Differentiation of estradiol receptors in rat uterine cytosol by sensitivity to tamoxifen.

Analysis of rat uterine cytosol by means of immobilized antibody discloses the presence of two distinct high affinity, low capacity estradiol binding components. One of these is readily saturable by the antiestrogen tamoxifen (TS = tamoxifen sensitive), the other is tamoxifen insensitive (TI). Only TS-estradiol binding shows positive cooperativity at low ligand concentration. TS but not TI is lost when frozen tissue is thawed at 4°C then refrozen and stored for an additional 12 hours. Experiments with ovariectomized rats show that TI is formed only in the presence of estradiol. In the estrus cycle TS increases in the order: metestrus, diestrus, proestrus and estrus. The quantity of TI is the same in metestrus and diestrus and also the same but fractionally higher in proestrus and estrus.     

Nuclear progesterone receptors and characterization of cytosol receptors in the rat hypothalamus and anterior hypophysis

Progesterone receptors in nuclei and cytosols from the hypothalamus and anterior hypophysis of oestrogen-primed immature and mature female rats were investigated. In the hypothalamic and hypophysial nuclei the binding and exchange of [³H]-R5020 with progesterone or R5020 was achieved after 2 h at 0–10°C, but rapidly degraded at 30°C. In addition, when unlabelled R5020 was added to the incubation tubes previously incubated with [³H]-R5020 at 0–10°C, unlabelled R5020 was found to exchange with [³H]-R5020 bound to nuclei, confirming that [³H]-R5020 binding is due to an exchange reaction. Scatchard analysis of the specific binding curves revealed high-affinity and low capacity binding. Progesterone receptor complexes extracted with 0.4 M KCl from purified and crude (800 g pellet) nuclei prepared from the hypothalamus and anterior hypophysis of the oestrogen-primed adult female rats incubated with [³H]-R5020 were identified in the vicinity of 5S by gradient centrifugation. From these results it is concluded that nuclear progesterone receptors exist in the hypothalamus and anterior hypophysis. Moreover, it is interesting to note that progestin binding sites resistant to extraction with 0.4 M KCl exist even in the purified hypothalamic and hypophysial nuclei.In the hypothalamic and anterior hypophysial cytosols an exchange reaction was observed at 0–10°C as in the nuclei. The 7S cytosol receptors at low ionic strength sedimented in the 4S region in a high salt medium (0.4 M KCl), both in the hypothalamus or hypophysis, suggesting a possible relationship between aggregate- and subunit receptors. Moreover, progesterone receptors in the hypothalamic and hypophysial cytosols were separated on polyacrylamide agarose gels electrophoretically from oestrogen- and androgen-receptors labelled with [³H]-R2858 and [³H]-R1881, respectively.The existence of nuclear progesterone receptors in the hypothalamus and anterior hypophysis, together with the cytosol receptors, provide further evidence for a possible role of the steroid-receptor interaction in the mechanism of the central action of progesterone.     

Accumulation of 5 alpha-dihydrotestosterone in purified plasma membranes and in the myelin of male rat hypothalamus

The lack of 5 alpha-reductase activity in purified membranes of hypothalamic cells or in myelin was demonstrated. On the other hand, it was shown that incubation of hypothalamic slices with tritiated testosterone resulted in the accumulation and retention of 5 alpha-dihydrotestosterone in purified plasmic membranes and in myelin. The biological significance of such a retention remains unknown.     

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.     

Effect of 5 alpha-dihydrotestosterone on sexual receptivity and neural progestin receptors in ovariectomized rats given pulsed estradiol

Experiments were carried out to examine the mechanism whereby 5 alpha-dihydrotestosterone (DHT) antagonizes the stimulatory effects of estrogen plus progesterone (P) on sexual receptivity (lordosis) in the ovariectomized rat. Estradiol (E2; 1 microgram s.c. in 10% ethanol) was administered in a discontinuous (pulsed) treatment regimen thought to mimic phase requirements of estrogen action; two injections of E2 were given either 6 or 12 h apart (first injection, Hour 0). Progesterone (0.5 mg in oil) was injected at Hour 20, and behavioral testing occurred at Hour 24. Dihydrotestosterone (2.5 mg s.c. in 10% ethanol/propylene glycol) inhibited lordosis when it was given before (-12 or -3 h), between (+3, or -3 and +3 h), or after (+8 h) the two E2 injections, but was not effective when given at +20 h. Significant inhibition of E2 + P-induced lordosis was achieved by 2.5 but not 1.0 or 0.2 mg DHT at -3 h, while uterine weights in the same animals were reduced significantly by 2.5 and 1.0 mg DHT. Serum E2 and DHT concentrations peaked rapidly after injection, declining to near baseline by 3 and 12 h, respectively. Induction of cytosolic progestin receptors (cPR) in the preoptic area and medial basal hypothalamus by estrogen was not prevented by DHT when animals were given the two pulsed E2 injections or daily injections of estradiol benzoate, although P was able to override the inhibitory behavioral effects of DHT in the latter but not the former group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of corticosterone on the negative feedback action of testosterone, 5 alpha-dihydrotestosterone and estradiol in the adult male rat

