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N P Illsley

Publications and source records attributed to N P Illsley.

At least 19 recordsLinked to original sources
PubMed2006

Effects of chronic hypoxia in vivo on the expression of human placental glucose transporters.

Birth weight is reduced and the risk of preeclampsia is increased in human high altitude pregnancies. There has been little work to determine whether hypoxia acts directly to reduce fetal growth (e.g. reduced blood flow and oxygen delivery), or via changes in functional capacities such as nutrient transport. We therefore investigated the expression of a primary nutrient transporter, the GLUT1 glucose transporter and two in vitro markers of hypoxia (erythropoietin receptor, EPO-R, and transferrin receptor, TfR) in the syncytial microvillous (MVM) and basal membrane fractions (BMF) of 13 high (3100 m) and 12 low (1600 m) altitude placentas from normal term pregnancies. Birth weight was lower at 3100 m than at 1600 m despite similar gestational age, but none of the infants were clinically designated as fetal growth restriction. EPO-R, TfR and GLUT1 were examined by immunoblotting and maternal circulating erythropoietin and transferrin by ELISA. EPO-R was greater on the MVM (+75%) and BMF (+25%) at 3100 m. TfR was 32% lower on the MVM at 3100 m. GLUT1 was 40% lower in the BMF at 3100 m. Circulating EPO was greater at high altitude, while transferrin was similar, and neither correlated with their membrane receptors. BMF GLUT1 was positively correlated with birth weight at high, but not low altitude. In this in vivo model of chronic placental hypoxia, syncytial EPO-R increased as expected, while nutrient transporters decreased, opposite to what has been observed in vitro. Therefore, hypoxia acts to reduce fetal growth not simply by reducing oxygen delivery, but also by decreasing the density of nutrient transporters.

Adult↗
PubMed2002

Polar expression and phosphorylation of human leptin receptor isoforms in paired, syncytial, microvillous and basal membranes from human term placenta.

The hormone leptin (OB) and its receptor (OB-R) are key homeostatic regulators of mammalian body weight. Two predominant isoforms of OB-R are expressed by alternative splicing: the long form, OB-RL, with full signalling capacity is highly expressed in the hypothalamus and the short, signalling-defective form, OB-Rs, is ubiquitously expressed. In a previous study we detected expression of OB-RL and OB-Rs in human syncytiotrophoblast cells using in situ hybridization and immunohistochemistry (Bodner et al., 1999). The aim of this study was to investigate leptin receptor isoform expression and phosphorylation in paired, syncytial, microvillous and basal membranes from human term placenta by Western blot analysis. Both the OB-RL and the OB-Rs isoforms were detected in the syncytial membrane preparations. The OB-RL isoform was observed exclusively in microvillous membranes, whereas the OB-Rs isoform was found in both microvillous and basal membrane preparations. No significant differences were observed between syncytial membranes from normal and type 1 diabetic pregnancies. To test the phosphorylation capacity of the OB-R isoforms, microvillous and basal membrane vesicles loaded with ATP were stimulated with leptin and the phosphorylation status of the OB-R at the tyrosine 985 (Y985) was determined. A single band at the molecular weight corresponding to the molecular weight of the OB-RL isoform was detected exclusively in the ATP-loaded microvillous vesicles. We conclude that the long form OB-RL is expressed exclusively in the microvillous membrane of the syncytiotrophoblast and is capable of being phosphorylated, suggesting that it has signal transduction capacity.

Adolescent↗
PubMed2000

Glucose transporters in the human placenta.

