Human Placental Cytosol Research Articles

Methylation of Catechins and Procyanidins by Rat and Human Catechol-O-Methyltransferase: Metabolite Profiling and Molecular Modeling Studies

Catechins and procyanidins are major polyphenols in plant-derived foods. Despite intensive studies in recent years, neither their biochemical nor their toxicological properties have been clarified sufficiently. This study aimed to compare the methylation of catechins and procyanidins by the enzyme catechol-O-methyltransferase (COMT) in vitro. We conducted incubations with rat liver cytosol and human placental cytosol including S-adenosyl-l-methionine. The set of substrates comprised the catechins (-)-epicatechin (EC) and (+)-catechin (CAT), the procyanidin dimers B1, B2, B3, B4, B5, and B7 as well as procyanidin trimer C1. After extraction, metabolites were analyzed by means of liquid chromatography-electrospray ionization-mass spectrometry and liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. EC and CAT were converted to two monomethylated metabolites each by human and rat COMT, with the 3'-O-methyl derivatives being consistently the main metabolites. Furthermore, the flavanyl units of procyanidins were methylated consecutively, leading to monomethylated and dimethylated dimeric metabolites as well as monomethylated, dimethylated, and trimethylated C1 metabolites. The methylation status of each flavanyl unit was determined by means of mass spectrometric quinone-methide fragmentation patterns. In addition, molecular modeling studies were performed with the aim to predict the preferred site of methylation and to verify the experimental data. In conclusion, our results indicate that the degree and position of methylation depend clearly on the three-dimensional structure of the entire substrate molecule.

Characterization of a high-affinity, low-capacity androgen binding protein in human term placental cytosol.

A high-affinity, low-capacity androgen binding component in human placental cytosol was characterized. (3H)-R1881, a synthetic androgen, was used as radioactive ligand for characterization and quantification of this placental androgen binding component. Various naturally occurring as well as synthetic steroids were first investigated for their in vitro stability under receptor assay conditions (i.e., an overnight incubation at 4 degrees C). Only the synthetic steroids were found to be metabolically stable. The rate constant of dissociation was found to be 6.42 X 10(-4) s-1 (on the basis of k-1 the rate constant of association was 1.27 X 10(5) M-1 s-1). The concentration of androgen binding protein was measured in 50 human term placentae. The mean concentration was 169 +/- 132 fmol/mg protein, and the average Kd was 5.07 +/- 2.2 X 10(-9) M. Placentae of female fetuses contained significantly higher concentrations (219 +/- 140 fmoles/mg protein) than those of male fetuses (115.2 +/- 101 fmoles/mg protein). The (3H)-R1881-androgen binding protein complex exhibited a sedimentation coefficient of 4S under low salt conditions. The binding site was only specific for androgens and progestins but not for steroids of other classes.

Synthesis of Thyroid Hormone Binding Proteins Transthyretin and Albumin by Human Trophoblast

Mechanisms regulating materno-fetal transfer of thyroid hormone are not well understood. Modulation of trophoblast type 3 iodothyronine deiodinase (D3) may play an important role. The objective of this study was to investigate trophoblast thyroid hormone binding proteins that may modulate interactions between D3 and T4. Placentas were obtained by informed consent from women delivering normal infants by repeat cesarean section at 38-40 wk gestation. T4 and T3 binding was examined in human placenta. Serum thyroid hormone binding proteins were identified by Western blotting, and their mRNA was examined by RT-PCR. Presence of these proteins in trophoblast was determined by immunocytochemistry and immunofluorescence. Cytosol was progressively purified to reveal additional thyroid hormone binding proteins that were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Effects of mefenamic acid on placental deiodination were examined by HPLC. We detected high-affinity T4 and T3 binding in human placental cytosol. All three major serum-binding proteins, T4 binding globulin (TBG), transthyretin (TTR), and albumin, were present in cytosol. TTR mRNA and albumin mRNA were detected in human placenta, and TTR and albumin were identified histochemically in syncytiotrophoblasts. Neither TBG mRNA nor TBG was detected, suggesting that plasma TBG had contaminated the cytosol preparation. Low-affinity thyroid hormone binding proteins alpha-1-antitrypsin and alpha-1-acid glycoprotein were also identified. Addition of mefenamic acid, a potent inhibitor of thyroid hormone binding, to placental cytosol significantly enhanced deiodination of T4 by D3. Placenta produces a series of thyroid hormone binding proteins that may modify thyroid hormone deiodination and materno-fetal thyroid hormone transport.

