RESUMO
INTRODUCTION: Abnormally invasive placenta (AIP, aka placenta accreta spectrum; PAS) is an increasingly common pregnancy pathology, which, despite significant morbidity risk to the mother, is often undiagnosed prior to delivery. We tested several potential biomarkers in plasma from PAS mothers to determine whether any were sufficiently robust for a formal, diagnostic accuracy study. METHODS: We examined hyperglycosylated hCG (h-hCG), decorin and IL-8, based on biological plausibility and literature indications that they might be altered in PAS. These analytes were assayed by ELISA in maternal plasma from five groups, comprising (1) normal term controls, (2) placenta previa controls, and cases of (3) placenta increta/percreta without placenta previa, (4) placenta previa increta/percreta and (5) placenta previa accreta. RESULTS: There were no differences in h-hCG, ß-hCG or the h-hCG/ß-hCG ratio between the groups. Mean decorin levels were increased in previa controls (Group 2) compared to the other groups, but there was substantial overlap between the individual values. While an initial multiplex assay showed a greater value for IL-8 in the placenta previa increta/percreta group (Group 4) compared to placenta previa controls (Group 2), the subsequent validation ELISA for IL-8 showed no differences between the groups. DISCUSSION: We conclude that the absence of differences and the extent of overlap between cases and controls does not justify further assessment of these biomarkers.
Assuntos
Gonadotropina Coriônica/sangue , Decorina/sangue , Interleucina-8/sangue , Placenta Acreta/diagnóstico , Adulto , Biomarcadores/sangue , Feminino , Humanos , Placenta Acreta/sangue , Placenta Prévia/sangue , Placenta Prévia/diagnóstico , GravidezRESUMO
Decreased placental oxygenation and increased oxidative stress are implicated in the development of preeclampsia. Oxidative stress arises from imbalance between pro-versus anti-oxidants and can lead to biological oxidation and apoptosis. Because pregnant women living at high altitude (3100 m, HA) have lowered arterial PO2 and an increased incidence of preeclampsia, we hypothesized that HA placentas would have decreased anti-oxidant enzyme activity, increased oxidative stress (lipid peroxidation, protein oxidation and nitration) and greater trophoblast apoptosis than low-altitude (LA) placentas. We measured enzymatic activities, lipid and protein oxidation and co-factor concentrations by spectrophotometric techniques and ELISA in 12 LA and 18 HA placentas. Immunohistochemistry (IHC) was used to evaluate nitrated proteins and specific markers of apoptosis (activated caspase 3 and M30). Superoxide dismutase activity was marginally lower (p=0.05), while glutathione peroxidase activity (p<0.05), thioredoxin concentrations (p<0.005) and thioredoxin reductase activity p<0.01 were all reduced in HA placentas. Decreased anti-oxidant activity was not associated with increased oxidative stress: lipid peroxide content and protein carbonyl formation were lower at HA (p<0.01). We found greater nitrotyrosine residues in the syncytiotrophoblast at 3100 m (p<0.05), but apoptosis did not differ between altitudes. Our data suggest that hypoxia does not increase placental oxidative stress in vivo. Nitrative stress may be a consequence of hypoxia but does not appear to contribute to increased apoptosis. Lowered placental concentrations of anti-oxidants may contribute to the susceptibility of women living at HA to the development of preeclampsia, but are unlikely to be etiological.
Assuntos
Estresse Oxidativo , Placenta , Humanos , Hipóxia/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Trofoblastos/metabolismoRESUMO
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.