The availability of antibodies and cDNA probes specific for the various members of the facilitated-diffusion glucose transporter (GLUT) family has enabled researchers to obtain a much clearer picture of the mechanisms for placental uptake and transplacental transport of glucose. This review examines studies of human placental glucose transport with the aim of providing a model which describes the transporter isoforms present in the placenta, their cellular localization and functional significance. The GLUT1 glucose transporter, present on both the microvillous and basal membranes of the syncytial barrier, is the primary isoform involved in the transplacental movement of glucose. Although GLUT3 mRNA is widely distributed, GLUT3 protein is localized to the arterial component of the vascular endothelium, where it may play a role in enhancing transplacental glucose transport. This data is in contrast to the situation in other mammalian species, such as the mouse, rat and sheep, where GLUT3 protein is not only present in those epithelial cells which carry out transplacental transport but becomes an increasingly prominent isoform as gestation progresses. The asymmetric distribution of GLUT1 in the human syncytiotrophoblast (microvillous>basal) means that basal GLUT1 acts as the rate limiting step in transplacental transfer. Changes in basal GLUT1 therefore have the potential to cause alterations in transplacental transport of glucose. Although there appear to be no changes in syncytial GLUT1 expression in intrauterine growth retardation, in diabetic pregnancies increases in basal GLUT1 expression and activity have been observed, with significant consequences for the maternal-fetal flux of glucose. Little is known of glucose transporter regulation in the placenta save for the effects of hyper- and hypoglycemia. GLUT1 expression and activity appear to be inversely related to extracellular glucose concentration, however within the physiological range, GLUT1 expression is relatively refractory to glucose concentration. Information is still needed on gestational development, on the expression and activity in well-defined conditions of intrauterine growth retardation, on the mechanisms and consequences of the changes observed in diabetic pregnancy and on the role of external agents other than glucose in regulating placental glucose transport.

Animals↗
PubMed2000

Episialin acts as an antiadhesive factor in an in vitro model of human endometrial-blastocyst attachment.

Episialin, which is found on the apical membrane of human endometrial epithelium, has been postulated to act as an antiadhesive factor through the steric hindrance generated by its extensively glycosylated structure. The present studies were designed to test this hypothesis in an in vitro model of endometrial-blastocyst attachment. Episialin was expressed in human endometrial carcinoma cells (HEC-1A > RL95-2), and attachment of JAr choriocarcinoma cells to the endometrial cell monolayers was inversely related to episialin expression. Treatment of endometrial monolayers with type III sialidase increased JAr binding, and this increase was suppressed by HMFG1, a monoclonal antibody specific for episialin. The effects of sialidase appear to have resulted from a contaminant protease rather than from a loss of sialic acid residues, because sialidase preparations other than type III were ineffective. After sialidase treatment, conditioned medium from cells treated with type III sialidase contained more episialin than medium from cells treated with other sialidase preparations. Similar attachment-assay results were obtained using O-sialoglycoprotein endopeptidase; after treatment, the increase in JAr binding (>50%) was suppressed by the antiepisialin antibody. These results demonstrate for the first time that episialin acts as an antiadhesive agent in a model of human endometrial-blastocyst attachment.

Blastocyst↗
PubMed1999

Composition and permeability of syncytiotrophoblast plasma membranes in pregnancies complicated by intrauterine growth restriction.

The objective of this study was to determine placental membrane permeabilities to water, urea and mannitol in intrauterine growth restriction (IUGR) and compare them to normal gestational age matched controls. Further, we wished to investigate whether potential changes in permeability were related to changes in membrane fluidity, cholesterol or phospholipid fatty acid content of the membranes. Syncytiotrophoblast microvillous (MVM) and basal membranes (BM) were isolated from normal and IUGR placentas at term. Passive permeability to water, urea, and mannitol showed no significant alterations in IUGR compared to controls. Cholesterol content in BM, but not in MVM, was lower in placentas from pregnancies complicated by IUGR. However, membrane fluidity did not change in these pregnancies. The phospholipid fatty acid composition of the plasma membranes isolated from all placentas showed a predominance of unsaturated fatty acid species in the BM and saturated species in the MVM. In the MVM from IUGR, mead acid (20:3), behenic acid (22:0) and nervonic acid (24:1) constituted higher percentages of the total when compared to normally grown controls. In the BM from IUGR, mead acid (20:3) was increased relative to the total phospholipid fatty acid content. In conclusion, the syncytiotrophoblast membranes exhibit only minor changes in passive permeability and composition when the pregnancy is complicated by IUGR.

