Receptors In Human Placenta Research Articles

Nondetectable concentrations of human placental ah receptors are associated with potent induction of microsomal benzo[ a]pyrene hydroxylase in individuals exposed to polychlorinated biphenyls, quaterphenyls, and dibenzofurans

Human exposure to polychlorinated biphenyls, quaterphenyls, and dibenzofurans occurred in an accidental food poisoning episode in Taiwan in 1979. Our objective was to evaluate the role of Ah receptors in the production of biochemical effects detected in placentas from exposed subjects. Placentas were obtained from pregnant women who were identified from a registry of exposed individuals. Benzo[ a]pyrene (BaP) hydroxylase was quantified in placental microsomes and Ah receptor was investigated in placental cytosolic and nuclear fractions by a variety of receptor-binding techniques using 2,3,7,8-[1,6- 3H]tetrachlorodibenzo- p-dioxin ([ 3H]TCDD) as the ligand. Microsomal BaP hydroxylase was dramatically elevated in placentas of exposed subjects compared to those of nonexposed, nonsmoking control subjects. However, concentrations of displaceable [ 3H]TCDD binding in placental preparations from exposed or control subjects were uniformally low; approximately 1.0 fmol/mg cytosolic protein. Further evaluation of displaceable TCDD binding by sucrose-gradient sedimentation and hydroxylapatite column chromatography revealed that binding properties were different than those for the Ah receptor. These data suggest that extremely low concentrations of Ah receptors in human placenta may be sufficient to markedly elevate microsomal BaP hydroxylation. Alternatively, induction in human placenta may be mediated by a mechanism not requiring Ah receptor.

Use of multiple radioligands to characterize adenosine receptors in human placenta

To identify and characterize fully the adenosine receptor of human placenta, particulate preparations of placental parenchyma were examined using several adenosine receptor ligands, [3H]2-chloradenosine ([3H]2-Cl-ADO), [3H]N6-ethylcarboxamideadenosine ([3H]NECA), and [3H]N6-(L-phenylisopropyl)-adenosine ([3H]PIA). Each of the three ligands bound to the particulate preparations with unique binding characteristics. Binding of [3H]PIA characterized a high-affinity low-capacity receptor while the [3H]NECA displayed lower-affinity, higher-capacity binding. Saturation isotherms for [3H]2-Cl-ADO appeared intermediate in terms of affinity and binding capacity. Saturation isotherms of binding data also disclosed displaceable non-receptor binding at high labelled ligand concentrations. Competitive binding studies supported the observation that PIA and NECA bind to sites of high and low affinity, respectively. However, the multicomponent competition curves for [3H]2-Cl-ADO binding suggest binding to the receptors with differential affinity. These studies confirm the presence of adenosine receptors in the human placenta and suggest that these receptors are of both the high-and low-affinity subtypes.

Disulphide reduction alters the immunoreactivity and increases the affinity of insulin-like growth-factor-I receptors in human placenta.

We previously identified two forms of the insulin-like growth-factor-I (IGF-I) receptor in human placenta: a lower-affinity form reactive with an autoantiserum (B-2) to the insulin receptor and a higher-affinity non-immunoreactive form [Jonas & Harrison (1985) J. Biol. Chem. 260, 2288-2294]. Evidence is now presented that the lower-affinity immunoreactive forms are convertible into higher-affinity non-immunoreactive forms via reduction of receptor disulphide bonds. Treatment of placental membranes with increasing concentrations of dithiothreitol (DTT): (1) converted native Mr-290 000 heterotetrameric IGF-I receptors into Mr-180 000 dimers (determined by chemical cross-linking of 125I-IGF-I with disuccinimidyl suberate); (2) increased 125I-IGF-I binding, owing to an increase in receptor affinity; and (3) abolished the reactivity of Triton-solubilized IGF-I receptors with antiserum B-2 and transformed the curvilinear plot of IGF-I binding to a linear form. In isolated complexes between receptor and B-2 antibody, DTT increased 125I-IGF-I binding and released a single class of higher affinity IGF-I receptors of Mr 180,000. Thus DTT-treated IGF-I receptors have similar properties to the higher-affinity non-immunoreactive forms of the native receptor, except that reduced dimeric forms are not detected by cross-linking of 125I-IGF-I to native membranes. Cleavage of the inter-dimeric disulphide bonds is therefore not a prerequisite for higher-affinity binding or loss of immunoreactivity. These observations suggest that the thiol redox state of the IGF-I receptor in vivo is an important determinant of receptor conformation and therefore of the biological responses to IGF-I.

