Comparative binding specificity of methyltrienolone in human and rat prostate.

The binding of methyltrienolone (R 1881) in crude human hyperplastic prostate cytosol was determined by a charcoal assay. Maximum binding was observed after 2-3 h of incubation at 0 degrees C. This binding decreased steadily thereafter and reached 41% of the 2-hour values after 96 h of incubation. In human hyperplastic prostate, the binding of 3H-R 1881 was competed by low concentrations of R 1881, R 5020 and progesterone and by high concentrations of dihydrotestosterone (DHT) and 17 alpha-methyl-DHT. In the rat prostate, on the other hand, this binding was competed by low concentrations of DHT and 17 alpha-methyl-DHT and only by high concentrations of progesterone and R 5020. The apparent association constant (Ka) of R 1881 was determined in three human prostates and found to be 0.2-0.4 X 10(9) liters/mol; the number of binding sites ranged from 101 to 158 fmol per mg of protein. These findings constitute further evidence for the existence of relatively large amounts of a progesterone-binding component in human hyperplastic prostate.     

Effect of 17β estradiol on [3H] progesterone binding in rat brain

A progesterone-binding component is reported in the cerebral hemispheres of immature female rat. [³H] progesterone binding in the brain cytosol is increased following two weeks of estradiol administration. The [³H] progesterone binding by this component can be reduced by pretreatment with unlabeled steroid. In addition, the binder from both control and estradiol-treated groups shows inter-action with ATP immobilized on columns of ATP-Sepharose.     

Affinity chromatography of estrogen- and progesterone-binding proteins of human uterus

The purification of estrogen- and progesterone-binding proteins of human uterus by employing affinity resins coupled with steroid-bovine serum albumin conjugates, led to the isolation of preparations with estrogen- and progesterone-binding sites havingK _d values in the range of 0.96 to 1.20 × 10^-9 M. These were different from theK _d values of 10^-10 M and 10^-8 M obtained for two types of binding sites present in the crude cytosolic and nuclear fractions. The purified proteins sedimented on sucrose gradient withS values in the range of 3.6–4.4. The cytosolic and nuclear estrogen- and progesterone-binding proteins, thus purified, showed differences in specificity of binding to the hormone. While the cytoplasmic proteins were more specific in their binding to estradiol or progesterone, the nuclear proteins bound Cortisol with equal or moderate affnity. These results demonstrate the presence of distinct physiological forms of estrogen- and progesterone-binding proteins in the cytoplasm and nucleus, thus pointing to the importance of both these compartments in hormone action.     

Effect of protease inhibitors on androgen receptor analysis in rat prostate cytosol

Prostate androgen receptors are liable to proteolytic digestion during in vitro analysis; thus, various proteolytic enzyme inhibitors were tested for their ability to improve the androgen receptor assay. The serine (phenylmethylsulfonylflouride, aprotinin, p-aminobenzamidine) and thiol-senine (leupeptin, bacitracin) protease inhibitors individually present in the homogenization buffer significantly increased the measurable androgen binding sites by 30-35% in rat prostate cytosol as determined by saturation analysis with [3H]-17 beta-hydroxy-17-methyl- 4,9 11-estratrien-3-one (R-1881) for 20 hr at 4 degrees C. The apparent binding affinity was also increased by these compounds. Various combinations were tried and aprotinin/bacitracin was found to be additive in effect. This combination was also shown to prevent receptor degradation as determined by sucrose density gradient centrifugation. The carboxyl protease inhibitor, pepstatin A, was ineffective in improving the receptor assay. Rabbit bile, an inhibitor of seminin, interfered with receptor binding thus rendering it ineffective for use in saturation analysis. The results show that the use of serine-thiol protease inhibitors significantly improves the cytosol androgen receptor yield and assay sensitivity; therefore, we recommend routine inclusion of these compounds(s) in the homogenization buffer for androgen receptor assays.     

