Poly(I:C) source, molecular weight and endotoxin contamination affect dam and prenatal outcomes, implications for models of maternal immune activation.

The viral mimetic polyinosinic:polycytidylic acid (poly(I:C)) is increasingly used to induce maternal immune activation (mIA) to model neurodevelopmental disorders (NDDs). Robust and reproducible phenotypes across studies are essential for the generation of models that will enhance our understanding of NDDs and enable the development of improved therapeutic strategies. However, differences in mIA-induced phenotypes using poly(I:C) have been widely observed, and this has prompted the reporting of useful and much needed methodological guidelines. Here, we perform a detailed investigation of molecular weight and endotoxin variations in poly(I:C) procured from two of the most commonly used suppliers, Sigma and InvivoGen. We demonstrate that endotoxin contamination and molecular weight differences in poly(I:C) composition lead to considerable variability in maternal IL-6 response in rats treated on gestational day (GD)15 and impact on fetal outcomes. Specifically, both endotoxin contamination and molecular weight predicted reductions in litter size on GD21. Further, molecular weight predicted a reduction in placental weight at GD21. While fetal body weight at GD21 was not affected by poly(I:C) treatment, male fetal brain weight was significantly reduced by poly(I:C), dependent on supplier. Our data are in agreement with recent reports of the importance of poly(I:C) molecular weight, and extend this work to demonstrate a key role of endotoxin on relevant phenotypic outcomes. We recommend that the source and batch numbers of poly(I:C) used should always be stated and that molecular weight variability and endotoxin contamination should be minimised for more robust mIA modelling.

Computational modelling of placental amino acid transfer as an integrated system.

Placental amino acid transfer is essential for fetal development and its impairment is associated with poor fetal growth. Amino acid transfer is mediated by a broad array of specific plasma membrane transporters with overlapping substrate specificity. However, it is not fully understood how these different transporters work together to mediate net flux across the placenta. Therefore the aim of this study was to develop a new computational model to describe how human placental amino acid transfer functions as an integrated system. Amino acid transfer from mother to fetus requires transport across the two plasma membranes of the placental syncytiotrophoblast, each of which contains a distinct complement of transporter proteins. A compartmental modelling approach was combined with a carrier based modelling framework to represent the kinetics of the individual accumulative, exchange and facilitative classes of transporters on each plasma membrane. The model successfully captured the principal features of transplacental transfer. Modelling results clearly demonstrate how modulating transporter activity and conditions such as phenylketonuria, can increase the transfer of certain groups of amino acids, but that this comes at the cost of decreasing the transfer of others, which has implications for developing clinical treatment options in the placenta and other transporting epithelia.

Phenylalanine transfer across the isolated perfused human placenta: an experimental and modeling investigation.

Membrane transporters are considered essential for placental amino acid transfer, but the contribution of other factors, such as blood flow and metabolism, is poorly defined. In this study we combine experimental and modeling approaches to understand the determinants of [(14)C]phenylalanine transfer across the isolated perfused human placenta. Transfer of [(14)C]phenylalanine across the isolated perfused human placenta was determined at different maternal and fetal flow rates. Maternal flow rate was set at 10, 14, and 18 ml/min for 1 h each. At each maternal flow rate, fetal flow rates were set at 3, 6, and 9 ml/min for 20 min each. Appearance of [(14)C]phenylalanine was measured in the maternal and fetal venous exudates. Computational modeling of phenylalanine transfer was undertaken to allow comparison of the experimental data with predicted phenylalanine uptake and transfer under different initial assumptions. Placental uptake (mol/min) of [(14)C]phenylalanine increased with maternal, but not fetal, flow. Delivery (mol/min) of [(14)C]phenylalanine to the fetal circulation was not associated with fetal or maternal flow. The absence of a relationship between placental phenylalanine uptake and net flux of phenylalanine to the fetal circulation suggests that factors other than flow or transporter-mediated uptake are important determinants of phenylalanine transfer. These observations could be explained by tight regulation of free amino acid levels within the placenta or properties of the facilitated transporters mediating phenylalanine transport. We suggest that amino acid metabolism, primarily incorporation into protein, is controlling free amino acid levels and, thus, placental transfer.

Maternal obesity is associated with a reduction in placental taurine transporter activity.

