Optimal radioligand exchange conditions for measurement of occupied androgen receptor sites in rat ventral prostate

This study was undertaken to define optimal conditions for exchange of ³H-R-1881 with endogenous hormone bound to androgen receptor (AR) sites in homogenates of rat ventral prostate (RVP) of mature animals. To minimize inactivation of AR binding sites under exchange conditions, extracellular proteases present in RVP were removed by mincing and washing tissue fragments in a Ca²⁺-free cell culture medium (J-MEM) containing 1% casein, prior to homogenization in a TEDG buffer (50 mm Tris-maleate buffer, pH 7.4; 1.5 mm EDTA; 2.0 mm DTT; and 10% ($\text{v}\text{v}$) glycerol) containing 0.5 mm phenylmercuric sulfonyl fluoride (PMSF) and 1.0 mm sodium azide. Na₂MoO₄ (final concentration, 20 mm) was added to homogenate fractions which then were incubated at 0–4 °C for 1–5 days with a saturating concentration of ³H-R-1881 (20 nm) in the absence and presence of 2 μm radioinert R-1881. Heparin (200 μg/ml) was added to the incubation medium to “solubilize” nuclear chromatin. Free and bound R-1881 were separated by a hydroxylapatite (HAP) batch procedure. Using these conditions, it has been found that (i) incubation periods of 72–96 h at 0–4 °C were required to achieve maximal specific exchange binding of ³H-R-1881 (B_max) in total homogenates from normal intact rats. Heparin addition (200 μg/ml) did not change B_max and had little or no effect on the rate of exchange. Mean B_max was 6.7 ± 1.6 (SD) pmol ³H-R-1881/mg DNA. R-1881 exchange at 24 h of incubation was only about 40% of B_max. Nonspecific binding, a small fraction (<10%) of B_max, was near maximal at 2 h. Incubation at 15 °C gave similar R-1881 exchange values to those obtained at 0 °C during the first 24 h, but at 48 h and thereafter R-1881 binding markedly decreased. These B_max values in total homogenates of normal intact RVP are about 2.5 times greater than the AR values obtained in 1-day castrated rats, when compared on a DNA basis, (ii) Addition of gelatin (0.25%) to inhibit seminin activity had no effect on B_max. Deletion of either MoO₄^2? or PMSF from the standard exchange medium reduced B_max values ~20%; if both PMSF and MoO₄^2? were deleted, B_max was reduced to a greater extent (~35%). B_max was reduced (40%) when homogenates were prepared without preliminary J-MEM:casein pretreatment and incubated in standard exchange medium with PMSF and MoO₄^2?. (iii) Despite AR stabilization by MoO₄^2? and inhibition of protease activities during exchange incubation, AR inactivation increased exponentially, so that the maximal ³H-R-1881 binding value achieved at 96 h was estimated to represent about 50% of the AR sites originally present. (iv) The binding sites in total homogenates occupied by ³H-R-1881 at 24, 72, and 96 h of exchange exhibited steroid specificity characteristics of AR, as determined by competition studies with a wide variety of steroid hormones and analogues. Scatchard plots of ³H-R-1881 exchange binding in total homogenates of normal intact RVP incubated for 72 or 96 h indicated a single class of affinity sites with apparent K_d of 5 to 6 nm. (v) Sucrose density gradient centrifugation of homogenates incubated for 72 or 96 h showed that the specific ³H-R-1881 binding sites were distributed in two broad peaks associated with low-molecular-weight components. One with S value ~3.5 may be “activated” AR; the other near the top of the gradient (S < 1.6) may include meroreceptor forms of AR.     

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.     

Purification and characterization of the androgen receptor from rat ventral prostate

