Relations between 3H-estradiol uptake and receptor content of estrogen responsive tissues of castrated female rat

The time course of 3H-Estradiol-17 beta (3H-E2) uptake, and estrogen receptor content in estrogen responsive tissues were studied between 0 and 12 h after injection of 0.5 microgram/kg of 3H-E2 or cold E2 injection to castrated adult female rats. The plasma concentration of 3H-E2 between 10 min and 2 h after injection was in the range of the plasma E2 level of cyclic rat. The total 3H-E2 uptake was well correlated with the receptor content in all tissues. The rank order of 3H-E2 uptake was: uterus (Ut) greater than anterior pituitary (Ap) greater than hypothalamus (Ht) greater than plasma. The cytosol 3H-E2 uptake showed its maximal level 10 min after injection in all tissues. Parallel time course between plasma 3H-E2 and cytosol uptake was obtained for each separate tissue. The nuclear 3H-E2 uptake showed its maximal values 2 h after injection with a subsequent decline. Cytosolic estrogen receptor (Rc) content showed a depletion-replenishment cycle after cold E2 injection in all tissues. Nuclear estrogen receptor (Rn) content in Ut increased progressively from 0 to 14 h after injection, but in Ap it showed its maximal level 2 h after injection, declining afterwards. In Ap, nuclear 3H-E2 uptake and Rn level showed parallel time courses. The maximal level of both parameters coinciding with the time of maximal Rc depletion. However, the Rn level in Ut increases more slowly at greater length than the nuclear 3H-E2 uptake, both processes being divergent. These findings are interpreted as the expression of tissular differences in the rate of nuclear receptor formation from the Rc-E complex previously translocated into nucleus and attached to chromatin.     

Kinetics of catechol estrogen-estrogen receptor dissociation: a possible factor underlying differences in catechol estrogen biological activity

The mechanisms underlying the differences in uterotrophic potency between 2- and 4-hydroxyestrogens were explored. Doses of estradiol (E2)(10 micrograms/kg), 2-OHE2 (500 micrograms/kg) and 4-OHE2 (100 micrograms/kg) sufficient to induce near maximal cell nuclear estrogen receptor (ERn) binding were injected subcutaneously into 26 day old female rats. Uterine ERn concentrations declined more rapidly after 2-OHE2 than after E2 or 4-OHE2. E2 and 4-OHE2 both elicited a significant increase in uterine wet weight, measured at 24-36 hrs after injection. 2-OHE2 had no significant effect and neither synergized with nor antagonized the effects of simultaneously administered E2 or 4-OHE2. Under in vitro conditions at 25 degrees C, 2-hydroxyestrone (2-OHE1) and 2-OHE2 both dissociated from the receptors more rapidly than either their parent monophenolic estrogens or the corresponding 4-hydroxyestrogens. These results suggest that differences in estrogenic potency between 2- and 4-hydroxyestrogens may partly be a function of the dissociation kinetics of their estrogen receptor complexes.     

Effect of estradiol and other endocrine factors on the level and dynamics of estrogen receptors in the liver cells of rats

The authors studied the effects of different doses of estradiol (E2) on the level of estrogen receptors (ER) in female rat hepatocytes and the dynamics of ER distribution between the cytosol and nuclear cell fractions and compared the changes in the ER level in the liver in different endocrine states of the body. It was shown that the ER level in hepatocytes was far lower than in uterine cells and drastically increased during puberation and after ovariectomy. It was also found that the ER level in hepatocytes was dependent on pituitary functions. After a single injection of E2, the ER level in cytosol descended and that in the nucleus rose. The degree of ER reduction in cytosol and increase in the nucleus correlated with the dose of E2. The dynamics of intercombined changes in the levels of cytosol and nuclear ER was studied at different time intervals following the injection of different doses of the hormone. Some essential organ-specific features of E2 reception in the liver were revealed. Different mechanisms of the control of ER content in the liver and uterus are postulated.     