Human placental GnRH-like factors: II. Inhibition of enzymatic degradation of GnRH-II and [D-Trp6]GnRH ethylamide tracers by human term placental cytosol fractions reveals the presence of GnRH-binding protein(s).

We describe the preliminary characterization of GnRH-binding protein(s) in human placental cytosol. Samples were analysed by chromatography on Sephadex G25. Radiolabelled GnRH and its analogues elute significantly later than the total column volume (V(t)) on Sephadex G25 column chromatography. However, incubation of GnRH II or GnRH agonist tracers with human placental cytosol reduced the intact tracer peak, with the concomitant appearance of a new peak eluting in the total column volume (V(t)). This peak increased with increasing cytosol concentration and duration of incubation, and probably represented degraded GnRH tracer, since (i) degradation-resistant GnRH agonist tracer, [D-Trp(6)]GnRH EtA, was inactivated more slowly than GnRH II, (ii) boiling of cytosol fractions abolished formation of this peak and (iii) peptidase inhibitors blocked its formation. A second new tracer peak eluted in the column void volume (V(o)) and was largely unaffected by peptidase inhibitor concentrations that blocked tracer degradation. The magnitude of this high molecular weight peak depended on the GnRH tracer employed, cytosol concentration, and the pH, duration and temperature of incubation. Tracer associated with this third peak appeared similar to intact GnRH tracer by TLC. Unlabelled GnRH analogues and isoforms decreased both tracer degradation and formation of the V(o) peak, but their specificity and affinity for the two processes differed. Ligand blots identified several bands that were abolished by inclusion of unlabelled agonist during incubation. Our data indicate the presence of specific GnRH binding protein(s) and GnRH peptidases that may modulate local actions of GnRH in the human placenta.

Identification of the 49-kDa autoantigen associated with lymphocytic hypophysitis as alpha-enolase.

Lymphocytic hypophysitis is part of the spectrum of organ-specific autoimmune diseases, and although its histopathology is well documented, its pathogenesis is unclear. Serum autoantibodies directed against a 49-kDa cytosolic protein are detected by immunoblotting in 70% of patients with biopsy-proven lymphocytic hypophysitis. Here we report the purification and identification of this first target autoantigen in lymphocytic hypophysitis. The autoantigen has a molecular mass of 49 kDa, a cytosolic localization, and a ubiquitous tissue distribution. The 49-kDa protein was purified from monkey brain and human placental cytosol. Limited amino acid sequencing after proteolytic digestion of the human placental protein showed identity with alpha-enolase. The identification was confirmed using sera from patients with pituitary autoimmunity, which strongly reacted with recombinant human alpha-enolase and yeast enolase, but not with rabbit muscle beta- enolase. This indicates that the immunoreactive epitopes are largely conserved from yeast to human, but are not present in beta-enolase. alpha-Enolase autoantibodies are not specific to pituitary autoimmune disease and have been reported in other autoimmune diseases. However, this study is the first to indicate a role for alpha-enolase as an autoantigen in lymphocytic hypophysitis.

Evaluation of 7-hydroxy-flavones as inhibitors of oestrone and oestradiol biosynthesis.

A series of 4-aryl substituted 7-hydroxy-flavones were prepared using the three-step Baker-Venkataraman synthesis in good overall yields. The flavones were all evaluated in vitro for inhibitory activity against aromatase (P450AROM, CYP19), using human placental microsomes, and for inhibitory activity against 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD-1) using human placental cytosol. The phenyl, 4-fluoro-phenyl and 4-bromo-phenyl derivatives displayed moderate inhibitory activity against P450AROM (IC50 17.2, 13.5 and 10.1 microM, respectively), none of the flavones, including the standard genistein, displayed any inhibitory activity against 17beta-HSD type 1 at 100 microM concentration.

