Placenta
Volume 31, Issue 4 , Pages 289-294, April 2010

Aldosterone and Cortisol Acutely Stimulate Na+/H+ Exchanger Activity in the Syncytiotrophoblast of the Human Placenta: Effect of Fetal Sex

Maternal and Fetal Health Research Group, School of Clinical and Laboratory Sciences, University of Manchester, Manchester Academic Health Sciences Centre, St Mary's Hospital, Oxford Road, Manchester M13 9WL, UK

Accepted 23 December 2009. published online 03 February 2010.

Article Outline

Abstract 

Na+/H+ exchanger (NHE) activity regulates intracellular pH (pHi) in the placental syncytiotrophoblast. In other tissues aldosterone and cortisol have been shown to up-regulate NHE activity via an acute, non-genomic effect. Here we tested the hypothesis that these corticosteroids stimulate NHE in the syncytiotrophoblast. Villous fragments from term placentas were loaded with 1 μM BCECF (pH sensitive fluorescent dye) and the syncytiotrophoblast acidified with a pre-pulse of 20 mM NH4Cl. The Na+-dependent recovery of pHi from this acid load was taken as a measure of NHE activity (pH units/sec, mean ± SEM, n = number of placentas). In placental villi from female babies aldosterone significantly increased the rate of recovery of pHi from an acid load (0.0087 ± 0.0005 versus 0.0056 ± 0.0009 pH units/s, n = 8 p < 0.05 Paired Student's t-test) which was inhibited by the mineralocorticoid receptor antagonist, spironolactone (1 μM) but not the glucocorticoid antagonist mifepristone (1 μM). There was no effect on the rate of recovery from an acid load in villi from placenta from male babies. Alone, neither cortisol (1 μM, n = 5) nor carbenoxolone (100 μM, n = 9), an inhibitor of 11-β-hydroxysteroid dehydrogenase-2 (11-β-HSD-2), altered the rate of recovery from an acid load. However, simultaneous application of cortisol with carbenoxolone significantly increased the rate of recovery from an acid load but again only in placentas from female babies (0.0080 ± 0.0017 versus control 0.0037 ± 0.0005, p < 0.05 pH units/s, n = 9 Paired Student's t-test). Stimulation by cortisol in female tissue was inhibited by mifepristone but not spironolactone. In conclusion, syncytiotrophoblast NHE activity is increased acutely by aldosterone and, when 11-β-HSD-2 is blocked, by cortisol. These non-genomic effects are only evident in placentas from female babies and are mediated by classical mineralocorticoid and/or glucocorticoid receptors.

Keywords: Placenta, Aldosterone, Cortisol, Na+/H+ exchanger

 

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1. Introduction 

The Na+/H+ exchangers (NHEs) are a widely expressed family comprised of nine isoforms. NHE1-5 and 8 mediate the electroneutral exchange of one extracellular Na+ for one intracellular H+ and subserve a number of different cellular functions including maintenance of intracellular pH [pH]i, transepithelial Na+ transport, cell volume homeostasis and cell proliferation [1]. NHE 6, 7 and 9 are localized to the intracellular organelles and modulate intraorganelle pH, most notably of the Golgi and post Golgi network [2]. At least three NHE isoforms have been identified in the human placenta, localized to both the microvillous (MVM) and basal plasma membranes (BM) of the human placental syncytiotrophoblast (the transporting epithelium of this organ), although reported NHE isoform distribution to these membranes varies between studies [3], [4], [5], [6]. Previously, we have shown by immunoblotting that NHE 1 protein is localized to the MVM and BM, whilst NHE 3 is only found on the MVM [6]. However, NHE 1 activity predominates on MVM, with no evidence of NHE 3 activity detectable under our control experimental conditions [6]. pH recovery from an acid load by the human placental syncytiotrophoblast, is predominantly mediated by NHE activity [7]. Furthermore, NHE activity is decreased in MVM vesicles isolated from the syncytiotrophoblast of placentas from babies who have suffered intrauterine growth restriction [5]. There are previous data suggesting that NHE in the syncytiotrophoblast can be regulated by phosphorylation [8]. However, there are no data on what hormones might regulate NHE activity in this human tissue.

