Control of estrogen and androgen receptors in the rat pineal gland by catecholamine transmitter

Sucrose gradient studies of rat pineal cytosol incubated with ³H-estradiol (female pineals) or ³H-5 α -dihydrotestosterone (male pineals) revealed a radioactivity peak in the 8 S region which disappeared after superior cervical ganglionectomy or incubation with excess unlabeled hormone. Ganglionectomy decreased significantly estradiol and testosterone uptake by the pineal gland $\text{in vitro}$ as well as high affinity binding to pineal cytoplasmic and nuclear components. Norepinephrine treatment counteracted all the effects of ganglionectomy but was unable to modify hormone uptake and binding by the pineal gland of sham-operated controls. Pre-treatment with actinomycin D or propranolol but not with phentolamine impaired norepinephrine effects; propranolol blockage however was only partial. Administration of isoproterenol, L-dopa or phentolamine increased hormone uptake by denervated pineals. The effects of isoproterenol were also observed $\text{in vitro}$ and were blocked by propranolol. These results indicate that sex steroid receptors in the pinealocytes are controlled by norepinephrine via beta-adrenergic receptors and that depletion of neural norepinephrine enhanced responsiveness of pineal hormone receptors to exogenous catecholamines.     

The effect of ether on formation and dissociation of rat uterine estradiol-receptor complex

The addition of 2% v/v ethyl ether to uterine cytosol incubation mixtures produced a 25% increase in the rat of association of 0 degrees with 3H-estradiol and almost a 3-fold increase in the rate of exchange of estradiol-receptor complex with 3H-estradiol at 20 degrees. Uterine cytosol saturated with non-radioactive estradiol required over 8 hours for complete exchange with 3H-estradiol at 20 degrees while in the presence of 2% ether exchange was complete in 2 hours and revealed about 15% more specific binding sites. The presence of ether in reaction mixtures had no effect on dextran coated charcoal separation on bound from free 3H-estradiol nor on the stability of the receptor on uterine cytosol.     

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.     

Effect of estradiol-17 beta and prostaglandins on rat myometrial gap junctions

The effects of estradiol-17 beta and indomethacin on myometrial gap junction development, plasma estradiol levels and uterine PGF2 alpha content were evaluated in immature and/or ovariectomized, mature rats. High doses of estradiol stimulated the development of gap junctions in the myometrium of animals from both groups. Concomitant injections of estradiol and indomethacin to ovariectomized rats potentiated the estradiol stimulation of gap junctions. Plasma estradiol levels were lower in ovariectomized rats treated with both estradiol and indomethacin than in animals treated with estradiol alone. Indomethacin also enhanced the uptake and retention of 3H-estradiol into uterine tissues. Uterine PGF2 alpha content of ovarectomized rats was stimulated with the initial injection of estradiol but thereafter, the PGF2 alpha content declined with repeated injections to values lower than that observed in controls. Prostaglandin F2 alpha content in tissues from rats treated with estradiol plus indomethacin were also higher than that observed in rats treated with indomethacin alone, however, the values obtained in both groups were significantly lower compared to those from control animals. These results are consistent with the hypothesis that steroid hormones and prostaglandins regulate myometrial gap junction formation. Regulation of myometrial gap junctions by prostaglandins is discussed with respect to a down regulation of the steroid-receptor mechanism and effects on cyclo-oxygenase or lipoxygenase products.     

Iodohexestrols. II. Characterization of the binding and estrogenic activity of iodinated hexestrol derivatives, in vitro and in vivo.

