Antagonism of estrogen-induced prolactin release by dihydrotestosterone

Previous work from our laboratory has demonstrated that progesterone can inhibit estrogen-induced prolactin release in female rats. Since androgens have been reported to mimic progesterone actions in certain systems, and to antagonize estrogen action in rat uteri, the purpose of this study was to determine whether androgens, like progestins, can inhibit estrogen-induced prolactin release. The ovariectomized (26 days of age) immature rat was used as the model for analysis of this question. Dihydrotestosterone (DHT) was chosen to be used throughout the study since it does not undergo aromatization to estrogens. In response to estradiol exposure (2 micrograms/rat), prolactin release reached peak values at 12 h and returned to control levels by 24 h. A second injection of estradiol 13 h after its initial injection stimulated a second increase in serum prolactin at 25 h. A single injection of DHT (0.8 mg/kg BW) 1 h before the second estradiol injection blocked the increase in serum prolactin. DHT had no effect on basal serum prolactin levels. The DHT inhibition of estrogen-induced prolactin release required estrogen priming. A dose dependency for the DHT effect was demonstrated, with low doses effective and high doses ineffective, in inhibiting estrogen action. This effect of DHT seemed to be androgen receptor-mediated, since flutamide blocked the effect. However, the possibility of progestin receptor mediation could not be ruled out since RU486 also blocked DHT's effect. Flutamide was also effective in blocking progesterone's inhibition of estrogen-induced action. This is perhaps consistent with an overlap of activities in androgens and progestins reported by several investigators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple estrogen binding sites in the uterus: stereochemistry of receptor and non-receptor binding of diethylstilbestrol and its metabolites

Indenestrol A (IA), an oxidative metabolite of the synthetic estrogen diethylstilbestrol (DES), has high binding affinity for estrogen receptor in mouse uterine cytosol but possesses weak biological activity. Racemic mixture of optically active [3H]indenestrol A (IA-Rac) was separated and purified into individual enantiomers on a semi-preparative scale by HPLC with a Chiralpak OP(+) column. The structure-activity relationship was investigated among the [3H]IA enantiomers (IA-R and IA-S) and [3H]DES through direct saturation binding assays using mouse uterine cytosol. Specific binding curves and Scatchard plots were obtained for each [3H]ligand; DES, IA-Rac, IA-R and IA-S. IA-S enantiomer (Kd = 0.67) binds to the estrogen receptor with the same affinity as DES (Kd = 0.71) and four times higher affinity than IA-R (Kd = 2.56). The number of binding sites for IA-S is approximately the same as estradiol, DES and IA-Rac while IA-R binds far fewer sites than the other ligands. Saturation binding assays indicated that [3H]DES and [3H]IA enantiomers exhibited a higher level of non-specific binding to the cytosol receptor compared to estradiol which has a low level of non-specific binding. These binding studies led to the detection of an additional binding component for the stilbestrol compounds in estrogen target tissue cytosol preparations. Sucrose density gradient separation assays under low salt conditions showed that both [3H]DES and [3H]IA compounds bound to the 8S form of the receptor, the same as E2. But, in addition both DES and IA bound to another binding component in 4S region. The binding to the 4S component were partially displaced by the addition of excess unlabeled E2 and DES. Further characterization of the 4S component is described.     

Regulation of uterine progesterone receptors by the nonsteroidal anti-androgen hydroxyflutamide

