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The outer epithelial cell layer of human placenta, the syncytiotrophoblast, is a specialised terminally differentiated multinucleate tissue. It is generated and renewed from underlying cytotrophoblast cells that undergo proliferation, differentiation and fusion with syncytiotrophoblast. Acquisition of fresh cellular components is thought to be balanced by apoptosis and shedding of aged nuclei. This process of trophoblast cell turnover maintains a functional syncytiotrophoblast, capable of sufficient nutrient transfer from mother to foetus. Foetal growth restriction (FGR) is a pregnancy complication associated with aberrant trophoblast turnover and reduced activity of certain amino acid transporters, including the taurine transporter (TauT). Taurine is the most abundant amino acid in human placenta implying an important physiological role within this tissue. Unlike other amino acids, taurine is not incorporated into proteins and in non-placental cell types represents an important osmolyte involved in cell volume regulation, and is also cytoprotective. Here, we investigated the role of taurine in trophoblast turnover using RNA interference to deplete primary human trophoblast cells of TauT and reduce intracellular taurine content. Trophoblast differentiation was compromised in TauT-deficient cells, and susceptibility of these cells to an inflammatory cytokine that is elevated in FGR was increased, evidenced by elevated levels of apoptosis. These data suggest an important role for taurine in trophoblast turnover and cytoprotection.

Na(+)/H(+) exchanger (NHE) activity regulates intracellular pH (pH(i)) 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 muM BCECF (pH sensitive fluorescent dye) and the syncytiotrophoblast acidified with a pre-pulse of 20 mM NH(4)Cl. The Na(+)-dependent recovery of pH(i) 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 pH(i) 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 microM) but not the glucocorticoid antagonist mifepristone (1 microM). There was no effect on the rate of recovery from an acid load in villi from placenta from male babies. Alone, neither cortisol (1 microM, n = 5) nor carbenoxolone (100 microM, n = 9), an inhibitor of 11-beta-hydroxysteroid dehydrogenase-2 (11-beta-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-beta-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., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Synthetic glucocorticoids (GCs) are given to women with threatened preterm labour but their administration has been linked to reduced infant birthweight. The underlying mechanisms are unknown, but impaired placental development and/or function has been implicated. The activity of the system A amino acid transporter is decreased in placentas from pregnancies complicated by fetal growth restriction. Whether GCs adversely affect placental amino acid transport is unknown. The objective of this study was to determine the regulatory effects of GCs on system A transport using a human in vitro placental explant model. Term explants (n=7) were treated with dexamethasone (DEX 10(-8)M or 10(-6)M) or vehicle for 48 h. System A activity was measured by the uptake of (14)C-N-methylated aminoisobutyric acid by explants. Explants were also processed for electron microscopy (EM), immunohistochemistry, and qRT-PCR. Lactate dehydrogenase (LDH), human chorionic gonadotropin (hCG) and human placental lactogen (hPL) release into the culture medium was measured. DEX (10(-6)M) stimulated system A activity compared to vehicle (p<0.05). System A transporter proteins were localized to the newly regenerating syncytiotrophoblast layer, but mRNA levels were unchanged with DEX treatment. DEX did not adversely affect explant viability as determined by reduced LDH release (p<0.05). DEX treatment was associated with morphological (accelerated apical microvilli formation, nuclear maturation, and increased cell organelle number) and functional (elevated hCG secretion, increased 11beta-HSD2 mRNA expression and reduced cytotrophoblast proliferation (p<0.05 for all)) markers of syncytiotrophoblast differentiation. These findings suggest that DEX stimulates system A activity and promotes syncytiotrophoblast differentiation and maturation., (2009 Elsevier Ltd. All rights reserved.)