The affinity of ortho-iodinated hexestrols for the estrogen binding protein from rat uterus, determined by competitive binding assay, decreases with progressive iodine substitution; 3-iodohexestrol (I-Hex) has a binding affinity 42% that of estradiol. Analysis of [3-H]-I-Hex binding in rat uterine cytosol by sucrose density gradient centrifugation shows both an estrogen-specific binding component (8 S) and a more abundant component (4 S) that is not estrogen specific. Scatchard analysis indicates that this latter binding is of high affinity (Kd equals to 3.7-8.3 times 10- minus-9 M) but is not uterine specific. Polyacrylamide gel electrophoresis shows that most of the [3-H]-I-Hex binding activity in serum and uterine cytosol is distinct from and anodic to the principal protein component (albumin), and that is comigrates with [14-C]thyroxine binding activity. In in vitro incubation of rat uteri, I-Hex can block the specific uptake of [3-H]estradiol into the nuclear fraction; it itself causes a translocation of estrogen-specific binding capacity (as measured by exchange) from cytoplasm to nuclei, and can induce the synthesis of an estrogen-specific uterine protein, all under conditions where it is not metabolically deiodinated to hexestrol. The uterotrophic activities of the iodohexestrols are in most cases comparable to that expected on the basis of their competitive binding affinities. However, selective, estrogen-specific uptake of [3-H]-I-Hex into rat uterus, either in vitro or in vivo, cannot be demonstrated.     

Differential binding of antiestrogens by rat uterine and chick oviduct cytosol

Analysis of the interactions of two synthetic estrogen antagonists, tamoxifen and CI 628, with rat uterine and chick oviduct cytosol revealed significant differences in the antiestrogen binding properties of these tissues. In the rat uterus CI 628, tamoxifen and estradiol were bound to a similar number of saturable binding sites and estradiol could completely inhibit the binding of tritiated antiestrogens to these sites. In contrast, high affinity, saturable antiestrogen binding sites in chick oviduct were present at three times the concentration of estradiol binding sites and estradiol could only partially inhibit the binding of tritiated antiestrogens to these sites. It is concluded that antiestrogens bind to the estrogen receptor in both tissues and that chick oviduct has an additional saturable antiestrogen binding site distinct from the classical estrogen receptor site.     

Uptake of estradiol by the rat pineal organ. Effects of cervical sympathectomy, stage of the estrous cycle and estradiol treatment

Rat pineal organs of spayed rats took up and retained estradiol $\text{in vitro}$ up to 32-fold the concentration present in the incubation media. This phenomenon was maximum at 37°C and after 2-h incubations. Most (86–91%) of [³H] radioactivity recovered from the incubated pineals was identified as estradiol by thin-layer chromatography. Treatment with dextran-coated charcoal of nuclei-free pineal homogenates incubated with [³H] estradiol of different SA uncovered a high affinity, low capacity binding of estradiol to cytosol components. Uptake of estradiol by the nuclear fraction also proceeded in a saturable fashion. Similar findings were made in uterine homogenates of spayed rats.Estradiol uptake by the pineal organ and the uterus of cycling rats varied as a function of the stage of the estrous cycle, maxima being observed in diestrus and minima in proestrus. The administration of a priming dose of estradiol benzoate to spayed rats caused high affinity binding components of the pineal cytosol to increase by about 150%. Nuclear binding of estradiol was also increased by the estradiol priming dose. Pineal denervation, i.e., by superior cervical ganglionectomy, caused pineal estradiol uptake to decrease significantly by about 20%. These data suggest that the early steps of estradiol action on the pineal organ may resemble those of the uterus.     

Effect of estradiol-17ß and prostaglandins on rat myometrial gap junctions

The effects of estradiol-17ß and indomethacin on myometrial gap junction development, plasma estradiol levels and uterine PGF_2α content were evaluated in immature and/or ovariectomized, mature rats. High doses of estradiol stimulated the development of gap junctions in the myometrium of animals from both groups. Concomitant injections of estradiol and indomethacin to ovariectomized rats potentiated the estradiol stimulation of gap junctions. Plama estradiol levels were lower in ovariectomized rats treated with both estradiol and indomethacin than in animals treated with estradiol alone. Indomethacin also enhanced the uptake and retention of ³H-estradiol into uterine tissues. Uterine PGF_2α content of ovarectomized rats was stimulated with the initial injection of estradiol but thereafter, the PGF_2α content declined with repeated injections to values lower than that observed in controls. Prostaglandin F_2α content in tissues from rats treated with estradiol plus indomethacin were also higher than that observed in rats treated with indomethacin alone, however, the values obtained in both groups were significantly lower compared to those from control animals. These results are consistent with the hypothesis that steroid hormones and prostaglandins regulate myometrial gap junction formation. Regulation of myometrial gap junctions by prostaglandins is discussed with respect to down regulation of the steroid-receptor mechanism and effects on cyclo-oxygenase or lipoxygenase products.     