In transporting epithelia other than the placenta, such as renal epithelia, the steroid hormones, cortisol and aldosterone, regulate transcellular sodium transport by modulating the activity of sodium transport proteins, including NHEs [9]. The classical actions of aldosterone and cortisol are through genomic pathways leading to upregulation of protein synthesis. These are mediated by mineralocorticoid receptors (MRs) for aldosterone and by both MR and glucocorticoid receptors (GRs) in the case of cortisol [10]. However, aldosterone and cortisol also have a rapid (non-genomic) effect on the activity of NHE in uterine and placental chorionic plate arteries [11] as well as in other epithelial cells [12] and a number of cell lines [13]. Both MR and GR have affinity for cortisol at physiological concentrations. However, in mineralocorticoid target tissues the MR is usually protected from the effects of cortisol by the presence of an enzyme, 11-β-hydroxysteroid dehydrogenase-2 (11-β-HSD-2), which converts cortisol to inactive cortisone [10], [14].

The components of the synthesis and secretion of both cortisol and aldosterone are present in placental tissue [15] although there are no data to show whether they are produced by the human placenta. However, using immunohistochemistry, qPCR and Western blotting, GR, MR and 11-β-HSD-2 have been shown to be expressed in the placenta throughout gestation [16], [17], [18], [19], [20]. Interestingly, in placentas from normal pregnancies there is a greater expression of mRNA for MR and GR in placentas from female babies compared to male babies [21]. However, in the placentas of female babies of asthmatic women there was a reduction in the mRNA expression of MR, GR and 11-β-HSD-2 and the babies exhibited an increased incidence of intrauterine growth restriction (IUGR, [21]). The reduced expression of mRNA for these proteins along with the birth weight reduction were not observed when the asthma was controlled by the use of glucocorticoid inhalers [22], suggesting a gender specific role for glucocorticoids in modulating growth during pregnancy, possibly via altering the placental transport of nutrients.

Here we used a previously reported in vitro technique for measuring intracellular pH ([pH]i) recovery in the syncytiotrophoblast [6] to test the hypothesis that aldosterone and cortisol regulate the activity of NHE in the syncytiotrophoblast via an acute, non-genomic effect. We examined whether the effects we observed were via MR or GR and the involvement of 11-β-HSD-2 in modulating cortisol activity. Finally, we examined the possibility that effects of aldosterone and cortisol on NHE in the placenta are gender specific.

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2. Methods 

2.1. Chemicals 

Ethanol, magnesium chloride, potassium chloride, potassium hydroxide, sodium chloride and sodium hydroxide were purchased from Merck (Poole, Dorset, UK.). Aldosterone was purchased from Fluka, supplied through Sigma (Poole, Dorset, UK.). Amiloride, ammonium chloride, carbenoxolone, choline (chloride), dimethyl sulfoxide (DMSO), glucose, cortisone, mifepristone, 3-N-(morpholino)propane-sulfonic acid (MOPS), nigericin, poly-l-lysine, spironolactone were purchased from Sigma (Poole, Dorset, UK.). 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF AM) was purchased from Molecular Probes, Cambridge Bioscience, Cambridgeshire, UK.

2.2. Tissue collection 

Placental tissue was obtained, with informed consent and the approval of the Local Research Ethics Committee, from patients admitted to the Central Delivery Unit at St Mary's Hospital, Manchester. Placentas were collected at term, following vaginal delivery or caesarean section (due to breech or for previous section), from women who had uncomplicated singleton pregnancies.

2.3. Tissue preparation and visualisation 

Samples of placental villous tissue were taken from midway between the chorionic and basal plate of the placenta and finely chopped to produce small fragments as described previously [6] and stored at 37 °C in Tyrode's buffer (pH 7.4) until experimentation for no longer than 4 h post partum [23].

