Receptors In Human Placenta Research Articles

Expression of leukaemia inhibitory factor (LIF) receptor in human placenta: a possible role for LIF in the growth and differentiation of trophoblasts.

Leukaemia inhibitory factor (LIF) is a cytokine that displays multiple activities in various tissues and is essential for blastocyst implantation in mice. In the human uterus, LIF is expressed in endometrial tissue and the decidua. To elucidate the role it plays, the mRNA levels for two LIF receptor (R) subunits, LIF-R and gp130, were examined in human endometrium, placenta and decidua by Northern blot hybridization. The expression of LIF-R gene was detected in the chorionic villus during the first trimester, in term placenta, and at lower levels in the decidua. The expression of LIF-R gene was not detectable in non-pregnant endometrium. The expression of the gp130 gene was detected in all tissues examined. During pregnancy, there was no significant change in the mRNA concentration of LIF-R in the placenta, while that of gp130 increased after the second trimester. The human choriocarcinoma cell line, BeWo, was found to express LIF-R and gp130. LIF inhibited forskolin-induced human chorionic gonadotrophin (HCG)-beta production by BeWo in a dose-dependent manner, and it ameliorated forskolin-induced growth suppression. These findings suggest that LIF plays a regulatory role in trophoblast growth and differentiation during pregnancy in human placenta.

Type II and type IIB activin receptors in human placenta

The expression of type II activin receptor(ActRII) and type IIB activin receptor(ActRIIB) messenger RNA(mRNA) in different stages of development of the human placenta was examined by Northern blot analysis. In our experiments, the first trimester placenta showed the highest levels of ActRIIB expression and human placenta from different stages expressed both type II and IIB activin receptor mRNAs. These observations support the hypothesis that activin might act as a paracrine/autocrine factor in human placenta and activin might have stage specific functions through the different types of receptors.

Expression of leukaemia inhibitory factor (LIF) receptor in human placenta: a possible role for LIF in the growth and differentiation of trophoblasts

Expression of leukaemia inhibitory factor (LIF) receptor in human placenta: a possible role for LIF in the growth and differentiation of trophoblasts

Colocalisation of Vascular Endothelial Growth Factor and Its Flt-1 Receptor in Human Placenta

Vascular endothelial growth factor (VEGF) is an angiogenic protein which acts on both endothelial and trophoblast cells. In first trimester placenta, VEGF immunoreactive protein was detected in cytotrophoblast shell suggesting a role in the regulation of cytotrophoblast growth and differentiation as they also expressed VEGF receptor (flt-1) protein. VEGF and flt-1 immunoreactive proteins were expressed in Hofbauer cells within the villous mesenchyme, macrophages and in maternal decidual cells while weak VEGF immunoreactive protein was seen in syncytiotrophoblast surrounding the placental villi in first and second trimester placentae. At term, there was relatively weak VEGF and flt-1 immunostaining in the syncytiotrophoblast while intense VEGF immunostaining was seen in the Hofbauer and maternal decidual cells. Extravillous trophoblast showed immunostaining for flt-1 but no staining for VEGF. Both amnion and chorion expressed strong VEGF immunoreactivity throughout gestation. Smooth muscle cells surrounding the vein and arteries of the umbilical cord showed weak VEGF immunoreactivity while no immunoreactivity was localised in endothelial cells. VEGF stimulated parathyroid hormone-related protein (PTHrP) release (mean (+/- SD): basal, 0.96 +/- 0.03; 10 ng/ml VEGF165, 2.07 +/- 0.18 and 20 ng/ml VEGF165, 2.43 +/- 0.18 pmol/l/well of PTHrP1-86) in condition medium from immortalised first trimester trophoblast cell line. These results suggest that VEGF in addition to acting as an autocrine mitogen for trophoblast proliferation may also function as a paracrine mediator of vascular tone by releasing vasorelaxants from trophoblasts.