Case-Control Studies↗
PubMed1999

Diabetes alters the expression and activity of the human placental GLUT1 glucose transporter.

This study was designed to investigate the effects of maternal diabetes on glucose transporter expression and glucose transport activity in the human placenta. Syncytiotrophoblast microvillous and basal membranes were prepared from placental tissue obtained at term from pregestational diabetics (White class B) and gestational diabetics controlled either by diet alone (class A1) or by diet and insulin (class A2). These membranes were used to measure GLUT1 glucose transporter expression and D-glucose transport activity. Diabetic groups showed no differences in placental weights or neonatal birth weights compared to controls, although 8 of 25 diabetic fetuses were macrosomic. Glycemic control in the diabetics at term, as assessed by maternal glycosylated hemoglobin, was within normal limits. Basal membrane GLUT1 density was about 2-fold higher in all diabetic groups compared to that in controls, as measured by immunoblotting, whereas no changes were found for the microvillous membranes. D-Glucose uptake across the basal membrane was increased by 40% in the diabetic groups; no changes were observed for the microvillous membrane. These results demonstrate that diabetes causes an increase in basal membrane GLUT1 expression and activity that persists despite a lack of evidence for current or recent maternal hyperglycemia. This suggests the potential for an extended increase in transplacental glucose flux in the absence of maternal hyperglycemia, which may contribute to fetal macrosomia and the other consequences of diabetic pregnancy.

Adolescent↗
PubMed1998

Glycaemic regulation of glucose transporter expression and activity in the human placenta.

To determine whether the expression and activity of glucose transporters in human trophoblast are regulated by glucose, syncytiotrophoblast cells, choriocarcinoma cells, and villous fragments were incubated with a range of glucose concentrations (0-20 mM, 24 h). Expression of GLUT1 and GLUT3 glucose transporters was measured by immunoblotting, while glucose transporter activity was determined by [3H]2-deoxyglucose uptake in the cultured cells. GLUT1 expression in syncytial cells was enhanced following incubation in absence of glucose, reduced by incubation in 20 mM glucose but was not altered by incubation at 1 or 12 mM glucose. Transporter activity was inversely related to extracellular glucose over the entire range of concentrations tested (0-20 mM). Incubation of villous fragments in 20 mM glucose produced a limited suppression of GLUT1 expression, but no effects were noted following incubation at 0 or 1 mM glucose. Neither GLUT1 expression in JAr and JEG-3 choriocarcinoma cells nor transport activity in JEG-3 cells was affected by extracellular glucose concentration. Unlike syncytial cells, JAr, JEG-3 and BeWo all expressed GLUT3 protein in addition to GLUT1. These results show that while syncytiotrophoblast GLUT1 expression is altered at the extremes of extracellular glucose concentration, it is refractory to glucose alone at lower concentrations. By contrast, an inverse relationship exists between glucose transporter activity and extracellular glucose. This suggests that there are post-translational regulatory mechanisms which may respond to changes in extracellular glucose concentration.

Adult↗
PubMed1997

Guanidine transport in a human choriocarcinoma cell line (JAR).

PURPOSE: Many endogenous substances and xenobiotics are organic cations. Transplacental transport of organic cations is an important determinant of the delivery of these compounds to the fetus. The aim of this study was to determine the mechanisms of organic cation transport using the human choriocarcinoma cell line (JAR) as a model system with [14C]guanidine as a ligand. METHODS: Uptake studies of [14C]guanidine were carried out in JAR cell monolayers on day 2 after plating. RESULTS: [14C]guanidine uptake was temperature dependent, saturable (Km = 167 microM) and inhibited by many organic cations including amiloride, cimetidine, quinine, quinidine and nicotine. [14C]guanidine uptake exhibited a counterflux phenomenon indicative of a carrier-mediated process. The uptake of [14C]guanidine was sodium and pH-independent and could be driven by an inside-negative membrane potential difference. CONCLUSIONS: This is the first demonstration of an electrogenic guanidine transporter in a human cell culture model. This transporter may play a role in the transplacental transport of many clinically used drugs and xenobiotics.

