Characterization of a [3H]methyltrienolone (R1881) binding protein in rat liver cytosol

The binding of radiolabelled methyltrienolone 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [3H] R1881 macromolecular complex which sediments in the 8-9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [3H] R1881 was recovered by thin layer chromatography. Characteristics of this [3H] R1881-8S binding protein include high affinity (Kd = 2.3 +/- 41 nM) and low binding capacity (18.8 +/- 3.3 fmol/mg cytosol protein), precipitability in 0-33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [3H] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a Kd = 2.8 nM for [3H] R1881, and is androgen specific (testosterone greater than 5 alpha-dihydrotestosterone greater than estradiol greater than progesterone greater than cyproterone acetate greater than diethylstilbestrol greater than dexamethasone greater than triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.     

Characterization of a unique corticosterone-binding protein in Candida albicans

This paper further characterizes a protein we have demonstrated in Candida albicans which has the ability to bind corticosterone and related steroid hormones. Fungal cells are disrupted and cytosol is incubated with [3H]corticosterone for 3 h at which time peak steady state binding is achieved. Bound hormone is separated from free using Sephadex G-50 minicolumns or dextran-coated charcoal. Binding was found to be a linear function of protein concentration. The bound hormone co-migrates with authentic corticosterone in thin layer chromatographic systems indicating no metabolism of the radioprobe. Scatchard analysis of the binding in the pseudohyphal form of C. albicans yielded values of 6.3 nM for the Kd and a binding capacity of about 650 fmol/mg of cytosol protein; both determinations are comparable to our findings in the yeast form of this organism. A series of sterols were tested for their ability to displace [3H]corticosterone from the yeast binder, and the results show that the binder is remarkably selective and stereo specific. Physical-chemical studies show the binder to be degraded at high temperatures and that binding is destroyed by trypsin and sulfhydryl blockers. The protein sediments at 4 S on sucrose gradients and does not exhibit ionic dependent aggregation. The molecular weight is estimated to be approximately 43,000 daltons by gel chromatography. We hypothesize that this intracellular protein may represent a primitive form of either the mammalian glucocorticoid receptor or the plasma corticosteroid-binding globulin.     

A hybrid ligand method for androgen receptor measurement

Existing techniques for androgen receptor (AR) assay are complicated by cross-reactivity of ligand binding affinities that can lead to incorrect estimation of receptor concentration. Two most frequently used ligands are [3H]dihydrotestosterone [( 3H]DHT) and [3H]methyltrienolone [( 3H]R1881), which in addition to binding to AR also bind to sex hormone binding globulin (SHBG; Kd = 1.5 nM) and progesterone receptors (PgR; Human Kd = 1 nM, rat Kd = 6 nM) respectively. Triamcinolone acetonide (TMA) is commonly used to block binding of [3H]R1881 to PgR, however at high concentrations TMA itself will bind AR (Kd = 7 microM). We have developed a hybrid ligand method for the measurement of AR in the presence of SHBG and PgR. This method used [3H]R1881 as the high specific activity labelled tracer and DHT as the unlabelled competitor of specific AR binding. Using this assay, 20% of human colorectal carcinomas were found to contain AR.     

Demonstration of an androgen receptor in rat pancreas.

[1,2,6,7-3H]Testosterone (250 muCi) was administered to castrated male rats; after 30 min a labelled testosterone-receptor protein complex with a pI of 5.1 was recovered from the pancreatic cytosol. A labelled testosterone-receptor complex with an identical pI was also extracted from the nuclear fraction of rat pancreas after incubation of minced pancreatic tissue with 0.1 muM-]1,2,6,7-3H]testosterone for 30 min at 37 degrees C. Studies in vitro showed that [1,2,6,7-3H]testosterone was bound to a receptor protein focusing at a pI of 5.1 and with a Kd of 2 nM and a number of binding sites of 4.7 fmol/mg of protein in castrated male rats. The testosterone-receptor complex sedimented at 3.5 S in high-salt sucrose-density gradients, was excluded from Sephadex G-200 and Ultragel ACA-34, was stable towards treatment with dextran-coated charcoal, was relatively sensitive to heat, and was stable to treatment with deoxyribonuclease and ribonuclease, but was sensitive to treatment which proteinase. It is suggested that the pancreatic androgen receptor, which was also present in castrated female rats, may play a role in sex-steroid regulation of pancreatic function.     