Assuntos
Regulação da Expressão Gênica , Transportador de Glucose Tipo 1/metabolismo , Hipóxia/metabolismo , Placenta/metabolismo , Adulto , Altitude , Doença da Altitude/metabolismo , Peso ao Nascer , Doença Crônica , Feminino , Humanos , Gravidez , Receptores da Eritropoetina/metabolismo , Receptores da Transferrina/metabolismoRESUMO
The microvillous membrane of the human placental syncytiotrophoblast contains an amiloride-inhibitable, electroneutral, Na+/H+ antiporter. The kinetic characteristics of this antiporter have been investigated to determine its response to alterations in intracellular and extracellular H+ and Na+ concentrations. Antiporter activity was measured using a pH-sensitive fluorescent probe entrapped in placental microvillous vesicles. We report here on the kinetic characterization of the antiporter, a transporter which displays simple, saturable kinetics for the external site but complex kinetics at the internal site. Measurement of the external Na+ and H+ dependences demonstrated that Na+ and H+ compete for binding to a single external binding site which displays saturation kinetics. The external Km determined for Na+ was 8.2 +/- 4.0 mM, while the external pK was 7.29 +/- 0.02. The Vmax calculated from these experiments was 0.57 +/- 0.10 nequiv./s per mg membrane protein. By contrast, the internal dependences for both Na+ and H+ showed significant deviations from simple linear kinetics. Decreasing internal pH to 6.0 stimulated Na+/H+ exchange to a greater degree than predicted for a single-site saturable binding model, in a manner which suggested allosteric activation. At the other extreme, Na+/H+ exchange ceased above an internal pH of 7.1, despite the existence of an inwardly-directed Na+ gradient. Increasing intracellular Na+ caused inhibition of Na+/H+ exchange but the intracellular Na+ dependence showed that the effect is due to a mechanism more complex than simple, competitive inhibition between Na+ and H+. These results show that the microvillous Na+/H+ antiporter is insensitive to changes in extracellular Na+ and H+ concentrations in the physiological range. Changes in intracellular Na+ and H+ however are likely to cause marked changes in antiporter activity. These characteristics suggest that cellular Na+ and H+ concentrations are tightly controlled in the placental syncytiotrophoblast and that the Na+/H+ antiporter may play a significant role in their regulation.
Assuntos
Proteínas de Transporte/análise , Placenta/metabolismo , Amilorida/farmacologia , Sítios de Ligação , Proteínas de Transporte/fisiologia , Cloretos/metabolismo , Feminino , Homeostase , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Microvilosidades/metabolismo , Placenta/ultraestrutura , Gravidez , Sódio/metabolismo , Trocadores de Sódio-HidrogênioRESUMO
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.
Assuntos
Membrana Celular , Microvilosidades , Placenta/ultraestrutura , Fosfatase Alcalina/análise , Feminino , Humanos , Immunoblotting , Queratinas/análise , Gravidez , ATPase Trocadora de Sódio-Potássio/análise , Vimentina/análiseRESUMO
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.
Assuntos
Membrana Celular/metabolismo , Retardo do Crescimento Fetal/metabolismo , Complicações na Gravidez/metabolismo , Trofoblastos/metabolismo , Estudos de Casos e Controles , Membrana Celular/química , Permeabilidade da Membrana Celular , Colesterol/metabolismo , Ácidos Graxos/análise , Feminino , Humanos , Técnicas In Vitro , Manitol/metabolismo , Fluidez de Membrana , Lipídeos de Membrana/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Gravidez , Trofoblastos/química , Ureia/metabolismo , Água/metabolismoRESUMO
Enzymological and endocrine studies of rat serum cholinesterase (CHE) suggest that this enzyme is subject to a complex and specific form of regulation. Adult CHE activity levels are 3-fold higher in adult females than in males. Gonadectomy and/or hypophysectomy abolish these sex differences. Androgen and estrogen replacement to gonadectomized but not to hypophysectomized animals reverses this action. Adrenalectomy produces no significant changes in serum CHE levels in either male or female rats. An ectopic pituitary plus the appropriate steroid cannot reverse the effect of hypophysectomy. The catalytic properties of CHE alter concurrently with isozyme changes and are reflected in the changes in the ratio of hydrolysis of the butyryl and acetyl substrates. Androgens and estrogens, acting through the hypothalamic-hypophyseal axis, appear to modulate the synthesis of specific CHE isozymes.
Assuntos
Adrenalectomia , Castração , Colinesterases/sangue , Hormônios Esteroides Gonadais/farmacologia , Hipofisectomia , Envelhecimento , Animais , Animais Recém-Nascidos , Dietilestilbestrol/farmacologia , Di-Hidrotestosterona/farmacologia , Estradiol/farmacologia , Feminino , Isoenzimas/sangue , Masculino , Hipófise/transplante , Ratos , Fatores Sexuais , Testosterona/farmacologia , Transplante HomólogoRESUMO
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.
