[Neurotrophins and pain in endometriosis]. / Neurotrophines et douleur: étude d'expression et de corrélation dans l'endométriose.

OBJECTIVES: To evaluate the expression of five members of the neurotrophins family in ovarian endometriotic cyst (endometrioma) (OMA), compared to eutopic endometrium (EE) and to examine the correlation between the levels of induction and the pain intensity. PATIENTS AND METHODS: Twelve Caucasian women in luteal phase, operated for painful stage IV endometriosis were assigned to 2 groups according to a total Visual Analog Scale (tVAS) score above 15 or below 10. tVAS takes into account all VAS scores for dysmenorrhea, deep dyspareunia, non cyclic chronic pelvic pain, gastrointestinal and lower urinary symptoms. Samples of OMA and EE were processed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) for NGF, BDNF, NT-3, NT-4/5 and NTRK2 mRNA expression. Expression levels in OMA were compared to those in EE on one hand and between two groups of 6 mild painful and 6 highly painful patients on the other. RESULTS: All neurotrophins were significantly higher expressed in OMA than in EE, in particular NGF and BDNF (induction ratios: 20.6 and 9.7, respectively). In contrast, no correlation was observed between induction ratios and pain intensity. CONCLUSION AND DISCUSSION: This is the first study reporting an over-expression of all neurotrophins in endometriosis, as only NGF was previously documented. It confirms the central role of this family in the genesis and modulation of pain in endometriosis. Anti-neurotrophin selective therapy might be a promising way of analgesia in the future.

Cullins in human intra-uterine growth restriction: expressional and epigenetic alterations.

Intra-uterine growth restriction (IUGR) is defined by a restriction of fetal growth during gestation. It is a prevalent significant public health problem that jeopardizes neonatal health but also that can have deleterious consequences later in adult life. Cullins constitute a family of seven proteins involved in cell scaffold and in selective proteolysis via the ubiquitin-proteasome system. Most Cullins are critical for early embryonic development and mutations in some Cullin genes have been identified in human syndromes including growth retardation. Our work hypothesis is that Cullins, particularly CUL4B and CUL7, are involved in placental diseases and especially in IUGR. Thus, expression of Cullins and their cofactors was analyzed in normal and pathological placentas. We show that they present a constant significant over-expression in IUGR placentas, whose extent is dependent on the position of the interrogated fragment along the cDNAs, suggesting the existence of different isoforms of the genes. Particularly, the CUL7 gene is up-regulated up to 10 times in IUGR and 15 times in preeclampsia associated with IUGR. The expression of cofactors of Cullins participating to functional complexes has also been evaluated and showed a similar significant increase in IUGR. Promoters of Cullin genes appeared to be under the control of the SP1 transcription factor. Finally, methylation levels of the CUL7 promoter in placental tissues are modulated according to the pathological conditions, with a significant hypomethylation in IUGR. These results concur to pinpoint the Cullin family as a new set of markers of IUGR.

A hierarchical analysis of transcriptome alterations in intrauterine growth restriction (IUGR) reveals common pathophysiological pathways in mammals.

Intra-uterine growth restriction (IUGR) is a frequent disease, affecting up to 10% of human pregnancies and responsible for increased perinatal morbidity and mortality. Moreover, low birth weight is an important cause of the metabolic syndrome in the adult. Protein depletion during the gestation of rat females has been widely used as a model for human IUGR. By transcriptome analysis of control and protein-deprived rat placentas, we were able to identify 2543 transcripts modified more than 2.5 fold (1347 induced and 1196 repressed). Automatic functional classification enabled us to identify clusters of induced genes affecting chromosome structure, transcription, intracellular transport, protein modifications and apoptosis. In particular, we suggest the existence of a complex balance regulating apoptosis. Among repressed genes, we noted several groups of genes involved in immunity, signalling and degradation of noxious chemicals. These observations suggest that IUGR placentas have a decreased resistance to external aggression. The promoters of the most induced and most repressed genes were contrasted for their composition in putative transcription factor binding sites. There was an over-representation of Zn finger (ZNF) proteins and Pdx1 (pancreatic and duodenal homeobox protein 1) putative binding sites. Consistently, Pdx1 and a high proportion of ZNF genes were induced at the transcriptional level. A similar analysis of ZNF promoters showed an increased presence of putative binding sites for the Tata box binding protein (Tbp). Consistently again, we showed that the Tbp and TBP-associated factors (Tafs) were up-regulated in IUGR placentas. Also, samples of human IUGR and control placentas showed that human orthologous ZNFs and PDX1 were transcriptionally induced, especially in non-vascular IUGR. Immunohistochemistry revealed increased expression of PDX1 in IUGR human placentas. In conclusion, our approach permitted the proposition of hypotheses on a hierarchy of gene inductions/repressions leading to massive transcriptional alterations in the IUGR placenta, in humans and in rodents.