We have recently reported that the anti-androgen hydroxyflutamide causes delayed implantation and exhibits antideciduogenic activity in the rat. The present experiments were conducted to examine whether hydroxyflutamide binds to the uterine progesterone receptors and/or alters the progesterone binding sites in the uterus. Cytosol and nuclear fractions from decidualized rat uterus were incubated with [3H]-R5020 without or with increasing concentrations of radioinert R5020, RU486, dihydrotestosterone, or hydroxyflutamide. From the log-dose inhibition curves, the relative binding affinity of both hydroxyflutamide and dihydrotestosterone was less than 0.1% and 2%, compared with R5020 (100%) for displacing [3H]-R5020 bound to uterine cytosol and nuclear fractions, respectively. Injection of estradiol-17 beta (1 microgram/rat) to ovariectomized prepubertal rats induced a 1.85-fold increase in uterine weight by 24 h. Hydroxyflutamide at 2.5 or 5.0 mg did not significantly alter the estrogen-induced increase in uterine weight. Compared to vehicle alone, estrogen induced an approximately 5-fold increase in uterine cytosolic progesterone binding sites. Hydroxyflutamide at both 2.5- and 5.0-mg doses significantly attenuated the estrogen-induced elevation in uterine progesterone binding sites. These studies demonstrate that hydroxyflutamide does not bind with high affinity to progesterone receptors, but suppresses the estrogen-induced elevation in progesterone receptor levels in the uterus.     

Preliminary characterization of an endogenous inhibitor of [3H]estradiol binding in rat uterine nuclei

The rat uterus contains two classes of specific nuclear estrogen-binding sites which may be involved in estrogen action. Type I sites represent the classical estrogen receptor (Kd = 1 nM) and type II sites (Kd = 10-20 nM) are stimulated in the nucleus by estrogen under conditions which cause uterine hyperplasia. Dilution of uterine nuclear fractions from estrogen treated rats prior to quantitation of estrogen binding sites by [3H]estradiol exchange results in an increase (3- to 4-fold) in the measurable quantities of the type II site. Estimates of type I sites are not affected by dilution. These increases in type II sites following nuclear dilution occur independently of protein concentration and result from the dilution of a specific endogeneous inhibitor of [3H]estradiol binding to these sites. The inhibitor activity is present in cytosol preparations from rat uterus, spleen, diaphragm, skeletal muscle, and serum. Preliminary characterization of the inhibitor activity by Sephadex G-25 chromatography shows two distinct peaks which are similar in molecular weight (300). These components (alpha and beta) can be separated on LH-20 chromatography since the beta-peak component is preferentially retained on this lipophilic resin. Partial purification of the LH-20 beta inhibitor component by high performance liquid chromatography and gas-liquid chromatography-mass spectrometric analysis suggests the putative inhibitor activity is not steroidal in nature and consists of two very similar phenanthrene-like molecules (molecular weights 302 and 304). Analysis of cytosol preparations on LH-20 chromatography shows that non-neoplastic tissues (uterus, liver, lactating mammary gland) contain both and inhibitor components whereas estrogen-induced rat mammary tumors contain very low to nonmeasurable quantities of the beta-peak inhibitor activity.     

ZK98299, a novel antiprogesterone that does not interact with chicken oviduct progesterone receptor

Steroid antagonists, at receptor level, are valuable tools for elucidating the mechanism of steroid hormone action. We have examined and compared the interaction of avian and mammalian progesterone receptors with progestins; progesterone and R5020, and a newly synthesized antiprogesterone ZK98299. In the chicken oviduct cytosol, [3H]R5020 binding to macromolecule(s) could be eliminated with prior incubation of cytosol with excess radioinert steroids progesterone or R5020 but not ZK98299. Alternatively, [3H]ZK98299 binding in the chicken oviduct was not abolished in the presence of excess progesterone, R5020, or ZK98299. In the calf uterine cytosol, [3H]R5020 or [3H]ZK98299 binding was competeable with progesterone, R5020 and ZK98299 but not estradiol, DHT or cortisol. Furthermore, immunoprecipitation and protein A-Sepharose adsorption analysis revealed that in the calf uterine cytosol, the [3H]R5020-receptor complexes were recognized by anti-progesterone receptor monoclonal antibody PR6. This antibody, however, did not recognize [3H]ZK98299-receptor complexes. When phosphorylation of progesterone receptor was attempted in the chicken oviduct mince, presence of progesterone resulted in an increased phosphorylation of the known components A (79 kDa) and B (110 kDa) receptor proteins. Presence of ZK98299 neither enhanced the extent of phosphorylation of A and B proteins nor did it reverse the progesterone-dependent increase in the phosphorylation. The avian progesterone receptor, therefore, has unique steroid binding site(s) that exclude(s) interaction with ZK98299. The lack of immunorecognition of calf uterine [3H]ZK98299-receptor complexes, suggests that ZK98299 is either interacting with macromolecule(s) other than the progesterone receptor or with another site on the same protein. Alternatively, the antisteroid binds to the R5020 binding site but the complex adopts a conformation that is not recognized by the PRG antibodies.     