Prostatic cytosol binding of 5 alpha-androstane-3 beta, 17 beta-diol and estradiol-17 beta

The goal of the present research was characterization of the interaction of 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) with prostatic estradiol-17 beta(E2) binding sites to address the role of this 5 alpha-dihydrotestosterone(DHT)a metabolite in prostatic regulation. Using dextran-charcoal assay we demonstrated specific 3 beta-diol and E2 binding sites in rat ventral prostate cytosol (RVPC) and dog prostate cytosol (DPC). In both cytosols, E2 binding is of high affinity (Ka congruent to 10(9) M-1; RVPC:68 fmol/mg protein), DPC:170 fmol/mg protein), and 3 beta-diol binding is of moderate affinity (Ka congruent to 10(8) M-1; RVPC:62 fmol/mg protein, DPC:165 fmol/mg protein). Unlabeled 3 beta-diol competes effectively for cytosolic 3H-E2 binding sites, whereas unlabeled DHT, 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and testosterone (T) are poor competitors for 3H-E2 binding sites. Using DNA-cellulose column chromatography, we separated prostatic androgen and estrogen binding activities. The E2 binding activity which adhered to DNA-cellulose was displaced by 100-fold excess 3 beta-diol but not by DHT. Thus data from two assay procedures show competition of 3 beta-diol for 3H-E2 binding sites in rat and dog prostate.     

The nuclear binding of dexamethasone-receptor complex from fast and slow skeletal muscle in rats

1. The binding to isolated muscle nuclei of the complex of dexamethasone with cytosol receptors from rat soleus (Sol) and extensor digitorum longus (EDL) muscles was measured. 2. The ratio of bound to total amount of complex was higher in Sol. 3. The binding of complex per mg of cytosol protein was also higher in Sol. 4. These results suggest that activation and nuclear binding of the steroid-receptor complex are not the sites of the different sensitivity of the two muscle types to glucocorticoid.     

Assay of androgen binding sites by exchange with methyltrienolone (R 1881).

Methyltrienolone (R 1881 - 17beta-hydroxy-17alpha-methyl-estra-4,9,11-trien-3-one) binds specifically to androgen receptor in rat prostate cytosol where, unlike androstanolone, it is not metabolized. By exchanging bound endogenous hormone in rat prostate cytosol with labelled R 1881, it is possible to measure total (free anc occupied) binding sites. This assay method has also been applied to the measurement of androgen receptor sites in human benign prostatic hypertrophy where R 1881 has the added advantage of not being bound by any contaminating plasma protein (sex hormone binding protein).     

Corticosteroid receptor in type II pneumocytes of the rat

Glucocorticoid receptor in rat type II pneumocytes has been characterized. The Scatchard plot analysis of 3H-dexamethasone binding to type II cells showed a single class of binding sites. The apparent Kd of 3H-dexamethasone binding by a whole cell assay was 9.1 nM and the maximal binding capacity was 78.0 f mol/10(6) cells (0.31 pmol/mg cytosol protein).     

Structure and refinement of the oxidized P21 form of uteroglobin at 1.64 A resolution

One of the monoclinic P21 forms of uteroglobin, a progesterone-binding protein secreted by the rabbit uterus, was crystallized and subjected to X-ray diffraction analysis at 1.64 A resolution. The analysis was refined to an R factor of 0.19 and the 1096 non-hydrogen atomic positions are known to an accuracy of about 0.18 A. The average isotropic temperature factor B was 10.4 A2. Uteroglobin is a dimer of two independent polypeptide chains of 70 residues linked by two disulfide bridges and related by a pseudo binary axis. Each monomer is folded into four alpha-helices. An oblong hydrophobic pocket is observed inside the dimer, and the possibility that it represents a progesterone-binding site is discussed. The present model includes 165 possible sites for water molecules, of which six are located in the hydrophobic pocket. Polar groups are involved in hydrogen bonding (intramolecular, intermolecular or with water molecules).     