Recent studies show that apoptosis, programmed cell death, plays an important role in the normal development of the human placenta and that an altered balance between proliferation and apoptosis of villous trophoblasts is associated with abnormal pregnancies. The TNF-related apoptosis-inducing ligand (TRAIL) is a molecule belonging to TNF superfamily. The role of TRAIL and its Death Receptor 5 (DR5) in regulating villous trophoblast cell turnover in normal and pathologic pregnancies remains to be explored. In order to elucidate the role of TRAIL in the regulation of placental growth, primary cytotrophoblast cells were isolated from normal term placentas (n=13) and cultured for 18 and 66h to generate mononucleate and multinucleate trophoblasts, respectively. The protein expression and localisation of TRAIL and DR5 were determined by Western blotting and immunofluorescence. Secreted sTRAIL was also measured by ELISA. Trophoblast apoptosis was measured by TUNEL in the presence of recombinant TRAIL (rTRAIL), and DR5 relocalisation was assessed by immunostaining after 18h exposure to TNFalpha. We demonstrated that TRAIL protein expression and the secretion of soluble TRAIL (sTRAIL) were down-regulated in syncytialised villous trophoblasts and that sTRAIL was independent of biochemical differentiation, as TRAIL-neutralizing antibody (2E5) failed to influence hCG production. TRAIL immunoreactivity was detected in mono- and multinucleated trophoblast cells and localised to the cytoplasm and cellular membranes -- more intense staining was associated with apoptotic nuclei. rTRAIL failed to induce apoptosis in trophoblasts cells owing to the nuclear localisation of DR5. However, TNFalpha treatment caused the redistribution of intracellular DR5 to the cell surface, potentiating apoptotic susceptibly to exogenously administered rTRAIL. These findings highlight a mechanism by which TRAIL and DR5 serve to protective trophoblasts in normal development, but may be activated in conditions of excessive TNFalpha.

Maintaining placental syncytiotrophoblast, a specialized multinucleated transport epithelium, is essential for normal human pregnancy. Syncytiotrophoblast continuously renews through differentiation and fusion of cytotrophoblast cells, under paracrine control by syncytiotrophoblast production of human chorionic gonadotropin (hCG). We hypothesized that K(+) channels participate in trophoblast syncytialization and hCG secretion in vitro. Two models of normal-term placenta were used: 1) isolated cytotrophoblast cells and 2) villous tissue in explant culture. Cells and explants were treated with K(+) channel modulators from 18 h, and day 3, onward, respectively. Culture medium was analyzed for hCG, to assess secretion, as well as for lactate dehydrogenase (LDH), to indicate cell/tissue integrity. hCG was also measured in cytotrophoblast cell lysates, indicating cellular production. Syncytialization of cytotrophoblast cells was assessed by immunofluorescent staining of desmosomes and nuclei. Over 18-66 h, mononucleate cells fused to form multinucleated syncytia, accompanied by a 28-fold rise in hCG secretion. 1 mM Ba(2+) stimulated cytotrophoblast cell hCG secretion at 66 h compared with control, whereas 5 mM tetraethylammonium (TEA) inhibited hCG secretion by >90%. 0.1-1 mM 4-aminopyridine (4-AP) reduced cytotrophoblast cell hCG secretion and elevated cellular hCG; without altering cellular integrity or syncytialization. In villous explants, hCG secretion was not altered by 1 mM Ba(2+) but inhibited by 5 mM 4-AP and 5/10 mM TEA, without affecting LDH release. Anandamide, pinacidil, and cromakalim were without effect in either model. In conclusion, 4-AP- and TEA-sensitive K(+) channels (e.g., voltage-gated and Ca(2+)-activated) regulate trophoblast hCG secretion in culture. If these K(+) channels participate in hCG secretion in situ, they may regulate trophoblast turnover in health and disease.

Changes in tissue architecture and ultrastructure in the cat placenta during long-term explant culture have been examined over an 11-day period. Pieces of cat placenta, dissected from the lamellar region, were cultured in CMRL-1066 medium and tissue was fixed for electron microscopy at 2, 5, 7, and 11 days' culture, as well as before culture was initiated (day 0). Four samples were examined at each time point. After two days, the trophoblast basal lamina and attached cytotrophoblast cells had begun to separate from the syncytium and the cytotrophoblasts were spreading over the surface of the exposed fetal stromal compartment, and by five days were showing signs of growth. At seven days' culture, cytotrophoblast multilayering was common, and vascular and stromal components were also well preserved with collagen biosynthesis evident. By 11 days, the centre of the culture was compacted and degenerate with loss of tissue architecture, but on the outside polyp-like growths could be seen, with a well-developed covering of trophoblast containing fat and secretory droplets, lining a connective tissue matrix and stromal components. The cat placenta, therefore, like the human, has the capacity for regrowth in explant culture, including both trophoblast and stromal components.