BACKGROUND/OBJECTIVES: Maternal obesity increases the risk of poor pregnancy outcome including stillbirth, pre-eclampsia, fetal growth restriction and fetal overgrowth. These pregnancy complications are associated with dysfunctional syncytiotrophoblast, the transporting epithelium of the human placenta. Taurine, a ß-amino acid with antioxidant and cytoprotective properties, has a role in syncytiotrophoblast development and function and is required for fetal growth and organ development. Taurine is conditionally essential in pregnancy and fetal tissues depend on uptake of taurine from maternal blood. We tested the hypothesis that taurine uptake into placental syncytiotrophoblast by the taurine transporter protein (TauT) is lower in obese women (body mass index (BMI)⩾30 kg m(-)(2)) than in women of ideal weight (BMI 18.5-24.9 kg m(-)(2)) and explored potential regulatory factors. SUBJECTS/METHODS: Placentas were collected from term (37-42-week gestation), uncomplicated, singleton pregnancies from women with BMI 19-49 kg m(-)(2). TauT activity was measured as the Na(+)-dependent uptake of (3)H-taurine into placental villous fragments. TauT expression in membrane-enriched placental samples was investigated by western blot. In vitro studies using placental villous explants examined whether leptin or IL-6, adipokines/cytokines that are elevated in maternal obesity, regulates TauT activity. RESULTS: Placental TauT activity was significantly lower in obese women (BMI⩾30) than women of ideal weight (P<0.03) and inversely related to maternal BMI (19-49 kg m(-)(2); P<0.05; n=61). There was no difference in TauT expression between placentas of ideal weight and obese class III (BMI⩾40) subjects. Long-term exposure (48 h) of placental villous explants to leptin or IL-6 did not affect TauT activity. CONCLUSIONS: Placental TauT activity at term is negatively related to maternal BMI. We propose that the reduction in placental TauT activity in maternal obesity could lower syncytiotrophoblast taurine concentration, compromise placental development and function, and reduce the driving force for taurine efflux to the fetus, thereby increasing the risk of poor pregnancy outcome.

Computational modelling of amino acid exchange and facilitated transport in placental membrane vesicles.

Placental amino acid transport is required for fetal development and impaired transport has been associated with poor fetal growth. It is well known that placental amino acid transport is mediated by a broad array of specific membrane transporters with overlapping substrate specificity. However, it is not fully understood how these transporters function, both individually and as an integrated system. We propose that mathematical modelling could help in further elucidating the underlying mechanisms of how these transporters mediate placental amino acid transport. The aim of this work is to model the sodium independent transport of serine, which has been assumed to follow an obligatory exchange mechanism. However, previous amino acid uptake experiments in human placental microvillous plasma membrane vesicles have persistently produced results that are seemingly incompatible with such a mechanism; i.e. transport has been observed under zero-trans conditions, in the absence of internal substrates inside the vesicles to drive exchange. This observation raises two alternative hypotheses; (i) either exchange is not fully obligatory, or (ii) exchange is indeed obligatory, but an unforeseen initial concentration of amino acid substrate is present within the vesicle which could drive exchange. To investigate these possibilities, a mathematical model for tracer uptake was developed based on carrier mediated transport, which can represent either facilitated diffusion or obligatory exchange (also referred to as uniport and antiport mechanisms, respectively). In vitro measurements of serine uptake by placental microvillous membrane vesicles were carried out and the model applied to interpret the results based on the measured apparent Michaelis-Menten parameters Km and Vmax. In addition, based on model predictions, a new time series experiment was implemented to distinguish the hypothesised transporter mechanisms. Analysis of the results indicated the presence of a facilitated transport component, while based on the model no evidence for substantial levels of endogenous amino acids within the vesicle was found.

Placental-specific Igf2 knockout mice exhibit hypocalcemia and adaptive changes in placental calcium transport.