Androgen receptor in human placental villi

In studies with a synthetic androgen, R 1881, an androgen-binding component was found in the cytosol of human placental villi. Kinetic analysis indicated that the Kd value of this component was 1.4 nM at 0-4 degrees C and that binding of R 1881 amounted to 277 +/- 73 fmol/mg protein. glycerol density gradient ultracentrifugation showed a peak of binding activity in the 8S region in a medium of low ionic strength, but in the 4.5S region in a medium containing 9.5 M KCl. The R 1881-binding component was inactivated by mild heat- or trypsin-treatment, but not by treatment with DNase or RNase. Most of the R 1881-binding activity was sedimented at 20 to 40% saturation of ammonium sulfate. These findings indicate that the R 1881-binding component in human placental cytosol is quite similar in its characteristics to androgen receptors, which are present in various androgen-responsive organs. Testosterone was a more potent competitor of R 1881-binding than DHT or cyproterone acetate. Scatchard plots indicated that the binding site of testosterone was identical with that of R 1881. These findings suggest that the androgen receptor in placental cytosol is specific for testosterone. The Kd value for testosterone was calculated to be 3.2 nM.     

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 (Kd 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 testosterone 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 the placental binding site is stable at elevated temperatures with no loss of binding after 4 h at 45 degrees 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.     

Characterization of estrogen-induced progestin binding in cytosol of the R3327 prostatic carcinoma of the rat

High affinity binding of the synthetic steroids methyltrienolone (R1881) and promegestone (R5020) to cytosol protein from the Dunning (R3327) experimental prostatic carcinoma of the rat was investigated. Animals bearing tumours of approx 1.5 cm mean diameter were either left untreated, or were administered diethylstilbestrol diphosphate (DESP) in the drinking water in doses close to those used clinically for the treatment of human prostatic carcinoma. Tumours were excised after 10-40 days, and binding of [3H]R1881 and [3H]R5020 to tumour cytosol was characterized using Scatchard analysis, sucrose density gradient centrifugation, and steroid competition, under conditions optimal for the conservation and assay of progesterone receptor. Both ligands were bound in much higher concentrations by cytosol from DESP-treated tumours than from untreated tumours. Binding was of high affinity (Kd congruent to 1 nM), was specific for progestins, and sedimented in peaks at approximately 8S and approximately 4S in sucrose density gradients. We conclude the DESP treatment of rats bearing the R3327 prostatic carcinoma induces synthesis of progesterone receptor in this tumour.     

Atypical Androgen Receptor in the Human Melanoma Cell Line IIB-MEL-J

To evaluate the presence of androgen receptors in the human melanoma cell line IIBMEL-J, a Scatchard plot analysis was performed. Cells in culture revealed a single binding component with an apparent dissociation constant (K_D) at 37°C of 11 nM and a binding capacity of 326 fmol/mg protein when measured with [³H]-R1881. Competition analysis revealed an atypical relaxation of specificity, since not only androgen (testosterone, dihydrotestosterone [DHT], R1881) and antiandrogen (hydroxy-flutamide [OH-FLU]) competed for [3H]-R1881 binding, but also estradiol, progesterone, and cortisol at 500-fold excess concentration. Binding of [3H]-estradiol and [3H]-R5020 in the absence of unlabeled DHT were completely suppressed in its presence. Immunohistochemistry of androgen receptor with a monoclonal antibody showed that nuclei were vigorously stained. Different doses of flutamide (FLU) and OH-FLU tested on cultured IIB-MEL-J cells in the presence of serum inhibited significantly cell proliferation in a dose-dependent manner. When cells were incubated with 10 nM DHT and 1%charcoal-adsorbed serum, a significant stimulation of growth that was observed was inhibited by 4 μM OH-FLU. DHT stimulation was completely reversed by the antiestrogen tamoxifen. In addition, male nude mice transplanted with IIB-MEL-J tumor were treated with FLU when tumors were palpable. FLU was effective in diminishing tumor growth and increasing survival rate of the animals. As a conclusion, the presence of functional androgen receptors in these cells has been demonstrated by growth inhibition in vitro and in vivo with antiandrogens, and their atypical nature is suggested by binding cross-reactivity and competition studies.     

