the differentiation of tissues and maturation of organs in the developing fetus. Both fetal and maternal serum levels of cortisol increase during gestation and can vary from 20 to 50 nM in the fetal and 200 to 800 nM in the maternal circulations (8, 11). Under stress, this can increase two- to threefold (28), and increased cortisol has been associated with problems in fetal growth (7), leading to irreversible reprogramming of the hypothalamic-pituitary-adrenal axis. In pigs in late gestation, increased fetal plasma cortisol levels are also associated with growth-retarded fetuses (1, 24). Given the associations between both altered placental system A activity, altered cortisol levels, and IUGR, it is essential to identify any regulation of system A amino acid transport in the placenta by cortisol.

In early studies, Karl et al. (15) showed that cortisol increased MeAIB uptake in isolated trophoblasts. However, this ignores the physiologically important efflux step into the fetal circulation and also does not consider how the increase is regulated, whether through translocation of protein, through increased protein expression, or through increased levels of mRNA. This paper remedies that deficit and further shows that regulation can take place at all three levels of transcellular transport. We also test whether cortisol treatment can alter the physical structure of the cells, altering passive permeability properties and hence making the cells more generally leaky to nutritional and other solutes.

Passive diffusion may be responsible for ~50% of the unidirectional flux of solutes across the human placenta (31). Therefore, any change to syncytiotrophoblast permeability and integrity would affect fetal nutrient supply. By measuring the transepithelial electrical resistance of a polarized monolayer, it is possible to determine any changes to its integrity. Cortisol did not affect the integrity of the monolayer. The [3H]mannitol data indicated that the permeability properties of the monolayers remained the same as control. Therefore, any changes in transcellular [14C]MeAIB transfer were because of altered specific transport systems, primarily amino acid transport system A, and not altered cell permeability or monolayer integrity. The specificity of the transport system was also confirmed using inhibition studies. Inhibition of MeAIB transfer by "cold" MeAIB and alanine occurred in both control cells and treated cells to the same degree, indicating cortisol-stimulated transfer occurred via system A.

As discussed above, previous studies in placental cells (14, 15) demonstrated that increased cortisol (>1 µM) levels stimulated amino acid uptake via amino acid transport system A. This study supports and extends these results, not only demonstrating that cortisol stimulates transepithelial transfer of the system A substrate MeAIB at concentrations >1 µM but also at more physiological levels (50 nM) and within much shorter time periods. The dose-dependent increase in [14C]MeAIB transcellular transfer, however, was not reflected in the expression of the system A transporters at the mRNA or protein level. Previous studies (28) demonstrated that increased system A activity caused by cortisol was abolished by cycloheximide and actinomycin D. However, these studies used high levels of exposure of cortisol and were carried out before the cloning of the system A transporters. They were, therefore, unable to identify which transporters, at what level, were regulated by cortisol.

Cortisol had no effect on the levels of SNAT1 mRNA. SNAT1 protein levels could not be measured but, in light of the mRNA data, it is unlikely that SNAT1 protein expression would change. The involvement of SNAT1 in the cortisol-mediated increase of system A activity remains unknown, and further knockdown experiments will be required to establish its role.

SNAT2 mRNA and protein expression levels remain at control levels after exposure to lower levels (20 to 50 nM) of cortisol. However, after incubation of cells in 1,000 nM cortisol, both SNAT2 mRNA and protein expression were increased. In contrast, lower cortisol levels regulate SNAT2 localization. After exposure to either 50 or 1,000 nM cortisol for 24 h, relocalization of preformed SNAT2 transporters occurred from intracellular stores to the plasma membrane. This occurred within 2 h of exposure to 50 nM cortisol and was maintained throughout the 24-h exposure period compared with control cells. The mechanism underlying this relocalization is unknown. Boehmer et al. (2) demonstrated regulation of the closely related SN1 transporter (SLC38A3) by the serum- and glucocorticoid-dependent kinase 1 (SGK1) that is related to protein kinase B (PKB), which plays a role in the trafficking of the glucose transporter GLUT4. It is possible that cortisol regulates SGK1 in the BeWo cells and stimulates trafficking of the SNAT2 transporter to the plasma membrane. It may also be possible that the low levels of cortisol are stimulating system A transport indirectly, perhaps through alteration of cytokine expression that in turn regulates PKB and protein trafficking.

The data may have some limitations. We do not, for example, consider the role of other cell layers in regulation of transport nor do we consider how external endocrine stimuli may modulate the response to cortisol. These shortcomings could, perhaps, be avoided by using placental perfusion methods, but these in turn would be far more difficult to interpret than this comparatively simple and well-defined system. In balance, therefore, we believe that our findings provide a clear and well-defined role for cortisol in the transplacental transfer of amino acids and of the function of system A.

These findings may also have wider implications regarding placental transport and uterine development. Many studies have shown that increased fetal cortisol levels are associated with adverse intrauterine conditions such as hypoxemia and undernutrition (4) and that increased cortisol levels in late gestation may be related to intrauterine growth retardation (25). In contrast, both Klemcke et al. (17) and Nwagwu et al. (22) showed that in early gestation smaller fetuses had reduced levels of cortisol. Our data show that physiologically relevant levels of cortisol increase system A transcellular transfer in a placental cell line and provides novel insight into the mechanism of regulation of amino acid transport system A in the trophoblast by cortisol. Our data raise the question of whether a certain level of cortisol in early gestation is required to increase system A activity and whether inadequate cortisol in smaller fetuses would cause insufficiency in placental system A activity.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was funded by SEERAD flexible fund, Framework V QLK-1999-00337, and the International Copper Association.


   ACKNOWLEDGMENTS
 
We thank Dr. A. L. Schwartz (St. Louis Children's Hospital, St. Louis, MO) for the supply of the BeWo cells and Dr. P. Samientos (PRIMM, Milan, Italy) for the production of the SNAT2 antibody.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. J. McArdle, Maternal-Fetal Physiology, Rowett Research Institute, Aberdeen, AB21 9SB UK (E-mail: H.McArdle{at}rowett.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.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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