Biological Transport↗
PubMed1996

A novel technique for studying cellular function in human placenta: gestational changes in intracellular pH regulation.

This paper presents a new method for the study of cell function in primary human placental syncytiotrophoblast cells. Chorionic villous tissue fragments from term and first trimester placenta were loaded with fluorescent pH sensitive indicator dye HPTS and made adherent to a microscope cover-slip. The fragments were superfused and intracellular pH (pHi) was studied by microfluorimetry. We used this new methodology to examine the role of the Na+/H+ antiporter in pHi regulation. Syncytial cells demonstrated homeostatic pHi regulation, recovering back to basal pHi after intracellular acidification. In the absence of HCO3-, the Na+/H+ antiporter was the primary means by which syncytiotrophoblast cells recovered from an intracellular acid load in both term and first trimester samples. The rate of recovery from intracellular acidification showed a strong correlation to degree of acidification, confirming allosteric modification of antiporter activity by intracellular protons. The transporter was regulated by phosphorylation mediated by protein kinase C (PKC) at both gestational ages. This methodology represents a powerful new technique for the study of syncytiotrophoblast cell ionic regulation.

Female↗
PubMed1996

Differential expression of protein kinase C isoforms in the human placenta.

The extensive role played by protein kinase C (PKC) in signal transduction prompted this study of the expression and localization of PKC isoforms in human placental syncytiotrophoblast. Membranes prepared from these cells and samples of villous tissue were analysed by immunoblotting and immunocytochemistry using isoform-specific antibodies. PKC beta 2, gamma, epsilon and zeta were found to be present in both microvillous and basal membranes from term placenta. The alpha isoform was observed only on the basal membrane while the beta 1 isoform was confined to the microvillous membrane. The basal microvillous ratios for beta 2, gamma, epsilon and zeta ranged between 0.3 and 0.5, demonstrating a substantial asymmetry in plasma membrane localization. Immunocytochemistry supported the isoform identification and localization observed in the immunoblotting experiments. Moreover the cellular distribution showed that the majority of syncytical PKC was bound to the plasma membranes, in contrast to the other villous cell types. Immunoblotting experiments demonstrated significant increases in PKC beta 2 and epsilon on the microvillous membrane and PKC gamma and epsilon on the basal membrane between 16 and 40 of weeks gestation. This is the first detailed mapping of PKC isoform distribution in an epithelial cell type and demonstrates the potential for selectivity in signal transduction through phosphorylation of isoform specific and spatially-separated substrates.

Amino Acid Sequence↗
PubMed1995

Beta-adrenergic regulation of cyclic AMP synthesis in cultured human syncytiotrophoblast.

Isolated elements of the beta-adrenergic/adenyl cyclase signal transduction system have been studied previously using purified membranes. We used cultured syncytiotrophoblast cells to identify components of this signalling system and the interactions which regulate syncytial adenyl cyclase. Generation of cyclic AMP (cAMP) was stimulated in these cells by both forskolin and isoproterenol but not by dopamine, adenosine, carbachol or prostaglandin E1. Synthesis was also stimulated by treatment with cholera toxin, indicating the involvement of the G-protein, Gs. Somatostatin inhibited isoproterenol- or forskolin-stimulated cAMP generation, an effect which could be blocked by pretreatment of the cells with pertussis toxin, demonstrating the mediation of somatostatin action by Gi. Furthermore, secretion of human chorionic gonadotrophin (hCG) was increased significantly by isoproterenol while somatostatin blocked the isoproterenol-stimulated release of hCG. These results clearly demonstrate that adenyl cyclase in syncytiotrophoblast is controlled by a stimulatory pathway operating through Gs and inhibitory pathway acting through Gi.

Adenylyl Cyclases↗
PubMed1995

Cellular localization of glucose transporter messenger RNA in human placenta.