Identification and partial characterization of the cytoplasmic androgen receptor in bovine ovarian capsule

[3H]Dihydrotestosterone (DHT) binding to a specific protein in the cytosol of bovine ovarian capsule was studied in vitro. The specific androgen-binding protein in the cytosol was analyzed by chromatographic and ultracentrifugal techniques. From Scatchard analysis, the dissociation constant was 7.4 nM and the number of binding sites was 58.8 fmol/mg protein. Testosterone and 17 alpha-methyltrienolone (R1881) compete for [3H]DHT binding. In the presence of sodium molybdate and at low salt concentrations, the steroid-protein complex sediments as a 9S form, while in the presence of high salt, it sediments at 3.5S. In the absence of molybdate or in the presence of high salt, the 9S form dissociates in a temperature-dependent manner into smaller units. These properties are consistent with the presence of a typical androgen receptor in the bovine ovarian capsule.     

19-hydroxyandrostenedione does not modulate [3H]aldosterone binding to human mononuclear leucocytes and rat renal cytosol

To verify the aldosterone amplifying action of 19-hydroxyandrostenedione (19-OH-AD), we investigated [3H]aldosterone and [3H]19-OH-AD binding to type I (mineralocorticoid) receptor in the renal cytosol of adrenalectomized and ovariectomized rat, and human mononuclear leucocytes (MNL). In the [3H]aldosterone binding study, the cytosol was incubated with [3H]aldosterone and 200-fold RU28362 (11 beta,17 beta-dihydroxy-6-methyl,17 alpha-(1-propynyl)-androsta-1,4,6- trien-3-one), a pure glucocorticoid, with or without 19-OH-AD. Scatchard plots of [3H]aldosterone binding to cytosol with 0.2 or 20 nM 19-OH-AD or without 19-OH-AD were linear. Dissociation constants (Kd) and maximum bindings (Bmax) without 19-OH-AD, and with 0.2 and 20 nM 19-OH-AD were: 0.71 +/- 0.03 nM and 23.0 +/- 3.4 fmol/mg protein (mean +/- SD, n = 3), 0.72 +/- 0.05 nM and 23.1 +/- 2.3 fmol/mg protein (n = 3), and 0.77 +/- 0.04 nM and 22.9 +/- 4.8 fmol/mg protein (n = 3), respectively. 19-OH-AD did not significantly change the Kd and Bmax of [3H]aldosterone binding. A high concentration of 19-OH-AD slightly displaced 0.2 or 5 nM [3H]aldosterone bound to cytosol. In human MNL, Scatchard plots of [3H]aldosterone binding with both 0.2 and 20 nM 19-OH-AD and without 19-OH-AD were linear. Kd and Bmax were, respectively, 1.00 nM and 780 sites/cell in the absence of 19-OH-AD, and 1.07 nM and 774 sites/cell in the presence of 0.2 nM 19-OH-AD. Without 19-OH-AD they were, respectively, 0.95 nM and 551 sites/cell, and 1.10 nM and 560 sites/cell with 20 nM 19-OH-AD. A high concentration of 19-OH-AD slightly displaced 0.2 or 5 nM of [3H]aldosterone bound to MNL. In both tissues, there was no obvious specific binding of [3H]19-OH-AD within the range of 1-60 nM. The above results suggest that the amplifying effect of 19-OH-AD on aldosterone mineralocorticoid action may not occur at the binding site of aldosterone to type I receptor, and that 19-OH-AD itself may not have any direct or indirect mineralocorticoid actions on the steroid receptor-mediated process in the rat kidney and human MNL.     

Specific progesterone receptors in dimethylbenzanthracene (DMBA)-induced mammary tumors.

Properties of a progesterone receptor present in the cytosol (105,000 xg supernatant) of dimethylbenzanthracene (DMBA)-induced mammary tumors were studied using the highly potent progestin [3H]R 5020 (17, 21-dimethyl-19-nor-pregna-4, 9-diene-3,20-dione). As shown by sucrose gradient analysis, specific binding of [3H] R 5020 is associated with components migrating at 7-8S and 4S. Low affinity binding of the synthetic progestin is eliminated by treatment with dextran-coated charcoal. [3H] R 5020 binding is highly progestin-specific since it is easily displaced by unlabelled norgestrel, R 5020 and progesterone while estradiol-17beta, dihydrotestosterone, testosterone, testosterone and diethylstilbestrol have much lower activity. Dexamethasone and cortisol have little, if any, effect on [3H] R 5020 binding.     