Regulation of low density lipoprotein receptors by adrenocorticotropin in the adrenal gland of mice and rats in vivo

Membranes prepared from the adrenal gland of mice and rats possess high affinity binding sites that recognize 125I-labeled human low density lipoprotein (LDL). These binding sites resemble the functional LDL receptors that mediate the uptake of LDL by cultured mouse and bovine adrenal cells. The number of LDL binding sites per mg of membrane protein increased 2- to 5-fold over 24 h when mice or rats were treated with adrenocorticotropin (ACTH). In rats, this increase was accompanied by a similar ACTH-induced increase in the adrenal uptake of intravenously administered 125I-LDL, suggesting that the LDL binding sites mediate the uptake of LDL by the adrenal in the intact animal. The number of LDL binding sites on adrenal membranes rose by 5-fold when animals were rendered lipoprotein-deficient, either by treatment of mice with 4-aminopyrazolopyrimidine or by treatment of rats with 17 alpha-ethinyl estradiol. This increase was prevented when endogenous ACTH secretion was blocked by administration of dexamethasone, suggesting that ACTH was required. The current experiments suggest that LDL receptors provide one source of cholesterol for the mouse and rat adrenal in vivo and that the number of LDL receptors of this organ is regulated by ACTH.     

Estradiol receptors in the rat uterus under long-term hormone administration at different doses

Multiple administration of estrogens in doses of 1 and 10 micrograms reduces specific binding of 3H-estradiol by subcellular fractions of rat uterus tissues. The cytoreceptor apparatus of the uterus of androgen-sterile rats is marked by the decreased response to a prolonged (7-8 days) hormone administration as compared to responses seen in normal and ovariectomized animals. The depletion of estrogen receptors is not linked with a rise of the endogenous steroid level in blood plasma and cytosol from uterine tissues.     

Estradiol-17β receptors in the immature rat ovary

Estradiol binding components in the cytosol and nuclear fractions of the ovary from immature rats (22–28 days old) were characterized by $\text{in vitro}$ methods. Several of the biochemical characteristics of the estradiol binding components in the ovarian tissue were compared with the estradiol receptor from the uterus. The results suggest that the ovarian estradiol binding components are similar to the specific high affinity estradiol receptors in the uterus. In the cytosol of intact rat ovary a significant fraction of the total binding sites was found to be occupied, presumably by the endogenous estrogen. Following hypophysectomy there was a significant increase in the available cytosol binding sites. Evidence for translocation of cytosol receptor-estrogen (RE) complex to the nucleus was obtained for the ovary. The sedimentation properties of the RE complex of the ovary and the uterus are similar. The ovarian cytosol RE complex sediments at 7-8S in glycerol gradients at low ionic strength and at 4S in sucrose gradients at high ionic strength. Following extraction with 0.4 M KCl the ovarain nuclear RE complex sediments at 5S in sucrose gradients which is identical to that of the uterine nuclear receptor.     

Effect of estradiol and progesterone on long chain fatty acyl-coenzyme A levels in the rat uterus

