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.     

Androgen binding as evidenced by a whole cell assay system using cultured canine prostatic epithelial cells

The androgen receptor content in the prostate has been usually evaluated using subcellular fractions without taking into account cellular and functional heterogeneity of the gland. Using enriched populations of immature canine prostatic epithelial cells cultured in primary monolayers, a whole cell assay system was developed to measure androgen receptors. Tritiated dihydrotestosterone (DHT) and/or methyltrienolone (R1881) in serum-free medium were used as ligands and Triamcinolone acetonide (0.5 microM) was added to prevent the binding of R1881 to other types of receptors. The amount of radiolabelled ligand specifically bound to the cells was determined at equilibrium. Specific binding was proportional to the number of cells seeded. Scatchard analysis revealed the presence of at least two types of binding sites. The Kd for the high affinity binding site was 2 x 10(-9) M. Competition studies indicated that this component was specific for androgens; Methyltrienolone, Mibolerone and the antiandrogen RU 23908 were the most efficient competitors. They were followed by DHT, 5 alpha-androstane-3 alpha, 17 beta-diol, testosterone, estradiol and estrone. Progesterone, 5 alpha-androstane-3 beta, 17 beta-diol and epitestosterone were not inhibitors. The level of specific binding was 11.0 +/- 7.6 fmol of bound R1881 per 10(6) cells (n = 34) or 2075 +/- 1434 fmol per mg of DNA; these values correspond to an average of 6624 +/- 4577 sites per cell. Thus, using this whole cell assay system, specific and androgen receptors were detected in immature prostatic epithelial cells in culture. This assay will therefore be useful to study the interrelationship between androgen binding activity and specific cell functions.     

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.     

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.     

Effects of repeated androgen treatments on metabolism and nuclear binding of androgen in the infant murine submandibular gland

1. Androgen responsiveness of esteropeptidase of the murine submandibular gland developed rapidly in normal males compared with in normal females and castrated males. 2. Repeated treatments of infant mice of both sexes with testosterone (T), 5 alpha-dihydrotestosterone (DHT) or 5 alpha-androstane-3 alpha, 17 beta-diol increased androgen responsiveness of this enzyme, but did not affect those of 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDase; androgen metabolizing enzymes) of the gland. 3. Exchange assay of nuclear androgen receptor using 3H-DHT showed that in both sexes, amounts of binding in animals pretreated with T were higher than those in animals pretreated with sesame oil. 4. These results suggest that there is parallelism between the androgen responses and amounts of nuclear androgen binding, not androgen responses of 5 alpha-reductase and 3 alpha-HSDase.     

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.     

Quantification of cytosolic steroid receptors in secretory and non-secretory epithelial cells of the canine prostate

Radiolabelled methyltrienolone, dihydrotestosterone and estradiol were used as ligands to identify and quantify androgen and estrogen receptors in freshly dispersed cells from the canine prostate. Soluble extracts (cytosols) were obtained from secretory and non-secretory epithelial cells separated on the basis of their density in Percoll gradients. For both cell types, as well as for the whole prostate, Scatchard plot analyses were linear and showed a single class of high affinity binding sites: Kd values of 3.6 +/- 2.2 X 10(-9) M and 3.0 +/- 1.2 X 10(-10) M were measured for the androgen and estrogen receptors, respectively. The number of binding sites for the cytosolic androgen receptor, expressed per mg of protein or per mg of DNA, was 2.4- to 6.7-fold higher in the non-secretory cells compared to the secretory cells. However, these two cell types contained a similar number of specific sites for the estrogens. The specificities of the androgen and estrogen receptors were shown to be identical for the two cell types: the binding of [3H]R1881 was strongly inhibited by unlabelled R1881, 5 alpha-androstane-3 alpha, 17 beta-diol and dihydrotestosterone, while 5 alpha-androstane-3 beta, 17 beta-diol, estradiol and estrone did not displace bound R1881. The addition of triamcinolone acetonide did not alter the binding of R1881 in extracts of either cell type or in the whole prostate. The binding of [3H]estradiol to the estrogen receptor was highly specific since a strong displacement was only observed with estradiol (83%).     

Effect of male accessory glands autoaggression on androgenic cytosolic and nuclear receptors of rat prostate.