Androgen receptors in ventral prostate glands of zinc deficient rats

Androgen binding has been studied in the prostate cytosol of zinc deficient rats by charcoal assays. Rats were housed individually in plastic cages and maintained on a zinc deficient diet for 3 months. The cytosol fraction of prostate gland was incubated with various concentrations of tritiated methyltrienolone (3H-R1881, a synthetic androgen) alone or in the presence of 500-fold excess of radioinert R1881. Scatchard analysis of the data revealed that the number of androgen binding sites in the cytosol fraction of the zinc deficient rat prostate was 31 +/- 5.2 fmol/mg cytosol protein. This was significantly lower than that (84 +/- 11.5 fmol/mg protein) of the controls. Their dissociation constant (Kd = 1.6 +/- 0.6 nM) on the other hand was not different from that (1.7 +/- 0.7 nM) of control animals. This decrease in the concentration of cytosol receptor sites in the zinc deficient state suggests that this metal is involved in the androgen-binding process in the target cells.     

Androgen receptors in brain and pituitary of female rats: cyclic changes and comparisons with the male

The in vitro binding of a synthetic androgen, methyltrienolone ([3H]-R1881), to brain and pituitary (PIT) cytosol and nuclear extracts was determined in male and female rats. Purified cytosol was prepared from PIT or hypothalamic-preoptic area-amygdala (HPA) and incubated in the presence of 0.1 to 10 nM [3H]-R1881. Scatchard analysis revealed the presence of a single, saturable, high-affinity binding site in PIT cytosol with a dissociation constant (Kd) of 0.42 X 10(-10) M in females and 0.95 X 10(-10) M in intact males. The Kd of HPA cytosol was much less in castrated males [0.47 +/- 0.05 (SEM) X 10(-10)M, n = 7] and females (0.63 +/- 0.1 X 10(-10) M, n = 4) than in intact males (5.8 +/- 1.1 X 10(-10) M, n = 8). Treatment of castrated males with dihydrotestosterone (DHT) for 24 h (250 micrograms/100 g of body weight) increased the Kd of HPA cytosol only slightly (1.6 X 10(-10) M, mean of two replicates). Scatchard analysis of salt-extracted nuclear androgen receptor (ARn) showed a single, high-affinity binding site with similar Kd values in PIT and HPA of intact and castrated, DHT-treated male rats (PIT Kd = 7.3 X 10(-10) M, 9.3 X 10(-10) M; HPA Kd = 1.5 X 10(-9) M, 1.3 X 10(-9) M, respectively). Competition studies involving a range of several radioinert steroids revealed that the binding of [3H]-R1881 to cytosol (ARc) and nuclear extract was specific for androgen receptor when triamcinolone acetonide (10 microM) was added. The ARc and ARn levels were quantified in PIT, preoptic area (POA), hypothalamus (HT), amygdala, hippocampus, and cortex by single point estimation. Significantly (p less than 0.01) greater amounts of ARc were detected in PIT of ovariectomized females (32.7 +/- 2.9 fmol/mg of protein) than in that of orchidectomized males (22.33 +/- 1.6 fmol/mg of protein). The highest levels in the brain were seen in HT and POA. Pituitary ARc in females varied throughout the estrous cycle. Significantly (p less than 0.01) greater amounts were detected on estrus (45.8 +/- 2.2 fmol/mg of protein) and proestrus (39.0 +/- 1.9 fmol/mg of protein) than on diestrus (29.2 +/- 1.5 fmol/mg of protein). These data confirm the existence of specific receptors for androgen in male and female brain and PIT, and suggest an important role for androgen in the control of PIT hormone secretion in the female.     

Androgen binding in peripheral tissues of fetal rhesus macaques: effects of androgen metabolism in liver