High levels of expression of the glucose transporter (GLUT) isoforms 1 and 3 have been demonstrated in the human placenta by Northern blotting. However, the cellular localization of placental GLUT mRNA has not been described. Furthermore, recent preliminary kinetic data indicate that GLUT 2 might be present in syncytiotrophoblast. Human placental tissue from preterm (16-22 weeks) and term pregnancies was collected for identification and localization of glucose transporter mRNA. Following paraffin embedding, sections were cut and in situ hybridization was performed with fluorescein-labelled cRNA. In addition, immunoblotting and immunocytochemistry were carried out using an anti-GLUT 2 antibody. GLUT 1 mRNA was highly expressed in syncytiotrophoblast cells at term. GLUT 1 expression was much less abundant in non-syncytial cells. In contrast, GLUT 3 mRNA was present in lower amounts and more evenly distributed between syncytial and other placental cells. GLUT 1 mRNA was also highly abundant in preterm syncytiotrophoblast. The cellular distributions of GLUT 1 and GLUT 3 mRNA in the preterm placentas were similar to those in term tissue. With regard to GLUT 1, these findings correlate well with cellular localization and gestational development of GLUT 1 protein. No GLUT 2 protein was detected. It is concluded that GLUT 1 is the main isoform involved in transplacental glucose transport in the human.

Female↗
PubMed1993

Osmotic water permeabilities of human placental microvillous and basal membranes.

Literature data suggest that water accumulation by the human fetus is driven by osmotic gradients of small solutes. However, the existence of such gradients has not been supported by prior measurements. Attempts to estimate the size of the gradient necessary to drive net water movement have been seriously hampered by the lack of permeability data for the syncytiotrophoblast membranes. Stopped-flow light scattering techniques were employed to measure the osmotic water permeability (Pf) of microvillous (MVM) and basal membrane (BM) vesicles isolated from human term placenta. At 37 degrees C, the Pf was determined to be 1.9 +/- 0.06 x 10(-3) cm/sec for MVM and 3.1 +/- 0.20 x 10(-3) cm/sec for BM (mean +/- SD, n = 6). At 23 degrees C, Pf was reduced to 0.7 +/- 0.04 x 10(-3) cm/sec in MVM and 1.6 +/- 0.05 x 10(-3) cm/sec in BM. These Pf values are comparable to those observed in membranes where water has been shown to permeate via a lipid diffusive mechanism. Arrhenius plots of Pf over the range 20-40 degrees C were linear, with activation energies of 13.6 +/- 0.6 kcal/mol for MVM and 12.9 +/- 1.0 kcal/mol for BM. Water permeation was not affected by mercurial sulfhydryl agents and glucose transport inhibitors. These data clearly suggest that water movement across human syncytiotrophoblast membranes occurs by a lipid diffusion pathway. As noted in several other epithelial tissues, the basal membrane has a higher water permeability than the microvillous membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Non-electrolyte solute permeabilities of human placental microvillous and basal membranes.

1. Permeability to non-electrolytes of isolated microvillous and basal membranes from human term placenta was measured using stopped-flow light-scattering techniques. The studied solutes were urea, ethylene glycol, glycerol, creatinine, erythritol, arabitol and mannitol. 2. At 37 degrees C, permeability of the microvillous membrane to mannitol and urea was 0.30 +/- 0.02 x 10(-6) cm/s (mean +/- S.E.M.) and 3.2 +/- 0.2 x 10(-6) cm/s, respectively. The corresponding permeabilities for the basal membrane were 1.2 +/- 0.1 x 10(-6) cm/s (mannitol) and 4.4 +/- 0.3 x 10(-6) cm/s (urea). The basal membrane was substantially more permeable to hydrophilic solutes than the microvillous membrane. This is probably due to differences in lipid composition, as illustrated by membrane cholesterol content, which was found to be approximately 50% lower in the basal as compared to the microvillous membrane. 3. Similarities between permeabilities in placental membranes and lipid bilayers and the linear relationship noted between solute hydrophobicity and placental permeability suggested that solutes permeate both human syncytiotrophoblast membranes by a solubility/diffusion mechanism. In the microvillous membrane this was supported by data obtained for activation energies (> 10 kcal/mol) and reflection coefficients (close to 1). In the basal membrane, low activation energies for glycerol and urea and a low reflection coefficient for urea indicated that these solutes may, in part, share a common pathway with water. 4. It was estimated that the placental permeability to molecules with a molecular weight under 200 observed in vivo can, to a great extent, be accounted for by transcellular permeation.