Antagonism of estrogen-induced prolactin release by progesterone

Previous work from our laboratory has shown that during the process of nuclear occupancy of the progesterone receptor complex (1-2 h), nuclear estradiol receptors of the anterior pituitary are depleted. The purpose of this study was to determine whether the depletion of nuclear estradiol receptors by progesterone had functional biological significance. The ovariectomized (26 days of age) immature rat was used as the model for analysis of this question. The ability of estradiol to release prolactin from the anterior pituitary was the function chosen to determine the biological significance of the progesterone and estradiol interactions. In response to estradiol exposure (2 micrograms/rat), prolactin release reached peak values from 8 h to 12 h and returned to control levels by 24 h. A second injection of estradiol 13 h after the initial injection stimulated a second increase in serum prolactin at 25 h. This model of two injections of estradiol 13 h apart served to provide adequate levels of anterior pituitary progesterone receptors and elevated serum prolactin levels upon which superimposed progestin modulation could be examined. A single injection of progesterone (0.8 mg/kg BW) 1 h before the second estradiol injection blocked the increase in serum prolactin. This action was a receptor-mediated event because progesterone had no effect without estrogen priming or when the progesterone antagonist RU486 was used. Finally, when the interval between the progesterone and second estradiol injection was extended to 4 h, a time period when progesterone does not deplete pituitary nuclear estrogen receptors, the estrogen-induced increase in serum prolactin was not blocked.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased LH pulse frequency and estrogen secretion associated with termination of anestrus followed by enhancement of uterine estrogen receptor gene expression in the beagle bitch

The relationships among pulsatile LH secretion pattern, estrogen secretion, and expression of the uterine estrogen receptor gene were examined throughout the estrous cycle in beagle bitches. In Experiment 1, blood samples were collected from 30 bitches every 10 min for 8 h from a cephalic vein during different phases of the estrous cycle. An increase in the mean plasma levels of LH occurred from mid to late anestrus (P < 0.01). The LH pulse frequency increased (P < 0.01) from late anestrus to proestrus, and was strongly correlated (r = 0.96, P < 0.001) with the mean plasma level of estradiol-17 beta (E2). In Experiment 2, middle uterine samples, including the myometrium and endometrium, from 18 bitches were taken at 6 stages of the estrous cycle. The total number of estrogen receptors and nuclear estrogen receptor and its mRNA levels in the uterus also increased (P < 0.01) from late anestrus to proestrus. Mean plasma E2 level and the number of uterine estrogen receptor were positively correlated (r = 0.81, P < 0.05). In Experiment 3, nine bitches were ovariectomized in mid anestrus. Two weeks later they received a single injection of 10 or 50 micrograms/kg, i.m., estradiol benzoate. The number of uterine estrogen receptor and their mRNA levels for ovariectomized bitches were low, but increased (P < 0.05) after treatment with a low dose of estradiol benzoate. These results suggest that increases in LH pulse frequency and estrogen secretion are associated with termination of anestrus and that subsequent enhancement of uterine estrogen receptor expression may be up-regulated by estradiol.     

Estrogen receptors in the rat uterus. Retention of hormone-receptor complexes.

The retention pattern and biochemical characteristics of estrogen receptors in the nuclei of uterine cells were studied as a function of time after the in vivo injection of estradiol (E2) to immature female rats. One hour after the injection of 0.1 mug of tritiated E2, approximately 0.20 pmol per uterus of receptor bound hormone is retained in uterine nuclei. This dose of E2 produces a maximal uterotrophic response. Six hours after E2 administration, uterine nuclei retain 0.04-0.08 pmol of hormone per uterus. Hormone receptor complexes extracted from uterine nuclei 1, 3, and 6 h after in vivo injection of hormone have similar structural and binding characteristics. Receptors extracted at all three times sediment at 5S in high salt gradients and have a dissociation binding constant of approximately 3 nM for E2. The wash-out curves of receptors as a function of salt concentration are identical for uterine nuclei from animals treated for 1 or 6 h with estradiol, suggesting that the nature of the nuclear binding of receptors is not altered during this time interval. Experiments utilizing the injection of unlabeled estradiol, followed by an in vitro exchange procedure with tritiated estradiol, indicated that the total nuclear estrogen receptor sites, i.e., filled and vacant, decreased similarly.     