Fragments were immobilised, loaded with the pH sensitive dye BCECF AM and NHE activity measured as the recovery from an acid load as described previously [6]. Briefly fragments were immobilised onto poly l-lysine (Sigma) coated glass cover slips and loaded with 1 μM BCECF at 37 °C, pH 7.4 for 5 min in control Tyrode's buffer (containing in mM; NaCl 135, KCl 5, CaCl2 1.8, MgCl2 1, MOPS 10 and glucose 5.6). BCECF loaded villous fragments were washed with BCECF-free Tyrode's buffer for 5 min prior to experimentation. The villous fragments were visualised using a Nikon TE300 inverted microscope and excited using light from a xenon arc lamp passed through an Optoscan Monochromator at 440 nm and 490 nm (Cairn Research Limited, Faversham, Kent, UK) and images for analysis acquired every 5 s using a C-Coolsnap HQ Cooled digital CCD camera (Roper Photometrics via Cairn Research Limited). Emission data at 530 nm from five selected areas of syncytiotrophoblast around a single terminal villous was acquired using MetaFlour software (Universal Imaging Corporation, Pennsylvania, USA) and analysed through Excel (Microsoft Corporation, Redmond, Washington, USA) and PRISM graphics packages (version 3.0, GraphPad Software Incorporated, San Diego, California).

2.4. Measurement of [pH]i in human placental syncytiotrophoblast 

Calibration of the BCECF fluorescence ratio (490:440 nm) to obtain [pH]i was performed using a high K+/nigericin (10μM) method as described previously in refs. [6], [24]. The activity of NHE was measured in intact syncytiotrophoblast by monitoring the recovery from an acid load imposed by an ammonium chloride (20 mM) pulse (Fig. 1); this is both Na+-dependent and blockable by amiloride [6], consistent with this being attributable to the activity of NHEs. Experiments were performed using control Tyrode's buffer, ammonium chloride Tyrode's buffer (containing in mM; NaCl 115, NH4Cl 20, KCl 5, CaCl2 1.8, MgCl2 1, MOPS 10, glucose 5.6), and Na+-free Tyrode's buffer (containing in mM; CholineCl 135, KCl 5, CaCl2 1.8, MgCl2 1, MOPS 10, glucose 5.6) at 37 °C and pH 7.4. The experimental protocol (see Fig. 1) involved a 5 min ammonium chloride pulse, followed by a 3 min washout of ammonium chloride in Na+-free Tyrode's buffer, followed by a 5 min recovery phase in the presence of Na+ (control Tyrode's buffer). Aldosterone, cortisol and carbenoxolone (an inhibitor of 11-β-HSD-2 [25]) used here only at 100 μM, a concentration shown previously to be maximally effective in both adult and fetal arteries [11], were dissolved in the Na+-free and control Tyrode's buffer so the tissue could be exposed to them during the Na+-free washout period and recovery phase (Fig. 1). Mifepristone (1 μM [26]), spironolactone (1 μM; [27]), 0.1% ethanol (vehicle for aldosterone, mifepristone and spironolactone) and DMSO (vehicle for BCECF and amiloride) were dissolved in the ammonium chloride, Na+-free and control Tyrode's buffer so the tissue could be exposed to the inhibitors prior to the steroid additions, during the Na+-free washout period and during the recovery phase (Fig. 1). Neither 0.1% ethanol nor DMSO alone had an effect on baseline pHi or NHE activity (data not shown). At the end of each phase of the protocol the bath solution change was completed within 20 s. The rate of recovery from acidification was quantified by fitting the initial portion (first 30 s) of the [pH]i recovery with a linear regression [6], [8] and the data expressed as pH recovery in pH units/s.

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  • Fig. 1 

    (A) Experimental protocol for the measurement of recovery from an acid load in the syncytiotrophoblast of the isolated human placental villous. All procedures were performed at 37 °C and arrows indicate solution changes. Tissue was loaded with BCECF (see methods) and incubated in control Tyrode's buffer then switched to Tyrode's buffer containing 20 mM NH4Cl (substituted for 20 mM NaCl) for 5 min. The incubating medium was then changed to a Na+-free solution (choline substituted for Na+) for 3 min before being returned to normal Na+ containing Tyrode's buffer. Compounds (aldo = aldosterone, cort = cortisol, carbenx = carbenoxolone) under investigation were added to the medium for the Na+ free period and the recovery phase when the tissue was returned to normal Na+ Tyrode's buffer as indicated by the bars above the experimental trace. MR = mineralocorticoid receptor, GR = glucocorticoid receptor (B) To calculate the initial recovery rate, a linear regression was applied to the first 30 s of the recovery phase post acid load and Na+ free period dashed line.

2.5. Statistics 

All data are expressed as mean ± SEM, n = number of placenta from which the fragments were isolated. Statistical analysis was by Student's t-test, paired or unpaired or one-way analysis of variance (ANOVA) as appropriate (see text) followed by Bonferroni multiple comparison test. Differences of p < 0.05 were considered significant.