The purpose of this study was to compare Na+/H+ exchanger (NHE) activity in the microvillous (MVM) and basal (BM) plasma membrane of the human placental syncytiotrophoblast and to determine the relative contribution of various NHE isoforms to this activity. Uptake of 22Na into isolated MVM vesicles in the presence of a H+ gradient, at initial rate, was four- to fivefold higher than that by BM vesicles (214+/-28 vs. 49+/-9 pmol/mg protein per 30 s, respectively, means+/-SEM, n=8, 6, P<0.001). The 22Na uptake by MVM, but not by BM, was reduced in the absence of a H+ gradient and in the presence of 500 microM amiloride. To determine the contribution of NHE1, NHE2 and NHE3 isoforms to NHE activity in MVM, we investigated the effect of amiloride analogues which show isoform selectivity. HOE 694, an analogue selective for NHE1 at low concentrations, inhibited 22Na uptake with an EC50 of 0.13+/-0.05 microM (n=6), whereas S3226, an analogue selective for NHE3 at low concentrations had an EC50 of 3.01+/-0.85 microM (n=5). To investigate this further, we measured recovery of syncytiotrophoblast intracellular pH (pHi) from an acid load using a H+-selective, fluorescent dye (BCECF) loaded into isolated intact placental fragments. This recovery was blocked in the absence of Na+ and the presence of amiloride (500 microM) and concentrations of HOE 694 and S3226 were comparable to those used in vesicle experiments. Overall these data show that under the conditions used NHE activity in the term placental syncytiotrophoblast is absent from BM. NHE activity in the MVM is attributable predominantly to NHE1.

We localized alkaline phosphatase and plasma membrane calcium-ATPase (PMCA) in the cat placental syncytiotrophoblast to address their polarized distribution and their potential as markers for specific plasma membrane purification. We used enzyme- (alkaline phosphatase) and immuno- (PMCA) histochemistry and, for alkaline phosphatase, compared data to observations on the human placenta. Alkaline phosphatase activity in the cat was localized to the decidual cell membranes, to within the associated interstitial space and on the subjacent apical (maternal facing) plasma membrane of the syncytiotrophoblast. Occasional maternal capillaries were positive on their basal surface and there was focal staining within the syncytiotrophoblast. This widespread distribution is less specific than in the human placenta where alkaline phosphatase was restricted to the apical and basal plasma syncytiotrophoblast membranes, with much greater density on the apical membrane. Expression of PMCA in the cat was restricted to the basal membrane of the syncytiotrophoblast only. This specific localization of PMCA is identical to the human placenta and all other species in which its placental localization has been studied. We conclude that the plasma membranes of the cat syncytiotrophoblast show a broadly similar functional polarization to the human and that PMCA would prove a useful marker in isolation of the cat syncytiotrophoblast basal plasma membrane.