Evidence of the existence of cholinergic muscarinic receptors in human placenta

The action of some nicotinic acetylcholine receptor agonists was re-examined on the surface field potentials (N-waves) evoked by electrical stimulation of the lateral olfactory tract in guinea-pig olfactory cortical brain slices. Bath superfusion of nicotine or the nicotinic stimulants dimethylphenylpiperazinium (DMPP), lobeline, cytisine, tetramethylammonium or suberyldicholine (up to 100 μM) had little or no effect on the extracellular N-wave amplitude, or the membrane potential, input resistance or excitability of olfactory neurones recorded intracellularly. In contrast, the muscarinic agonists, carbachol or oxotremorine-M consistently depressed the field in a reversible dose-dependent manner. Interestingly, in the presence of the ganglionic stimulants DMPP (n = 6 slices) or lobeline (n = 5 slices) (10–50 μM), the effects of carbachol or oxotremorine-M were antagonized in a week competitive-type manner (pA2 values = 5.58 and 5.63 respectively, estimated from Schild plots, constrained to unity slope). This anti-muscarinic action was unaffected by d-tubocurarine or hexamethonium. Nicotine, cytisine, tetramethylammonium and suberyldicholine showed much weaker and inconsistent carbachol-blocking effects. Combination of DMPP with atropine produced dose ratio shifts close to those predicted for a common-site interaction of two competitive antagonists. In conclusion, consistent pre- or postsynaptic nicotinic agonist actions could not be detected in olfactory cortex slices; however, some ganglionic nicotinic agonists were shown to exhibit significant anti-muscatinic effects on this preparation. We suggest this action might be due to a direct atropine-like mechanism.

Labelling of D2-dopaminergic and 5-HT2-serotonergic binding sites in human trophoblastic cells using [3H]-spiperone.

We previously reported that dopamine (DA) inhibited the release of human placental lactogen (hPL) from human placental cells. We also demonstrated the presence of D2-dopamine receptors in membrane preparations of human term placenta. The aim of the present study was to characterize these D2 receptors on freshly isolated human trophoblastic cells. The binding of [3H]-spiperone to these cells showed a curvilinear Scatchard plot suggesting the presence of two classes of binding sites (Kd1 = 1.26nM; Kd2 = 44.3nM). Competition experiments showed the following inhibitory binding potencies: serotonin-2 (5-HT2) > or = D2 >>> alpha-adrenergic, beta-adrenergic, D1-dopamine, thus suggesting the presence of 5-HT2 binding sites. We have examined this possibility by blocking [3H]-spiperone binding to 5-HT2 receptors in the presence of 50nM ketanserin, a selective antagonist of 5-HT2 sites. Under this condition, the linear Scatchard plot obtained suggested a single population of homogeneous binding sites for [3H]-spiperone with a Kd of 0.55nM. To further characterize placental D2 receptors we conducted binding experiments with [3H]-raclopride, an more selective D2 antagonist. The linear Scatchard plot obtained with this ligand suggested one class of binding sites for [3H]-raclopride (Kd = 6nM) with the following inhibitory potencies: D2 >>> beta-adrenergic >> 5-HT2, D1, alpha-adrenergic. These results suggest an important paracrine function for DA in human placenta and show for the first time that [3H]-spiperone binds putative 5-HT2 receptors in human placenta.

Growth-stimulatory monoclonal antibodies against human insulin-like growth factor I receptor.

Monoclonal antibodies (mAbs) against purified human placental insulin-like growth factor I (IGF-I) receptors were prepared and characterized. Three IgG mAbs were specific for the human IGF-I receptor and displayed negligible crossreactivity with the human insulin receptor. They stimulated 125I-labeled IGF-I (125I-IGF-I) or 125I-IGF-II binding to purified human placental IGF-I receptors and to IGF-I receptors expressed in NIH 3T3 cells in contrast to the well-studied mAb alpha IR-3, which inhibits 125I-IGF-I or 125I-IGF-II binding to both forms of IGF-I receptors. The mAbs introduced in this study stimulated DNA synthesis in NIH 3T3 cells expressing human IGF-I receptors approximately 1.5-fold above the basal level and the IGF-I- or IGF-II-stimulated level. In contrast, alpha IR-3 inhibited both basal and IGF-I or IGF-II-stimulated DNA synthesis by approximately 30%. Inhibition of IGF-II-stimulated DNA synthesis by alpha IR-3 was as potent as its inhibition of IGF-I-stimulated DNA synthesis, although IGF-II binding to the IGF-I receptors was not inhibited by IGF-II as potently as was IGF-I. With the purified IGF-I receptors, both inhibitory and stimulatory mAbs were shown to activate autophosphorylation of the IGF-I receptor beta subunit and to induce microaggregation of the receptors. These results suggest that conformational changes resulting from receptor dimerization in the presence of either type of mAb may affect the signal-transducing function of the IGF-I receptor differently. These additional mAbs and alpha IR-3 immunoprecipitated nearly 90% of IGF-I binding activity from Triton X-100-solubilized human placental membranes, indicating that IGF-I receptor reactive with these mAbs is the major form of the IGF-I receptor in human placenta.