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3. Results 

The basal [pH]i under control conditions for the experiments reported here was 7.36 ± 0.02 (n = 86), and the control rate of recovery from an acid load was 0.00483 ± 0.00026 pH units/s (n = 86). The basal [pH]i in placentas from male and female babies was not different (7.34 ± 0.03 (n = 46) compared with 7.37 ± 0.03 (n = 40) respectively). Fig. 1 is an example of the real time changes in [pH]i during the experimental protocol; there was no effect of gender on these changes. The agonists and antagonists had no effect on the magnitude of the alkalinisation imposed by the addition of NH4Cl or on the magnitude of the acid load elicited by the NH4Cl pulse (data not shown). The rate of recovery from an acid load was unaffected by mode of delivery (data not shown) and there was no difference in the rate of recovery from an acid load under control conditions in the placentas from male (0.00475 ± 0.00032 pH units/s, n = 46) compared to those from female (0.00493 ± 0.00044 pH units/s, n = 40) babies.

In investigating aldosterone effects, we initially analysed data pooled without consideration of the sex of the baby and used a 10 nM concentration, a concentration known to be effective in similar experiments in other tissues [11], [13] when used to investigate rapid activation of NHEs. At this concentration, aldosterone significantly increased the rate of pHi recovery from an acid load from 0.00504 ± 0.00052 to 0.00663 ± 0.00067 pH units/s (n = 23, p < 0.01 Paired Student's t-test). This effect was completely inhibited in the presence of amiloride (500 μM, n = 3, 0.00070 ± 0.00081 pH units/s; p < 0.05 Student's t-test) a non-specific inhibitor of NHE activity [28]. In further experiments we found that 1 nM (n = 3) aldosterone had no effect on pHi recovery and that 100 nM (n = 4) had no further effect than that seen at 10 nM (data not shown) and we therefore used 10 nM for all further studies. Considering the effect of gender there was no significant change in the rate of recovery from an acid load with aldosterone compared to control in tissue from male babies, but there was a significant increase in the rate of recovery from an acid load in tissue from female babies (Fig. 2). This stimulation of recovery from an acid load by aldosterone in female babies was inhibited by 1 μM spironolactone, but not by 1 μM mifepristone (Fig. 3).

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  • Fig. 2 

    Recovery of syncytiotrophoblast [pH]i following acid loading by a 20 mM NH4Cl pulse in the absence (control) or in the presence of aldosterone (10 nM). Open bars represent placenta from male babies, closed bars represent placenta from female babies and the bars are mean + SEM with n (in brackets) = number of placentas. *p < 0.05 versus control: paired Student's t-test.

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  • Fig. 3 

    Recovery of syncytiotrophoblast [pH]i in placentas from female babies following acid loading by a 20 mM NH4Cl pulse in the absence (control), or presence of aldosterone (10 nM) and also in the presence of spironolactone (spir, 1 μM) or mifepristone (mif, 1 μM). Bars are mean + SEM with n (in brackets) = number of placentas. *p < 0.05, **p < 0.01 versus control, #p < 0.05 versus aldosterone alone: ANOVA followed by Bonferroni's post hoc test.

In investigating cortisol effects, we also initially analysed data pooled without consideration of the sex of the baby. Neither cortisol (1 μM) nor carbenoxolone (100 μM) alone had an effect on the rate of pHi recovery by the syncytiotrophoblast from an acid load (Fig. 4). However when applied in combination these compounds significantly increased the rate of recovery from an acid load (Fig. 4). The recovery from an acid load in the presence of the combined dose of cortisol with carbenoxolone was completely inhibited in the presence of amiloride (cortisol with carbenoxolone 0.00656 ± 0.00074 pH units/s, compared to cortisol, carbenoxolone with amiloride 0.0002 ± 0.0009 pH units/s, n = 5). In further experiments we found that 100 nM cortisol (n = 3) had no effect on pHi recovery and that 10 μM (n = 3) had no further effect than that seen at 1 μM (data not shown) and we therefore used 1 μM cortisol for all further studies. Next, the data set was divided into that from placentas of male and female babies. There was no significant effect of combined cortisol and carbenoxolone on rate of recovery from an acid load in placentas from male babies but this combination markedly increased the rate of recovery from an acid load in placentas from female babies (Fig. 5). This effect could not be attributed to differences in mode of delivery. In preliminary experiments the effect of a range of concentrations of spironolactone on the cortisol/carbenoxolone stimulation in female syncytiotrophoblast was investigated. For comparison with the aldosterone stimulation (Fig. 4) we report the effect of 1 μM spironolactone (Fig. 6). The stimulation of recovery from an acid load by the combined application of cortisol and carbenoxolone in tissue from placentas of female babies was not reduced by spironolactone (1 μM); however in two further experiments (data not shown) 10 μM spironolactone completely abolished the cortisol and carbenoxolone stimulation. The cortisol and carbenoxolone stimulation was completely inhibited by mifepristone (1 μM) (Fig. 6).