Opioid receptors in human placenta may be affected by methadone and propoxyphene treatment.

Opioid receptors in human placenta may be affected by methadone and propoxyphene treatment.

The human placenta contains two distinct binding and immunoreactive species of insulin-like growth factor-I receptors.

Two species of insulin-like growth factor-I (IGF-I) receptors in human placenta have been delineated on the basis of their immunoreactivity with an autoantiserum (B-2) to the insulin receptor. When all the IGF-I binding sites in solubilized human placenta were assayed by polyethylene glycol precipitation, a curvilinear Scatchard plot was obtained which could be resolved into two single classes of binding sites: one immunoprecipitable by B-2 IgG and the other, nonimmunoprecipitable. The B-2 reactive sites bound IGF-I with lower affinity (Kd = 7.1 X 10(-10) M) than the B-2 nonreactive sites (Kd = 2.1 X 10(-10) M) and cross-reacted more readily with insulin, the IGF-I/insulin-binding potencies being congruent to 120 and congruent to 1100, respectively. Both receptor subtypes bound IGF-I with congruent to 30-fold higher affinity than multiplication-stimulating activity, and, after affinity cross-linking with 125I-IGF-I, migrated as specific reduced bands of Mr = 138,000 during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit sizes of the B-2 reactive IGF-I receptor were similar to those of the insulin receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I-labeled receptors immunoprecipitated by autoantiserum B-2 or autoantiserum B-10 (which recognizes only insulin receptors) revealed, in both cases, specific reduced bands of Mr = 130,000 and 90,000; the same bands were also seen after sequential precipitation with B-10 and B-2 antisera to enrich the proportion of IGF-I receptors recovered. The presence of two distinct binding and immunoreactive species of IGF-I receptors in human placenta raises the possibility that cell- or tissue-specific isotypes of the IGF-I receptor could mediate the different biological actions of IGF-I.

Ontogenesis and characteristics of epidermal growth factor receptors in human placenta

Epidermal growth factor promotes growth in many cell types. The role of epidermal growth factor during gestation and fetal development is unknown. This study investigates the presence and binding characteristics of epidermal growth factor receptors in placentas throughout gestation. Cell membrane preparations were obtained from first-, second-, and third-trimester placentas and hydatidiform moles. Specific epidermal growth factor receptors were observed as early as 6 weeks of gestation. Throughout gestation, Scatchard analysis of epidermal growth factor binding was curvilinear, and dissociation studies were consistent with site-to-site interaction with negative cooperativity. Affinity constants at high (Ke = 9.9 ± 0.79 × 109 L/mol) and low (Kf = 3.0 ± 0.25 × 109 L/mol) receptor occupancy were unchanged throughout normal gestation. However, the number of receptors per milligram of protein significantly increased with advancing normal gestation (correlation coefficient, r = 0.81). In hydatidiform moles, the number of receptors was reduced when compared to that of normal tissue of similar gestational age. Our study provides a quantitative basis for further evaluation of epidermal growth factor as a possible factor in embryogenesis and fetal development.

Antireceptor antibodies as probes of insulinlike growth factor receptor structure.