Evidence is emerging that the ability of the placenta to supply nutrients to the developing fetus adapts according to fetal demand. To examine this adaptation further, we tested the hypothesis that placental maternofetal transport of calcium adapts according to fetal calcium requirements. We used a mouse model of fetal growth restriction, the placental-specific Igf2 knockout (P0) mouse, shown previously to transiently adapt placental System-A amino acid transporter activity relative to fetal growth. Fetal and placental weights in P0 mice were reduced when compared with WT at both embryonic day 17 (E17) and E19. Ionized calcium concentration [Ca(2+)] was significantly lower in P0 fetal blood compared with both WT and maternal blood at E17 and E19, reflecting a reversal of the fetomaternal [Ca(2+)] gradient. Fetal calcium content was reduced in P0 mice at E17 but not at E19. Unidirectional maternofetal calcium clearance ((Ca) K (mf)) was not different between WT and P0 at E17 but increased in P0 at E19. Expression of the intracellular calcium-binding protein calbindin-D(9K), previously shown to be rate-limiting for calcium transport, was increased in P0 relative to WT placentas between E17 and E19. These data show an increased placental transport of calcium from E17 to E19 in P0 compared to WT. We suggest that this is an adaptation in response to the reduced fetal calcium accumulation earlier in gestation and speculate that the ability of the placenta to adapt its supply capacity according to fetal demand may stretch across other essential nutrients.

Facilitated transporters mediate net efflux of amino acids to the fetus across the basal membrane of the placental syncytiotrophoblast.

Fetal growth depends on placental transfer of amino acids from maternal to fetal blood. The mechanisms of net amino acid efflux across the basal membrane (BM) of the placental syncytiotrophoblast to the fetus, although vital for amino acid transport, are poorly understood. We examined the hypothesis that facilitated diffusion by the amino acid transporters TAT1, LAT3 and LAT4 plays an important role in this process, with possible effects on fetal growth. Amino acid transfer was measured in isolated perfused human placental cotyledons (n = 5 per experiment) using techniques which distinguish between different transport processes. Placental TAT1, LAT3 and LAT4 proteins were measured, and mRNA expression levels (measured using real-time quantitative-PCR) were related to fetal and neonatal anthropometry and dual-energy X-ray absorptiometry measurements of neonatal lean mass in 102 Southampton Women's Survey (SWS) infants. Under conditions preventing transport by amino acid exchangers, all amino acids appearing in the fetal circulation were substrates of TAT1, LAT3 or LAT4. Western blots demonstrated the presence of TAT1, LAT3 and LAT4 in placental BM preparations. Placental TAT1 and LAT3 mRNA expression were positively associated with measures of fetal growth in SWS infants (P < 0.05). We provide evidence that the efflux transporters TAT1, LAT3 and LAT4 are present in the human placental BM, and may play an important role in the net efflux of amino acids to the fetus. Unlike other transporters they can increase fetal amino acid concentrations. Consistent with a role in placental amino acid transfer capacity and fetal growth TAT1 and LAT3 mRNA expression showed positive associations with infant size at birth.

The contribution of SNAT1 to system A amino acid transporter activity in human placental trophoblast.

UNLABELLED: System A-mediated amino acid transport across the placenta is important for the supply of neutral amino acids needed for fetal growth. All three system A subtypes (SNAT1, 2, and 4) are expressed in human placental trophoblast suggesting there is an important biological role for each. Placental system A activity increases as pregnancy progresses, coinciding with increased fetal nutrient demands. We have previously shown SNAT4-mediated system A activity is higher in first trimester than at term, suggesting that SNAT1 and/or SNAT2 are responsible for the increased system A activity later in gestation. However, the relative contribution of each subtype to transporter activity in trophoblast at term has yet to be evaluated. The purpose of this study was to identify the predominant subtype of system A in cytotrophoblast cells isolated from term placenta, maintained in culture for 66h, by: (1) measuring mRNA expression of the three subtypes and determining the Michaelis-Menten constants for uptake of the system A-specific substrate, (14)C-MeAIB, (2) investigating the contribution of SNAT1 to total system A activity using siRNA. RESULTS: mRNA expression was highest for the SNAT1 subtype of system A. Kinetic analysis of (14)C-MeAIB uptake revealed two distinct transport systems; system 1: K(m)=0.38+/-0.12mM, V(max)=27.8+/-9.0pmol/mg protein/20min, which resembles that reported for SNAT1 and SNAT2 in other cell types, and system 2: K(m)=45.4+/-25.0mM, V(max)=1190+/-291pmol/mg protein/20min, which potentially represents SNAT4. Successful knockdown of SNAT1 mRNA using target-specific siRNA significantly reduced system A activity (median 75% knockdown, n=7). CONCLUSION: These data enhance our limited understanding of the relative importance of the system A subtypes for amino acid transport in human placental trophoblast by demonstrating that SNAT1 is a key contributor to system A activity at term.