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  • Fig. 4 

    Recovery of syncytiotrophoblast [pH]i following acid loading by a 20 mM NH4Cl pulse (a) in the absence (control) or in the presence of carbenoxolone (100 μM) or (b) cortisol (1 μM), or (c) combined application of cortisol (1 μM) and carbenoxolone (100 μM). Bars represent mean + SEM with n (in brackets) = number of placentas. *p < 0.05 versus control: paired Student's t-test.

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  • Fig. 5 

    Recovery of syncytiotrophoblast [pH] i following acid loading by a 20 mM NH4Cl pulse in the absence (control) or in the presence of a combined application of cortisol (1 μM) and carbenoxolone (100 μM). Open bars represent placenta from male babies, closed bars represent placenta from female babies and the bars are mean + SEM with n (in brackets) = number of placentas. *p < 0.05 versus control: paired Student's t-test comparing experiments performed on the same placentas (n = 9).

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  • Fig. 6 

    Recovery of syncytiotrophoblast [pH]i in placentas from female babies following acid loading by a 20 mM NH4Cl pulse in the absence (control), in the presence of a combined application of cortisol (1 μM) and carbenoxolone (100 μM) and also in the presence of spironolactone (spir, 1 μM) or mifepristone (mif, 1 μM). Bars are mean + SEM with n = 8 placentas. *p < 0.05 versus control, #P < 0.05 versus cortisol/carbenoxolone: ANOVA followed by Bonferroni's post hoc test.

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4. Discussion 

We have investigated the effect of short-term incubation of term placental tissue with corticosteroids on the activity of NHE (measured as the recovery of pHi from an acid load) in the syncytiotrophoblast. Our data show that under these experimental conditions, aldosterone and cortisol (in the presence of carbenoxolone) have a stimulatory effect on the activity of NHE but only in placentas from female babies. We assume the effects are non-genomic because of the acute nature of the experiments.

Using pharmacological inhibitors of either MR (spironolactone) or GR (mifepristone) we have shown that the increase in the activity of NHE in the presence of aldosterone was modulated by MR which are expressed in the syncytiotrophoblast [17]. The observation that exogenous cortisol only stimulated NHE activity in the presence of carbenoxolone (used at concentrations previously reported by others to block 11-β-HSD-2 [29]) suggests that placental 11-β-HSD-2 activity is normally great enough to metabolise high concentrations of this steroid. In regard to receptors, the effect of the combination of cortisol/carbenoxolone on NHE activity was clearly blocked by the GR inhibitor mifepristone. Spironolactone at 1 μM did show a trend towards partially reducing the effect of the combined application of cortisol/carbenoxolone (Fig. 6) and in a small set of further experiments 10 μM abolished the cortisol/carbenoxolone stimulation (data not shown). These data suggest that, in the placenta, cortisol modulates NHE activity predominantly through GR but at least a portion of the response could be due to interaction with MR.

In a number of human and rodent epithelial cells and cell lines, the rapid effect of corticosteroids on NHE activity has been investigated [12], [13]. This rapid response was not via protein synthesis routes as cyclohexamide, an inhibitor of protein transcription, did not affect the response nor was the response blocked by the MR (spironolactone) or the GR inhibitor RU486 (mifepristone). In the cortical collecting duct cell line M-1, the stimulation of NHE activity by aldosterone was inhibited by chelerythrine-chloride (an inhibitor of PKC activity) as was the aldosterone activation of ERK 1/2 [12]. The effect of aldosterone was also attenuated by the MAPK inhibitor PD98059, suggesting that the rapid activation of NHE by aldosterone in this cell line was via a PKC/MAPK pathway that functions independently of the classical (genomic) MR.