In rhesus monkeys sexual differentiation of the brain and reproductive tract (RT) is androgen-dependent. Presumably these effects are mediated through the androgen receptor (AR). The AR has not been characterized in fetal tissues such as liver, kidney, heart, spinal cord and RT in this species. We characterized AR binding using [3H]R1881 as the ligand in cytosols from tissues obtained on days 100-138 of gestation. Scatchard analyses revealed a single, saturable, high affinity AR in liver, kidney, heart, spinal cord and RT. The apparent dissociation constant (Kd) ranged from 0.52 to 0.85 nM with no significant tissue differences. The number of AR (Bmax; fmol/mg protein) differed significantly (P less than 0.01) between tissues (liver greater than RT much greater than kidney greater than or equal to heart greater than or equal to spinal cord). Radioinert testosterone (T) and 5 alpha-dihydrotestosterone (DHT) but not androstenedione, progesterone, estradiol-17 beta, estrone or cortisol in a 50-fold molar excess inhibited [3H]R1881 binding to the AR in spinal cord, heart, kidney and RT. However, in liver only DHT competed significantly (P less than 0.01) for binding. This difference in binding of DHT vs T in the liver was further investigated by incubating liver and kidney cytosols with [3H]DHT and [3H]T at 4 degrees C. We identified the metabolic products by mobility on Sephadex LH-20 columns and reverse isotope dilution. Liver cytosols metabolized [3H]DHT to 5 alpha-androstane- 3 alpha,17 beta-diol (5 alpha-diol) and [3H]T to 5 beta-androstane-3 alpha, 17 beta-diol (5 beta-diol) at 4 degrees C. In contrast, kidney cytosols metabolized [3H]DHT while [3H]T remained unchanged. Further studies indicated that a 50-fold molar excess of 5 alpha-diol inhibited the binding of [3H]R1881 in liver cytosols by about 50% whereas the same molar concentration of 5 beta-diol had no effect. These data demonstrate the presence of AR in peripheral tissues of fetal rhesus monkeys and suggest that androgens through their receptors may affect development of these tissues. Liver cytosols are capable of metabolizing T and DHT at 4 degrees C at conditions similar to those used for measuring cytosolic AR. However, T and DHT are metabolized differently, generating different isomers which have different affinities for hepatic AR.     

Interaction of medroxyprogesterone acetate with cytosol androgen receptors in the rat hypothalamus and pituitary

The binding of medroxyprogesterone acetate (MPA) with cytosol androgen receptors from rat pituitary and hypothalamus was studied. The pituitary and hypothalamic cytosol androgen receptors from adult castrated female rats were in vitro labeled using 3H natural (testosterone (T) and 5 alpha-dihydrotestosterone (DHT] and [3H]synthetic (methyltrienolone) androgens as radioligands. The [3H]androgen-receptor complexes sedimented with a coefficient of 8S in linear sucrose gradients. When incubated with an excess of radioinert MPA, specific binding was abolished indicating interaction of MPA with androgen receptors. Furthermore specific [3H]MPA-androgen cytosol receptor complexes could be identified in these neuroendocrine tissues when a post-gradient receptor labeling technique was used in the absence or presence of radioinert MPA, DHT, and triamcinolone acetonide. A study of binding kinetics disclosed that the equilibrium dissociation constant and saturation binding capacity for the MPA binder, were similar to those exhibited by DHT binding to androgen receptors in both studied tissues under identical experimental conditions. The overall results were interpreted as demonstrating that MPA interacts with cytosol steroid receptors other than those of progesterone in the rat hypothalamus and anterior pituitary. The data are consistent with MPA binding to androgen receptors.     

An androgen-dependent pilosebaceous tumor spontaneously developed in the Japanese house musk shrew Suncus murinus

Following administration of [3H]testosterone to castrated male Japanese house musk shrews (Suncus murinus), radioactive metabolites were detected in sidegland nuclei and the major one of them was dihydrotestosterone (DHT). The androgen binding capacity of the cytoplasmic fraction of sideglands was measured in vitro by the use of [3H]R1881 as a ligand. The binding showed a high affinity for R1881 (Kd = 6.2 X 10(-10) M) and a low capacity (Bmax = 22 fmol/mg protein). Sucrose density gradient centrifugation brought about a peak of [3H]R1881 in the 7S region in low ionic strength buffer. Their characteristics as described above are consistent with those of other androgen target organs. A cutaneous pilosebaceous tumor, which spontaneously developed on the sidegland of old male S. murinus, was transplanted to nude athymic mice. It grew in males only and failed to grow in females and castrated males. A specific androgen binding was found in this tumor (Kd = 7.8 X 10(-10) M, Bmax = 100 fmol/mg protein). Therefore, this transplantable pilosebaceous tumor is androgen-dependent and can be utilized as a new suitable model in the study of the mechanism of androgen on tumor development.     

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.     

A receptor-like binding macromolecule for 1 alpha, 25-dihydroxycholecalciferol in cultured mouse bone cells.

