Sydney A. Shain

Estradiol-17 beta affects estrogen receptor distribution and elevates progesterone receptor content in baboon aorta.

We used the synthetic estrogen R2858 (moxestrol) and estradiol-170, respectively, to characterize the estrogen receptor in baboon (Papio sp.) aortic or myocardial cytoplasmic and nuclear preparations. We observed regional differences in the cytoplasmic fraction estrogen and progesterone receptor content of aortic arch, thoracic aorta, and abdominal aorta when tissues from either oophorectomized or oophorectomized estradiol-170-treated subjects were compared. The estrogen receptor content was highest in the abdominal aorta and lowest in the aortic arch. In contrast, the cytoplasmic fraction progesterone receptor content was highest in the aortic arch and lowest in the abdominal aorta. The nuclear fraction estrogen receptor could not be demonstrated in preparations from cardiovasculature of oophorectomized female baboons. The use of Silastic implants to administer a physiologic concentration of estradiol-170 to oophorectomized female baboons caused a 20% to 50% reduction in cytoplasmic fraction estrogen receptor content, which was quantitatively accounted for by the appearance of estrogen receptor in the corresponding nuclear aortic or myocardial preparation. Estrogen administration caused a 20% to 40% increase inc cytoplasmic fraction progesterone receptor content in both myocardium and aorta; however, differences were significant only for abdominal aorta (p < 0.05). Estradiol- 170 treatment caused a tenfold increase in uterine cytoplasmic fraction progesterone receptor content in treated as compared to oophorectomized control females, suggesting that baboon cardiovasculature is less sensitive to changes in endogenousestrogen concentration than is uterus. The ability of estradiol-170 to affect apparent intracellular distribution of baboon cardiovascular estrogen receptors and to elevate cytoplasmic fraction progesterone receptor content suggests that these estrogen receptors are physiologically functional and indicates that estrogen may directly regulate primate cardiovascular cell function.

Hormone receptors of the baboon cardiovascular system. Biochemical characterization of aortic and myocardial cytoplasmic progesterone receptors.

We used the synthetic progestin R5020 (17α,21-dimethyl-19-norpregna-4,9-diene- 3,20-dione) to characterize cytoplasmic progesterone receptors in baboon(Pupio sp.) aorta and myocardium. The relative ability of selected steroids to inhibit binding of radiolabeled R5020 to aortic cytoplasmic progesterone receptors was radioinert R5020, 1.0; progesterone, 0.31; triamcino- lone acetonide, 0.06; testosterone, 0.002; estradiol-17β, 0.01; and cortisol, 0.001. The relative ability of these same steroids to inhibit binding of radiolabeled R5020 to myocardial cytoplasmic progesterone receptors was, respectively, 1.0, 0.58, 0.21, 0.01, 0.01, and 0.001. Both aortic and myocardial cytoplasmic progesterone receptors migrated as macromolecules with a sedimentation coefficient of 8–9S on low ionic strength linear sucrose gradients. Cytoplasmic binding of R5020 was inactivated by incubation at 37°C. Saturation analysis at 2°C showed aorta and myocardium, respectively, contained 41.6 ± 16.1 (mean ± SD) and 14.0 ± 2.8 fmol R5020 binding sites/mg cytosol protein. The dissociation constant for R5020 was 2.8 ± 1.2 nm, aorta, and 2.0 ± 1.1 nM, myocardium. The presence of progesterone receptors in baboon cardiovascular tissues suggests that progestins may directly influence cardiovascular tissue function.

Androgen regulation of androgen receptor content and distribution in the ventral and dorsolateral prostates of aging AXC rats.

Total androgen receptor content of ventral or dorsolateral prostate of intact, aged (730-740 day old) rats is decreased 50% when compared to intact, young mature (150-170 day old) rats. Treatment with exogenous testosterone increased ventral and dorsolateral prostate androgen receptor content per cell in aged rats to values identical to those of prostates of young mature rats. The increase in prostate receptor content was not attributable to testosterone mediated cellular hypertrophy or hyperplasia. At 24 hr post-orchiectomy ventral prostate cytoplasmic androgen receptors are depleted of endogenous androgen, without any decrease in number of receptors per cell, and nuclear androgen receptors are undetectable. During 30 to 60 min after a single 200 microgram testosterone injection, ventral prostate nuclear receptor content increased to the level of intact control rats without producing any reduction in total cytoplasmic androgen receptor content. Although dorsolateral prostate is devoid of cytoplasmic androgen receptor, the effects of orchiectomy and testosterone treatment upon nuclear androgen receptor are comparable to those seen in ventral prostate. These effects of orchiectomy and testosterone injection upon prostatic receptor content and distribution were identical in prostates of young and aged rats. Our studies show that receptor processing in prostates of young and aged rats does not involve a process by which nuclear receptor is derived by depletion of cytoplasmic receptor. Moreover, our studies of the effect of short-term (48 hr) exogenous testosterone treatment upon androgen receptor content in prostates of aged rats are the first demonstration that androgen receptor content may be enhanced independent of generalized androgen mediated anabolic effects in prostate.