Ligand-binding properties of estrogen receptor proteins after interaction with surface-immobilized Zn(II) ions: evidence for localized surface interactions and minimal conformational changes

The site- or domain-specific immobilization of steroid receptor proteins with preserved structure and function would facilitate the identification and purification of receptor-associated regulatory components and nucleic acids. We have demonstrated previously that restricted surface regions of the estrogen receptor protein contain high affinity binding sites for immobilized Zn(II) ions. Possible conformational changes in receptor at the stationary phase immobilized metal ion interface were evaluated by monitoring alterations in the equilibrium dissociation constant (Kd) for [3H]estradiol. Soluble estrogen receptor proteins (unliganded) present in immature calf uterine cytosol were immobilized via surface-exposed Zn(II)-binding sites to beads of agarose derivatized with iminodiacetate (IDA)-Zn(II) ions. The IDA-Zn(II) bound receptor was incubated with increasing concentrations of [3H]estradiol (0.01-20 nM) in the presence and absence of unlabeled competitor (diethylstilbestrol) to determine the level of specific hormone binding. Steroid-binding experiments were performed in parallel with identical aliquots of soluble receptor. Analyses of the equilibrium binding data revealed the presence of a single class of high-affinity (Kd = 2.44 +/- 1.5 nM, n = 10) steroid-binding sites which were only marginally affected by receptor immobilization via surface-exposed Zn(II) bindings sites (Kd = 2.58 +/- 0.56 nM, n = 4). These data are consistent with the location of surface accessible Zn(II) binding site(s) on the receptor at or near the DNA binding domain which, upon occupancy, do not influence the steroid binding domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Androgen receptor-mediated inhibition of estrogen-induced uterine peroxidase

Flutamide, an anti-androgen known to act through the androgen receptor, abolished the inhibitory action of testosterone on the induction of peroxidase in immature rat uteri without affecting inhibition produced by progesterone. The time course of the androgen effect on estrogen-induced uterine peroxidase, uterine weight and glucose 6-phosphate dehydrogenase activity was also determined together with the effect of flutamide on these steroid hormone-sensitive parameters. The possible mechanism of action of these compounds is discussed, particularly in the light of estrogen-induced eosinophilia. It is proposed that the observed interaction between testosterone and estradiol is mediated through their own specific receptors and not by illicit occupation of the estrogen receptor by the androgen. 5-Androstene-3 beta, 17 beta-diol (Adiol), an androgen known to exert estrogenic effects through the estrogen receptor, induced uterine peroxidase and was without significant effect on the action of estradiol, in contrast to testosterone.     

High affinity estrogen binding by rabbit liver

Macromolecular binding components for [3H]estradiol-17beta are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [3H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4--5 S and the other had a sedimentation coefficient of 8--9 S. The two components differed from each other regarding steroid specificity and various physiocochemical parameters. [3H]estradiol binding to the 4--5 S component was not inhibited by estrogens, 5alpha-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appear to be saturable and label was rapidly stripped from it by charcoal. Estradiol binding to the 8--9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4--5 S moiety. The specific binding protein has a Kd of 3.05 . 10(-10) M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incubation of [3H]estradiol with mature male liver cytosol at 0--5 degrees C polar metabolites of estradiol are produced.     