The aim of this study was to determine whether extracellular ATP ([ATP](o)) stimulated a Ca(2+)-activated K(+) efflux in trophoblast cells that was dependent on extracellular Ca(2+) ([Ca(2+)](o)). Cytotrophoblast cells, isolated from human placenta, were examined following 18 h (relatively undifferentiated) and 66 h (multinucleate cells) of culture. Potassium efflux was measured using (86)Rb as a trace marker. Intracellular Ca(2+) ([Ca(2+)](i)) was examined by microfluorometry using fura 2. [ATP](o) significantly increased (86)Rb efflux to a peak that declined to control (18-h cells) or an elevated plateau (66-h cells) and was inhibited by 100 nM charybdotoxin. Removing [Ca(2+)](o) significantly reduced (86)Rb efflux in both groups as did application of 150 microM GdCl(3). [ATP](o) significantly increased [Ca(2+)](i) in both groups of cells. The response was reduced by removing [Ca(2+)](o) and applying 150 microM GdCl(3). For both (86)Rb efflux and microfluorometry experiments, the response to [ATP](o) was more dependent on [Ca(2+)](o) in 66-h cells compared with 18-h cells (approximately 70% greater). Cytotrophoblast cells exhibit an [ATP](o)-stimulated Ca(2+)-activated K(+) efflux. The dependency of this pathway on [Ca(2+)](o) is greater in the 66-h multinucleate syncytiotrophoblast-like cells, suggesting that the mechanism for Ca(2+) entry may be altered during differentiation of trophoblast cells.

Ion transport is important for driving nutrient transport across the syncytiotrophoblast and yet is poorly understood. We have examined K(+)currents under basal conditions in cultured cytotrophoblast cells, at various stages of differentiation, using the whole cell patch clamp technique. Cytotrophoblast cells were isolated from human term placenta and maintained in culture for up to 3 days. Cells were studied at four stages of progressive morphological differentiation: (i) mononuclear cells, (ii) mononuclear cells in aggregates, (iii) small multinucleate cells and (iv) large multinucleate syncytiotrophoblast-like cells. In the conditions of whole cell recording the only K(+) selective current identified in all cell types was a strong inwardly rectifying current which was sensitive to Ba(2+) and Cs(+). This current was unaffected by intracellular ATP whereas intracellular GTPgammas caused either run down of the current or activated a linear current. The characteristics of the current described are consistent with those of the inwardly rectifying K(+) channel Kir2.1. The inwardly rectifying K(+) current was observed in three out of 19 (16 per cent ) mononuclear cells, seven out of 21 (33 per cent ) mononuclear aggregates, eight out of 21 (38 per cent ) small multinucleate cells and 16 out of 19 (84 per cent ) large multinucleate cells. This inwardly rectifying K(+) current is likely to have an important role in determining net K(+) diffusion across the syncytiotrophoblast cell membrane, perhaps increasing in importance as the cells terminally differentiate., (Copyright 2001 Harcourt Publishers Ltd.)

We have investigated the functional characteristics of term human placental villous explants kept in long-term (7-11 days) culture. Fragments of placental villous tissue (approximately 5-10 mg wet wt) were cultured in supplemented CMRL-1066 culture medium for up to 11 days. After the first day of culture, the syncytiotrophoblast appeared vacuolated and eventually degenerated. However, a new syncytiotrophoblast developed by day 4, being indistinguishable from that of a fresh placenta by 11 days. Release of human chorionic gonadotrophin increased and activity of lactate dehydrogenase in culture medium decreased with culture time. Transport variables were measured over the first 7 days of culture. Basal (86)Rb efflux was reduced with time in culture and was inhibited by Ba2+, suggesting the efflux was mediated by K+ channels. At all stages of culture, (86)Rb efflux was stimulated by ATP, hyposmotic medium, and ANG II. A complex pattern of efflux changes with culture time and type of stimulator was observed, suggesting that several compartments of the tissue contributed to stimulated efflux. This culture system provides opportunities for studies of chronic regulation of placental function.