The androgen receptor has been purified from rat ventral prostate cytosol by a combination of differential DNA-Sepharose 4B chromatography and testosterone 17 beta-hemisuccinyl-3,3'-diaminodipropylamine-Sepharose 4B affinity chromatography. Approximately 8 micrograms of protein was obtained from 38 g of rat ventral prostate, with a yield of 24%. The receptor was purified approximately 120 000-fold. Silver nitrate staining of a sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel revealed a major polypeptide band migrating at 86 000 daltons. Affinity labeling of a partially purified receptor preparation with either 17-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one or 17 beta-hydroxy-[1,2,4,5,6,7,16,17-3H8]-5 alpha-androstan-3-one 17-(2-bromoacetate) produced a major band of radioactivity migrating at 86 000 daltons on a NaDodSO4 gel. Under nondenaturing conditions, a Mr of 85 000 was determined by gel filtration (42 A) and sucrose gradient sedimentation analysis (4.5 S). The purified receptor had an isoelectric point of 6.3 [3H]-4,5 alpha-Dihydrotestosterone, bound to the purified receptor, was displaced with 4,5 alpha-dihydrotestosterone greater than testosterone much greater than progesterone greater than 5 alpha-androstane-3 alpha, 17 beta-diol greater than 17 beta-estradiol greater than cortisol. A number of physicochemical properties of the purified receptor were similar to those of the receptor in crude cytosol.     

Characteristics of cytosol androgen receptor in rat thymus

Male and female rat thymic cytosol contained specific androgen receptor. The apparent dissociation constants (Kd) were 2.4 nM in males and 2.5 nM in females, and the number of binding sites (NBS) were 23.7 fmol/mg protein in males and 34.2 fmol/mg protein in females. Transformation of receptor to the DNA binding state was achieved by heat or KCl treatment of [3H]R1881-receptor complex, and the characteristics of transformed and nontransformed receptors were investigated. The nontransformed androgen-receptor complex eluted at 0.20-0.25 M KCl from DEAE-Sephacel and sedimented at 9.1 S and its molecular weight was 255,000 on agarose gel chromatography, while the transformed receptor complex eluted at 0.03-0.15 M KCl with a broad peak and sedimented at 4.5 S and its molecular weight was 80,000-85,000. The minicolumn binding assay revealed that approximately 57% of the total receptor complexes bound to DNA-cellulose following heat treatment (20 degrees C, 1 h). Castration exerted no effect on the physicochemical properties of cytosol androgen receptor, but it increased the number of binding site to the female level.     

Association of androgen binding sites with the endoplasmic reticulum of rat ventral prostate

Microsomes from ventral prostate of 24-h castrated rats contain a single set of tissue-specific high-affinity, low-capacity androgen binding sites. These sites are indigenous to the endoplasmic reticulum, as shown by purification procedures associated with marker enzymes and electron microscopic analyses. When prostatic microsomal membranes are separated from plasma membranes using the nuclear or the mitochondrial pellets as the source of fractionation in sucrose gradients, the androgen binding activity is selectively associated with fractions rich in rough endoplasmic reticulum and ribosomes. Eighty-four percent of the total content of Na+/K+ adenosine triphosphatase (ATPase) and only 27% of the total binding capacity were concentrated in fractions rich in smooth-surfaced vesicular membranes, when nuclear suspensions constituted the membrane source. In contrast, the region of the same gradient when enriched in rough endoplasmic reticulum and deficient in plasma membrane content contained 73% of the androgen-binding capacity and only 14% of the ATPase. For fractions collected using mitochondrial suspensions as starting material, the ratio (total glucose-6-phosphatase/total binding capacity) was closer to 1.0 than similar ratios of ATPase/binding capacity, indicating co-sedimentation of binding sites with microsomal membranes and not with plasma membranes. Na+/K+ ATPase, but not 5' nucleotidase, is a valid plasma membrane marker for ventral prostate. Microsomal androgen receptors may constitute a new level of regulation of androgen action in target cells.     

Aging in the rat prostate. Reduction in detectable ventral prostate androgen receptor content.