Expression and localization of alpha-fetoprotein mRNA and protein in human early villous trophoblasts.

Alpha-fetoprotein (AFP) is a major plasma protein produced during human fetal life. It is a good marker for several possible disorders affecting gestation. We previously reported that afp gene expression, which takes place mainly in yolk sac and fetal liver, also occurs in normal human placenta, specifically in early pregnancy. The aim of the present study was to determine the precise location of AFP synthesis sites within the placental villi. In situ hybridization and immunohistochemical experiments were performed on sections obtained from placentas of first-trimester and full-term pregnancies. We found that the pattern of afp gene expression was restricted to specific villous trophoblastic areas in early placentas. Both afp transcripts and AFP protein were mainly located in discontinuous regions, at junctions between two villi and at budding sites. In contrast, no AFP expression was detected in the cytotrophoblastic extravillous proliferative zone or in other placental cell types. According to the earlier studies, no AFP synthesis was detected in placental villous tissue from full-term pregnancies, using in situ hybridization and immunohistochemistry.

Alpha-fetoprotein gene expression in early and full-term human trophoblast.

Alpha-fetoprotein (AFP) is a major serum glycoprotein synthesized during fetal life mainly by the yolk sac and the fetal liver. At term, it reaches high concentrations in the maternal intervillous blood, which is in direct contact with the placental trophoblastic microvillous membrane, and this suggests the placental origin of the AFP at the fetal-maternal interface. We used several experimental approaches to investigate the expression of AFP gene and fetal protein production in early gestation and term placentas. RT-PCR and immunological studies clearly identified AFP messenger RNA and AFP protein in the placental villi from first trimester of pregnancy. The AFP gene was also expressed in highly purified cytotrophoblasts from early placentas, and enzymo-immunoassay showed that AFP protein was synthesized and secreted by early cytotrophoblasts. AFP was also detected in the cytoplasm of these cells by immuno-cytochemistry. However, none of these methods detected any expression of the AFP gene in full-term placental villi or in cultured trophoblasts. These findings demonstrate that both AFP mRNA and protein are present in trophoblastic cells early in pregnancy. The absence of AFP gene expression in term placental villi also suggests, that the AFP at the fetal-maternal interface is attributable to a notable transplacental passage of AFP from fetal blood in late pregnancy.

Corticosteroid-binding globulin status at the fetomaternal interface during human term pregnancy.

The status of the corticosteroid-binding globulin (CBG) at the fetomaternal interface, especially in the maternal intervillous blood space (I), was investigated and compared to that of CBG in the maternal (M) and fetal (umbilical arteries [A] and vein [V]) peripheral circulations at term. Immunoquantitation of plasma CBG showed that the CBG concentration in I was 30% less than that in M (P < 0.001) and threefold higher than that in umbilical cord blood (P < 0.001). The microheterogeneity of CBG studied by immunoaffinoelectrophoresis in the presence of concanavalin A and Western blotting indicated that the CBG in I was mainly of maternal origin and different from fetal CBG. A CBG mRNA, but no classic 50- to 59-kDa CBG, was found in isolated term trophoblastic cells. The steroid environment of the CBG in I differed greatly from that in the peripheral maternal and fetal circulations, because the progesterone:cortisol molar ratio in I was 75-fold higher than that in M and 7- to 10-fold higher than that in the fetal circulation. Binding studies revealed that the affinity constants of CBG for cortisol in I, A, and V were significantly lower than that in M plasma (P < 0.02) in their respective hormonal contexts. The binding parameters for I-CBG stripped of endogenous steroids and lipids were close to those for M-CBG but different from those of fetal CBG (P < 0.001). These data reflect the physiological relevance of the CBG-steroid interaction, especially with very CBG-loaded progesterone at the fetomaternal interface during late pregnancy.