Insulin receptors and Type I insulinlike growth factor (IGF) receptors have a similar structure with a major binding subunit of Mr approximately 130,000 linked by disulfide bonds to other membrane proteins to form a Mr greater than 300,000 complex. Both insulin and Type I IGF receptors also interact with both insulin and IGF, although with different binding affinities. We used a panel of human and rabbit sera containing antibodies to insulin receptors to determine whether these sera also interact with Type I IGF receptors. Immunoglobulins from five of five human sera inhibited binding of 125I-insulin and 125I-IGF-I to insulin receptors and Type I IGF receptors in human placenta and human lymphocytes. The rank order of reactivity with both receptors was the same; two sera, however, appeared to be selectively less reactive with the Type I IGF receptor, especially in placenta. Sera from five of seven patients and from a rabbit immunized with purified insulin receptor effectively immunoprecipitated both placental insulin receptors and Type I IGF receptors. Of the remaining sera, one had only a low titer against the insulin receptor and did not immunoprecipitate the IGF receptor, whereas the second serum effectively immunoprecipitated cross-linked and surface-iodinated insulin receptors, but had negligible reactivity against the Type I IGF receptor. These results suggest that most antisera to the insulin receptor also contain antibodies to Type I IGF receptors. Whether both specificities are inherent in the same or different antibody molecules remains to be determined. These data support the hypothesis that the insulin and IGF-I receptors are separate but related molecules, although there remains a small possibility that both receptors are domains on the same protein.

Binding characteristics of an adenosine receptor in human placenta.

The binding characteristics of human placental microsomes were evaluated for properties related to an adenosine receptor using 2-chloro[8-3H]adenosine as the ligand. Saturation of binding sites occurred with 0.75 pmol of ligand/mg of protein. Analysis of these data by Scatchard plot indicates a single class of binding sites with a Kd of 56 nM with 1.1 pmol of ligand bound/mg of protein. The specificity of 2-chloro[8-3H] adenosine binding was assessed by the ability of adenosine analogs to compete for binding sites. Using this approach, the Kd for 5'-N-ethylcarboxamideadenosine was estimated to be 0.3 microM and for N6-(L-2-phenylisopropyl)adenosine or N6-cyclohexyladenosine to be greater than 100 microM. Isobutylmethylxanthine and theophylline, receptor antagonists, have Kd values of 19 microM and 150 microM, respectively. Chloroadenosine binding to placental microsomes was time-dependent and reached equilibrium at approximately 20 min. The kob was 0.24 min-1 and the k1 was 0.71 X 10(8) min-1 M-1. Reversibility of chloroadenosine binding at equilibrium was completed at 5 min with a k2 value of 1.17 min-1. The Kd calculated from the reverse rate constant was 17 nM. Our observations suggest that 2-chloro[8-3H]adenosine identifies binding sites in human placenta with characteristics of an adenosine receptor. The binding properties conform to the previous description of a low affinity stimulatory receptor (Ra or A2 receptor).

Glucocorticoid receptor in human placenta: Studies of concentration and functional differences of preterm and term tissue

Glucocorticoids have been suspected of being a factor in premature parturition. This study identifies and characterizes a glucocorticoid receptor in human placentas at various gestational ages. Placental segments in culture translocate 3H-labeled steroid to the nucleus. Scatchard analysis of placental cytosol reveals a relatively high-affinity (dissociation constant = 10−8) and low-capacity binding of dexamethasone to a protein. An 8S-to-4S shift with low-to-high salt treatments is shown by sucrose density gradient analysis. The binding protein is precipitated by 35% ammonium sulfate. This ammonium sulfate fraction of cytosol displays a steroid specificity and a capacity of the receptor to bind to nuclear acceptor sites in cell-free assays. Thus, the presence of a glucocorticoid receptor is supported. A sevenfold to twelvefold increase in concentration of receptor was found between samples of placental tissue from the first and second trimesters; no significant difference was detected between the second and third trimesters. The capacities of the “receptors” from mature and premature placental tissues to bind to chromatin were not markedly different; thus, the biologic activities of the receptors among the different trimesters appear equivalent. The placenta thus appears to be a target tissue for glucocorticoids. The pronounced differences in receptor concentrations in placentas from different trimesters may reflect differential responsiveness of the organ to the systemic steroid.