Evidence for the expression of growth hormone receptors in human placenta

Since human placenta produces a growth hormone variant, it seemed important to search for evidence of GH receptors in that organ. Evidence for the expression of the GH receptor (GHR) gene was obtained by northern blot analysis. In addition, GHR poly A+RNA was detected in RNA from cultured trophoblastic cells, but not from placenta fibroblasts. There was a low but significant specific binding of pituitary GH-N and placental GH-V to placenta plasma membranes. Both variants apparently bound to the same receptor, which is present in the first trimester as well as in the term placenta. These results suggest that placental GH may have paracrine or autocrine functions in the placenta.

Localization of three subtypes of Fcγ receptors in human placenta by immunohistochemical analysis

We investigated the localization of the three subtypes of (Fc gamma R) in the normal human placenta using immunohistochemical and immunocytochemical techniques with specific monoclonal antibodies. The analysis revealed that Fc gamma RI was expressed on Hofbauer cells, that Fc gamma RII was expressed on Hofbauer cells and endothelial cells of fetal vessels, while Fc gamma RIII was expressed on trophoblasts, especially syncytiotrophoblasts. Moreover, we demonstrated that the expression of Fc gamma RI and Fc gamma RII on Hofbauer cells and endothelial cells of the fetal vessels in the 1st trimester placenta was different from that in the 3rd trimester placenta. These results, therefore, indicate that the three subtypes of Fc gamma R in the human placenta may contribute to maintenance of placental functions, because each Fc gamma R molecule displays unique biological functions similar to those on leukocytes.

Scintillation Proximity Assay

Scintillation proximity assay (SPA) technology has been used to investigate the competitive binding of [125I]endothelin-1 (ET-1) by ET-1, endothelin-2 (ET-2), endothelin-3 (ET-3), sarafotoxin 6b (SFTX-b), and big endothelin (big ET) to endothelin receptors in human placenta and porcine lung. Specific binding of [125I]ET-1 to high-affinity receptors was detected in membranes coupled to wheat germ agglutinin (WGA)-coated beads impregnated with scintillant (fluomicrospheres). The binding characteristics of ET-1 were similar in both of the tissues studied. In contrast, the receptor-binding properties of ET-2, ET-3, and SFTX-b were different in human placental and porcine lung membranes. This suggests that different endothelin receptor populations exist in these tissues. In addition, the binding characteristics of ET-3 and SFTX-b differed markedly to those of ET-1 and ET-2 in both tissues. This may be due to the four amino acid residue substitution in the N-terminal loop of both ET-3 and SFTX-b. We have also demonstrated that the ET-1 precursor, big ET, competitively inhibits [125I]ET-1 binding in these tissues, but is 100-fold less potent than ET-1.

Gene Expression of Macrophage Colony‐Stimulating Factor and its Receptor in Human Placenta and Decidua

Macrophage colony-stimulating factor (M-CSF) induces proliferation of monocyte/macrophage progenitor cells and can also activate some functions of mature cells including fetally derived placental cells. To study the role of M-CSF in the pregnant female reproductive tract, the expression of M-CSF mRNA and its receptor, c-fms proto-oncogene, in human placenta and decidua was identified. M-CSF and c-fms mRNAs, 4.7Kb and 3.9Kb respectively, were detected by Northern blotting in the early stage placenta and subsequently increased during pregnancy. These mRNAs were not detected in the nonpregnant endometrium but were strongly induced in maternal decidua with the same mRNA size as in the placenta. Northern blot hybridization on the endometrium of a pseudopregnant uterus revealed that the expression of endometrial M-CSF and c-fms mRNAs is regulated by synergistic action of female sex steroid hormones. These findings indicate that, in an autocrine and/or paracrine manner, M-CSF is deeply involved in the local proliferation and differentiation of cells at the materno-fetal interface, and support the placental immunotrophism hypothesis.

Endothelin receptors in human placenta: relationship to vascular resistance and thromboxane release

This investigation was performed to study the potential role of endothelin in the modulation of fetoplacental vascular resistance in the human placenta. Full-term placentas from uncomplicated pregnancies were studied within 30 min of delivery. The umbilical artery and vein to a single placental cotyledon were cannulated and the artery perfused with RPMI media (0.82 ml/min). Endothelin 1 caused a sustained dose-dependent increase in perfusion pressure. Infused endothelin 1 (50 nM) stimulated thromboxane release 2.3-fold compared with basal values. Thromboxane release persisted for 15 min after discontinuation of endothelin. Properties of human placental endothelin 1 receptors were defined in binding studies performed on a crude membrane fraction of placental cotyledons. Binding was saturable, reached steady state by 3 h at 25 degrees C, and was linear with protein concentration. Scatchard analysis of binding data indicated a single class of high-affinity binding sites with a Kd of 36.1 +/- 9.7 pM and a density of 185.4 +/- 9.6 fmol/mg protein (n = 5). The potency order for competitive inhibition of the binding of 125I-labeled endothelin 1 was endothelin 1 greater than endothelin 2 = endothelin 3 = sarafotoxin S6b greater than big endothelin (human) = big endothelin (porcine). Phenylephrine, bradykinin, norepinephrine, atrial natriuretic factor, diltiazem, U46619, and angiotensin II did not displace 125I-endothelin 1 from its receptors. These experiments demonstrate that endothelin 1 is a potent pressor substance in the human fetoplacental cotyledon. Pressor effects of endothelin may be mediated by a combination of direct effects and stimulation of vasoconstrictor prostanoids.