A simple and efficient method for hemoglobin removal from mammalian tissue cytosol by zinc sulfate and its application to the study of lipoxygenase

A simple and efficient method is described to remove hemoglobin (Hb) from human term placental cytosol to study dioxygenase and co-oxidase activities of lipoxygenase. In the untreated samples, 70% – 80% of the linoleic acid-dependent dioxygenase and co-oxidase activities were found to be associated with the pseudo-lipoxygenase activity of Hb. Zinc sulfate (0.5 mM) precipitated ⪢97% of the Hb present in the cytosol. The dioxygenase activity of the ZnSO4treated cytosol exhibited a Vmaxvalue of 313 nmoles linoleic acid hydroperoxide formed/min/mg protein and a KMof 1.4 mM for linoleic acid. The ZnSO4treated cytosol displayed co-oxidase activity toward benzidine, dimethoxybenzidine, guaiacol, pyrogallol, tetramethylbenzidine and tetramethyl- p -phenylenediamine. Nordihydroguaiaretic acid, 5,8,11-eicosatriynoic acid, butylated hydroxyanisole, butylated hydroxytoluene and gossypol caused concentration dependent inhibition of dioxygenase and co-oxidase activities. These results suggest ZnSO4precipitation of Hb from cytosol does not alter the functional characteristics of the human term placental lipoxygenase.

Human placental gonadotrophin-releasing hormone-like factors: an artefact of human placental peptidases?

Non-denatured human placental cytosol fractions displaced tracer binding in parallel with gonadotrophin-releasing hormone (GnRH) isoform and agonist peptides in GnRH-specific radioimmunoassays and radioreceptor assays. However, placental immuno- and receptor binding-GnRH-like activity was highly correlated with inactivation of GnRH tracers, suggesting that placental GnRH-like factors may be an artefact of ligand degradation during assay. The properties and inhibitor sensitivities of the major (125)I-labelled GnRH-degrading enzymes of term placental cytosol were studied using a dextran-coated charcoal (DCC) adsorption assay as a rapid screen for GnRH tracer inactivation. Three different activities were demonstrable: (i) a cathepsin D-like enzyme (M(r) 55 kDa), active against all radiolabelled GnRH isoforms and agonists tested, optimal at acid pH, and inhibited specifically by pepstatin; (ii) a metallo-thiol endopeptidase activity (M(r) 70 kDa) optimal at alkaline pH (7-9) which degraded GnRH isoforms to a greater extent than GnRH analogues, inhibited dose-dependently by low concentrations of thiol reagents (N-ethylmaleimide, thimerosal), chelating agents (o-phenanthroline, EDTA), and by tosyl-phenylalanyl-chloromethyl ketone but not by other serine protease inhibitors; and (iii) a bacitracin-sensitive enzyme optimal at physiological pH. These observations permitted the development of a robust radioreceptor assay which minimized GnRH tracer degradation. Under these assay conditions, the GnRH-like radioreceptor assay activity of human placental cytosol fractions was markedly reduced.

Human placental GnRH-like factors: parallel displacement in GnRH immuno- and receptor-binding assays can be caused by degradation of radiolabelled GnRH tracers

Human term placental cytosol fractions decreased the specific binding of gonadotrophin-releasing hormone (GnRH) isoform tracers to placental membranes (and to rat pituitary GnRH receptors and anti-GnRH antibodies) in a dose-dependent manner, and in parallel to GnRH standard curves. However, cytosol fractions had little or no effect on the binding of two GnRH superagonist tracers. The specificity of placental binding sites for a range of GnRH-like and unrelated peptides was shown to be similar with GnRH isoforms or GnRH agonists as binding ligands, suggesting that isoforms and agonists did not bind to different forms of the GnRH-receptor. Inclusion of a cocktail of protease inhibitors during the preparation of placental cytosol significantly reduced immuno- and receptor-binding activity. Moreover, incubation of radiolabelled chicken GnRH II with placental cytosol led to marked inactivation of tracer, as assessed by radioreceptor and radioimmunoassays for GnRH, high resolution liquid chromatography, thin layer chromatography and adsorption to dextran-coated charcoal and other matrices. There was a good negative correlation between tracer degradation and apparent GnRH immuno- and receptor-binding activities. These results emphasize the important effects which proteases in un-denatured tissue extracts can have on radioreceptor and radioimmunoassays due to inactivation of peptide tracers, and suggest that previous measurements of receptor- and immuno-active GnRH-like factors may have been over-estimated due to peptidase action during the GnRH assay.