1alpha, 25-Dihydroxycholecalciferol (1,25-(OH)2D3), the active form of vitamin D, like other steroid hormones, initiates its action by binding to cytoplasmic receptors in target cells. Although the 1,25-(OH)2D3 receptor has been well studied in intestine, little information beyond sucrose gradient analyses is presently available from mammalian bone. We, therefore, employed primary cultures of mouse calvarial cells to characterize the mammalian receptor in bone. A hypertonic molybdate-containing buffer was found to protect receptor binding. On hypertonic sucrose gradients, the 1,25-(OH)2-[3H]D3 binder sedimented at 3.2 S. Scatchard analysis of specific 1,25-(OH)2[3H]D3 binding sites at 0 degrees C yielded an apparent Kd of 0.26 nM and an Nmax of 75 fmol/mg of cytosol protein. Competitive binding experiments revealed the receptor to prefer 1,25-(OH)2D3 greater than 25-(OH)-D3 = 1 alpha-(OH)-D3 greater than 24R,25-(OH)2D3; vitamin D3, dihydrotachysterol, sex steroids, and glucocorticoids exhibited negligible binding. As shown in other systems, the receptor could be distinguished from a 25-(OH)-[3H]D3 binder which sedimented at approximately 6 S. In summary, cultured mouse calvarial cells possess a macromolecule with receptor-like properties. This system appears to be an ideal model for the investigation of 1,25-(OH)2D3 receptor binding and action in mammalian bone.     

ZK91587: a novel synthetic antimineralocorticoid displays high affinity for corticosterone (type I) receptors in the rat hippocampus

In vitro cytosol binding assays have shown the properties of binding of a novel steroid, ZK91587 (15 beta, 16 beta-methylene-mexrenone) in the brain of rats. Scatchard and Woolf analyses of the binding data reveal the binding of [3H] ZK91587 to the total hippocampal corticosteroid receptor sites with high affinity (Kd 1.9 nM), and low capacity (Bmax 17.3 fmol/mg protein). When 100-fold excess RU28362 was included simultaneously with [3H] ZK91587, the labelled steroid binds with the same affinity (Kd 1.8 nM) and capacity (Bmax 15.5 fmol/mg protein). Relative binding affinities (RBA) of various steroids for the Type I or Type II corticosteroid receptor in these animals are: Type I: ZK91587 = corticosterone (B) greater than cortisol (F); Type II: B greater than F much greater than ZK91587. In the binding kinetic study, ZK91587 has a high association rate of binding in the rat (20.0 x 10(7) M-1 min-1). The steroid dissociates following a one slope pattern (t 1/2 30 h), indicating, the present data demonstrate that in the rat hippocampus, ZK91587 binds specifically to the Type I (corticosterone-preferring/mineralocorticoid-like) receptor.     

Evidence for an androgen receptor in the seminiferous tubules of the human testis

Androgen receptors (AR) were studied in seminiferous tubule cytosol and testicular nuclear extracts prepared from testes of previously untreated elderly men undergoing orchiectomy as therapy for prostatic carcinoma. Cytosol exhibited high affinity (Kd = 0.8 nM), saturable binding of [3H]methyltrienolone; however, the synthetic progestin, promegestone was a stronger competitor for MT binding sites than were 5 alpha-dihydrotestosterone (DHT) or testosterone (T), suggesting the presence of progesterone-like binding sites. Addition of triamcinolone acetonide (TA) produced the usual relative steroid specificity for AR binding and reduced the measured AR binding capacity by 19 +/- 8% (Mean +/- SD, n = 3). The umber of MT binding sites was 30-40 fmol/mg protein, or an average of 65 fmol/g testis, and the equilibrium dissociation constant at 0 degrees C was 0.6-1.4 nM. In the presence of sodium molybdate, binding was stable for 40 h at 0 degrees C and the half-time of dissociation of the MT-AR complex was 12-16 h. The binding of salt extractable (600 mM KCl) nuclear sites to MT was saturable and was specific for androgens. The number of binding sites in nuclear extracts was 170 fmol/g testis and the apparent equilibrium dissociation constant was 4.2 nM. Thus, the binding of MT to human seminiferous tubule cytosol and testicular nuclear extract exhibits properties which are nearly identical to those of the prostate AR. Further study of this androphilic protein may provide insight into the role of androgen in normal and abnormal spermatogenesis in man.