Fatty acyl-CoAs are potential in vivo inactivators of glucose-6-phosphate dehydrogenase (G6PD). Ovariectomized mature rats (n = 74) were given 5 micrograms of estradiol intravenously, then killed 0, 24, 36, 48 and 72 h later. Control levels of myristoyl-, palmitoyl-, stearoyl-, arachidonoyl-, oleoyl- and linoleoyl-CoA were 0.6, 3.2, 4.7, 3.4, 2.4 and 3.0 micrograms/uterus and were increased 39, 110, 146, 100, 84 and 69% at 36-48 h, respectively. Levels of fatty acyl-CoAs in the rat uterus become elevated 36 h after estradiol treatment. At the same time G6PD changes from a stable enzyme to one that is irreversibly inactivated, possibly due to being rapidly degraded. Progesterone (2 mg subcutaneously every 12 h, n = 30), administered beginning at either 24 or 36 h after estradiol treatment, had no effect on estradiol-induced changes in myristoyl-, palmitoyl-, or stearoyl-CoA. Compared to the groups of rats treated with estradiol alone, animals treated with combinations of estradiol and progesterone exhibited higher levels of arachidonoyl-CoA after 48 h, and oleoyl-CoA and linoleoyl-CoA were greater after 72 h. Progesterone increased the estradiol-induced levels of unsaturated fatty acyl-CoAs suggesting that progesterone may induce uterine fatty acid desaturase activity and/or uptake of dietary fatty acids. Addition of fatty acyl-CoAs, at concentrations seen in vivo at 36-48 h after estradiol, to purified G6PD, causes irreversible G6PD inactivation.     

Preliminary characterization of an endogenous inhibitor of [3H]estradiol binding in rat uterine nuclei

The rat uterus contains two classes of specific nuclear estrogen-binding sites which may be involved in estrogen action. Type I sites represent the classical estrogen receptor (Kd = 1 nM) and type II sites (Kd = 10-20 nM) are stimulated in the nucleus by estrogen under conditions which cause uterine hyperplasia. Dilution of uterine nuclear fractions from estrogen treated rats prior to quantitation of estrogen binding sites by [3H]estradiol exchange results in an increase (3- to 4-fold) in the measurable quantities of the type II site. Estimates of type I sites are not affected by dilution. These increases in type II sites following nuclear dilution occur independently of protein concentration and result from the dilution of a specific endogeneous inhibitor of [3H]estradiol binding to these sites. The inhibitor activity is present in cytosol preparations from rat uterus, spleen, diaphragm, skeletal muscle, and serum. Preliminary characterization of the inhibitor activity by Sephadex G-25 chromatography shows two distinct peaks which are similar in molecular weight (300). These components (alpha and beta) can be separated on LH-20 chromatography since the beta-peak component is preferentially retained on this lipophilic resin. Partial purification of the LH-20 beta inhibitor component by high performance liquid chromatography and gas-liquid chromatography-mass spectrometric analysis suggests the putative inhibitor activity is not steroidal in nature and consists of two very similar phenanthrene-like molecules (molecular weights 302 and 304). Analysis of cytosol preparations on LH-20 chromatography shows that non-neoplastic tissues (uterus, liver, lactating mammary gland) contain both and inhibitor components whereas estrogen-induced rat mammary tumors contain very low to nonmeasurable quantities of the beta-peak inhibitor activity.     

Translocatable estrogen receptors in rat splenocytes

Estradiol has previously been shown to suppress the response of the cellular immune system of the rat while enhancing the production of IgM antibodies. Analysis of the cytosol from rat splenocytes showed saturation of specific binding sites at concentrations of between 80 and 160 nM [3H]-estradiol with an approximate Kd of 12 nM. Competitive binding studies showed a dose-dependent decrease in the binding of [3H]-estradiol to the receptor in the presence of increasing concentrations of unlabeled estradiol. Dexamethasone, progesterone and R1881 (synthetic androgen) had no effect on the binding of [3H]-estradiol. The in vivo administration of estradiol resulted in increased nuclear binding of [3H]-estradiol as compared to vehicle treated controls. These results indicate that rat splenocytes possess specific, translocatable estrogen receptors which may be responsible for the observed modulation of the immune system.     

CDRI-85/287: studies on competition to estrogen binding sites in the immature rat uterus.

Ability of compound CDRI-85/287, a new nonsteroidal antiestrogen with negligible inherent estrogenicity, to inhibit uptake of 3H-estradiol (3H-E2) by the immature rat uterus in vivo was investigated. Different doses of 85/287 were administered either intraperitoneally 30 min before 3H-E2 or orally 1 and 6 hr before 3H-E2. A dose dependent inhibition in 3H-E2 uptake was observed after administration of the compound by either route and was 69% at 50 micrograms/rat ip dose and 80% at 2.5 mg/kg po dose. In in vitro competitive binding assay, however, the compound showed poor affinity (RBA 0.42% of estradiol-17 beta) for cytosolic estrogen receptors. Considering the potent anti-estrogenic as well as anti-implantation efficacy of the compound, its action in vivo appears to be mediated via its active metabolite(s).     