Aging in the rat is associated with a reduction in the detectable androgen receptor content of the ventral prostate. The reduction in cytoplasmic receptor content did not appear to be attributable to an aging-associated production of a receptor-inactivating factor or to an aging-associated change in the sedimentation properties of the androgen receptor of young and aged animals.Saturation analysis of cytoplasmic extracts prepared from two different breeds of similar albino rats and a genetically distinct strain of inbred brown rats demonstrated quantitative aging-associated reductions in the androgen-receptor content per cell of the ventral prostate. The reduction in receptor content per cell appeared to increase progressively in magnitude with increasing age. The mean value for the cytoplasmic androgen receptor sites per cell for the oldest animals (mean age 884 days) was only 14% of the mean value for the young mature animals (mean age 185 days) of the same breed. The binding affinities of the detectable androgen receptor of the young mature and aged animals were essentially identical. This observation does not eliminate the possibility that the observed reduction results from an aging-associated production of defective receptor. Evaluation of the total DNA content of the ventral prostate did not provide evidence for an aging-associated selective loss of receptor-containing cells. These data in toto were consistent with the interpretation that aging is associated with a mean reduction in the androgen-receptor content per receptor-containing cell.Both cytoplasmic and nuclear androgen retention were evaluated in vivo. These experiments provided qualitative confirmation of the in vitro saturation analyses as there was a highly significant aging-associated reduction in the amount of androgen specifically bound by these prostatic compartments. Total specific androgen retention by the ventral prostate of aging adults was reduced by 55% relative to young mature animals. This result was nearly identical to that obtained for the same breed and age category of animals when evaluated by in vitro saturation analysis.Preliminary in vitro experiments revealed a diminution in the uptake of androgen receptor by purified nuclei from aged animals relative to purified nuclei from young mature animals. The magnitude of the diminution in nuclear acceptor capacity was insufficient to account for the reduction in nuclear retention of androgen determined in vivo. The data were consistent with the interpretation that the cytoplasmic receptor is the major determinant of nuclear androgen retention in the ventral prostate.     

Primary structure and androgen regulation of a 20-kilodalton protein specific to rat ventral prostate

Nuclear and cytosolic forms of a 20-kdalton rat ventral prostate protein were purified and partially sequenced from their N-termini. Isolated nuclei were treated with micrococcal nuclease and extracted in 0.6 M NaCl, and proteins were separated by affinity chromatography on Matrex gel green A, ammonium sulfate fractionation, and fast protein liquid chromatography on Superose 12. The 43 amino acid N-terminal sequence of the nuclear 20-kdalton protein was identical with the cytosolic protein except it lacked 7 N-terminal amino acids present in the cytosolic form. The DNA sequence of a full-length complementary DNA clone isolated from a ventral prostate gt11 library extended the N-terminal sequence of the cytosolic form by an additional nine amino acids from the predicted initiation methionine. The cDNA included the nucleotide sequence for the 43 amino acid N-terminal sequence of the purified 20-kdalton protein and predicted molecular weights of 16,686, 17,521, and 18,650, respectively, for the nuclear, cytoplasmic, and nonprocessed proteins. Northern blot analyses of reproductive tract tissue RNAs using the 20-kdalton protein cDNA as probe revealed a single mRNA species of 0.92 kb detectable only in extracts of rat ventral prostate. Expression of the 0.92-kb mRNA was androgen dependent since the mRNA was undetectable in extracts obtained 4 days after castration and was restored 16 h after restimulation with androgen.     

Effects of steroidal and non-steroidal antiandrogens on the androgen binding properties of the rat ventral prostate androgen receptor

Steroidal (cyproterone acetate) and non-steroidal (RU23908 and hydroxyflutamide) antiandrogens are able to block testosterone-induced increases in nuclear androgen receptor (AR) in the prostate of 1-day orchidectomized rats, but when given alone, RU23908 and hydroxyflutamide increase nuclear AR (RU23908 greater than hydroxyflutamide) in the same animal model. The increases in nuclear AR induced by antiandrogen alone or with testosterone alone are blocked by cycloheximide 1 h after administration, suggesting that androgen or antiandrogens induce de novo AR synthesis. Concomitant to nuclear AR accumulation, testosterone is able to induce depletion of cytosol and microsomal AR. Blockade of testosterone-induced depletion of microsomal AR, but not of cytosol AR, occurs in the presence of antiandrogens. Cyproterone acetate has a higher relative binding affinity (RBA) for microsomal AR and cytosol AR than RU23908 or hydroxyflutamide. This phenomenon is in good agreement with the degree of inhibition by these compounds of the association rate of androgen for the microsomal AR. This correlation between RBA and inhibition of the initial rate of hormone binding to the receptor is not found for cytosol AR. The results show that antiandrogens are not 'pure' antagonists of androgen action and they are potent agonists in the absence of testosterone. Furthermore, testosterone alone or antiandrogens per se regulate AR levels acutely by protein-synthesis dependent mechanisms of action, in rat ventral prostate.     