In contrast, there are recent suggestions of aldosterone evoking an acute response through the classical MR including a rapid phosphorylation of ERK1/2 and JNK1/2 proteins [30]. In other non-placental tissues there are complex expression patterns of a variety of GR isoforms [31], [32] along with the transient attachment of GR to the membrane bound G protein β subunit [33]. Therefore complex interactions with GR isoforms and the possibility of interaction with membrane bound proteins may be responsible for the rapid non-genomic response of NHE to activation by cortisol and aldosterone in the placental syncytiotrophoblast. The mechanism of acute effects of corticosteroids through MR and GR which we observe here in syncytiotrophoblast now need further characterization.

Because previous studies had shown differential expression of MR and GR and 11-β-HSD-2 in placentas from male and female babies, we examined possible gender effects in our data set. We found that the effect of aldosterone and cortisol on NHE activity was only observed in placentas from female babies. The reasons for this gender effect on the activity of NHE require further investigation. However, it is known that the expression of MR, GR and 11-β-HSD-2 mRNA in the placenta at term is dependent on gender [22]. Placentas from female babies have a greater activity of 11-β-HSD-2 than those from males and, consistent with this, there is a tendency towards lower fetal plasma cortisol in girls [34], although gender effects on aldosterone circulating concentrations are unclear. This higher activity of 11-β-HSD-2 in placentas from female babies may mean that GR and MR are normally more protected from the effects of cortisol in vivo and so become more sensitive, in terms of NHE activity, when the enzyme activity is blocked in vitro as in our experiments here. By contrast, placentas from male babies were not responsive to exposure to aldosterone or cortisol, in terms of NHE activity; perhaps there is downregulation of MR and GR following more continuous cortisol exposure in vivo. Interestingly, there are increasing numbers of reports which suggest gender differences in handling of many ions and drugs by membrane transporters, both under normal physiological and pathological conditions [35].

Circulating levels of cortisol [36] and aldosterone [37] comparable to those utlised in these experiments have been reported during pregnancy. Human fetuses are exposed to excess glucocorticoids when their mothers are stressed [38], through therapeutic use in preterm labour [39] and when there are alterations in metabolism [40]. In normal pregnancies fetal cortisol is expressed in first trimester and increases throughout gestation however at term fetal cortisol levels [41] are much lower than maternal [42], [43], [44]. In adults cortisol and aldosterone have a role in the regulation of fluid balance through action on the kidney. The function of the fetal kidney is to maintain amniotic fluid electrolyte balance [45] and, as it does not play a major part in fetal electrolyte balance, it is possible that cortisol and aldosterone play a role in regulating electrolyte transfer across the placenta and therefore fetal fluid balance.

Altering the ability of a cell to maintain its intracellular pH can have a considerable number of consequent effects on other processes such as cell death and division and modulation of transporter function. We have recently shown that dexamethasone can directly increase System A transport into term placental explants [46]. In addition cortisol stimulates the expression of SNAT2, expression and System A transport in a human placental cell line (BeWo) [47]. Interestingly, knockout of 11-β-HSD-2 results in an upregulation of System A transport by the mouse placenta [48]. The System A amino acid transporter, intimately involved in the provision of short chain amino acids by the human placenta for fetal growth [49], is highly pH sensitive [50], [51]. It will be interesting to examine, in future experiments, how the modulation of NHE by corticosteroids impacts on synctriotrophoblast functions in this regard.

In conclusion, as with other transporting epithelia, acute exposure of the human placental syncytiotrophoblast to aldosterone and cortisol stimulates NHE activity. This rapid, non-genomic NHE activation occurs via pathways which are fetal sex dependent and which involve the classical MR and/or GR.

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Acknowledgements 

This work was funded by The Wellcome Trust with infrastructure support from the Manchester NIHR Biomedical Research Centre. We would like to thank the midwives of the Central Delivery Unit of St Mary's Hospital, Manchester, for their help in obtaining normal placentas.

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PII: S0143-4004(10)00003-2

doi:10.1016/j.placenta.2009.12.025

Placenta
Volume 31, Issue 4 , Pages 289-294, April 2010

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