Enucleation of human endometrial cells: nucleo-cytoplasmic distribution of DNA polymerase alpha and estrogen receptor

Nucleo-cytoplasmic distribution of estrogen receptors and DNA polymerase alpha activity in human endometrial adenocarcinoma cells (HEC-50 line) was evaluated after separation of nuclei following either homogenization or enucleation with cytochalasin B. About 30% of the estrogen receptor was found in the nuclear fraction after homogenization whereas 86% was found in the karyoplasts after enucleation. The total amounts of estrogen receptor per cell after homogenization and enucleation were not significantly different (14,000-17,000 binding sites/cell). Receptor measurements were carried out using the hydroxylapatite method after labeling with [3H]estradiol (5 nM [3H]E2 +/- 500 nM E2) at 30 degrees C for 3 h. About 20% of the DNA polymerase alpha activity was found in the nuclear fraction after homogenization, whereas 96% was found in the karyoplasts after enucleation. The average total activity (0.84 Units/10(6) cells) in homogenized cells was about 1/8 of the activity in karyoplasts. These results indicate that estrogen receptor and DNA polymerase alpha activity reside in the nucleus in intact HEC-50 cells. DNA polymerase alpha is translocated to the cytoplasmic fraction and inactivated after homogenization.     

Sexual receptivity in hamsters: brain nuclear estrogen and cytosolic progestin receptors after single and multiple steroid treatments and during the estrous cycle

This series of experiments investigated the relationship between various treatments consisting of estradiol benzoate (EB) and progesterone (P) on sexual receptivity and on concentrations of nuclear estrogen receptors (NER) and cytosolic progestin receptors (CPR) in the hypothalamus-preoptic area in female hamsters. The injection of 1 microgram EB at 0 and 24 hr resulted in higher levels of receptivity (after 0.25 or 0.5 mg P), NER and CPR compared to those obtained after a single injection of 2 micrograms EB. Animals treated with 5 micrograms EB at 0 and 24 hr displayed greater levels of receptivity (after 0.5 mg P) and had higher NER concentrations than animals given a single injection of 10 micrograms EB. Groups treated with either 1 microgram EB at 0 hr or 0.5 microgram EB at 0 and 24 hr did not differ and showed low levels of receptivity, NER and CPR, NER and CPR were also measured on each day of the estrous cycle. NER levels rose between Days 1 and 2, again between Days 2 and 3, and remained elevated on Day 4. CPR levels increased between Days 2 and 3, and there was no difference between Days 3 and 4. Taken together, these data suggest that receptivity in hamsters after estrogen exposure is correlated with the accumulation and maintenance of relatively high NER levels and on the induction of CPR. This can be accomplished by a single large injection of EB or by smaller split doses.     

脑室注射白介素-1β引起的热痛敏作用

实验在SD大鼠上应用脑室微量注射和辐射热测痛的方法,研究了脑内微量注射白介素－1β对大鼠痛阈的影响。实验大鼠分为给药组和对照组,在给药组大鼠脑室注射不同剂量的白介素－1β（5、50和500pg/kg),对照组大鼠脑室注射配药液。白介素－1受体拮抗剂（IL－1ra,50ng/kg)在脑室注射白介素－1β前20min给予。实验以大鼠对光热刺激引起的缩爪反射潜伏期为痛阈指标。结果表明,脑室注射白介素－1β可显著缩短大鼠对光热刺激的缩爪反射潜伏期,并具有剂量依赖性关系。脑室给予500pg/kg的白介素－1β 20min后,大鼠对光热刺激的缩爪反射潜伏期显著缩短,40min时达峰值,然后逐渐恢复。该作用可被白介素－1β受体拮抗剂阻断。结果提示脑中白介素－1β可通过作用于白介素－1受体引起热痛敏作用。     

Effect of the aromatase inhibitor, 4 hydroxyandrostenedione, on the endotoxin-induced changes in steroid hormones in male rats.