The effect of immunization against male accessory gland (MAG) homogenates over androgenic cytosolic and nuclear receptors of rat prostate was studied. In the MAG-immunized rats the Bmax of cytosolic receptors was significantly increased (120.3 +/- 44.3 vs 47.7 +/- 24.9 fmol/mg protein, p less than 0.01, mean +/- SD). In contrast, the Bmax of nuclear receptors in the MAG-immunized rats showed no significant difference as regarded controls (kidney immunized rats) when expressed as fmol/100 micrograms DNA (196.1 +/- 84.8 vs 148.3 +/- 88.9) but it show to slight differences (p less than 0.1) when data were reported as percent of weight of tissue (2,189 +/- 918.6 vs 1,303 +/- 611.2 fmol/g wet issue). Results (mean +/- SD) on binding affinity of cytosolic receptors showed no significant differences in MAG-immunized rats as compared with controls (Kd: 1.98 +/- 0.66 vs 1.92 +/- 0.20 nM). Likewise, only a slight difference between both groups was attained for Kds of nuclear receptors (2.34 +/- 0.28 vs 1.80 +/- 0.62 nM, p less than 0.2). On the other hand, 5 alpha 1-dihydrotestosterone (DHT) values obtained in prostate homogenates were significantly decreased in MAG-immunized rats as compared with controls (17.4 +/- 2.0 vs 7.1 +/- 0.9 ng/g tissue, mean +/- SD, p less than 0.01). However, testosterone (T) levels in gland tissue showed no significant differences between both groups (2.4 +/- 0.5 vs 2.6 +/- 0.3 ng/g tissue) with an increase in the T: DHT ratio from 0.14 to 0.37.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estradiol binding components in intestinal mucosa of female rats.

The presence of estrogen binding components (EBC) in intestinal mucosa of female rats was investigated by competitive-binding assay using radiolabelled and nonlabelled estradiol 17 beta (E2). EBC were found exclusively in the nuclear fraction and were absent from the cytosolic and from the microsomal fractions. Two types of nuclear EBC with different binding characteristics and capacities were found: Kd1 = 4.8 +/- 0.8 nM, n1 = 18.4 +/- 4.2 fmol/mg protein (n1 = 83.4 +/- 12.5 fmol/mg DNA) and Kd2 = 31.1 +/- 6.8 nM, n2 = 91.1 +/- 18.5 fmol/mg protein (412.7 +/- 80.0 fmol/mg DNA). Type 1 component showed slightly greater affinity for estrogens as compared to progesterone and dexamethasone whereas type 2 component bound other competitors with even greater affinity than E2.     

Effects of aromatizable and nonaromatizable androgen treatments on luteinizing hormone receptors and ovulation induction in immature rats

The effects of androgen pretreatment on follicle-stimulating hormone (FSH)-stimulated luteinizing hormone (LH) receptor induction in ovarian granulosa cells was examined. Immature female rats were treated with various doses (0.1-5 mg/rat) of testosterone (T), 5 alpha-dihydrotestosterone (DHT), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol), or 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol). Subsequent follicular development was stimulated by treatment with ovine FSH. LH receptor induction in granulosa cells and ovulatory responses to 10 IU human chorionic gonadotropin (hCG) were examined. Since LH receptor induction requires the synergistic action of both FSH and estradiol, the effects of the androgen pretreatment on FSH-stimulated estradiol production were also examined. Dihydrotestosterone treatment at doses greater than 1 mg inhibited LH receptor induction by approximately 70%, which resulted in absent ovulatory responses. Treatment with 1 mg or more of T or 3 alpha-diol had no effect on LH receptor induction, yet the hCG-stimulated ovulation rate was reduced to 40% of that seen in vehicle-treated controls. 3 beta-Diol, at a dose of 1 mg/rat, did not affect LH receptor induction but did reduce hCG-stimulated ovulation responses. No significant effects of androgen treatment on ovarian or uterine weight or FSH-stimulated estradiol production were observed. These results suggest that androgens can act at multiple sites to inhibit ovarian follicular development and function. In addition these studies demonstrate that, although LH receptor induction is necessary, it may not be a sufficient condition to ensure ovulation of ovarian follicles.     

Androgen concentration and partial characterization of 5alpha reductase in the epididymis of the rhesus monkey.