Effects of ATD on male sexual behavior and androgen receptor binding: a reexamination of the aromatization hypothesis

The aromatization hypothesis asserts that testosterone (T) must be aromatized to estradiol (E2) to activate copulatory behavior in the male rat. In support of this hypothesis, the aromatization inhibitor, ATD, has been found to suppress male sexual behavior in T-treated rats. In our experiment, we first replicated this finding by peripherally injecting ATD (15 mg/day) or propylene glycol into T-treated (two 10-mm Silastic capsules) or control castrated male rats. In a second experiment, we bilaterally implanted either ATD-filled or blank cannulae into the medial preoptic area (MPOA) of either T-treated or control castrated male rats. With this more local distribution of ATD, a lesser decline in sexual behavior was found, suggesting that other brain areas are involved in the neurohormonal activation of copulatory behavior in the male rat. To determine whether in vivo ATD interacts with androgen or estrogen receptors, we conducted cell nuclear androgen and estrogen receptor binding assays of hypothalamus, preoptic area, amygdala, and septum following treatment with the combinations of systemic T alone. ATD plus T, ATD alone, and blank control. In all four brain areas binding of T to androgen receptors was significantly decreased in the presence of ATD, suggesting that ATD may act both as an androgen receptor blocker and as an aromatization inhibitor. Competitive binding studies indicated that ATD competes in vitro for cytosol androgen receptors, thus substantiating the in vivo antiandrogenic effects of ATD. Cell nuclear estrogen receptor binding was not significantly increased by exposure to T in the physiological range. No agonistic properties of ATD were observed either behaviorally or biochemically. Thus, an alternative explanation for the inhibitory effects of ATD on male sexual behavior is that ATD prevents T from binding to androgen receptors.     

Regulation of uterine progesterone receptors by the nonsteroidal anti-androgen hydroxyflutamide

We have recently reported that the anti-androgen hydroxyflutamide causes delayed implantation and exhibits antideciduogenic activity in the rat. The present experiments were conducted to examine whether hydroxyflutamide binds to the uterine progesterone receptors and/or alters the progesterone binding sites in the uterus. Cytosol and nuclear fractions from decidualized rat uterus were incubated with [3H]-R5020 without or with increasing concentrations of radioinert R5020, RU486, dihydrotestosterone, or hydroxyflutamide. From the log-dose inhibition curves, the relative binding affinity of both hydroxyflutamide and dihydrotestosterone was less than 0.1% and 2%, compared with R5020 (100%) for displacing [3H]-R5020 bound to uterine cytosol and nuclear fractions, respectively. Injection of estradiol-17 beta (1 microgram/rat) to ovariectomized prepubertal rats induced a 1.85-fold increase in uterine weight by 24 h. Hydroxyflutamide at 2.5 or 5.0 mg did not significantly alter the estrogen-induced increase in uterine weight. Compared to vehicle alone, estrogen induced an approximately 5-fold increase in uterine cytosolic progesterone binding sites. Hydroxyflutamide at both 2.5- and 5.0-mg doses significantly attenuated the estrogen-induced elevation in uterine progesterone binding sites. These studies demonstrate that hydroxyflutamide does not bind with high affinity to progesterone receptors, but suppresses the estrogen-induced elevation in progesterone receptor levels in the uterus.     