The SNAT4 isoform of the system A amino acid transporter is functional in human placental microvillous plasma membrane.

Placental system A activity is important for the supply of neutral amino acids needed for fetal growth. There are three system A isoforms: SNAT1, SNAT2 and SNAT4, but the contribution of each to system A-mediated transport is unknown. Here, we have used immunohistochemistry to demonstrate that all three isoforms are present in the syncytiotrophoblast suggesting each plays a role in amino acid transport across the placenta. We next tested the hypothesis that the SNAT4 isoform is functional in microvillous plasma membrane vesicles (MVM) from normal human placenta using a method which exploits the unique property of SNAT4 to transport both cationic amino acids as well as the system A-specific substrate MeAIB. The data show that SNAT4 contribution to system A-specific amino acid transport across MVM is higher in first trimester placenta compared to term (approx. 70% and 33%, respectively, P < 0.01). Further experiments performed under more physiological conditions using intact placental villous fragments suggest a contribution of SNAT4 to system A activity in first trimester placenta but minimal contribution at term. In agreement, Western blotting revealed that SNAT4 protein expression is higher in first trimester MVM compared to term (P < 0.05). This study provides the first evidence of SNAT4 activity in human placenta and demonstrates the contribution of SNAT4 to system A-mediated transport decreases between first trimester and term: our data lead us to speculate that at later stages of gestation SNAT1 and/or SNAT2 are more important for the supply of amino acids required for normal fetal growth.

Increased maternofetal calcium flux in parathyroid hormone-related protein-null mice.

The role of parathyroid hormone-related protein (PTHrP) in fetal calcium homeostasis and placental calcium transport was examined in mice homozygous for the deletion of the PTHrP gene (PTHrP-/- null; NL) compared to PTHrP+/+ (wild-type; WT) and PTHrP+/- (heterozygous; HZ) littermates. Fetal blood ionized calcium was significantly reduced in NL fetuses compared to WT and HZ groups at 18 days of pregnancy (dp) with abolition of the fetomaternal calcium gradient. In situ placental perfusion of the umbilical circulation at 18 dp was used to measure unidirectional clearance of (45)Ca across the placenta in maternofetal ((Ca)K(mf)) and fetoplacental ((Ca)K(fp)) directions; (Ca)K(fp) was < 5% of (Ca)K(mf) for all genotypes. At 18 dp, (Ca)K(mf) across perfused placenta and intact placenta ((Ca)K(mf(intact))) were similar and concordant with net calcium accretion rates in vivo. (Ca)K(mf) was significantly raised in NL fetuses compared to WT and HZ littermates. Calcium accretion was significantly elevated in NL fetuses by 19 dp. Placental calbindin-D(9K) expression in NL fetuses was marginally enhanced (P < 0.07) but expression of TRPV6/ECaC2 and plasma membrane Ca2+-ATPase (PMCA) isoforms 1 and 4 were unaltered. We conclude that PTHrP is an important regulator of fetal calcium homeostasis with its predominant effect being on unidirectional maternofetal transfer, probably mediated by modifying placental calbindin-D(9K) expression. In situ perfusion of mouse placenta is a robust methodology for allowing detailed dissection of placental transfer mechanisms in genetically modified mice.

Altered activity of the system A amino acid transporter in microvillous membrane vesicles from placentas of macrosomic babies born to diabetic women.