Aging-associated diminished rat prostate androgen receptor content concurrent with decreased androgen dependence

Abstract As inbred AXC rats aged from 137 to 976 days or randomly-bred Sprague-Dawley rats aged from 105 to 610 days, ventral prostate cytoplasmic androgen receptor content decreased 70 and 46%, respectively, and nuclear androgen receptor content decreased 41 and 26%, respectively. Identical evaluations employing dorsolateral prostate failed to provide evidence for a cytoplasmic androgen receptor; however, a 50% decrease in dorsolateral prostate nuclear androgen receptor content was demonstrated in these rats. Prostatic involution subsequent to orchiectomy was employed as a measure of androgen regulation of prostate function in aging rats. Both ventral and dorsolateral prostate evidenced an aging-associated decrease in either the absolute or relative loss in prostate RNA and protein content and wet weight subsequent to orchiectomy. By contrast, the effect of orchiectomy upon cell number was both tissue- and age-dependent. Orchiectomy did not diminish dorsolateral prostate cell number in any of the rats aged 3.5 to 21 months, whereas orchiectomy resulted in profound decreases ventral prostate cell number in rats 3.5 to 16 months of age and did not decrease ventral prostate cell number in rats 21 months of age. The data demonstrate an acquired marked androgen independence for ventral prostate cell number occurring concurrent with an aging-associated diminution in cytoplasmic androgen receptor content and thus suggest a associated diminution in cytoplasmic androgen receptor content and thus suggest a proufound role for cytoplasmic receptor in those processes regulating ventral prostate cell number.

Saturation analysis of the binding of androgens. Antiandrogens and estrogens by the cytoplasmic high affinity androgen receptor of the rat ventral prostate.

Ammonium sulfate fractionation of the cytoplasmic extract obtained from rat ventral prostate yields a protein fraction for which the contribution of nonspecific binding of 5α-dihydrotestosterone to the measured binding of this steroid is markedly reduced. The fractionated binding activity is suitable for utilization in a protocol which permits sensitive, facile and highly reproducible saturation analysis of the high affinity binding of 5α-dihydrotestosterone. The apparent binding constant for this prostatic androgen was 6.9 × 108 l/mol. The number of 5α-dihydrotestosterone binding sites per cell and concentration per 100 mg cytosol protein were 9500 and 8.55 ± 0.36 × 10−12 moles respectively. The apparent binding constants for a number of androgens, antiandrogens and estrogens were determined by evaluation of their ability to inhibit binding of 5α-dihydrotestosterone. Testosterone and all of the common prostatie metabolites of testosterone possessed measurable affinity for the binding component. Our data also demonstrate that the presence of a 3-keto or 17β-hydroxy function on the steroid nucleus is a strong determinant of binding affinity for the prostatic receptor.

Androgen Receptor Content of the Normal and Hyperplastic Canine Prostate

A procedure was developed for measurement of androgen receptors in cytoplasmic extracts of prostates from intact dogs. The protocol utilized exchange saturation analysis at 15 degrees C employing the synthetic androgen R1881 (17beta-hydroxy-17alpha-methylestra-4,9,11-trien-3-one) as the ligand probe and quantitatively detected total cytoplasmic androgen receptor (R(c), androgen-free receptor, and R(c)A, androgen-occupied receptor) present at the initiation of the assay. This protocol was employed in conjunction with a tissue mince saturation analysis procedure (for quantitation of nuclear androgen receptor) to quantitate total androgen receptor content of normal and hyperplastic prostates obtained from young (2.5- or 4.6-yr old) and aged (12.5-yr old) purebred dogs of known birth date. The total cytoplasmic androgen receptor content (picomoles per prostate) of hyperplastic prostates was 4.6-fold greater than that of normal prostates. The total nuclear androgen receptor content of hyperplastic prostates (picomoles per prostate measured in crude nuclear preparations) was either 5.0- (4.6-yr-old dogs) or 7.8-fold (2.5-yr-old dogs) greater than that of normal prostates. However, androgen receptor content per cell was identical for hyperplastic and normal canine prostates, with the exception that nuclear androgen receptor was diminished in prostates from 2.5-yr-old dogs. The cell content per gram dry weight was identical for hyperplastic and normal canine prostates. We conclude that canine prostate hyperplasia is characterized by coordinate proliferation of androgen receptor-positive and androgen receptor-negative cells and is not a consequence of increased accumulation of 5alpha-dihydrotestosterone due to proliferation of androgen receptors per prostate cell.