Heterogeneity of [3H]estradiol binding sites in the rat prostate: properties and distribution of type I and type II sites

In order to assess the rat prostate as a target tissue for receptor-mediated estrogen action, we have studied the properties and distributions of estrogen binding sites in the dorsolateral (DLP) and ventral (VP) prostate. Saturation analyses over a wide range of [3H]estradiol ([3H]E2) concentrations (0.5-100 nM) revealed two distinct types of binding sites in the cytosol and nuclear fractions of DLP of intact rats. The high affinity (type I) estrogen binding sites saturated at 2-4 nM of [3H]E2 and had a capacity of 170 fmol/mg DNA in the cytosol and 400 fmol/mg DNA in the nuclei. DLP type I sites had ligand specificity similar to that described for the classical estrogen receptors (ERs) found in female target tissues. The moderate affinity (type II) estrogen binding sites saturated at 15-30 nM of [3H]E2 and had a capacity of 850 fmol/mg DNA in the cytosol and 1600 fmol/mg DNA in the nuclei. DLP type II sites shared some characteristics of the type II ERs described for the rat uterus; they were estrogen specific, heat labile, and sensitive to reducing agents such as dithiothreitol. Saturation analyses on VP cytosols and nuclear fractions revealed only high affinity sites but no moderate affinity sites in the tissue preparations. Our finding that prostatic type II estrogen binding sites are present exclusively in the DLP supports the concept that basic biological differences exist between the two major prostatic lobes of the rat. Furthermore, our findings may help elucidate the observed differences in susceptibility between these two lobes to the hormonal induction of proliferative prostatic lesions.     

Development of an [3H] glucocorticoid exchange assay in rat liver cytosol

An exchange assay for the assessment of glucocorticoid binding sites both free and steroid bound, in rat liver cytosol has been developed. The procedure, which is straight forward and noncumbersome involves the utilization of two sulfhydryl interacting agents, $\text{p}$-hydroxymercuribenzoate and dithiothreitol. The former in low concentration dissociates the steroid from the glucocorticoid receptor complex in relatively short time. The latter regenerates, quite rapidly and with good yield, the glucocorticoid binding activity of the receptor treated with the mercurial compound. The high recovery (99%) of the hormone binding activity of the receptor may be due to the few steps involved in the procedure. The exchange assay when applied to a physiological experimental situation was found to be valid and gave a true measure of total receptor content in rat hepatic cytosolic preparations.     

Alcohol-induced changes in hepatic estrogen-binding proteins: A mechanism explaining feminization in alcoholics

Chronic alcoholic men frequently display an apparent hyperestrogenization manifested by enhanced hepatic synthesis of estrogen-responsive proteins as well as many other estrogen-linked tissue alterations. Because of these clinical observations, we assessed the effect of chronic alcohol ingestion, using a rat model, on the levels of two estrogen-binding proteins of male rat liver cytosol. These two estrogen-binding proteins, the estrogen receptor, and an unusual male-specific estrogen binder, differ in specificity for the non-steroidal estrogen diethylstilbestrol (DES), permitting development of an assay for each using unfractionated cytosol. The estrogen receptor was labeled with [³H]DES, and the male-specific estrogen binder with [³H]estradiol in the presence of unlabeled DES, since the latter protein does not recognize DES. The specificity of labeling under these conditions was verified by gel filtration chromatography. The livers of rats fed either an alcohol-containing (AF) or isocalorically matched control diet were assayed for the levels of both proteins. The livers of the AF animals had twice the content of estrogen receptor, as compared to the isocalorically matched control group (105 vs 52 fmol/mg cytosol protein). Conversely, the livers of the AF animals had only one-third as much male-specific estrogen binder as did those of the isocalorically matched control group (22 vs 62 pmol/mg cytosol protein). Alcohol feeding also resulted in those animals having smaller testes, seminal vesicles, and prostates, as well as decreased serum testosterone levels. No change in serum estradiol levels occurred after 34 days of alcohol feeding; however, 61 days of alcohol feeding resulted in an increase in serum estradiol levels in the AF animals. These results are incorporated into a proposed model of feminization of the chronic alcoholic male.     