Cell Membrane↗
PubMed1993

Glucose transporter protein expression in human placenta throughout gestation and in intrauterine growth retardation.

Despite the importance of glucose for fetal growth, gestational development of placental glucose transport capacity has not been studied in the human. Furthermore, inadequate glucose transport has been implicated as a pathophysiological mechanism in intrauterine growth retardation (IUGR). We studied glucose transporter (GLUT) protein expression in sections of normal term placental tissue (immunocytochemistry) and in syncytiotrophoblast microvillous (MVM) and basal membranes (BM) isolated from normal term, preterm, and IUGR placentas (immunoblotting). GLUT 1, but not GLUT 3, protein was abundantly present in syncytiotrophoblast membranes. MVM had approximately 3-fold higher GLUT 1 density than BM at term. MVM GLUT 1 density was maintained from 16 weeks of gestation to term. BM GLUT 1 density increased 2-fold in late second trimester and remained unaltered thereafter to term. GLUT 1 densities in term and preterm IUGR placentas were unaltered. Net D-glucose uptake rates corresponded to the GLUT 1 densities. These data suggest that 1) GLUT 1 is the main glucose transporter protein isoform in human syncytiotrophoblast; 2) the glucose transport capacity for MVM is potentially approximately 20-fold higher than that of BM; 3) GLUT 1 densities may be regulated independently in MVM and BM; 4) the increase in surface area and the maintenance of a high GLUT 1 density can account for the increase in placental glucose transport in the latter part of pregnancy; and 5) fetal hypoglycemia in IUGR is not due to a decrease in placental glucose transporter density.

Adult↗
PubMed1990

Simultaneous preparation of paired, syncytial, microvillous and basal membranes from human placenta.

A method for the simultaneous preparation of microvillous and basal membrane vesicles from human placental syncytiotrophoblast is described. Mg2(+)-aggregated basal membranes are separated from microvillous membranes by low-speed centrifugation after initial homogenization and centrifugation steps. Microvillous membranes (MVM) are obtained from the low speed supernatant while basal membranes (BM) contained in the Mg2(+)-aggregated material are resuspended and further purified on a sucrose step gradient. MVM and BM prepared by this method were enriched 20-fold and 11-fold as determined by the membrane marker enzymes, alkaline phosphatase (MVM) and adenylate cyclase (BM). There was minimal cross-contamination of the two isolated plasma membrane fractions and the yields obtained were 26% (MVM) and 21% (BM) compared to the initial homogenate. The MVM and BM fractions were free from contamination by mitochondrial or lysosomal membranes and showed only minor contamination by microsomal membranes. The two membrane fractions were also tested for the presence of non-syncytial plasma membranes by electrophoretic immunoblotting. Contamination of both MVM and BM by fibroblast, endothelial, macrophage and cytotrophoblast plasma membranes amounted to less than 15% of the total membrane protein as determined by immunoblotting. Vesicle orientation, determined from the latency of specific concanavalin A binding, was 88 +/- 4% right-side out for MVM and 73 +/- 12% right-side out for BM. This simple preparative procedure produces a high yield of both MVM and BM from human placenta. The analytical data demonstrates that 'paired' MVM and BM fractions derived from the same placental tissue have a high purity in terms not only of contamination by intracellular membranes, but also in terms of contamination by non-syncytial plasma membranes.

Alkaline Phosphatase↗