Bioflavonoid interaction with rat uterine type II binding sites and cell growth inhibition

Competition analysis with a number of known bioflavonoids demonstrated that these compounds (luteolin, quercetin, pelargonin) compete for [3H]estradiol binding to cytosol and nuclear type II sites in rat uterine preparations. The inhibition of [3H]estradiol binding to type II sites was specific and these bioflavonoids did not interact with the rat uterine estrogen receptor. Since estradiol stimulation of nuclear type II sites in the rat uterus is highly correlated with cellular hypertrophy and hyperplasia, we assessed the effects of these compounds on the growth of MCF-7 human breast cancer cells in culture and on estradiol stimulation of uterine growth in the immature rat. The data demonstrated that addition of quercetin (5-10 micrograms/ml) to MCF-7 cell cultures resulted in a dose-dependent inhibition of cell growth (DNA/flask). This effect was reversible by removal of quercetin from the culture medium, or by the addition of 10 nM estradiol-17 beta to these cell cultures containing this bioflavonoid. Since estradiol-17 beta (10 nM) stimulated nuclear type II sites and proliferation of MCF-7 cells, we believe bioflavonoid inhibition of MCF-7 cell growth may be mediated through an interaction with nuclear type II sites. This hypothesis was confirmed by in vivo studies which demonstrated that injection of luteolin or quercetin blocked estradiol stimulation of nuclear type II sites in the immature rat uterus and this correlated with an inhibition of uterine growth (wet and dry weight). These studies suggest bioflavonoids, through an interaction with type II sites, may be involved in cell growth regulation.     

The estradiol-stimulated lipoprotein receptor of rat liver. A binding site that membrane mediates the uptake of rat lipoproteins containing apoproteins B and E

Hepatic catabolism of lipoproteins containing apolipoproteins B or E is enhanced in rats treated with pharmacologic doses of 17 alpha-ethinyl estradiol. Liver membranes prepared from these rats exhibit an increased number of receptor sites that bind 125I-labeled human low density lipoproteins (LDL) in vitro. In the present studies, this estradiol-stimulated hepatic receptor was shown to recognize the following rat lipoproteins: LDL, very low density lipoproteins obtained from liver perfusates (hepatic VLDL), and VLDL-remnants prepared by intravenous injection of hepatic VLDL into functionally eviscerated rats. The receptor also recognized synthetic lamellar complexes of lecithin and rat apoprotein E as well as canine high density lipoproteins containing apoprotein E (apo E-HDLc). It did not recognize human HDL or rat HDL deficient in apoprotein E. Much smaller amounts of this high affinity binding site were also found on liver membranes from untreated rats, the number of such sites increasing more than 10-fold after the animals were treated with estradiol. Each of the rat lipoproteins recognized by this receptor was taken up more rapidly by perfused livers from estrogen-treated rats. In addition, enrichment of hepatic VLDL with C-apoproteins lowered the ability of these lipoproteins to bind to the estradiol-stimulated receptor and diminished their rate of uptake by the perfused liver of estrogen-treated rats, just as it did in normal rats. The current data indicate that under the influence of pharmacologic doses of estradiol the liver of the rat contains increased amounts of a functional lipoprotein receptor that binds lipoproteins containing apoproteins B and E. This hepatic lipoprotein receptor appears to mediate the uptake and degradation of lipoproteins by the normal liver as well as the liver of estradiol-treated rats. The hepatic receptor bears a close functional resemblance to the LDL receptor previously characterized on extrahepatic cells.     

Progesterone regulation of estrogen receptor in the rat uterus: A primary inhibitory influence on the nuclear fraction

The purpose of this study was to determine the short-term effects of progesterone action on estrogen receptor (Re) levels in the rat uterus. Ovariectomized, adrenalectomized rats were maintained on subcutaneous Silastic implants containing crystalline estradiol. Progesterone treatment with serum estradiol maintenance caused a rapid decrease (within 4 h) of total Re, attributable to loss of nuclear Re without a significant change in cytosol Re levels. Removal of estradiol implants resulted in an increase in total Re and cytosol Re at all time periods studied without a significant decrease in nuclear Re until 8 h. Combined estradiol withdrawal and progesterone treatment resulted in lower total Re levels and a more rapid decrease in nuclear Re than with estradiol withdrawal alone. These results demonstrate that progesterone rapidly and selectively decreases nuclear Re levels in rat uterus and suggest that this process is not dependent on cytosol Re or serum estradiol levels.