Translocatable estrogen receptors in rat splenocytes

Estradiol has previously been shown to suppress the response of the cellular immune system of the rat while enhancing the production of IgM antibodies. Analysis of the cytosol from rat splenocytes showed saturation of specific binding sites at concentrations of between 80 and 160 nM [3H]-estradiol with an approximate Kd of 12 nM. Competitive binding studies showed a dose-dependent decrease in the binding of [3H]-estradiol to the receptor in the presence of increasing concentrations of unlabeled estradiol. Dexamethasone, progesterone and R1881 (synthetic androgen) had no effect on the binding of [3H]-estradiol. The in vivo administration of estradiol resulted in increased nuclear binding of [3H]-estradiol as compared to vehicle treated controls. These results indicate that rat splenocytes possess specific, translocatable estrogen receptors which may be responsible for the observed modulation of the immune system.     

Bioflavonoid interaction with rat uterine type II binding sites and cell growth inhibition

Competition analysis with a number of known bioflavonoids demonstrated that these compounds (luteolin, quercetin, pelargonin) compete for [3H]estradiol binding to cytosol and nuclear type II sites in rat uterine preparations. The inhibition of [3H]estradiol binding to type II sites was specific and these bioflavonoids did not interact with the rat uterine estrogen receptor. Since estradiol stimulation of nuclear type II sites in the rat uterus is highly correlated with cellular hypertrophy and hyperplasia, we assessed the effects of these compounds on the growth of MCF-7 human breast cancer cells in culture and on estradiol stimulation of uterine growth in the immature rat. The data demonstrated that addition of quercetin (5-10 micrograms/ml) to MCF-7 cell cultures resulted in a dose-dependent inhibition of cell growth (DNA/flask). This effect was reversible by removal of quercetin from the culture medium, or by the addition of 10 nM estradiol-17 beta to these cell cultures containing this bioflavonoid. Since estradiol-17 beta (10 nM) stimulated nuclear type II sites and proliferation of MCF-7 cells, we believe bioflavonoid inhibition of MCF-7 cell growth may be mediated through an interaction with nuclear type II sites. This hypothesis was confirmed by in vivo studies which demonstrated that injection of luteolin or quercetin blocked estradiol stimulation of nuclear type II sites in the immature rat uterus and this correlated with an inhibition of uterine growth (wet and dry weight). These studies suggest bioflavonoids, through an interaction with type II sites, may be involved in cell growth regulation.     

Effect of estradiol and progesterone on phosphatidylinositol metabolism in the uterine epithelium of the mouse

The effect of estradiol and progesterone on uterine phosphatidylinositol (PtdIns) metabolism was examined in whole uteri and separated uterine luminal epithelium of ovariectomized mice. Incorporation of [3H]myo-inositol in vitro, into inositol-containing phospholipids extracted from whole uteri, increased in mice injected with estradiol, with maximal incorporation at 9-12 h. The breakdown of PtdIns to inositol polyphosphates was also stimulated in whole uteri by estrogen, with an abrupt increase between 6 and 9 h. Comparable increases in both processes occurred in the uterine epithelium after estrogen stimulation and were inhibited by progesterone pretreatment which by itself had little or no effect. These results suggest that PtdIns metabolism is involved in the stimulation of uterine epithelial cell proliferation by estrogens, and its inhibition by progesterone.     

Properties of the estrogen receptors contained in the MtTF4 tumor whose growth is inhibited by estradiol: 17 beta-estradiol, 17 alpha-estradiol and DNA bindings

The steroid and the DNA bindings of the estrogen receptor of the MtTF4 tumor whose growth is inhibited by estradiol where characterized and compared to those of uterine estrogen receptors. In the tumor cytosol: E protects its binding sites against thermal denaturation, depending on the effects of sodium molybdate upon the dissociation rate of [3H]E at 20 degrees C and the ability of receptor to bind to DNA, the activation (or transformation) process, supposed to be necessary for the full action of estrogen ligand, occurs on estrogen receptor complexes and the calf thymus DNA interacts with estrogen receptor with an affinity similar to that of uterine estrogen receptor. Kinetic and equilibrium studies with 17 alpha-[3H]E both in uterus and tumor indicate that this ligand is fast-associating, fast-dissociating and that its affinity for ER is 2- to 4-fold lower than that of 17 beta-[3H]estradiol one. Competition experiments between 17 beta-[3H]estradiol and the unlabelled 17 alpha epimer reveal, in both uterus and tumor, a time-dependent decrease of the apparent potency of 17 alpha-E to inhibit the binding of [3H]E. It is concluded that the estrogen receptors are very similar in MtTF4 tumor and uterus and the diversity of the response of cell growth to E is due rather to differences at the post-receptor level.     