Fetal macrosomia (FM) is a well-recognized complication of diabetic pregnancy but it is not known whether placental transport mechanisms are altered. We therefore studied the activity of the system A amino acid transporter, the system L amino acid transporter, and the Na+/H+ exchanger in microvillous membrane vesicles from placentas of macrosomic babies born to diabetic women (FM group), from placentas of appropriately grown babies born to diabetic women (appropriate for gestational age group) and from placentas of appropriately grown babies of normal women (control group). Sodium-dependent uptake of [14C]-methylaminoisobutyric acid at 30 s (initial rate, a measure of system A activity) was 49% lower into FM vesicles than into control vesicles (P < 0.02); this effect was due to a decrease in Vmax of the transporter with no change in Km. There was no significant difference in system A activity between the appropriate for gestational age group and control or FM group. There was also no difference between system L transporter or Na+/H+ exchanger activity between the three groups. We conclude that the number of system A transporters per milligram of membrane protein in the placental microvillous membrane is selectively reduced in diabetic pregnancies associated with FM.

Artificial perfusion of the fetal circulation of the in situ mouse placenta: methodology and validation.

Here we present methodology and validation (including measurement of unidirectional maternofetal clearance (Kmf) of (45)Ca and (14)C-mannitol) for in situ perfusion of the mouse placenta. On day 18 of gestation (term=19 days) mice were anaesthetised and the uterus delivered into a saline bath (40 degrees C). A fetus was selected, the umbilical artery and vein catheterised and perfused with Krebs Ringer (pH 7.4) at 60 microl/min. (45)Ca/(14)C-mannitol (2 microCi/5 microCi in 50 microl saline) was injected via maternal tail vein. Perfusate samples were collected every 5 min for 45 min. Maternal carotid artery pressure was monitored throughout perfusion. A terminal maternal cardiac blood sample was taken and analysed. Placentas were immersion fixed and processed for electron microscopy. Kmf for (45)Ca and (14)C-mannitol was calculated as perfusate [(45)Ca or (14)C-mannitol] x perfusion rate/maternal plasma [(45)Ca or (14)C-mannitol]xplacental weight. Maternal cardiac blood chemistry at termination (n=8-15, mean+/-SEM) was as follows: pH 7.153+/-0.016, PCO(2) 45.48+/-2.06 mmHg, PO(2) 66.47+/-7.10 mmHg, Na(+) 151.4+/-1.2 mmol/l, K(+) 5.54+/-0.17 mmol/l, Ca(2+) 1.15+/-0.03 mmol/l, glucose 7.2+/-0.5 mmol/l, and lactate 1.76+/-0.77 mmol/l. A successful 45 min perfusion in which perfusate recovery was >95% occurred in >50% of animals. Perfusion did not alter placental morphology or carotid pressure. Kmf (microl/min/g placenta) for (45)Ca (66.0+/-8.4 (n=7)) was significantly higher than Kmf for (14)C-mannitol (20.0+/-2.4 (n=5)) (p<0.01). These data demonstrate physiological perfusion of the mouse placenta in situ and its usefulness for measurement of solute transfer.

Purification and Na+ uptake by human placental microvillus membrane vesicles prepared by three different methods.

Three methods were used to prepare microvillus membrane vesicles from each of six human placentas. Two of these incorporated an agitation stage to preferentially remove microvilli and either Ca2+ (Method 1) or Mg2+ (Method 2) aggregation of non-microvillus membrane. The third method involved homogenisation of the tissue followed by Mg2+ aggregation of non-microvillus membrane (Method 3). Enrichment of alkaline phosphatase activity (27.6 +/- 1.9, 25.3 +/- 2.7) and ouabain binding (5.9 +/- 2.6, 5.3 +/- 2.2, respectively) was similar in vesicles prepared by Methods 1 and 2, respectively. Method 3 vesicles showed a significantly (P less than 0.01) lower alkaline phosphatase enrichment (18.1 +/- 1.2), but ouabain binding enrichment (6.3 +/- 1.3) was not different and vesicle protein recovery (mg/g placenta) was 5-fold greater. Na+ uptake in the presence of an outwardly directed proton gradient was significantly inhibited in all microvillus membrane vesicles by amiloride (0.5 mM). However, the amiloride sensitive component of Na+ uptake was 3-6-fold greater in Method 3 vesicles than in Method 1 and 2 vesicles, and showed overshoot above equilibrium in the former but not the latter. Further experiments using the pH sensitive dye, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein suggested that the proton gradient dissipated faster from Method 1 than from Method 3 vesicles. Thus methodological differences can have a marked effect on transport processes in microvillus membrane vesicles prepared from the human placenta.

Chloride transport by human placental microvillous membrane vesicles.