Evidence for progesterone receptors in the human fetoplacental vascular tree.

The presence of progesterone receptors (PR) throughout the human term fetoplacental vascular tree was investigated. By reverse transcription-polymerase chain reaction (RT-PCR), we showed expression of PR mRNAs in stem villi vessels, chorionic arteries and veins, and umbilical arteries and veins. Binding studies and Scatchard analysis revealed a single class of high-affinity binding sites for (3)H-R5020 (promegestone) in cytosolic extracts of all placental vessels, with K(d) values in the range of 2.5-4 nM. High levels of PR were detected in placental vessels compared to other vascular tissues. Thus, maximum binding capacities of stem villi vessels, chorionic arteries and veins, and umbilical arteries and veins were 247 +/- 25, 377 +/- 58, 295 +/- 40, 371 +/- 118, and 672 +/- 144 fmol/mg protein, respectively. Endothelial cell elimination in chorionic arteries did not significantly modify the number of PR. RT-PCR and binding studies also assessed PR expression in cultured placental vascular smooth muscle cells isolated from stem villi vessels. All these data suggested that most of the PR of fetoplacental vessels were from the media. In conclusion, we report here the first evidence of the presence of PR in the muscular layer of human term fetoplacental vessels. This finding, together with the high progesterone concentrations in cord blood, suggests that the interactions between the PR and its ligand may play a role in the physiology and physiopathology of human fetoplacental vascularization.

Endothelin receptor subtypes in the microvillous trophoblastic membrane of early gestation and term human placentas.

The 125I-labeled endothelin-1 ([125I]ET-1) binding sites in microvillous membranes from early gestation and term human placentas were investigated. The Kds for [125I]ET-1 binding to early gestation (68 +/- 15 pmol/l) and term (45 +/- 8 pmol/l) microvilli (n = 4) were not significantly different. The density of binding sites decreased significantly, from 243 +/- 80 fmol/mg protein in early gestation microvilli to 54 +/- 10 fmol/mg protein in term microvilli. The endothelin (ET) receptor (ET-R) subtype profiles were determined by competition binding studies with unlabeled ET-1, ET-3, and selective agonists and antagonists for ETA-R and ETB-R. In early gestation placental microvilli, we observed the presence of 72%) ETB-R, (mainly ETB2-R subtype), and 28%. ETA-R. Only ETB-R (mainly the ETB2-R subtype) was present in term placental microvilli. We suggest that the ETB-R on the placental microvillous membrane is involved in specific trophoblastic functions and may play a major role in ET clearance by modulating the amounts of ETs in the maternal intervillous blood space.

Endothelin-1 and ETA receptor expression in vascular smooth muscle cells from human placenta: a new ETA receptor messenger ribonucleic acid is generated by alternative splicing of exon 3.

Endothelin-1 (ET-1) is a potent vasoactive peptide in stem villi vessels, which are considered to be the major sites of placental vascular resistance. To investigate the influence of pregnancy-specific hormonal environment on ET and ET receptor (ET-R) expression, we first developed and characterized a culture of vascular smooth muscle cells from stem villi vessels. Secondly, we investigated whether the muscular layer of stem villi vessels could be a site of the ET expression described in the placenta, and we examined this expression in placental vascular smooth muscle cells (PVSMCs). Prepro-ET-1 and prepro-ET-3 messenger ribonucleic acid (mRNA) were identified in stem villi vessels, whereas only prepro-ET-1 mRNA was observed in PVSMCs. Third, with the goal of using PVSMCs as ET target cells, we characterized the ET-R expressed by these cells in comparison with the muscular layer of stem villi vessels. Whereas both ETA-R and ETB-R are present in stem villi vessels, we found that PVSMCs express exclusively ETA-R. In addition to the previously reported ETA-R spliced transcripts, we described a new ETA-R transcript, ETA-R delta 3, generated by exclusion of exon 3 in stem villi vessels and PVSMCs. Alternative splicing mechanisms of ETA-R mRNA could constitute a control of the abundance of active ETA-R in terms of contractility. PVSMCs will be a useful model to study the environmental stimuli involved in the regulation of ET and ET-R expression in the muscular layer of feto-placental vasculature.