Native form of endothelin receptor in human placental membranes

Little is known about the native form of endothelin receptor. To clarify its functional and structural properties, we solubilized the receptor from human placenta in an active form using mild detergents CHAPS and digitonin and showed that it is able to bind 125I-endothelin-1 in a specific manner, with a pH optimum between 6 and 8 in contrast to a reported pH optimum of 4. The molecular weight of the receptor was estimated as 340,000 by gel filtration of the solubilized membrane in the presence of 0.2% ( w v ) digitonin. When the solubilized membranes were labeled with 125I-endothelin-1 prior to gel filtration, the radioactive ligand also migrated in the position corresponding to a 340 kDa protein. These results indicated that the native form of endothelin receptor in human placenta is a 340 kDa protein.

Presence of D2-dofamine Receptors in Human Term Placenta

We studied the binding of [3H]-spiperone on human term placental membranes. This binding reached plateau level after 30 min incubation at 37 degrees C and was reversed (t1/2 approximately 5 min) by addition of an excess of unlabeled spiperone. Scatchard analysis of saturation experiments with increasing doses of [3H]-spiperone (0-25 nM) showed one class of high affinity binding sites with a dissociation constant (Kd) of 14 +/- 2 nM and a maximal binding capacity (Bmax) of 222 +/- 9 fmoles/mg protein. The affinity of 5 competitors was determined in competitive binding assays. The D2-dopamine antagonists were the most potent inhibitors: Ki for spiperone and haloperidol were 8 +/- 2 and 56 +/- 22 nM respectively. Dopamine inhibited [3H]-spiperone binding with a Ki of 570 +/- 50 microM whereas Schering 23390 (D1 antagonist) and propranolol (beta-adrenergic antagonist) were without effect. The binding was also inhibited by 100 microM GTP gamma S (38 +/- 8% inhibition), indicating that the dopamine receptor is coupled with a GTP binding protein. These results demonstrate for the first time the presence of D2-dopamine receptors in human placenta.

The presence of gonadotropin receptors in human placenta, amnion, chorion and decidua

The normal development of placenta relies essentially on a balanced proliferation, differentiation and apoptosis of cytotrophoblasts. These processes are tightly regulated by several hormones, cytokines, lipids and other molecules and anomalies in these events are associated with gestational complications. The cation channel transient receptor potential vanilloid 1 (TRPV1) is expressed in several organs and tissues and it participates in cellular events like nociception, inflammation and cell death. However, the expression and importance of this receptor in human placenta still remains unknown. In this work, we found that TRPV1 is expressed in human cytotrophoblasts and syncytiotrophoblasts. Furthermore, the TRPV1 agonists capsaicin and anandamide decreased cytotrophoblast viability and induced morphological alterations, such as chromatin condensation and fragmentation, which suggest the occurrence of apoptosis. Also, both TRPV1 agonists induced a loss of mitochondrial membrane potential and an increase of caspase 3/7 activity and production of reactive species of oxygen and nitrogen. Furthermore, capsaicin (10 μM) impaired the spontaneous in vitro differentiation of cytotrophoblasts into syncytiotrophoblasts by triggering TRPV1, as observed by the decrease in placental alkaline phosphatase activity and in human chorionic gonadotropin secretion. On the other hand, anandamide decreased placental alkaline phosphatase activity via a TRPV1-independent mechanism but did not influence the secretion of human chorionic gonadotropin. In conclusion, we showed that TRPV1 is expressed in human cytotrophoblasts and syncytiotrophoblasts and also reported the involvement of this receptor in cytotrophoblast apoptosis and differentiation.