Unidirectional uptake of chloride by microvillous membrane vesicles prepared from human term placentas was studied over a range of membrane potentials in the presence and absence of chloride transport inhibitors alone and in combination at maximally effective concentrations. At 0 mV, inhibition of chloride uptake by 0.1 mM DIDS, 0.5 mM DPC, and 0.5 mM DPC plus 0.1 mM DIDS was similar, suggesting a common action upon an anion exchanger; neither 0.1 mM furosemide nor 0.1 mM bumetanide alone had any effect. An inside-positive membrane potential was created by imposing an inwardly directed potassium ion gradient in the presence of valinomycin. Total chloride uptake increased with increasing membrane potential (0, 4.6, 17.3, 25.8 and 32.0 mV). The inhibition of uptake by DPC and DPC/DIDS increased with the membrane potential. The effect of DPC compared to DPC/DIDS was significantly different at 4.6, 17.3 and 25.8 mV, suggesting a degree of additivity of inhibition. Neither furosemide nor bumetanide had any effect at any potential. There was a significant increase in inhibition due to DIDS alone until the membrane potential reached 25.8 mV. But there was no significant difference between the level of inhibition at 32 mV as compared to 0 mV, providing evidence of a DIDS-sensitive conductance similar to that previously seen in patch clamp studies. We suggest that uptake of chloride across the microvillous membrane of the human placenta may be by at least three different pathways; an electroneutral, DIDS-sensitive anion exchanger, a DPC-sensitive chloride conductance and a DIDS-sensitive chloride conductance.

Gestational profile of Na+/H+ exchanger and Cl-/HCO3- anion exchanger mRNA expression in placenta using real-time QPCR.

The onset of maternal blood flow (10-12 weeks gestation) results in increased oxygenation of the placenta. We investigated whether the expressions of Na+/H+ exchanger (NHE) and Cl-/HCO3- anion exchanger (AE), thought to have an important role in maintaining intracellular pH of the syncytiotrophoblast and fetal pH homeostasis, are altered at the same time as this increase in blood flow. Real-time quantitative PCR was used to examine steady state levels of NHE (NHE1, 2, 3) and AE (AE1, 2) mRNA expression in early (6-9 weeks) and late (10-13 weeks) first trimester and full-term (38-40 weeks) placentas. beta-Actin, IF2B and GAPDH mRNA was also measured. None of the genes showed a significant difference in expression between the early and late first trimester groups. However, NHE2 (p < 0.001) and GAPDH (p < 0.05) mRNA expression significantly increased 18- and 3.7-fold between early first trimester and term. In conclusion, this study provides additional evidence that GAPDH is an unsuitable housekeeping gene for normalization of transcript levels in placenta. The expression of NHE and AE in the villous placenta is not altered concomitant with the onset of maternal blood flow. However, NHE2 transcripts appear to be gestationally regulated, which may contribute to changes in NHE activity.

Characterisation of long term cat placental explant cultures: uptake of taurine by system beta.

Dietary taurine is essential for cats and deficiency during pregnancy may lead to abortion, growth restriction or impaired neurological function of kittens. We previously described Na(+)- and Cl(-)-dependent taurine transport by system beta in fragments of freshly isolated cat placenta [Champion EE, Bailey SJ, Glazier JD, Jones CJP, Mann SJ, Rawlings JM, et al. Taurine uptake into cat placental tissue fragments. Placenta 2001;22:A.42]. Here we evaluate long term culture of cat placental explants as a model for the future study of chronic nutrient regulation of amino acid transport in this species. The cat placental explants displayed (i) Na(+)-dependent [(3)H]taurine uptake and (ii) taurine transporter protein on day 7 of culture, as observed in fresh cat placental fragments. The explants had preserved the ability to secrete PGF(2alpha) hormone until day 11 of culture and remained morphologically largely intact until day 7 of culture. This model of placental explant culture will provide an important in vitro method for the study of chronic regulation of amino acid transport in the cat.

Ultrastructural aspects of long-term cat placental explant culture.

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.

Characterization of cationic amino acid transporters and expression of endothelial nitric oxide synthase in human placental microvascular endothelial cells.