G protein expression in human fetoplacental vascularization. Functional evidence for Gs alpha and Gi alpha subunits.

GTP-binding proteins are key elements in coupling receptors to various effector systems. Using ADP-ribosylation by cholera (CTX) and pertussis (PTX) toxins and an immunodetection technique, we investigated the G protein expression profile in smooth muscle of stem villi vessels obtained from human term placentae. In placental vascular smooth muscle, we report the presence of two CTX-protein substrates of 42 and 45 kDa recognized by Gs alpha antibodies, and three Gi alpha isoforms, substrates of PTX, identified as Gi1 alpha, Gi3 alpha (two proteins of 41 kDa) and Gi2 alpha (a 40-kDa protein). We also characterized another target of PTX, a 40-kDa Go alpha-immunoreactive protein and detected the PTX-insensitive Gq-Gi1 alpha proteins. To assess the functional significance of the G alpha proteins identified in this tissue, we measured the adenylyl cyclase activity in the presence of guanyl nucleotides alone or with increasing concentrations of vasoactive intestinal peptide (VIP), and examined whether VIP-bound sites, in the presence of GTP gamma S, promote the release of G alpha proteins from the membranes of vascular smooth muscle. At low concentrations (0.1 nM to 0.01 microM), guanyl nucleotides stimulated adenylyl cyclase activity in a dose-dependent manner, while at higher concentrations (10 microM to 1 mM) the stimulation rate of cAMP production by guanyl nucleotides decreased. In a dose-dependent manner, VIP in the presence of GTP gamma S increased adenylyl cyclase activity and specifically promoted the release of both Gs alpha isoforms. In contrast, the release of Gi1 and Gi2 alpha isoforms was not significantly increased in the presence of VIP, while GTP gamma S alone stimulated their release. Our data show physical evidence of the activation of Gs proteins by VIP-bound membrane receptors, resulting in dissociation and release of Gs alpha subunits in the soluble fraction. They assess the specific coupling of the two Gi alpha isoforms to VIP receptors in smooth muscle wall of placental stem villi vessels. It would be of interest to investigate whether changes in Gs alpha expression and/or function are associated with the placental angiogenesis process during pregnancy.

Contractile proteins in human fetoplacental vessels.

OBJECTIVE: Our purpose was to compare the protein isoform composition of the contractile apparatus at different levels of the fetoplacental vessel musculature at term. STUDY DESIGN: Umbilical, chorionic, and stem villi vessel protein extracts were run on one- and two-dimensional gel electrophoresis; previously characterized human myometrium proteins were used as the smooth muscle proteins of reference. RESULTS: Fetoplacental vessel musculature exhibited a high actin/myosin ratio. The presence, in varying quantities, of myosin heavy chain and actin isoforms of smooth muscle type in the different vessels reflected their degree of differentiation. The presence of nonmuscle protein isoforms, particularly in stem villi vessels, indicated a certain degree of immaturity. CONCLUSIONS: The presence of smooth muscle contractile protein isoforms indicates that fetoplacental vessel musculature is highly differentiated. Regional modulation of fetoplacental blood flow could be, in part, the result of local differences in contractile apparatus protein composition.

Endothelin-induced phosphoinositide hydrolysis in the muscular layer of stem villi vessels of human term placenta.

In the present study, we examined the relationship between endothelin receptors and phosphoinositide breakdown in muscle explants of placental stem villi vessels. All peptides examined, i.e. endothelin-1 (ET-1), ET-3, sarafotoxin 6b (S6b) and S6c, were able to induce phosphoinositide hydrolysis in a dose-dependent manner: ET-1 was more potent than S6b and ET-3, with corresponding EC50 values of 44 +/- 16 pmol/l, 18 +/- 13 nmol/l and 33 +/- 24 nmol/l, respectively. Sarafotoxin induced only moderate stimulation of inositol phosphate accumulation. Both ET-1- and S6b-induced accumulation of inositol phosphate was almost totally (90%) inhibited by 100 mumol/l BQ 123, while the S6c response was not affected by the ETA receptor antagonist. In contrast, the ETB receptor antagonist IRL 1038 inhibited S6c-induced inositol phosphate accumulation by more than 80%, whereas inhibition was only about 30% for ET-1 and S6b stimulations. This indicates that both ETA and ETB receptors were coupled to the phospholipase C transducing system in the muscular layer of placental stem villi vessels, and there is evidence that the phosphoinositide hydrolysis response is obtained predominantly via ETA receptor activation.