Nuclear progesterone receptor in the hen pituitary and hypothalamus.

The existence of specific progesterone-binding sites with a high affinity and a limited capacity was demonstrated by [3H]progesterone exchange in the nuclear fractions of the pituitary and hypothalamus as well as in those of the oviduct magnum in the hen.     

The membrane-associated progesterone-binding protein 25-Dx is expressed in brain regions involved in water homeostasis and is up-regulated after traumatic brain injury

After traumatic brain injury, progesterone has important neuroprotective effects in the nervous system. There is better functional outcome and less oedema formation in pseudopregnant rat females (high levels of endogenous progesterone) than in males. In addition to intracellular progesterone receptors, membrane binding sites of the hormone such as 25-Dx may also be involved in neuroprotection. In the present study we investigated the distribution of the membrane-associated progesterone-binding protein 25-Dx in rat brain. Immunohistochemical analysis showed that 25-Dx is particularly abundant in the hypothalamic area, circumventricular organs, and ependymal cells of the lateral walls of the third and lateral ventricles. A strong signal was also detected in the meninges. Double immunofluorescence immunolabelling and confocal microscopy showed that 25-Dx is co-expressed with vasopressin in neurones of the paraventricular, supraoptic and retrochiasmatic nuclei. Levels of 25-Dx expression were higher in pseudopregnant females than in males. After traumatic brain injury, 25-Dx expression was up-regulated in neurones and induced in astrocytes, which play an important role in regulating water and ion homeostasis. The expression of 25-Dx in structures involved in CSF production (choroid plexus) and in osmoregulation (circumventricular organs, hypothalamus and meninges), and its up-regulation after brain damage, point to a novel and potentially important role of this progesterone-binding protein in the maintenance of water homeostasis after traumatic brain injury.     

Accelerated dissociation of estrogen receptor--ligand complexes by estradiol. Evidence for negative cooperativity of binding

The rate of dissociation of 17β-[³H]estradiol that had been previously equilibrated to a low degree of saturation of immature rat uterine cytoplasmic estrogen receptor was shown to increase over 40-fold in the presence of additional ligand. This effect was specific for either labeled or unlabeled estradiol, was observed under conditions in which the rebinding of dissociated ligand was shown not to occur, was distinguishable from the activation of cytoplasmic receptor, and was dependent upon the degree of saturation of the receptor by ligand. It occurred under conditions in which the receptor population was apparently uniform and stable and utilized an assay method that is particularly sensitive to low concentrations of cytosol protein. Once saturation of the receptor attained 15% of the available ligand binding sites, further increases of the dissociation rate of receptor-ligand complex could not be produced by the inclusion of additional estradiol. It was shown that exchange of ligand molecules in given binding sites was unlikely. Rather, support was given to the hypothesis that interactions were occurring between separate binding sites in the receptor population. The decrease of the apparent affinity of receptor for ligand when the fractional saturation of receptor increases has been defined as negative cooperativity. It is proposed that this phenomenon may be significant in the regulation of the response of target cells to estrogens.     

大白鼠妊娠早期(1—6天)子宫孕酮受体的变化

本实验中大鼠妊娠第三天(D_3)出现血浆孕酮含量和子宫细胞胞核中孕酮受体含量显著同步升高和胞质中孕酮受体含量明显下降的现象,为D_5胚泡着床准备了必要的条件。D_6时血浆孕酮,胞质和胞核中孕酮受体以及子宫重量均升高,标志胚泡着床后的生理变化。     

Effects of chlorpromazine on the inhibition and artifactual elevation of [3H]estradiol binding to estrogen receptors in rat uterine cytosol

Chlorpromazine acts to inhibit the specific binding of estradiol in rat uterine cytosol in vitro at concentrations between 0.1 and 0.75 mM. However, at higher concentrations (1.0-2.0 mM) it causes an apparent increase in binding that is due to free labeled estradiol in the assay buffer which is not adsorbed by the charcoal-dextran. This artifactual elevation can lead to misinterpretations of drug-induced potentiation of receptor sites.     