The 5alpha reductase activity ofthe monkey epididymis was studied. The enzyme was found in particulate subcellular fractions, its distribution closely resembling that of the microsomal marker enzyme NADPH: cytochrome c reductase, suggesting an association of 5alpha reductase with membranes of the endoplasmic reticulum. Maximal enzyme activity was found at pH 5.4 and at 32--37 C. The crude nuclear preparation had a Km: 0.315 x 10(-6)M and Vmax: 168 pmoles/mg protein/h. The microsomal enzyme had a Km: 0.243 x 10(-6)M and Vmax: 828 pmoles/mg protein/h. Neither enzyme preparation was affected by addition to the incubation media of dihydrotestosterone (DHT) or 5alpha-androstane-3alpha,17beta-diol. The endogenous androgen concentration in the epididymides of 2 different monkeys, in ng/g wet weight was: DHT 20.81 +/- 1.98; T: 9.0L +/- 2.83; diol: 3.03 +/- 0.41.     

Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affinity (Kd = 10(-9) M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [3H]tamoxifen binding capacity is thermolabile, being most stable at 4 degrees C and rapidly lost at 37 degrees C. More [3H]tamoxifen, however, is specifically bound at incubation temperatures of 25 degrees C and 37 degrees C than at 4 degrees C although prewarming nuclei has no effect, suggesting exchange of [3H]tamoxifen for an unidentified endogeneous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (Kd = 10(-9) M) [3H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78 +/- 0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000 X g, 30 min) contains high-capacity (955 +/- 405 (S.D.) fmol/mg protein), low-affinity (Kd = 10.9 +/- 4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000 X g, 60 min) contains low-capacity (46 +/- 15 (S.D.) fmol/mg protein), high-affinity (Kd = 0.61 +/- 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%-3.2%, and nuclear sites less than 0.5% of total sites.     

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.     

Androgen receptor in human skin fibroblasts. Characterization of a specific 17beta-hydroxy-5alpha-androstan-3-one-protein complex in cell sonicates and nuclei.

Cultured human skin fibroblasts were shown to contain an androgen binding activity (receptor) which was heat-labile and destroyed by trypsin. Specific binding was seen after incubations of these cells with 1,2-3-H-testosterone, 1,2-3-H17beta-hydroxy-5alpha-androstan-3-one (dihydrotestosterone, DHT) and 1,2-3-H-5alpha-androstane-3alpha, 17beta-diol. This receptor had a high affinity (Kd=0,2-1.6 nM) and a high degree of specificity for DHT. It was measured as a 3-H-DHT-protein complex by gel filtration chromatography using a method which distinguishes specific from nonspecific binding. Receptor activity was distributed about equally between nuclear and extranuclear components at all times studied and was present in both compartments when cell incubations were carried out at 4 degrees and 37 degrees. Saturation analysis indicated that there were 1250-18,600 binding sites per whole cell. By sucrose gradient centrifugation the receptor had a sedimentation coefficient (S20,w) of about 4. Cells grown for 8 days without serum in the medium maintained the same levels of 3-H-DHT binding. Within 15 hours puromycin (20 mug/ml) in serum-free medium caused a 40-60 percent decrease in binding for the same cell lines. Although the highest levels of 3-H-DHT binding were observed in fibroblasts from newborn foreskin, appreciable cytosol and nuclear binding were seen in cells from forearm, neck and abdominal skin. Receptor activity was stable during prolonged culture. Fibroblasts from several skin sites from patients with the androgen insensitivity syndrome (testicular feminization) had no detectable specific DHT binding. In this study it was demonstrated that skin fibroblasts can rapidly convert testosterone to its active form, DHT, bind DHT to a specific receptor protein and transport this complex to their nuclei. Therefore this may prove to be a convenient system for studying androgen action in vitro.     

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.     