Assuntos
Retardo do Crescimento Fetal/metabolismo , Proteínas de Transporte de Monossacarídeos/análise , Proteínas do Tecido Nervoso , Placenta/química , Adulto , Feminino , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 3 , Humanos , Immunoblotting , Imuno-Histoquímica , Lipídeos de Membrana/análise , Proteínas de Membrana/análise , GravidezRESUMO
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.
Assuntos
Expressão Gênica , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Placenta/metabolismo , Gravidez em Diabéticas/metabolismo , Adolescente , Adulto , Transporte Biológico , Glicemia/metabolismo , Membrana Celular/metabolismo , Diabetes Gestacional/metabolismo , Feminino , Glucose/metabolismo , Transportador de Glucose Tipo 1 , Hemoglobinas Glicadas/metabolismo , Humanos , Immunoblotting , Microvilosidades/metabolismo , GravidezRESUMO
Neonatal administration of o,p'-DDT [1, 1, 1-trichloro-2-(o-chlorophenyl-2-(chlorophenyl)ethane] or methoxychlor resulted in elevated levels of sex-differentiated hepatic monoamine oxidase activities in adult rats, but not in prepubertal animals. Exposure to these hormonally active xenobiotics may have changed the brain hormone environment during the critical period of development, resulting in endocrine alterations that were reflected by latent but permanent increases in hepatic monoamine oxidase activities, i.e. "altered imprinting". Hepatic glutathione S-transferases and cytochrome P-450 content also underwent sex differentiation, but neonatal treatment with o,p'-DDT or methoxychlor did not alter levels in adult rats. However, glutathione S-transferase activities and cytochrome P-450 content were higher in prepubertal animals treated neonatally with o,p'-DDT. In contrast to monoamine oxidase, effects on glutathione S-transferase activities and cytochrome P-450 content were attributed to induction by these xenobiotics.
Assuntos
DDT/toxicidade , Fígado/enzimologia , Metoxicloro/toxicidade , Monoaminoxidase/análise , Animais , Sistema Enzimático do Citocromo P-450/análise , Estrogênios/metabolismo , Feminino , Glutationa Transferase/análise , Fígado/efeitos dos fármacos , Masculino , Ratos , Ratos Endogâmicos , Fatores Sexuais , Maturidade SexualRESUMO
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.
Assuntos
Proteínas de Transporte de Monossacarídeos/metabolismo , Placenta/metabolismo , Animais , Feminino , Retardo do Crescimento Fetal/metabolismo , Humanos , Camundongos , Proteínas de Transporte de Monossacarídeos/genética , Placentação , Gravidez , Gravidez em Diabéticas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Especificidade da EspécieRESUMO
A simple method for determining unidirectional transplacental flux in the in vitro perfusion (transfer factor analysis) has been derived from a general treatment of non-compartmental analysis describing transfer between two accessible pools. This method was validated by comparison of the unidirectional transfer fractions for Evans Blue in a two-pool hydraulic model with the true transfer fractions determined from the pump flow rates in the model. There was excellent agreement between calculated and true transfer fractions. Transfer fractions obtained using this method were also compared to the fractions determined by a previously described technique, deconvolution analysis, for a hydraulic model in which a third, inaccessible pool was interposed between the two accessible pools. Good agreement was found between the two methods. Similar agreement was found for the fractional transfer of [14C]L-lactate in the in vitro perfused human placenta, calculated using transfer factor and deconvolution analysis. The sample collection and data processing are much simpler using the former method but the quality of information obtained is reduced accordingly.