Corticosterone acetate (10 mg/day) was administered to gonadectomized and adrenalectomized male rats bearing 5, 10 or 15 mm long testosterone filled silicone elastomer capsules. It was found that the serum testosterone levels induced by these capsules were not influenced by corticosterone treatment and that the weights of the prostates in the corticosterone treated rats were not different from their controls. In contrast, corticosterone acetate increased markedly the LH and FSH inhibitory effects of testosterone. Since several brain structures are able to convert testosterone into 17-beta-hydroxy-5-alpha-androstan-3-one (5-alpha-dihydrotestosterone) and/or estradiol, and these metabolites are probably involved in mechanisms controlling gonadotropin secretion, we studied also the effects of corticosterone on the feedback action of dihydrotestosterone and estradiol. 5 alpha-Dihydrotestosterone was administered by 5, 10 or 20 mm long elastomere capsules whereas estradiol was given by daily s.c. injections of 0.125, 0.25 or 0.50 micrograms estradiol benzoate. In the presence of corticosterone acetate the gonadotropin inhibitory action of testosterone, 5 alpha-dihydrotestosterone and estradiol increased more than 2 times.     

Identification of beta adrenergic receptors in rat hypothalamus.

(-)-[3H]-Dihydroalprenolol((-)[3H]DHA) binding in the rat hypothalamus appears to possess all the characteristics expected of physiologically relevant beta-adrenergic receptors. Binding of (-)-[3H]DHA to the hypothalamic sites was rapid (k1 = 1.3 X 10(-7) min-1) and also rapidly reversible. Binding was saturable at low concentrations of ligand (approximately 50-100 nM). The dissociation constant (KD) of (-)-[3H]DHA binding determined by equilibrium analysis was 19 nM. Binding displayed beta-adrenergic specificity. beta-Adrenergic agonists inhibited binding in the following order of potency: (-)-isoproterenol congruent to (-)-epinephrine greater than (-)-norepinephrine. Specific beta-adrenergic antagonists (-)-propranol and (-)-alprenolol inhibited binding at low concentrations (KD = 25-50nM) whereas the alpha-antagonist phentolamine inhibited binding at very high concentration (KD = 42 micron). Interactions of both agonists and antagonists with the sites showed stereoselectivity. The (-)-isomers of all beta-adrenergic agents tested were more potent than their respective (+)-isomers. These results suggest that specific receptor sites for beta-adrenergic catecholamines are present in rat hypothalamus.     

Purification and properties of the 5 alpha-dihydrotestosterone 3 alpha(beta)-hydroxysteroid dehydrogenase from human prostatic cytosol.

5 alpha-Dihydrotestosterone 3 alpha(beta)-hydroxysteroid dehydrogenase [3 alpha(beta)-HSDH] [EC 1.1.1.50/EC 1.1.1.51] which catalyses the conversion of 5 alpha-dihydrotestosterone (5 alpha-DHT) to both 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol was purified to an apparent homogeneous state using cytosol of three human hyperplastic prostates by a 4-step purification procedure. After each purification step 3 alpha-HSDH activity was coincident with 3 beta-HSDH activity. On average, specific 3 alpha-HSDH activity was enriched 856-fold, specific 3 beta-HSDH activity 749-fold compared to human prostatic cytosol using anion exchange, hydrophobic interaction, gel filtration and affinity chromatography. Examination of the purified enzyme by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) revealed a single protein band with silver staining. The molecular weight of the enzyme was estimated as 33 kDa by SDS-polyacrylamide gel electrophoresis and as 28 kDa by Sephacryl S-200 gel filtration indicating that the native 3 alpha(beta)-HSDH is a monomer. In the presence of the preferred co-factor, NADPH, the purified enzyme had a mean apparent Km for 5 alpha-DHT of 3.9 microM and a Vmax of 93.3 nmol (mg protein)-1 h-1 with regard to 3 alpha-HSDH activity, and a Km of 6.3 microM and a Vmax of 20.6 nmol (mg protein)-1 h-1 with regard to 3 beta-HSDH activity.     