Interactions of radiolabelled ligands with specific receptors: an analysis

The binding and displacement of beta-adrenoceptor blockers, [3H]propranolol ([3H]PRP) and [3H]dihydroalprenolol ([3H]DHA), were studied on isolated rat erythrocytes, their membranes and ghosts; the binding of [3H]DHA and a M-cholinoceptor blocker, [3H]quinuclidinylbenzylate ([3H]QNB), on cerebral cortex membranes. In all experiments, ligand-receptor interactions conformed to a model of two pools of receptors in the same effector system and the binding of two ligand molecules to the receptor. The results were similar for the displacement of [3H]PRP, [3H]DHA and [3H]QNB with propranolol, dihydroalprenolol and quinuclidinyl-benzylate, respectively. The parameters of [3H]PRP to beta-adrenoceptor binding for intact erythrocytes were: Kd1 = 0.74+/-0.07 nM, Kd2 = 14.40+/-0.41 nM, B1 = 24+/-2 unit/cell, B2 = 263+/-5 unit/cell; for ghosts, Kd1 = 0.70+/-0.17 nM, Kd2 = 19.59+/-2.59 nM, B1 = 9+/-1 fmol/mg protein, B2 = 39+/-4 fmol/mg protein. Receptor affinities were similar in erythrocytes and ghosts; on the ghost membrane, the number of receptors was considerably lower (B1 = 2 unit/cell, B2 = 6 unit/cell). The parameters of [3H]QNB to M-cholinoceptor binding of the cerebral cortex membrane were the following: Kd1 = 0.43 nM, Kd2 = 2.83 nM, B1 = 712 fmol/mg, B2 = 677 fmol/mg.     

Binding characteristics of [3H]delta(5)-androstene-3beta,17beta-diol to a nuclear protein in the rabbit vagina

In this study, we investigated the binding characteristics of [3H]Delta(5)-androstene-3beta,17beta-diol to rabbit vaginal cytosolic and nuclear extracts and in freshly excised intact tissue strips. [3H]delta(5)-Androstene-3beta,17beta-diol bound to a protein(s) in the vaginal nuclear extract with high affinity (K(d)=3-5 nM) and limited capacity (50-100 fmol/mg protein). No specific binding was detected in the cytoplasmic extracts. Competitive binding studies showed that binding of [3H]delta(5)-androstene-3beta,17beta-diol was effectively displaced with unlabeled delta(5)-androstene-3beta,17beta-diol but not with dehydroepiandrosterone, testosterone, dihydrotestosterone, triamcinolone acetonide, or progesterone. However, estradiol at high concentrations partially displaced bound [3H]delta(5)-androstene-3beta,17beta-diol. Incubation of freshly excised vaginal tissue strips with [3H]delta(5)-androstene-3beta,17beta-diol in the absence or presence of excess unlabeled delta(5)-androstene-3beta,17beta-diol for 1h at 37 degrees C resulted in specific binding to a soluble macromolecule in the nuclear KCl extracts. In addition, quantitative measurement of estrogen receptor, androgen receptor and delta(5)-androstene-3beta,17beta-diol binding protein was performed by equilibrium ligand binding assays using extracts of distal vaginal tissue from intact animals or ovariectomized animals treated for 2 weeks with vehicle, estradiol, testosterone, or estradiol plus testosterone. These changes in steroid hormone levels resulted in opposing trends between the estrogen receptor and delta(5)-androstene-3beta,17beta-diol binding protein, suggesting that delta(5)-androstene-3beta,17beta-diol binding protein is regulated differently by the hormonal milieu than the estrogen receptor. These data suggest that rabbit vaginal tissue expresses a novel binding protein which specifically binds delta(5)-androstene-3beta,17beta-diol and is distinct from the androgen and estrogen receptors.     

Explanation of the pseudohypoaldosteronism (PHA)-stress syndrome with an artificial aldosterone receptor model

A receptor for aldosterone was studied in the cytosol of rectal mucosa of two sisters (M.A., M.B.) with the clinical manifestations of pseudohypoaldosteronism (PHA). Compared to age matched controls the patients showed a decreased affinity for aldosterone (M.A. Kd1: 0.18 nM, Kd2: 4.55 nM; Nmax1: 0.185 fmol/mg cytosol protein (CP), Nmax2: 3.12 fmol/mg CP, respectively). In an attempt to find an explanation for the phenomenon of stress-induced electrolyte imbalance in PHA patients an experimental set up was designed, using aldosterone antibody material as artificial aldosterone receptor. Specific binding was evaluated in addition with and without a 25-100-fold molar excess of dexamethasone (DEX) in order to overcome the glucocorticoid affinity of the aldosterone receptor, a phenomenon proposed to be the cause for the severe consequences of stress in some patients with PHA. The aldosterone antiserum showed two binding sites, similar to the natural receptor (Kd1: 0.15 nM, Kd2: 1.30 nM; Nmax: 30 fmol/mg CP and 130 fmol/mg CP, respectively). Under the influence of DEX the high affinity binding site (Kd1) was occupied by the glucocorticoidanalogon (Kd: 1.30 nM; Nmax: 125 fmol/mg CP). In conclusion, in stress situations, with increased quantities of glucocorticoid circulating, the high affinity binding site of the aldosterone receptor might be occupied by the glucocorticoids, while the low affinity binding site in PHA patients might not have sufficient binding capacity to maintain the electrolyte balance.     