A specific cytosolic estrogen receptor in human term placenta

Administration of the antiestrogen ethamoxytriphetol (MER-25) during baboon gestation results in a marked decline in placental progesterone production. Since this effect in primates may be modulated via an estrogen receptor, the present study investigated the possible existence of an estrogen receptor in human placenta. Villous tissue of human, term placentas was homogenized in 0.01M Tris-HCl, ethylenediaminetetraacetic acid, dithiothreitol, glycerol buffer. Cytosol was incubated with 10−8M [3H] 17β-estradiol (E2) in the presence or absence of 10−6M diethylstilbestrol (DES). A single peak of [3H]E2 binding occurred in the 5.2 region after glycerol density gradient centrifugation, which was competed for by DES, E2, and enclomiphene. Scatchard analysis demonstrated E2 binding, which was saturable, of high affinity (Kd = 1.90 × 10−11M) and of low capacity (N = 0.13 × 10−14 moles/mg cytosolic protein). Competition for [3H]E2 binding was DES > E2 > estrone > MER-25 > enclomiphene, whereas androgens, progestins, and corticosteroids were ineffective. The results fulfill the criteria for a specific estrogen receptor. The influence of antiestrogen and, possibly, estrogen upon placental function in baboons may be modulated by an estrogen receptor.

Immunoautoradiographic detection of epidermal growth factor receptors after electrophoretic transfer from gels to diazo-paper

Several groups have attempted to identify the EGF receptor by a variety of biochemical techniques. Das et al. [l] identified the EGF receptor on 3T3 cells as a radioactive band of M, = 190 000 by photoaffmity labeling with a ‘251-EGF probe; Hock et al. [2] identified the EGF receptor in human placenta as a doublet of 160 000 and 180 000 M, by covalent cross-linking to ‘251-labeled EGF. The EGF receptor on A-43 1 cells was identified [3] as a radioactive band of M, 175 000 by ‘direct labeling’, which is accomplished by incubation of cells with 1251-EGF. Cohen and coworkers [4] have partially purified the EGF receptor of A-43 1 cells by affinity chromatography and analyzed the denatured proteins of such preparations by SDS-polyacrylamide gel electrophoresis. They found numerous protein bands in the affinity-purified preparations and suggested that a major protein band of M, 150 000 is the receptor for EGF in A-431 cells. More recently, Hunter and Cooper [S] isolated the EGF receptor from 32P-labeled A-43 1 cells by immunoprecipitation with an antiserum raised against crude plasma membrane from A-431 cells. They have estimated the M, of the EGF receptor present in the immunoprecipitates from A-43 1 cells as 155 000. The interaction of EGF with its membrane receptor, present in both crude plasma membranes from A-431 cells and in affinity-purified prepara-

Effect of gestational diabetes on insulin receptors in human placenta.

The aim of this work was the characterization of insulin binding to a partially purified preparation of plasma membranes from gestational diabetic placenta in order to establish a comparison with normal pregnancy. A decrease in the number of insulin receptors per mg protein in placenta from gestational diabetic mothers was found. Comparable inhibitory effects of unlabelled insulin suggest that the binding affinities were similar in both groups of patients. The ability of the placental preparation to degrade 125I-insulin was not significantly different in both situations. The existence of a negative feed-back regulation of the insulin receptors in placental membranes was suggested by the fact that plasma insulin levels were significantly higher in the gestational diabetic mothers.

Fcgamma receptors in human choroid plexus.

Crysotat sections of human choroid plexus adsorbed erythrocytes sensitized with IgG antibodies of human and rabbit origin. No adsorption occurred when the erythrocytes were sensitized with F(ab')2 or Facb fragments. The reaction was strongly inhibited by intact IgG and by Fc fragments and not inhibited by Facb and F(ab')2 or albumin. These properties are similar to those of corresponding receptors in human placenta. The presence of an Fcgamma receptor in choroid plexus may be of significance for the transfer of IgG from blood to cerebrospinal fluid.

Demonstration of Cells with IgG Receptor in Human Placenta

The presence of two types of cells with IgG surface receptors was demonstrated in mature and immature human placentas. One type corresponded morphologically to the Hofbauer cells and the second to the blood monocytes. Transitional forms between monocytes and Hofbauer cells were also observed.