Detection and cellular localization of plasma membrane-associated and cytoplasmic fatty acid-binding proteins in human placenta

The aim of this study was to investigate location and the types of membrane-associated and cytoplasmic fatty acid-binding proteins in human placental trophoblasts using monospecific polyclonal antibodies. Western blot analysis demonstrated the presence of multiple membrane and cytoplasmic fatty acid transport/ binding proteins in human placenta. In addition to previously reported placental membrane fatty acid-binding (p-FABPPm, 40 kDa), fatty acid translocase (FAT, 88 kDa) and fatty acid transport protein (FATP, 62 kDa) were detected in both microvillous and basal membranes of the human placenta. Among the cytoplasmic proteins, heart (H) and liver (L) type FABP were detected in the cytosol of the human placental primary trophoblasts as well as in human placental choriocarcinoma (BeWo) cells. The immunoreactivity of epidermal type (E)-FABP was not detected in trophoblasts or BeWo cells despite its presence in human placental cytosol. Location of FAT and FATP on the both sides of the bipolar placental cells may favour transport of free fatty acids (FFA) pool in both directions i.e. from the mother to the fetus and vice versa. However, p-FABPPm, because of its exclusive location on the microvillous membranes, may favour the unidirectional flow of maternal plasma long-chain polyunsaturated fatty acids present in the FFA pool to the fetus, due to binding specificity for these fatty acids. Although the roles of these proteins in placental fatty acid uptake and metabolism are yet to be understood fully, their complex interaction may be involved in the uptake of maternal FFA by the placenta for delivery to the fetus.

Protein phosphatase activity against protein kinase C-phosphorylated substrates in human placenta

The presence of endogenous modulators of protein kinase C (PKC) in human placenta has not been reported. The specific activity of PKC in human placental cytosol was 20.52 ± 1.8 pmol/min × mg protein. Partial purification of placental cytosol on diethylaminoethyl cellulose (DEAE) resulted in recovery of 145 per cent of original enzyme activity. Placental cytosol mixed with a control preparation of PKC significantly inhibited the control enzyme activity (control 42.42 ± 2.8 pmol/min; control + placental cytosol 27.44 ± 2.8 pmol/min, P < 0.05). The PKC-inhibitory activity was abolished by the addition of phosphatase inhibitors calyculin A (0.09 n m), microcystin LR (0.8 n m), and okadaic acid (0.4 n m). Protein substrates phosphorylated by PKC were rapidly dephosphorylated upon the addition of placental cytosol; this dephosphorylation was prevented by the presence of calyculin A and was removed by fractionation of placental cytosol on DEAE. Protein but not peptide substrate supported both the PKC-inhibitory activity and the dephosphorylation of PKC-phosphorylated substrates. The placental serine-threonine protein phosphatase was active against phosphorylase a, but not against substrate phosphorylated by cAMP-dependent protein kinase. These data indicate that the human placenta contains an endogenous inhibitor of PKC which interacts with substrate rather than with the PKC and that the inhibitor is a protein phosphatase.

Small and intermediate conductance chloride channels in HT29 cells.

Recently, it has been shown that intermediate conductance outwardly rectifying chloride channels (ICOR) are blocked by cytosolic inhibitor (C. I.) found in the cytosol of human placenta and epithelial cells. C. I. also reduced the baseline current in excised membrane patches of HT29 cells. In the present study, this effect of C. I. was characterized further. Heat treated human placental cytosol was extracted in organic solvents and dissolved in different electrolyte solutions. It is shown that the reduction of baseline conductance (g(o)) is caused by inhibition of small non-resolvable channels, which are impermeable to Na+ and SO4(2-), but permeable to Cl-. The regulation of these small Cl(-)-conducting channels (g(o)) and of ICOR was examined further. First, no activating effects of protein kinase A (PKA) on the open probability (Po) of the ICOR or on the g(o)) were observed. The Po of the ICOR was reduced by 22% in a Ca(2+)-free solution. g(o) was insensitive to changes in the Ca2+ activity. The effects of C. I. from a cystic fibrosis (CF) placenta and the CF pancreatic duct cell line CFPAC-1 were compared with the effects of corresponding control cytosols, and no significant differences between CF and control cytosols were found. We conclude that the excised patches of HT29 cells contain ICOR and small non-resolvable Cl(-)-conducting channels which are similarly inhibited by C. I.(ABSTRACT TRUNCATED AT 250 WORDS)