We investigated the expression and activity of arginine transporters and endothelial nitric oxide synthase (eNOS) in human placental microvascular endothelial cells (HPMEC). Using RT-PCR amplification products for eNOS, CAT1, CAT2A, CAT2B, CAT4, 4F2hc (CD98), rBAT and the light chains y+LAT1, y+LAT2, and b0+T1 were detected in HPMEC, but not B0+. Immunohistochemistry and Western blotting confirmed the presence of 4F2hc and CAT1 protein in HPMEC. 4F2hc-light chain dimers were indicated by a shift in molecular mass detected under nonreducing conditions. L-Arginine transport into HPMEC was independent of Na+ or Cl- and was inhibited by the neutral amino acid glutamine, but not by cystine. The Ki for glutamine inhibition was greater in the absence of Na+. Kinetic analysis supported a two-transporter model attributed to system y+L and system y+. Expression of eNOS in HPMEC was detectable by immunohistochemistry and ELISA but not by Western blotting. Activity of eNOS in HPMEC, measured over 48 h, either as the basal production of nitric oxide (NO) or as the accumulation of intracellular cGMP was not detectable. We conclude that HPMEC transport cationic amino acids by systems y+ and y+L and that basal eNOS expression and activity in these cells is low.

L-arginine transport by the microvillous plasma membrane of the syncytiotrophoblast from human placenta in relation to nitric oxide production: effects of gestation, preeclampsia, and intrauterine growth restriction.

Nitric oxide (NO) is an important regulator of placental perfusion, and its production is dependent on the activity of substrate (L-arginine) transporters. In the light of evidence for altered NO production in the feto-placental unit in preeclampsia and intrauterine growth restriction (IUGR), we investigated gestational changes in human placental L-arginine transport by systems y(+) and y(+)L in purified microvillous plasma membrane vesicles. We also examined the effect of preeclampsia and IUGR on the activity of these transport systems and the relationship between transporter activity and NO production (nitrate/nitrite concentrations) in the feto-placental unit. Between first trimester and term, there was a significant positive correlation between system y(+) activity and gestational age (r = 0.36; P = 0.013; n = 47), but a significant negative correlation between system y(+)L activity and gestational age (r = -0.6; P < 0.0001; n = 47). The activity of these transport systems was not altered in preeclampsia or IUGR. In placentas from normal term pregnancies, there was no correlation between the activity of microvillous plasma membrane L-arginine transporters and nitrate/nitrite concentrations in umbilical venous plasma or placental homogenate.

Activity and protein expression of the Na+/H+ exchanger is reduced in syncytiotrophoblast microvillous plasma membranes isolated from preterm intrauterine growth restriction pregnancies.

Regulation of syncytiotrophoblast intracellular pH is critical to optimum enzymatic and transport functions of the placenta. Previous studies of Na(+)/H(+) exchanger (NHE) activity in the placenta from pregnancies complicated by intrauterine growth restriction (IUGR) have produced conflicting results. The possible role of altered placental pH regulation in the development of acidosis in some fetuses subjected to IUGR remains to be fully established. We investigated the activity and protein expression of the NHE in syncytiotrophoblast microvillous (MVM) plasma membranes isolated from preterm and term placentas obtained from uncomplicated and IUGR pregnancies. Western blotting showed that the expression of NHE isoforms 1, 2, and 3 was approximately 10-fold greater in MVM than in basal plasma membrane (BM). Immunohistochemistry localized NHE-1 and NHE-2 to MVM and BM and NHE-3 to the MVM, BM, and cytoplasm of the syncytiotrophoblast. NHE-1 expression in MVM from preterm IUGR placentas was reduced by 55%, compared with gestational age-matched controls (P < 0.05, n = 6 and n = 16, respectively), whereas NHE-1 expression was unaltered in term IUGR placentas (n = 8). The activity (amiloride-sensitive Na(+) uptake) of NHE in MVM from IUGR preterm placentas was reduced by 48% (P < 0.05, n = 6). In contrast, MVM NHE activity was unchanged in term IUGR (n = 7). Using Northern blotting, no difference could be demonstrated in NHE-1 mRNA expression between IUGR and control groups. The reduced activity and expression of NHE in MVM of preterm IUGR placentas may compromise placental function and may contribute to the development of fetal acidosis in preterm IUGR fetuses.