Endothelial modulation of vasoconstrictor responses to endothelin-1 in human placental stem villi small arteries.

1. The aim of this study was to assess the role of endothelial cells in the modulation of vasocontractile responses to endothelin-1 (ET-1) of human placental vasculature. 2. Isolated stem villi small arteries (diameter = 170-250 microns) were obtained from healthy parturients who underwent caesarean surgery during the 39th week of pregnancy for cephalo-pelvic disproportion. Isometric tension was measured in vascular rings mounted in a myograph system and challenged with ET-1 (10(-12) to 10(-6) M). 3. The vasocontractile response to ET-1 was significantly (P < 0.001) increased in endothelial-denuded (active tension = 1156 +/- 214 mN mm-1) as compared with endothelial-preserved vascular rings (active tension = 458 +/- 48 mN mm-1). This difference was significantly (P < 0.05) but only partly abolished by the NO synthase inhibitor N omega-nitro-L-arginine (L-NOARG, 10(-4) M). 4. In endothelial-preserved rings submaximally precontracted with 5-hydroxytryptamine (10(-6) M), ET-1 (10(-12) to 10(-9) M) induced dose-dependent relaxation (maximum relaxation = 70 +/- 7%) at 10(-9) M, which was followed, at higher doses (10(-8) to 10(-6) M), by a contraction. In contrast, no relaxation was seen in endothelial-denuded rings. The relaxation in rings with endothelium was significantly (P < 0.001) reduced by L-NOARG (10(-4) M. Moreover, it was totally abolished by combined pretreatment with L-NOARG (10(-4) M) and the sulphonylurea glibenclamide (10(-5) M). 5. In conclusion, endothelial cells modulate the vascular responses to ET-1 through the release of NO and a substance acting on the ATP-sensitive K+ channel of smooth muscle of stem villi small arteries from healthy parturients.

In vitro contractile and relaxant responses of human resistance placental stem villi arteries of healthy parturients: role of endothelium.

As feto-placental vessels in humans are not innervated, regulation of vascular tone in the fetal extracorporeal circulation most likely depends on either circulating hormones or local paracrine mechanisms. However, the latter have not yet been fully investigated. The aim of our study was to characterize vasomotor behaviour of resistance stem villi arteries when challenged with various constrictor and dilator agents, with special emphasis on the physiological importance of endothelium. The latter is poorly characterized in this particular vascular bed in humans. Villous stem arterial rings (internal diameter 182 +/- 6 microns) were isolated under microscopy from term human placentae obtained after cesarean section. The vessels were mounted as ring preparations in an isometric myograph for tension measurements. Endothelium was removed from some of the rings by gentle insertion of a knotted human hair into the vascular lumen. Of the three vasoconstrictors tested, endothelin-1 (ET-1) showed the greatest potency, being 1,000 times more potent than serotonin and phenylephrine. The classical endothelium-dependent vasodilators, acetylcholine, adenosine diphosphate (ADP) and histamine, caused dose-dependent relaxation of the rings; an effect which was completely abolished by the removal of endothelium. Pre-treatment with the nitric oxide (NO) synthase inhibitor, N omega-nitro-L-arginine, also markedly reduced the endothelium dependent relaxant responses to ADP. By contrast, the vasodilatory response to sodium nitroprusside was not affected by endothelial removal. We conclude that i) ET-1 is a potent vasoconstrictor of the human placental vascular bed and ii) placental villous endothelial cells synthesize and release relaxing factor(s) which could possibly be NO.

Localization and production of immunoreactive endothelin-1 in the trophoblast of human placenta.