Delineation of atypical insulin receptors from classical insulin and type I insulin-like growth factor receptors in human placenta

Insulin-like growth factor (IGF)-binding sites copurifying with human placental insulin receptors during insulin-affinity chromatography consist of two immunologically distinct populations. One reacts with monoclonal antibody alpha IR-3, but not with antibodies to the insulin receptor, and represents Type I IGF receptors; the other reacts only with antibodies to the insulin receptor and is precipitated with a polyclonal receptor antibody (B-10) after labelling with 125I-multiplication-stimulating activity (MSA, rat IGF-II). The latter is a unique sub-population of atypical insulin receptors which differ from classical insulin receptors by their unusually high affinity for MSA (Ka = 2 x 10(9) M-1 compared with 5 x 10(7) M-1) and relative potencies for insulin, MSA and IGF-I (40:5:1 compared with 150:4:1). They represent 10-20% of the total insulin receptor population and account for 25-50% of the 125I-MSA binding activity in Triton-solubilized placental membranes. Although atypical and classical insulin receptors are distinct, their immunological properties are very similar, as are their binding properties in response to dithiothreitol, storage at -20 degrees C and neuraminidase digestion. We conclude that atypical insulin receptors with moderately high affinity for IGFs co-exist with classical insulin receptors and Type I IGF receptors in human placenta. They provide an explanation for the unusual IGF-II binding properties of human placental membranes and may have a specific role in placental growth and/or function.

Multiple forms of atrial natriuretic factor receptor in human placenta

Multiple forms of atrial natriuretic factor receptor have been identified in human placental membranes. Atrial natriuretic factor binds specifically to placental membranes and the binding activity could be solubilized using non ionic detergent, Triton X-100. Binding to the detergent solubilized preparation was inhibited 80% by the addition of 0.5 M sodium chloride. Affinity cross-linking analysis indicated that this binding was associated with a single protein band of molecular weight 170-kDa. On the other hand, if sodium chloride was added together with a chelator, o-phenanthroline, ANF binding to this preparation was stimulated 300%. Binding under these conditions was not to the 170-kDa protein but was associated with a broad band in the region of 100/110-kDa and a minor band at 200-kDa. These observations clearly indicated that in human placental membranes, atrial natriuretic factor binds to distinctly different molecular species depending on the presence or absence of certain ions and chelators. The two types of binding could be conveniently assayed in the presence of each other by elimination or inclusion of sodium chloride and o-phenanthroline in the assay system.

Characterization of the tumour necrosis factor receptor in human placenta

A specific receptor for tumour necrosis factor present in a purified plasma membrane preparation from the villous tissue of human placenta has been characterized. The data fit a one-class-of-sites model exhibiting a K D = 28.4 (± 0.002) n m, with 8.1 (±0. 05) × 10 11 receptors mg −1 membrane protein. These data provide evidence for the existence of a specific receptor for a major endotoxin-induced cytokine in the placenta.

Prenatal cocaine use associated with down regulation of receptors in human placenta

Cocaine use during pregnancy has been associated with abruptio placentae and spontaneous abortions. These effects may be secondary to the vasoconstrictive effects of cocaine or to other alterations. Since it has been demonstrated that the use of opiates during pregnancy alters placental receptors, the effects of cocaine on placental receptors was studied. Women who used cocaine during pregnancy showed a significant lowering of the total number of beta-adrenergic receptor binding sites, mu-opiate receptor binding sites and delta-opiate receptor binding sites. The decreases in Bmax for each of these receptors was not associated with a decrease in the K D. The potential causes for the receptor down regulation and effects are discussed.

Evidence for a subtype of insulin-like growth factor I receptor in brain

We examined the structure of receptors for insulin-like growth factor I (IGF-I), insulin, and epidermal growth factor (EGF) in human brain and human placenta using affinity cross-linking procedures and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In human brain, proteins specifically cross-linked to 125I-IGF-I, 125I-insulin, and 125I-EGF had apparent molecular weights of 120000, 115000 and 170000, respectively. In human placenta, proteins cross-linked to 125I-IGF-I and 125I-insulin were 10 kDa larger than the corresponding subunits in brain. The receptor labeled by 125I-EGF in placenta was indistinguishable from the EGF receptor in brain. The size discrepancy of IGF-I receptors in brain and placenta was no longer apparent after removing the carbohydrate moieties of the proteins with endo-β-N- acetylglucosaminidase F (EndoF). Furthermore, the brain IGF-I receptor was not cleaved by neuraminidase, whereas, the placental IGF-I receptor had increased mobility on SDS gels following neuraminidase treatment. The results indicate that (1) receptors for IGF-I and insulin in human brain are structurally distinct from the corresponding receptors in human placenta, (2) the structural heterogeneity of the receptors involves differences in N-linked glycosylation, particularly the terminal processing steps, and (3) EGF receptors are present in human brain and human placenta but are structurally similar in these tissues. We conclude that there is a selective modification in the glycosylation of receptors for IGF-I and insulin in brain.