NAD +-dependent 15-hydroxyprostaglandin dehydrogenase: Immunochemical characterization of the lung enzyme from pregnant rabbits

A polyclonal antibody was produced in guinea pig against the lung NAD +-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) purified from pregnant rabbits. Western blot analysis demonstrated that the protein identified by this antibody in the 105,000 g supernatant fraction of lung tissue from pregnant rabbits had a molecular mass of 30 kDa and comigrated with the purified PGDH. The specific activity of the lung PGDH in pregnant rabbits (25- to 28-day gestations) was 36.7 nmol NADH formed/min/mg protein compared to 0.3 nmol NADH formed/min/mg protein in nonpregnant rabbits. Although the PGDH activity in the lung cytosol of nonpregnant rabbits was inhibited by the anti-lung PGDH antibody, the 30-kDa protein was not detected by Western blot analysis. An examination of this 30-kDa protein during the gestational period indicated that the protein was present after 10 days and the amount of the protein increased from Day 10 to Day 28. This increase in the immunochemically reactive protein correlated with the marked increase in PGDH specific activity between 10 and 28 days. An immunochemically reactive protein also was observed in the ovary of 25- to 28-day pregnant rabbits and the specific activity of the ovary PGDH was 19.3 nmol NADH formed/min/mg protein. Only trace levels of the PGDH activity were detected in the ovaries of nonpregnant rabbits. A 30-kDa protein was not detected by the anti-rabbit lung PGDH in brain, kidney, bladder, uterus, liver, and heart tissue of pregnant or nonpregnant rabbits. When rabbit or human placental cytosol was examined with the anti-rabbit lung PGDH only faint 30-kDa bands were observed by Western blot analysis. A monoclonal antibody prepared against human placental PGDH did not recognize the 30-kDa band in the pregnant rabbit lung. Localization studies indicated a marked increase in immunochemical staining in pulmonary epithelial cells of pregnant rabbits as compared to nonpregnant rabbits. Lung epithelial cells but not endothelial cells were identified as containing the PGDH.

The Methyltrienolone Binding Protein of Human Placenta Requires Nicotinamide Adenine Dinucleotide Cofactor(s) for Steroid Binding*

The methyltrienolone binding protein (MTBP) found in human placental cytosol was found to require a low molecular weight modulator for steroid binding activity. Purification and characterization of the modulating activity showed that NAD+ is the endogenous substance responsible for activating MTBP to a form capable of steroid binding. The hierarchy of potency of exogenously added nucleotides is NADH greater than NAD+ = NADPH = NADP+. An investigation of the tissue distribution of human MTBP demonstrated that MTBP binding activity was present in placenta and chorion but absent from amnion and umbilical cord. Preliminary studies showed that rat, mouse, and rabbit placenta do not contain MTBP and suggest that MTBP may be a species-specific protein.

Characterisation and covalent labeling of the human placental methyltrienolone binding protein

Human placental cytosol contains an androgen binding protein which binds the synthetic androgen methyltrienolone (R1881) with high affinity ( K d 8.7 nM) and with an average binding capacity of 518 fmol/mg cytosol protein. This study provides further evidence that this protein is distinguishable from classical androgen receptors on the basis of steroid specificity and sulphydryl group sensitivity. Covalent labeling studies have shown this protein, which we have called “the methyltrienolone binding protein”, to have a mol. wt of 67,000 daltons.

Human placenta contains a high affinity R1881 binding site that is not the androgen receptor

Human placental cytosol was shown to contain a species that binds the synthetic androgen, methyltrienolone (R1881) with high affinity ( K d 6.5 nM). Major differences were found between this placental androgen binding species and the classical androgen receptor found in human foreskin cytosol. Competitive binding assays in the placental cytosol using [ 3H]R1881 as tracer showed a 200-fold excess of testosterane to compete poorly, while dihydrotestosterone and the synthetic androgen mibolerone did not compete at all. The placental R1881 binding component was found not to bind to hydroxylapatite, although all classes of steroid receptors are reported to do so. Temperature studies showed that he placental binding site is stable at elevated temperatures with no loss of binding after 4 h at 45° C. Ion exchange chromatography showed that the placental R1881 binding site eluted from DEAE cellulose at a lower salt concentration than foreskin androgen receptors. These results show that R1881 is not entirely specific for androgen receptors and that human placenta contains an androgen binding site that is not the classical androgen receptor.