Androgen-induced nuclear accumulation of the estrogen receptor.

The effect of androgens on the nuclear uptake of both tritiated estradiol (3H-E2) and the estrogen receptor was studied in immature rat uteri. It was demonstrated that in vitro preincubation of immature rat uteri with various androgens (1 muM to 50 muM) followed by incubation with 3H-E2 (20 nM) resulted in a greatly decreased specific nuclear uptake of 3H-E2. Non-androgenic steroids had no effect. It was also confirmed that 5alpha-dihydrotestosterone (DHT) causes the accumulation of the estrogen receptor in the nuclei of uterine tissue. In vitro incubations of rat uteri with DHT (1muM and 50muM) were found to cause maximal nuclear estrogen receptor accumulation to the same degree as caused by estradiol, i.e. the nuclear uptake of approximately 100% of the estrogen receptor. Antiandrogens, which block the binding of androgens to the testosterone receptor in various tissues, did not inhibit the DHT - induced decrease in the 3H-E2 uptake by the uterine nuclei or the DHT - caused accumulation of the estrogen receptor in nuclei. These results seem to indicate that the uterine testosterone receptor has no role in the androgen - induced nuclear uptake of the estrogen receptor. However, the non-steroidal antiestrogens inhibited the DHT - induced nuclear accumulation of the estrogen receptor. This would seem to indicate that the estrogen - and androgen - induced nuclear accumulation of the estrogen receptor share a common mechanism.     

Differential blockade of estrogen-induced uterine responses by the antiestrogen nafoxidine

We have used an experimental design described by Gardner et al. [6] for dissociating early and late uterine responses to estradiol, involving pretreatment of immature rats with 5 micrograms nafoxidine (Upjohn U-11, 100 A, UA) for 24 h, before administrating estradiol. In these conditions the authors showed that responses occurring 4-h after estradiol administration were not blocked, while 24-h responses were abolished. These findings were defined and extended in the present investigation which shows that: (1) The overall wet weight response of the uterus to estradiol in UA-pretreated animals is decreased when compared to saline pretreated rats. (2) The early increase in cGMP content induced at 2-4 h by estrogen is also decreased but not abolished by the pretreatment with UA, contrary to the late increase in cGMP, which is abolished. (3) The late estrogen-induced proliferative response, measured by the [3H]thymidine labeling index, in the myometrium, stroma and luminal epithelium is maintained after pretreatment with UA. It is remarkable that this occurs in the absence of any estrogen induced uterine hypertrophy as measured by the 24-h increase in uterine weight and RNA or protein content. These results strongly support the hypothesis proposed by Gardner et al. [6] that different control mechanisms might regulate early and late uterine responses to estrogen. Our data suggest the existence of the following dissociable groups of response: (1) Wet weight increase and early increase in cGMP content, (2) Late hypertrophy and second rise in cGMP content and (3) Proliferative response; which are respectively, moderately depressed, abolished or unaffected by UA pretreatment.     

Identification of immunoassayable estrogen receptor lacking hormone binding ability in tamoxifen-treated rat uterus

Using two different monoclonal antibodies to human estrogen receptor (ER), the enzymeimmunoassay was performed. The values of ER contents in human breast cancer and untreated rat uteri obtained by this procedure were correlated well with those by [3H] estradiol binding assay. When estradiol was injected to immature rats, the enzymeimmunoassay showed the uterine receptor dynamic pattern similar to those analyzed by exchange assays. In contrast, tamoxifen administration induced the immunoassayable but nonsteroid binding form of ER. This ER-like antigen was the heat-labile molecule with the sedimentation constant of 7 S while ER in untreated rat uterine cytosol sedimented at 9 S. These results suggest the presence of unique molecular state of ER induced by tamoxifen.     