Regarding their endocrine and paracrine activities, endothelins can be considered as peptide hormones and growth factors. The presence of endothelin-1 (ET-1)-binding sites on smooth muscle of placental villous vessels, on villous trophoblast and on purified trophoblast in culture raises the question of the origin of the peptide. In placenta, endothelin could derive from maternal, fetal and/or endogenous sources. Therefore, localization of ET-1 was investigated by use of immunohistochemistry in human term placenta and in cultured trophoblast using the avidin-biotin-peroxidase complex procedure. Specificity of immunostaining was demonstrated by applying ET-1 antibody that has been preabsorbed with excess peptide. In sections of placenta, ET-1 immunoreactivity (ET-1 IR) was specifically detected in the endothelium of the vessels and in the syncytiotrophoblast of the villi. ET-1 IR was also detected in the decidua-like cells and in the extravillous cytotrophoblast of the basal plate, a region where the maternal and fetal cells intermingle closely. The extravillous cytotrophoblast of the chorionic plate and of the placental septa also exhibited a strong ET-1 IR. For trophoblast culture cytotrophoblastic cells were obtained from placental villi by trypsin-DNase dispersion, further purified on Percoll gradient and enriched by employing a monoclonal anti-HLA class-I antibody. The trophoblastic origin of the cells was demonstrated by immunohistochemistry and by studying the secretion of gestational hormones during culture. After different periods of culture of purified cytotrophoblastic cells (1 to 5 days), ET-1 IR was observed in 95% of cells: cytotrophoblastic cells, trophoblast aggregates, and syncytiotrophoblast.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelin-1 binding sites and immunoreactivity in the cultured human placental trophoblast: evidence for an autocrine and paracrine role for endothelin-1.

In human placenta, endothelin (ET) could derive from maternal, fetal, and/or endogenous sources. Therefore, localization of ET-1 was investigated by immunohistochemistry in human term placenta and in cultured trophoblasts. In sections of placenta, ET-1 immunoreactivity (ET-1 IR) was specifically detected in the endothelium of the vessels and in the syncytiotrophoblasts of the villi. ET-1 IR was also detected in the decidual cells and in the extravillous cytotrophoblasts of the basal plate. The extravillous cytotrophoblasts of the chorionic plate and of the placental septa also exhibited strong ET-1 IR. For trophoblast culture, cytotrophoblastic cells were obtained from placental villi by trypsin-DNAse dispersion, further purified on a Percoll gradient, and enriched by employing a monoclonal anti-HLA class I antibody. After different periods of culture of purified cytotrophoblastic cells (1-5 days), ET-1 IR was observed in 95% of cells: cytotrophoblastic cells, trophoblast aggregates, and syncytiotrophoblasts. The presence of ET-1,2 immunoreactivity (ET-1,2 IR) in the culture media was demonstrated by radioimmunoassay. A uniform daily production of the peptide was observed over at least 5 days (approximately 50 fmol/10(6) cells/24 h). Furthermore, trophoblastic cells that had been cultured for 5 days contained significant amounts of ET-1,2 IR (24 fmol/10(6) cells). These results suggest a trophoblastic origin for ET-1 and support the hypothesis of a paracrine and autocrine action of the peptide in the physiology of the trophoblast and placenta.

Biochemical characterization and autoradiographic localization of [125I]endothelin-1 binding sites on trophoblast and blood vessels of human placenta.

The presence of endothelin binding sites in the human placenta raises the question of the precise localization of these receptors on well defined placental constituents. In order to find an answer to this problem various approaches were used. Specific binding sites for [125I] endothelin-1 (ET-1) were identified on human term placenta, not only on membranes of smooth muscles stem villi vessels, but also on trophoblastic plasma membranes prepared from trophoblast in culture. Scatchard analysis of binding data revealed a single class of high affinity binding sites with Kd values of 26 +/- 4 pmol/L for stem villi vessels and 126 +/- 4 pmol/L for trophoblast in culture, with maximum binding capacities of 681 +/- 61 and 224 +/- 53 fmol/mg protein, respectively. The anatomical localization of these binding sites was determined by in vitro autoradiography. Autoradiograms obtained from placental sections incubated with [125I]ET-1 indicate that [125I]ET-1 high affinity binding sites exist on placental stem villi vessels and on the trophoblastic layer of the villi. The latter localization was also found on autoradiograms of trophoblast in culture. The human placental syncytiotrophoblast is a polarized epithelium with the microvillous membrane, facing maternal blood space and the basal plasma membrane, facing fetal circulation. [125I]ET-1 high affinity binding sites are present on both membranes but the number of binding sites is higher on the basal plasma membrane. These findings lead to the suggestion that ET-1 may be involved in the regulation of the feto-placental circulation and may subserve specific trophoblastic functions.