Assuntos
Placenta/metabolismo , Feminino , Humanos , Técnicas In Vitro , Lactatos/metabolismo , Ácido Láctico , Modelos Biológicos , GravidezRESUMO
Experiments were performed to characterize the ionic conductances in microvillous and basal membranes from human placenta. Microvillous and basal membranes were prepared from term placental tissue by homogenization, magnesium precipitation, differential and sucrose density gradient centrifugation. The relative permeabilities of sodium, potassium and chloride were measured using the bi-ionic potential technique which employs a fluorescent probe [diS-C3-(5)] which partitions into membranes in a potential-dependent manner. The permeabilities of sodium and chloride relative to potassium were determined by measuring their effects on a known membrane potential produced by a potassium gradient. In microvillous membranes PNa/PK = 0.25 and PClPK = 0.19 while in basal membranes, PNa/PK = 1.31 and PCl/PK = 0.03. Measurements of chloride permeability relative to sodium confirmed these results. The cation conductances were inhibited by quaternary ammonium compounds. Addition of tetramethylammonium altered the relative permeabilities in a pattern suggesting a block of potassium conductance while tetraethylammonium appeared to block both sodium and potassium conductances.
Assuntos
Cloro/farmacocinética , Potássio/farmacocinética , Sódio/farmacocinética , Trofoblastos/fisiologia , Membrana Basal/fisiologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Canais Iônicos/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Microvilosidades/fisiologiaRESUMO
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.
Assuntos
Idade Gestacional , Trofoblastos/citologia , Trofoblastos/fisiologia , Feminino , Humanos , Concentração de Íons de Hidrogênio , Microscopia de Fluorescência , Fosforilação , Gravidez , Proteína Quinase C/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
The goal of this research was to investigate movement of sugars across placental plasma membranes. Changes in vesicle volume produced by solute uptake were measured by light scattering. Analysis, performed by fitting of the light scattering data to exponentials, revealed that for certain sugars such as glucose, a rapid component and a second, slower transport process were present. Measurements in the presence of the glucose transport inhibitor phloretin, comparison with the transport of mannitol and analysis of the concentration dependence of the two transport components were used to demonstrate that these two processes are consistent with protein-mediated and lipid-diffusional transport of glucose. Calculation of glucose flux rates using the time constants which define these processes provided values similar to those determined by radioisotopic methods. Glucose, 2-deoxyglucose and galactose were transported both by carrier-mediated and diffusional processes, while mannitol, fructose, ribose and 2-deoxyribose were transported solely by the latter process and not by a protein carrier. The rate of glucose transport across the syncytiotrophoblast basal membrane was slightly greater than that across the microvillous membrane, in contrast to that predicted previously by immunoblotting. In addition, measurements of hexose transmembrane diffusion showed that microvillous and basal transport rates were similar and lower than previously determined. We conclude that this new technique represents a simple and rapid method for investigating sugar transport across placental membranes.
Assuntos
Metabolismo dos Carboidratos , Luz , Placenta/metabolismo , Espalhamento de Radiação , Transporte Biológico , Desoxiglucose/metabolismo , Difusão , Feminino , Frutose/metabolismo , Galactose/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Transportador de Glucose Tipo 1 , Humanos , Metabolismo dos Lipídeos , Manitol/metabolismo , Troca Materno-Fetal , Proteínas de Transporte de Monossacarídeos/metabolismo , Gravidez , Rafinose , Ribose/metabolismoRESUMO
The dual (stimulatory and inhibitory) regulation of adenylyl cyclase was studied in syncytiotrophoblast basal membranes prepared from term human placenta. Stimulation of adenylyl cyclase activity with GTP, non-hydrolyzable GTP analogs, isoproterenol and PGE1 was observed, confirming the presence of an intact stimulatory pathway in these membranes. Investigations of the inhibitory pathway revealed tight coupling of the G-protein, Gi alpha, to catalytic adenylyl cyclase, with high doses of GTP producing 80 per cent inhibition of GTP/forskolin-stimulated activity. Confirming Gi alpha involvement, pertussis toxin (PTX) treatment of basal membranes augmented the responses of adenylyl cyclase to both GTP and forskolin. In addition, immunoblotting of basal membrane proteins revealed the presence of the G-protein subunits, Gs alpha, Gi alpha, and G beta/gamma. The response of adenylyl cyclase was measured to a series of agonists known to inhibit adenylyl cyclase in other tissues, however a reproducible inhibitory effect was produced only by somatostatin (approximately 80 per cent). Treatment of basal membranes with PTX caused a degree of reversal of the somatostatin-mediated adenylyl cyclase inhibition. However, the intoxication was insufficient to restore GTP/forskolin-stimulated activity.