Non-stoichiometric nuclear-cytoplasmic redistribution of estrogen receptor in adult rat uterus, following estradiol injection

In immature and ovariectomized rats acutely injected with estradiol (E2), accumulation of estradiol receptor complexes (E2R) from the uterine cytosol to the nucleus has been shown to be quantitative by numerous investigators. In the present study, translocation of E2R from the cytosol to the nuclear fraction in adult and ovariectomized estrogen prestimulated rats was analyzed. Twenty micrograms of E2, dissolved in saline containing 10% ethanol and 1 g% bovine serum albumin (B.S.A.) were injected intraperitoneally to the animals and 2 h later E2R in the cytosol and crude nuclear fractions were assayed by exchange techniques. Unlike a 91% recovery of the depleted cytosol E2R in the nuclear fraction of ovariectomized rats, only 39.2 and 27.5% were recovered in the adult and ovariectomized estrogen prestimulated rat uterus respectively. Moreover, depending on the temperature and duration of nuclear suspension incubation, from 18 up to 80% of the recovered nuclear E2R were solubilized in the incubation medium and nuclear post-incubation washes and could be measured by hydroxylapatite treatment (HAP). Saturation assays showed a plateau from 12 nM E2 3H onwards up to 80 nM. The Kd values computed for the receptors in the nucleus and HAP in all the three groups were of the order of 2 X 10(-9) M. In conclusion, after E2 administration to adult or ovariectomized estrogen prestimulated rats, a stoichiometric recovery of the depleted cytosol E2R in the nuclear fraction was not observed, even when leakage of nuclear receptor into the medium in course of exchange was taken into account. Chronic estrogenization appeared to modify the dynamics of uterine receptor.     

Effect of antibodies against the specific estrogen-binding protein in the rat liver on the hormone-binding activity of this protein and steroid hormone receptors

The effects of rabbit polyclonal antibodies (AB) against an unusual estrogen-binding protein (UEBP) isolated from rat liver by immunoadsorption on the interaction of [3H]estradiol with UEBP, estrogen receptors of uterus and other tissues, of [3H]dihydroxytestosterone with prostate androgen receptors, of [3H]progesterone with uterine progesterone receptors and of [3H]dexamethazone with rat thymus glucocorticoid receptors were investigated. It was shown that preincubation of cytosol of the above tissues with AB decreases the ability of UEBP, estrogen and androgen receptors to bind the corresponding ligands. The hormone-binding activity of progesterone and glucocorticoid receptors in the presence of AB remains thereby unchanged. The binding activity of UEBP in the presence of ABUEBP diminishes due to the decrease in the concentration of protein binding sites, whereas that of estrogen and androgen receptors decreases due to the diminution of affinity for their ligands which, in turn, is the result of reduction of the association rate constant. A cross influence of AB on the activity of uterine estrogen receptors of rabbit, guinea pig and mouse was observed. It was assumed that the structure of UEBP and sex steroid receptors has some similar elements.     

Steroid metabolism and binding activity in a murine renal tumor cell line

The purpose of this study was to partially characterize the steroid binding activity of murine renal tumor cells in continuous culture. The steroid receptor content of a cloned renal tumor cell line (RAG) and a subline RAG-2 was examined by sucrose gradient analysis, hydroxylapatite and dextran-coated charcoal methods. The RAG cells lacked estrogen- and progestin-binding activity, whereas specific 5 alpha-dihydrotestosterone (DHT) and dexamethasone (Dx) binding activities were detected as 8S peaks on low salt gradients. The specificity of DHT binding was examined by sucrose gradient analysis: DHT, R1881 and ORG2058 all completely inhibited [3H]DHT binding whereas diethylstilbestrol and Dx were ineffective. The androgen receptor content of the RAG cells was approx. 15 fmol/mg cytosol protein by the hydroxylapatite-filter assay, with an estimated Kd for methyltrienolone (R1881) of 5 nM at 0 degrees C. Scatchard analysis of [3H]Dx binding by RAG cytosol showed a Kd of 6 nM for Dx and 44 nM for corticosterone at 0 degrees C. Glucocorticoid receptor levels were estimated to be 182 fmol/mg cytosol protein by dextran-coated charcoal assay. Metabolism of [3H]testosterone and [3H]DHT by RAG cells was examined 1, 4 and 6 h after exposure to labeled hormone. Radioactive DHT was the primary intracellular metabolite recovered after exposure to [3H]testosterone. There was little conversion of DHT to androstanediol.     