Regional distribution and pharmacological characterization of [125I]endothelin-1 binding sites in human fetal placental vessels.

High-affinity binding sites for [125I]endothelin(ET)-1 have been detected in purified membrane preparations of the fetal arteries and veins of the chorionic plate and the stem villi vessels of the human term placenta. Regardless of the vessel type, the apparent dissociation constant was found to be in the picomolar range (26----45 pM), and the Bmax value close to 600 fmol/mg protein. In stem villi vessels, ET-1, ET-2, sarafotoxin S6b and vasocontractor intestinal peptide (VIC) were approximately equipotent in their competitive displacement of [125I]ET-1 binding. The endothelin precursors, human and porcine big-endothelin, recognized ET-1 sites with low affinity (nM range), a finding which reflects their low potency as recognized vasocontractant agents. Interestingly, [125I]ET-1 binding parameters and pharmacological profiles were identical in fetal veins and arteries of the chorionic plate. Similarly, a study carried out in rat aortic membranes, revealed the presence of high affinity [125I]ET-1 binding sites with pharmacological characteristics close to those of the human stem villi vessels. In all vessels investigated, the binding pattern of ET-3 against [125I]ET-1 was of a non-competitive nature. Thus, these results demonstrate the presence of specific [125I]ET-1 binding sites along the vascular tree of the fetal side of the placenta and would support evidence currently available, favouring the existence of distinct ET-1 and ET-3 receptors. Finally, ET-1 in the human placenta may play an important physiological role as regulator of vascular resistance and/or be implicated as a pathological factor in certain pregnancy-related diseases.

Ultrastructural visualization of the internalization of low density lipoprotein by human placental cells.

Low density lipoproteins (LDL) were conjugated to colloidal gold to visualize the route for internalization of LDL in the cultured cells of human term placenta. Cells were obtained from placental villi (caesarian section) by a standard trypsin-DNase dispersion method followed in some cases by a Percoll gradient centrifugation step. Employing electron microscopy it was observed that after 3 days of culture, cells obtained by trypsin-DNase dispersion were a mixture of macrophages, mononucleated cells and large multinucleated cells. When the cells were incubated for 3 days after the Percoll purification, essentially multinucleated cells identical to the syncytiotrophoblast were present. The number of LDL receptor was increased by preincubation in medium with lipoprotein depleted serum. In binding experiments cells incubated at 4 degrees C for 2 h with medium containing gold LDL conjugates showed gold LDL attached to the plasma membrane without characteristic localization. After incubation with gold LDL at 37 degrees C for various times, the three cellular types showed ligand internalization. Gold LDL endocytosis involved first coated pits but also uncoated plasmalemmal invaginations. Then gold LDL was further observed in coated and non coated vesicles, smooth walled endosomes, multivesicular bodies and tubular vesicles. Lastly free gold particles were observed in lysosome like dense bodies. These results prove the internalization of gold LDL conjugates by human cultured placental cells, particularly by syncytiotrophoblast like multinucleated cells. This accumulation of LDL (the major cholesterol carrying protein in humans) is recognised to be responsible for the exogenous cholesterol supply indispensable to the progesterone biosynthesis and cellular growth of the placenta.

Detection of distinct receptors for native and acetylated low-density lipoproteins in human placental microvilli by ligand-immunoblotting.

Ligand-immunoblotting was used to detect distinct receptors for native low-density lipoprotein and for acetylated low-density lipoprotein on microvillous membranes from human term placentas. Antisera directed against native and modified low-density lipoproteins were prepared in rabbits and their specificities were assessed by immunodiffusion and immunoelectrophoresis. The receptor for low-density lipoprotein was detected as a 160 kDa protein and that for acetylated low-density lipoprotein as a 200 kDa protein. These receptors were compared with their counterparts in cultured human skin fibroblasts, bovine adrenal cortex and J774 macrophage-like cells. This is the first investigation that visualizes the presence of receptors for both native and modified low-density lipoproteins in a steroidogenic tissue.