Androgen glucuronyl transferase activity in rat liver, evidence for the importance of hepatic tissue in 5 alpha-reduced androgen metabolism

To elucidate the role of the liver in 5 alpha-reduced androgen metabolism, we used a rat liver glucuronyl transferase assay to determine the conversion of 17 beta-hydroxy-5 alpha-androstane-3-one (DHT), 5 alpha-androstane-3 alpha, 17 beta-diol (androstanediol), and androsterone to their glucuronide metabolites. Serum levels of the two isomers of androstanediol glucuronide (androstanediol 3- and 17-glucuronide) were also measured. Using 5 microM unconjugated steroid as substrate, the production rate (pmol/mg/min) for each product from its respective unconjugated steroid was 6.9 +/- 0.4 for DHT glucuronide, 101 +/- 3.3 for androstanediol 3-glucuronide, 71 +/- 2.0 for androstanediol 17-glucuronide, and 181 +/- 11 for androsterone glucuronide. Production rates for androstanediol glucuronide were 800 times greater for rat liver than for rat prostate, when examined under similar conditions. In the presence of either 0 or 5 microM unlabeled androstanediol, about 60% of the androstanediol glucuronide formed by rat liver was androstanediol 3-glucuronide. In normal male rat serum, 69 +/- 8% (mean +/- SEM) of total androstanediol glucuronide was androstanediol 3-glucuronide. We have previously shown that rat prostate forms androstanediol 17-glucuronide, but not androstanediol 3-glucuronide. The results from the present study indicate that rat liver forms both androstanediol glucuronide isomers, and does so in about the same ratio as is found in rat serum. The rate of glucuronidation is also much greater in rat liver than in rat prostate. While other sites of glucuronidation are possible, these results are consistent with the hypothesis that DHT and other unconjugated androgens formed in rat prostate are conjugated to glucuronic acid mainly in the liver.     

ACTH receptors in nervous tissue. High affinity binding-sequestration of [125I]Phe2,Nle4]ACTH 1-24 in homogenates and slices from rat brain

We have demonstrated specific, high affinity binding of a biologically active Tyr23-monoiodinated derivative of ACTH, [125I][Phe2,Nle4]ACTH 1-24, in rat brain homogenates. Similarly, in metabolically inhibited and noninhibited rat whole brain slices there is a specific "binding-sequestration" process that is dependent on time, protein concentration, and pH. In homogenates, binding curves were best described by a two-site model and provided the following parameters: Kd1 = 0.65 +/- 0.47 nM, Bmax1 = 21 +/- 41 fmol/mg protein; Kd2 = 97 +/- 48 nM, Bmax2 = 3.5 +/- 1.8 pmol/mg protein. In metabolically viable brain slices, concentration-competition curves of [125I][Phe2,Nle4]ACTH 1-24 binding-sequestration can be described by three components (Kd1 = 14 +/- 24 nM, Bmax1 = 50 +/- 95 fmol/mg protein; Kd2 = 2.4 +/- 1.9 microM, Bmax2 = 44 +/- 49 pmol/mg protein; Kd3 = 0.16 +/- 1.0 mM, Bmax3 = 5.3 +/- 54 nmol/mg protein). Metabolic inhibition, by removal of glucose and addition of 100 microM ouabain, abolishes the lowest affinity, highest capacity binding-sequestrian component only (Kd1 = 7.1 +/- 14 nM, Bmax1 = 8.7 +/- 16 fmol/mg protein; Kd2 = 7.4 +/- 4.49 microM, Bmax2 = 37 +/- 27 pmol/mg protein). The two binding-sequestration parameter estimates obtained from metabolically inhibited tissue slices are not significantly different from those of the two higher affinity components obtained with noninhibited tissue. Thus, metabolic inhibition permits demonstration of ACTH receptor binding only, unconfounded by sequestration or internalization of ligand:receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)