Characterization of R5020 and RU486 binding to progesterone receptor from calf uterus

We have examined and compared the binding characteristics of the progesterone agonist R5020 [promegestone, 17,21-dimethylpregna-4,9(10)-diene-3,20-dione] and the progesterone antagonist RU486 [mifepristone, 17 beta-hydroxy-11 beta-[4-(dimethylamino) phenyl]-17 alpha-(prop-1-ynyl)-estra-4,9-dien-3-one] in calf uterine cytosol. Both steroids bound cytosol macromolecule(s) with high affinity, exhibiting Kd values of 5.6 and 3.6 nM for R5020 and RU486 binding, respectively. The binding of the steroids to the macromolecule(s) was rapid at 4 degrees C, showing saturation of binding sites at 1-2 h for [3H]progesterone and 2-4 h for both [3H]R5020 and [3H]RU486. Addition of molybdate and glycerol to cytosol increased the extent of [3H]R5020 binding. The extent of [3H]RU486 binding remained unchanged in the presence of molybdate, whereas glycerol had an inhibitory effect. Molybdate alone or in combination with glycerol stabilized the [3H]R5020- and [3H]RU486-receptor complexes at 37 degrees C. Although the rate of association of [3H]RU486 with the cytosolic macromolecule was slower than that of [3H]R5020, its dissociation from the ligand-macromolecule complex was significantly slower than [3H]R5020. Competitive steroid binding analysis revealed that [3H]progesterone, [3H]R5020, and [3H]RU486 compete for the same site(s) in the uterine cytosol, suggesting that all three bind to the progesterone receptor (PR). Sedimentation rate analysis showed that both steroids were bound to a molecule that sediments in the 8S region. The 8S [3H]R5020 and [3H]RU486 peaks were abolished by excess radioinert progesterone, RU486, or R5020.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogen receptors in the adrenal cortex of the possum (Trichosurus vulpecula)

1. Specific [3H]estradiol binding activity with characteristics of estrogen receptors was found in the cytosols and nuclear extracts of the adrenal cortex proper and special zone of the brushtail possum (Trichosurus vulpecula). 2. The specific estradiol receptor had a sedimentation coefficient on sucrose gradients of approximately 9S and a molecular weight on gel filtration of more than 200,000. The adrenal cortex cytosol binds [3H]estradiol with high affinity (Ka 5.5 X 10(9) M-1), and limited capacity (Bmax 62.7 fmol/mg cytosol prot). In competition experiments with different steroids the receptor showed a high affinity for four estrogens and a very low affinity to androgens, progesterone and cortisol. 3. There was no difference in the affinity and maximum binding capacity of the cytosols from cortex proper in male and female animals, but the binding capacity of the special zone of females was half that of cortex proper. Estradiol receptors were found in the kidney, liver, lung, testis and muscle but only in the adrenal and prostate was the binding capacity relatively high compared with the uterus. 4. The specific binding capacity of [3H]estradiol to cytosols of adrenal cortex at different stages of the estrus cycle and pregnancy was unrelated to that of the uterus. In the adrenal the receptor concentration was lowest at estrus, when uterine concentration was high, while in late pregnancy the binding of adrenal cortex and uterus cytosols was almost the same. 5. The possible physiological significance of the presence of a specific estrogen receptor in male and female possums is discussed.