Calcium-phospholipid enhanced protein phosphorylation in human placenta.

Calcium-activated, phospholipid-dependent protein phosphorylation has not been studied in placenta. Human placental cytosol was subjected to an endogenous protein phosphorylation assay using [gamma-32P]ATP in the presence of calcium and phosphatidylserine. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. When compared to basal levels, calcium (10(-6) M) in combination with phosphatidylserine (50 micrograms/ml) significantly enhanced (P less than 0.01) 32P incorporation into phosphoproteins having mol wt 47,000, 43,000, and 37,000. Half-maximal 32P incorporation was observed with 3.5 X 10(-7) M Ca2+ in the presence of phosphatidylserine (50 micrograms/ml). The effect of phosphatidylserine was biphasic. In the presence of Ca 10(-6) M, 32P incorporation increased to a maximum at 70 micrograms/ml of phosphatidylserine. The increase was suppressed at 150 micrograms/ml. Tetracaine caused a dose-dependent inhibition of calcium-activated, phospholipid-dependent enhancement of the three phosphorproteins. Calcium in the absence of phospholipid enhanced the phosphorylation of a protein of 98,000 mol wt. Phosphatidylserine suppressed this enhancement. Calmodulin (10(-6) M) had no detectable effect upon phosphorylation beyond that of calcium alone, but the calmodulin inhibitor R-24571 specificlly inhibited the calcium-stimulated 98,000 mol wt phosphoprotein. Calcium-activated, phospholipid-dependent phosphoproteins are present in human placental cytosol; whether calcium-activated, calmodulin-dependent phosphoproteins also are present remains a question.

Human placental nucleoside kinase activities.

Our studies have indicated that normal human placental cytosol contains a complex mixture of nucleoside kinase enzymes, some of which conform to previously characterized activities. Deoxyadenosine is phosphorylated by deoxycytidine kinase, adenosine kinase, and two as yet uncharacterized activities. Deoxyguanosine phosphorylation is associated with deoxycytidine kinase. More complete and detailed studies will be necessary to characterize these enzymes fully.

Inhibition of Platelet Aggregation by Human Placenta

Human placental cytosol inhibits platelet aggregation induced by high doses of collagen. The aim of this study was to investigate whether this anti-aggregating activity was caused only by the presence of various activities already described in the placenta (an ADP-consuming enzyme, a fatty acid cyclooxygenase inhibitor, and a thromboxane synthetase inhibitor) or whether another factor was present. Heating the cytosol at 50 degrees C for 6 min destroyed the inhibitor of collagen-induced aggregation. ADPase and the AA pathway inhibitors were not modified by this treatment. We therefore show the presence of an additional anti-aggregating factor: it is destroyed by heating at 50 degrees C. We also tested for the presence of an inhibitor of AA release in the placental cytosol using three different methods (rabbit platelets in PRP, washed rabbit platelets, and NRK fibroblasts) but no inhibition could be evidenced. We conclude that this new anti-aggregating factor, which is probably a protein, acts neither through AA release inhibition nor AA cascade inhibition.

The measurement of glucocorticoid receptors in human placental cytosol

Cytosol preparations of human term placenta in Tris-HCl buffer containing dithiothreitol were incubated with [3H]dexamethasone at 4 degrees C. Under these conditions the specific binding of [3H]dexamethasone was low when compared with cytosol preparations of sheep fetal lung or ovine placenta. The inclusion of sodium molybdate in the homogenization buffer and the removal of endogenous steroids by treating the cytosol with charcoal before incubation led to a faster and increased uptake of [3H]dexamethasone by human placenta. Scatchard plot analysis of the data showed that the placenta possesses a single class of high-affinity (Kd = 13 +/- 2 nM) low-capacity (193 +/- 34 fmol/mg protein) binding sites for [3H]dexamethasone. The binding of [3H]dexamethasone was reversible and was not due to plasma contamination. The specificity of the binding sites was confirmed by competition experiments in which the highest displacement of [3H]dexamethasone from the binding sites was caused by natural and synthetic corticosteroids. These findings indicate that the human placenta at term contains specific glucocorticoid receptors in high concentrations.