Assuntos
Adenilil Ciclases/metabolismo , Células Gigantes/enzimologia , Placenta/enzimologia , Toxina Adenilato Ciclase , Adenilil Ciclases/efeitos dos fármacos , Artefatos , Eletroforese em Gel de Poliacrilamida , Feminino , Proteínas de Ligação ao GTP/análise , Nucleotídeos de Guanina/farmacologia , Humanos , Immunoblotting , Toxina Pertussis , Gravidez , Somatostatina/antagonistas & inibidores , Fatores de Virulência de Bordetella/farmacologiaRESUMO
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.
Assuntos
Expressão Gênica , Isoenzimas/genética , Placenta/enzimologia , Proteína Quinase C/genética , Sequência de Aminoácidos , Animais , Bovinos , Membrana Celular/enzimologia , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Microvilosidades/enzimologia , Dados de Sequência Molecular , Gravidez , Proteína Quinase C/química , Ratos , Transdução de Sinais , Trofoblastos/enzimologiaRESUMO
In order to establish a gestational profile for placental transcellular permeabilities to water, urea and mannitol, syncytiotrophoblast microvillous (MVM) and basal membrane (BM) vesicles were isolated from human placentae obtained from 16 weeks of gestation to term. Using stop-flow/light-scattering techniques the rate of change in vesicle volume in response to an osmotic challenge was measured and osmotic water permeabilities (Pf) and solute permeabilities (Ps) calculated. Membrane fluidity was assessed by steady-state DPH anisotropy. Permeability of MVM to water and solutes increased by 20-30 per cent in mid-pregnancy and declined again after the 36th week of gestation. In BM, this pattern was apparent only for water permeability; solute permeabilities were not significantly altered. MVM cholesterol content was approx two-fold higher and membrane fluidity lower compared to BM. Cholesterol content in BM, but not in MVM, increased during the late third trimester. Membrane fluidity did not change consistently during gestational development. We conclude that syncytiotrophoblast plasma membranes exhibit small but significant changes in passive permeability to water and non-electrolytes from 16 weeks of gestation to term. It is suggested that an increased water permeability of the syncytiotrophoblast plasma membranes might contribute substantially to the gestational increase in water exchange across the human placenta observed in vivo.
Assuntos
Permeabilidade da Membrana Celular , Membrana Celular/metabolismo , Idade Gestacional , Trofoblastos/ultraestrutura , Água/metabolismo , Colesterol/análise , Feminino , Humanos , Manitol/metabolismo , Fluidez de Membrana , Microvilosidades/química , Microvilosidades/metabolismo , Concentração Osmolar , Gravidez , Ureia/metabolismoRESUMO
Respiratory parameters were studied in mitochondria from human placenta. Respiratory control and ADP/O ratios were low in this preparation. The adenine nucleotide content of placental mitochondria was found to be only one quarter of that found for adult uterine muscle tissue mitochondria prepared in the same way. Loading placental mitochondria with adenine nucleotides by incubation in the presence of ATP produced increased respiratory control ratios but no improvement in ADP/O ratios. Our evidence is consistent with the developmental changes shown to occur in rat liver, in which an increased concentration of adenine nucleotides is responsible for changes in respiratory parameters.
Assuntos
Nucleotídeos de Adenina/farmacologia , Mitocôndrias/metabolismo , Consumo de Oxigênio , Placenta/ultraestrutura , Difosfato de Adenosina/análise , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Mitocôndrias/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Gravidez , Útero/ultraestruturaRESUMO
The transfer of lactate across the human placenta was investigated using an in vitro dually-perfused placental preparation. Using a novel technique, the unidirectional flux of L-lactate was found to be linearly dependent on L-lactate concentration. In addition, unidirectional transfer rates were found to be the same in both maternal-to-fetal and fetal-to-maternal directions at the same lactate concentration. Transfer of [14C]L-lactate was decreased by approximately 15 per cent in competition with unlabelled L-lactate. Stereospecificity and permeability experiments demonstrated the existence of a transfer mechanism which could distinguish between the L- and D-isomers of lactate. Our data suggest that, while a stereospecific carrier for lactate exists in the perfused placenta, the bulk of transplacental lactate transfer takes place by non-carrier-mediated diffusion.