Antiestrogen specific,high affinity saturable binding sites in rat uterine cytosol

Analysis of rat uterine cytosol for Tamoxifen binding reveals that the saturable binding sites are only partially inhibited by estradiol-17β. Partial thermal denaturation of the cytosol at 30° C for 2 h 30 allows the characterization of a high affinity (Kd = 3.3 × 10^?9M) saturable Tamoxifen class of binding sites insensitive to estradiol-17β while remaining sensitive to the antiestrogens CI628 and Nafoxidine. The uterine concentration of these binding sites is lower in the uterus of immature or castrated animals, increases from metestrus to proestrus and reaches a peak on the day of estrus.     

A specific binding protein for 17beta-estradiol in retired breeder rat ventral prostate

A specific binding protein for 17β-estradiol has been detected in ventral prostate of normal retired breeder rats using sucrose density gradient techniques. The protein has an approximate sedimentation coefficient of 3. 5S. It is distinguishable from serum proteins which bind 17β-estradiol on the basis of binding specificity and sedimentation coefficient. It is also distinct from the cytoplasmic androgen binding protein known to be present in rat ventral prostate.     

Demonstration and characterization of cytosol androgen receptor in rat exorbital lacrimal gland

The presence of a macromolecule which binds androgen with a high affinity and a low capacity was demonstrated in the cytosol of the lacrimal glands of male and female rats. Evidence was found that this macromolecule was a protein by treatment with protease, trypsin or heat. A specific 8-8.5 S peak was obtained in both sexes by glycerol gradient centrifugation in low salt condition, whereas a specific 5.2 S peak was found in high salt condition. This protein could bind to DNA-cellulose after treatment of androgen-cytosol complexes by warming (25 degrees C 15 min) or exposure under high salt (0.4 M KCl). These results suggested that this protein was an androgen receptor.     

TNF-alpha-related apoptosis-inducing ligand decoy receptor DcR2 is targeted by androgen action in the rat ventral prostate

The apoptotic cell death process in the prostate is known to be under the control of androgens. Tumor necrosis factor-alpha (TNF-alpha)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF-alpha family of cytokines, known to induce apoptosis upon binding to its death domain-containing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2. Two additional TRAIL receptors, DcR1/TRAIL-R3 and DcR2/TRAIL-R4, lack functional death domains and act as decoy receptors for TRAIL. In this study, we examined whether TRAIL and cellular receptors expression was targeted by androgens during the apoptotic cell death process in the hormone sensitive ventral prostate. The role of androgens was investigated using two sets of experiment. (1) Androgen deprivation associated with an apoptotic process resulted in a decrease in DcR2 mRNA and protein expression in the ventral prostate 3 days after castration. Testosterone administration to castrated adult rats prevented the decrease in DcR2 mRNA and protein levels in the ventral prostate. In contrast, DcR2 expression was modified, neither in the dorsolateral nor in the anterior prostate following castration. No changes were observed in DR4, DR5, DcR1, and TRAIL mRNA and protein levels in prostate after castration. (2) A specific decrease in DcR2 expression was observed in the ventral prostate after treatment of rats with the anti-androgen flutamide. Together, the present results suggest that testosterone specifically controls DcR2 expression in the adult rat ventral prostate. Androgen withdrawal, by reducing DcR2 expression, might leave the cells vulnerable to cell death signals generated by TRAIL via its functional receptors.     

Postnatal development of lectin binding sites in the rat ventral prostate

Summary Five Fluorescein-isothiocyanate (FITC)-labelled lectins were used to study the postnatal development of carbohydrate constituents in the rat ventral prostate: Concanavalin A (Con A), wheat germ agglutinin (WGA), peanut agglutinin (PNA),Dolichos biflorus agglutinin (DBA) andRicinus communis agglutinin I (RCA-I) With all the lectins, tested, except RCA-I, specific binding sites could be shown for every stage of differentiation in the glandular epithelium. Binding sites for Con A, WGA, PNA and DBA were found from day 10 to 13 post partum onwards. Each lectin showed a characteristic localization. Binding sites for the lectins used changed to different extents during the following two weeks. After the 24th day post partum no further changes in the lectin binding pattern could be found. The development of the lectin binding properties showed that the changes in carbohydrate-containing constituents of the prostate correlate with the beginning of prostatic secretion and to prostatic epithelial differentiation. In the periacinar stroma the development of the lectin binding pattern was similar to that in the glandular epithelium. The changes of stromal binding sites for Con A and WGA during epithelial differentiation may reflect the changes of epithelial-stromal interactions in the prostate.