The regulation of placental anion transport influences fetal accretion and placental homeostasis. We investigated whether efflux of 125I- or 36Cl- from multinucleated cytotrophoblast cells derived from human term placenta is regulated by one of three stimuli: (a) the calcium ionophore A23187, (b) a 'cocktail' of agents designed to raise intracellular levels of cAMP, (c) a hyposmotic solution. After loading with the appropriate isotope for 2 h and thorough washing, cells were exposed to sequential aliquots of buffer applied and removed each minute. Following an equilibration period of 5 min one of the stimuli was applied at room temperature At the end of the experiment the cells were lysed to give a lysate count which was used to express the count obtained from each aliquot as percentage efflux of that possible for that minute. The cAMP 'cocktail' and A23187 were applied for 5 min; the hyposmotic solution was applied for 10 min. The results for 125I- at 7 min showed that the mean efflux in the presence of hyposmotic shock was greater than control (5.7 +/- 1.0% min-1 versus 2.2 +/- 0.1% min-1, respectively; mean +/- S.E.M., n = 4 placentas). Similarly mean efflux at 6 min in the presence of A23187 was also significantly greater than control (6.5 +/- 1.9% min-1 versus 2.6 +/- 1.0% min-1, respectively, n = 3 placentas). The mean efflux in the presence of the cAMP cocktail was not different from control at any time point. The results were qualitatively the same if 36Cl- was used in the place of 125I- and when the experiment was performed with 36Cl- in a HCO3- buffer gassed with CO2. Mean 125I- efflux at 6 min in response to hyposmotic challenge was 33% less (P < 0.01) in the presence of 1 mM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 37% less (P < 0.005) in the presence of 10 microM tamoxifen but no different if the hyposmotic solution was nominally calcium free. We conclude that there are differential effects of second messengers on anion efflux from the differentiated cytotrophoblast cells.

The aim of this study was to investigate whether the Kir2.1 gene is expressed by the human placenta throughout pregnancy and in cytotrophoblast cells at different stages of differentiation in culture. RNA was extracted from cytotrophoblast cells isolated from term placentas and maintained in culture for 18, 66 and 114 h and from first, second and third trimester placentas. Using the reverse transcriptase-polymerase chain reaction (RT-PCR) with gene-specific primers, a cDNA product of 1.2 kb, as expected for Kir2.1 gene expression, was detected in all the RNA samples from cytotrophoblast cells and from placentas. The RT-PCR products were verified by sequencing and by detection of the expected transcript size for the Kir2.1 mRNA at 5.6-5.7 kb on Northern blots, using the 1.2 kb cDNA generated by RT-PCR. Northern blot quantification, using a control 28S rRNA probe, showed no significant difference in Kir2.1 mRNA expression between any of the three stages of cytotrophoblast cell differentiation studied (ANOVA; n = 3 RNA samples from each stage). These data demonstrate that the Kir2.1 gene is expressed by the human placenta and, specifically, by cytotrophoblast cells, at all stages of development and differentiation.

1. To determine whether there is a change during differentiation, the activity and expression of Na(+)-K(+)-ATPase were studied in mononucleate cytotrophoblast cells (18 h culture) and syncytiotrophoblast-like cells (66 h culture). A choriocarcinoma-derived cell line (JAr) which, unlike the cytotrophoblast cells, divides in culture, was also studied for comparison. 2. Na(+)-K(+)-ATPase activity was assessed by measurement of ouabain-sensitive 86Rb+ uptake. Na(+)-K(+)-ATPase expression was determined by (i) measurement of [3H]ouabain binding and (ii) Northern hybridization to measure expression of alpha-1 and beta 1-subunit mRNA. 3. There was no significant difference in either activity or expression of Na(+)-K(+)-ATPase during differentiation of cytotrophoblast cells. However, expression of alpha 1- and beta 1-subunit mRNA was significantly lower in 66 vs. 18 h cultured cytotrophoblast cells. 4. Both Na(+)-K(+)-ATPase activity and [3H]ouabain binding was significantly greater in JAr cells than either cytotrophoblast cell groups, although expression of alpha 1- and beta 1-subunit mRNA was the same as cytotrophoblast cells cultured for 18 h. 5. It is concluded that N(+)-K(+)-ATPase activity and protein expression does not change during differentiation of cytotrophoblast cells but that there are changes in expression at the transcriptional or post-transcriptional level.