The protection,stabilization and reactivation of estradiol receptors in human myometrial cytosol

Protection of estradiol receptor binding sites in human mymetrial cytosol is achieved though the agency of dithiothreitol (Cleland's reagent) (lmM) at 4°C and ? 20°C storage temperatures. Prolonged storage of cytosol at 4° C without the protective reagent results in substantial loss of binding activity. This is partially restored after Cleland's reagent (lmM) addition. However, cytosol inactivated by heating at 60°C for 30 min cannot be reconstituted in this way.     

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

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

3H-estradiol binding to cytosol receptors of the rat olfactory epithelium

Two types of cytosol receptors of 3H-estradiol with high affinity and limited quantity of binding sites (KDI = 1-2 nM, BmaxI = 8 fmoles/mg protein; KDII = 10 nM, BmaxII = 8 fmoles/mg protein) were determined in the rat olfactory tissue. The amount of high affinity receptors of type I does not change with maturation of the rats, and has no sex difference. The role of estradiol receptors in the olfactory tissue of the rats is discussed.     

Estimation of 5 alpha-dihydrotestosterone receptors in human breast tumors by the immobilized antibody method.

A simple method for estimation of 5 alpha-dihydrotestosterone (DHT) receptors in the cytosol of human breast tumors is described. It is based on the competition of immobilized antibody with the receptors for the labeled hormone. Out of eleven patients with primary breast tumors, four had DHT-receptors, three were negative, and four patients were border line cases.     

Effect of 5 alpha-dihydrotestosterone and dexamethasone on estrogen receptors of the anterior pituitary and uterus.

The administration of 5 alpha-dihydrotestosterone (5 alpha-DHT) and dexamethasone has been shown to attenuate estrogen-induced prolactin release in the estrogen-primed rat. Therefore, the effect of these compounds was studied on anterior pituitary and uterine estrogen receptors. Injection of 0.8 mg/kg body weight of 5 alpha-DHT to ovariectomized adult rats treated with 2 micrograms estradiol/d for 4 days resulted in a significant decrease in occupied nuclear estrogen receptors of the anterior pituitary but not the uterus. Estrogen priming was essential for 5 alpha-DHT effect on occupied nuclear anterior pituitary estrogen receptors because this effect did not occur in ovariectomized vehicle-treated control animals. The administration of 1 mg/kg body weight of dexamethasone brought about a decrease in uterine but not anterior pituitary nuclear estradiol receptors. These results provide further evidence that the regulation of estrogen receptor dynamics is different in the anterior pituitary and the uterus and that different steroids can exert tissue-specific effects.     

Activation of cytosol aldosterone receptors in rat kidney

Cytosolic aldosterone-protein complexes are isolated from rat kidney slices after incubation with [3H]aldosterone and dexamethasone. Activated and unactivated forms of the complex are characterized by gel electrophoresis and hydroxyapatite chromatography after incubation at 4 degrees C and 25 degrees C respectively. It is found that the activated form reaches a maximum after 30 min at 25 degrees C and can be separated as an homogeneous peak by electrophoresis. Intermediate forms can also be identified. In the presence of 10 mM ATP, activation immediately occurs at 4 degrees C and is almost complete. In the presence of 10 mM molybdate, the activation is strongly enhanced and the increase in activated form may be about fifteen-fold whether molybdate is added during kidney homogenization or just before incubation at 25 degrees C. On the other hand molybdate reduces to one third the binding of the aldosterone-receptor complexes to nuclei. In the presence of the steroid RU 26988 which is a pure glucocorticoid, experiments done on aldosterone-receptors complexes and their binding to nuclei are confirmed. This proves that aldosterone is specific for mineralocorticoid sites. The general pattern of the mineralocorticoid receptor activation is discussed and its resemblance to the case of other steroid hormones is emphasized.     

Presence of three different estradiol binding proteins in rat prostate cytosol

Depending upon the experimental conditions, three distinct [³H]-17β-estradiol binding entities can be detected in rat prostate cytosol. A protein with characteristics similar to other estrogen receptors is found in prostate cytosol from intact rats. This protein has a sedimentation coefficient of 8S on sucrose gradients. It has a high affinity only for natural and synthetic estrogens. It decreases rapidly after castration (less than 20% of binding activity after 24 hr) and reappears progressively after 8–10 days.A second binding entity with a sedimentation coefficient of 4–5S on sucrose gradients is also observed. It is found both in intact and castrated rats. At low temperatures, the binding of estradiol increases progressively and reaches a maximum after 24–48 hr of incubation as determined by charcoal assay. This binding species is highly specific for natural estrogens but not for diethylstilbestrol and is probably identical with the 4–5S binder.Finally, a third binding entity is found in 24 hr castrated rats with short incubations at 0°C This binding is labile even at low temperatures. Natural and synthetic androgens compete more strongly than estradiol for this binding. This behaviour suggests that the third estradiol binding entity is identical with the androgen receptor.