Hormone receptors of the baboon cardiovascular system. Biochemical characterization of myocardial cytoplasmic androgen receptors

Using the synthetic androtfen R1881 (17β-hydroxy-17α-methyl-estra-4,9,ll-trien-3-one) as probe, we identified cytoplasmic androgen receptors in baboon myocardium. The relative binding affinity of selected steroids for the androgen receptor was R1881, 100%; 5α-dihydrotestosterone, 32.8%; testosterone, 29.6%, progesterone, 7.2%; R5020, 1.0%; and estradiol-17β, 5.8%. The androgen receptor migrated on low ionic strength linear sucrose density gradients as a macromolecule with a sedimentation coefficient of 8.5S. Saturation analysis performed at 2°C (available sites) showed that the androgen receptor content of baboon myocardial cytoplasmic extracts was 5.9 ± 1.4 μmol/mg protein and that the dissociation constant for R1681 was 1.16 ± 0.30 run. These cytoplasmic androgen receptors are indicated to be physiologically functional by previous autoradiographic studies (McGill et aL, 1980; McGill and Sheridan, 1981) that showed localization of radloisotope In nuclei of myocardial fibers following injection of baboons with 5α-dihydrotestosterone. Circ Res 49: 1010–1016, 1881

Testosterone metabolism by the prostate of the aging AXC rat.

Testosterone metabolism by the ventral prostate of inbred, senescent (36-month-old) AXC rats showed a shift to increased oxidative and diminished reductive metabolism when compared to young mature (3-month-old) or mature adult (6-month-old) AXC rats. The change was principally attributable to diminished production of 5 alpha-dihydrotestosterone and increased production of 4-androstenedione. By contrast, dorsolateral prostate testosterone metabolism in these same rats was always more reductive than that of ventral prostate and failed to show the post-maturation shift to oxidative testosterone metabolism which characterized ventral prostate. Co-incubation of either ventral or dorsolateral prostate from any of these rats with testosterone and estradiol showed that estradiol did not significantly affect prostate testosterone metabolism. Plasma testosterone content of senescent AXC rats was only 28% of that of mature adult rats. Chronic exogenous testosterone treatment enhanced reductive testosterone metabolism by ventral prostate and eliminated those changes in metabolite distribution which had differentiated testosterone metabolism by young and senescent AXC rats. The data support the interpretation that the aging-related diminution in AXC rat ventral prostate reductive metabolic capacity is primarily the consequence of diminished 5 alpha-dihydrotestosterone production secondary to diminished plasma testosterone content.

Sexual dimorphism characterizes baboon myocardial androgen receptors but not myocardial estrogen and progesterone receptors

Using biochemical methods we established that estrogen receptor content and distribution and progesterone receptor content in female and male baboon myocardium did not differ between sexes. In contrast, myocardial androgen receptor distribution between cytosolic and nuclear compartments was sexually dimorphic. Female baboon myocardial androgen receptors were restricted to the cytosolic compartment, whereas male myocardial androgen receptors were distributed between the cytosolic and nuclear compartments. Using human estrogen receptor cDNA we showed that baboon aorta, myocardium and uterus contain a 6.3 kb estrogen receptor transcript. Analyses performed with human progesterone receptor cDNA established that baboon aorta and uterus contain an 8 kb progesterone receptor transcript; however, progesterone receptor transcripts were not demonstrable in baboon myocardial RNA preparations. Because relative hybridization signal intensity reflected known uterine and aortic progesterone receptor content, failure to detect progesterone receptor transcripts in myocardial preparations may reflect sensitivity limitations and the fact that aortic progesterone receptor content is 5-fold greater than that of myocardium. Immunocytochemical analyses demonstrated that baboon myocardial progesterone receptors were present in greater than 25% of myocytes and generally absent from other myocardial cells. Our studies establish that: (1) gonadal steroid hormone receptor gene transcription occurs in cells of the baboon cardiovasculature, (2) these steroid hormone receptors may be physiologically functional, and (3) gonadal steroid hormone receptors may be restricted to specialized cells of the cardiovasculature.

IDENTIFICATION OF LIMITED CAPACITY ANDROGEN BINDING COMPONENTS IN NUCLEAR AND CYTOPLASMIC FRACTIONS OF CANINE PROSTATE

Nuclear and cytoplasmic androgen receptors were identified in canine prostate preparations. Saturation analysis employing 5α-dihydrotestosterone and tissue from intact dogs demonstrated that crude nuclear pellets contained 85 femtomoles of KCI-extractable and 106 femtomoles of ethanol-extractable receptor sites per 100 μg DNA. The apparent 5α-dihydrotestosterone association constant, 3-3.5 × 108 M−1, and steroid binding specificity of both receptor fractions were identical. 5α-Dihydrotestosterone, 19-nortestosterone, 17β-testosterone, and 5α-androstane-3λ,17β-diol were effective inhibitors of the nuclear binding of radiolabeled 5α-dihydrotestosterone, whereas 5α-androstane-3α,17α-diol, δ4-androstene-3,17-dione, progesterone, estradiol, and cortisol were not. The sedimentation constant of the KCl-extractable nuclear receptor on linear sucrose gradients was 4-5S. Cytoplasmic saturation analysis, employing tissue obtained from dogs 72 hr post-orchiectomy, demonstrated that 5α-dihydrotestosterone and the synt...