Whole-cell patch clamp experiments were performed on cultured human cytotrophoblast cells incubated for 24-48 hr after their isolation from term placentas. Cl--selective currents were examined using K+-free solutions. Under nonstimulated conditions, most cells initially expressed only small background leak currents. However, inclusion of 0.2 mM GTPgammaS in the electrode solution caused activation of an outwardly rectifying conductance which showed marked time-dependent activation at depolarized potentials above +20 mV. Stimulation of this conductance by GTPgammaS was found to be Ca2+-dependent since GTPgammaS failed to activate currents when included in a Ca2+-free electrode solution. In addition, similar currents could be activated by increasing the [Ca2+] of the pipette solution to 500 nM. The Ca2+-activated conductance was judged to be Cl--selective, since reversal potentials were predicted by Nernst equilibrium potentials for Cl-. This conductance could also be reversibly inhibited by addition of the anion channel blocker DIDS to the bath solution at a dose of 100 microM. Preliminary experiments indicated the presence of a second whole-cell anion conductance in human cytotrophoblast cells, which may be activated by cell swelling. Possible roles for the Ca2+-activated Cl- conductance in human placental trophoblast are discussed.

1. The electrochemical gradients for Na+ and K+ were assessed in a cell culture model of trophoblast differentiation. 2. Membrane potential difference (Em), intracellular water and Na+ and K+ contents were measured in choriocarcinoma cells (JAr cell line; 96% of which are undifferentiated trophoblast cells) and in mononucleate and multinucleate (differentiated) cytotrophoblast cells isolated from the human placenta at term. 3. There was a significant fall in Em from -57 mV in JAr cells, to -48 and -40 mV in mono-and multinucleate cytotrophoblast cells, respectively. Treatment with ouabain (1 mM for 15 min) depolarized the JAr cell membrane by 15 mV but did not affect cytotrophoblast cell membrane potential. 4. Intracellular K+ concentration was similar in JAr, mono- and multinucleate cytotrophoblast cells but Na+ concentration was higher in mononucleate cytotrophoblast cells compared with JAr cells. 5. Ouabain treatment (3 mM for 15 min) caused a small increase (4.5%) in cell water in mononucleate cytotrophoblast cells but lowered K+ (approximately 30%) and increased Na+ concentration (approximately 125%) in all the trophoblast cells studied. 6. The K+ equilibrium potential (EK) was more negative than Em in all cells and the difference between EK and Em was smaller in JAr cells (-25 mV) than in mono- and multinucleate cytotrophoblast cells (-33 and -43 mV, respectively). 7. The Na+ equilibrium potential (ENa) was positive in the trophoblast cells and the difference between ENa and Em was 122, 100 and 100 mV in JAr, mono- and multinucleate cytotrophoblast cells, respectively. 8. These results suggest that the electrochemical gradient for K+ is affected by the stage of trophoblast cell differentiation. In contrast, the electrochemical gradient for Na+ is similar in mono- and multinucleate cytotrophoblast cells.

Single mature intermediate villi from term placentas after normal gestation and vaginal or cesarian delivery were identified microscopically and mounted in a bath for conventional micro-electrode studies. With the application of strict selection criteria, the following observations on electrical potential difference (PD) were made. 1) With the tissue bathed in Earle's medium (37 degrees C) the PD across the syncytiotrophoblast microvillus membrane with respect to the bath was not normally distributed. The median PD was -22 mV (range -12 to -60 mV, n = 200). This fell to -6 mV after prior incubation for 4 h with cyanide (3 mM) or iodoacetate (2 mM) but was not altered by short-term application of these agents or by 0.1 mM ouabain. Substituting Na+ in Earle's medium with choline had no effect on PD, but replacing Cl- with gluconate caused a depolarization of 6 mV (P < 0.002). Increasing KCl in the bath fluid revealed an apparently low microvillus membrane K+ conductance. The low microvillus membrane PD may reflect a low Na(+)-K(+)-ATPase activity and/or a low membrane permeability to K+. 2) The transtrophoblast PD measured by insertion and withdrawal of the electrode was significantly different from zero [P < 0.003, median -3 mV (range 0 to -15 mV), n = 11]; and PD measured by insertion of the electrode into the villus core and beyond was -6 mV (significantly different from zero P < 0.003, range -2.5 to -10 mV, n = 6). If a similar PD were to exist in vivo, it could have a significant influence on ion transport across the placenta at term.