A microassay for the measurement of androgen receptors in human prostatic tissue.

A microassay utilizing R 1881 (methyltrienolone) has been developed for the measurement of androgen receptor sites in the cytosol and nuclear extract of human prostatic tissue. Binding of R 1881 to the progesterone binding molecule in cytosol was eliminated by the addition of triamcinolone acetonide. Utilizing a six tube, single point assay, the number of binding sites estimated in nuclear extract averaged 95% of the number measured by a full 7 point Scatchard analysis; the number estimated by the microassay in cytosol averaged 91%. When the single point assay was applied to needle biopsy specimens (200 mg of tissue), the estimated number of binding sites in nuclei averageed 83% of the number measured in bulk tissue (2 grams) utilizing a 7 point Scatchard analysis; the number in cytosol estimated by the microassay on needle biopsy specimens averaged 73%. It is hoped that this technique may be useful in correlating receptor content with hormonal responsiveness in men with metastatic carcinoma of the prostate.     

Evidence for a single steroid-binding protein in the rabbit progesterone receptor

The rabbit uterine progesterone receptor copurifies as two molecular weight (Mr) forms of about 105,000 and 78,000. To investigate whether these are different proteins, we have used protease digestion, reversible denaturation, and photoaffinity labeling in studies on the steroid-binding domain of the receptor. Digestion of the Mr 105,000 and 78,000 forms, photoaffinity labeled with [3H]R5020, with Staphylococcus aureus V8 protease revealed identical peptide fragments of Mr 43,000, 39,000, and 27,000-30,000. When receptor in cytosol was denatured, separated by electrophoresis, and then reconstituted, [3H]progesterone bound specifically to a single form at about Mr 105,000. After partial purification, the reversible denaturation procedure revealed both the larger and the smaller progesterone-binding species similar to the photoaffinity-labeled species in this preparation. Receptor in uterine cytosol prepared under mild conditions appeared as a predominant large molecular weight form on photoaffinity labeling with [17 alpha-methyl-3H]R5020, [6,7-3H]R5020, or [3H]RU27987. Further purification of this cytosol showed the generation of a smaller labeled species. These results from three different approaches reinforce the view that the rabbit progesterone receptor contains a single steroid-binding protein.     

Ginsenoside RG1 and corticosteroid receptors in rat brain

Old (28 months) male Wistar rats were treated chronically for two weeks with ginsenoside Rg1 or with vehicle delivered via sc implanted Alzet mini-pumps (rate of ginsenoside release 2.4 micrograms/0.5 microliter/h). The number of Type 1 corticosterone-preferring receptor sites (CR) and Type 2 glucocorticoid receptors (GR) was measured in the cytosol of hippocampus tissue of rat brain with an in vitro binding assay. In old rats the Bmax of Type 1 CR and Type 2 GR was reduced by 51.5% and 28.3% respectively. Following the two week treatment with Rg1 the Bmax of Type 1 CR increased by 60% and a receptor concentration was reached which was 21% lower than that observed in the young control animals. Minor differences in affinity of steroid binding to both receptor systems were observed in the groups of rats. The possible binding of ginsenosides to brain corticosteroid receptors in vitro was investigated as well. The inclusion of a 500 fold molar excess of Rg1 in hippocampus cytosol did not displace 3H-corticosterone from its soluble receptor sites. The affinity of Rg1 with these sites in vitro is therefore negligible. In conclusion, the binding capacity of Type 1 CR and Type 2 GR is reduced in the hippocampal brain region of aged rats. Upon chronic infusion of ginsenoside Rg1, only Type 1 CR capacity is restored towards the level observed in young control animals. This finding suggests that in old rats the ginsenoside enhances the CORT signal via Type 1 CR on the function of the hippocampus, which is a limbic brain structure involved in cognition, mood and affect.