The increase in circulating estrogen concentrations that follows injection of Escherichia coli endotoxin (Endo) may be due to increased aromatase activity. We have therefore analysed the effect of the aromatase inhibitor, 4 hydroxyandrostenedione (4OHA) on the steroid hormone response of male rats, particularly the dramatic increase in estrogens and decrease in androgens, induced by Endo. The concentrations of corticosterone (B), progesterone (P4), 17 alpha hydroxyprogesterone (17 alpha OHP4), androstenedione (delta 4), testosterone (T), estrone (E1) and estradiol (E2) were determined 2 hours after injection of increasing doses of 4OHA with and without Endo. The increase in serum estrogen concentrations and drop in serum androgen levels in response to Endo were blocked by a single dose of 4OHA. The effect of 4OHA appeared to be dose dependent. Low doses (30 mg/kg and 50 mg/kg) induced significant changes in the estrogen and androgen responses, but the high dose (100 mg/kg) blocked all changes in sex steroids induced by Endo. 4OHA did not alter the Endo-induced changes in other steroids.     

Detection of gamma-hydroxybutyrate in striatal microdialysates following peripheral 1,4-butanediol administration in rats

The illicit use and abuse of 1,4-butanediol (1,4-BD) results from its presumed conversion to gamma-hydroxybutyrate (GHB) and subsequent pharmacological effects via action on GABA-B and GHB-specific receptors. Using in vivo microdialysis we measured the appearance of GHB in the striata of rats after peripheral 1,4-BD administration. We developed and utilized an HPLC-UV (215 nm) detection of GHB that yielded a limit of quantification (S/N=10) of 2.0 micro g/mL (40 ng/injection) and a limit of detection (S/N=3) of 0.75 micro g/mL (15 ng/injection). GHB appeared in the striatal microdialysates within 20 min after intraperitoneal (i.p.) administration of varying doses of 1,4-BD. GHB concentrations reached dose-dependent maxima 80-100 min post-1,4-BD administration, with peak values of 10.6+/-2.9, 25.3+/-3.4 and 48.1+/-7.1 micro g/mL (mean+/-S.E.M.), corresponding to 1,4-BD doses of 250, 500 and 750 mg/kg, respectively. The conversion of 1,4-BD to GHB was completely prevented by the alcohol dehydrogenase inhibitor 4-methylpyrazole (4MP), administered prior to 1,4-BD, as evidenced by the failure of GHB to appear in the striatal microdialysates. Sleep times in animals were similarly correlated with GHB concentrations in the microdialysates.     

Psychotropic effects of glutamic acid diethyl ester in mice

In 1 hour after intraperitoneal injection glutamic acid diethyl ester (GED) in doses 200 and 500 mg/kg decreased locomotor activity and exploratory patterns of mice in "open field" test. GED in doses 100 and 200 mg/kg diminished the immobilization period of animals in forced swimming test, that proves the reversal interaction of glutamatergic and catecholaminergic systems in CNS. Glutamate receptors antagonist--GED in doses 100-200 mg/kg disrupted passive avoidance reaction at 30 min before acquisition and retrieval, therefore glutamate receptors are involved into fixation and retrieval of memory engram.     

Correlation between LH and estrogen receptor turnover in pituitary and hypothalamus of castrate rats following estrogen agonists and antagonists

A series of studies was undertaken to correlate the short-term dynamics of LH secretion and depletion-replenishment patterns of estrogen receptors (ER) in hypothalamic and pituitary cytosols of ovariectomized rats. Animals castrated for 2 weeks were administered various test compounds and analyzed at 1, 3, 5, 10 and 15 h post-treatment. A single injection of 10 micrograms 17 beta-estradiol (E2) to ovariectomized rats elicited a rapid depletion of ER in both pituitary and hypothalamus and a dramatic, though delayed, fall in serum LH. ER replenishment occurred in both tissues through 15 h and LH recovered in a similar manner. When cycloheximide was administered along with E2, ER replenishment was completely inhibited in both tissues; serum LH fell and failed to recover. Actinomycin D injected with E2 blocked replenishment in pituitary but not hypothalamus; serum LH recovered in parallel with the hypothalamic ER pattern. 17 alpha-E2 elicited only slight changes in ER and LH was suppressed 10-20% through 15 h. CI-628 caused a near total depletion of pituitary ER with no subsequent replenishment, whereas hypothalamic ER content was virtually unaltered; serum LH was suppressed and later recovered. Orchidectomized rats given 5 micrograms E2 demonstrated a less complete ER depletion in hypothalamus, and an earlier replenishment than that seen in pituitary or hypothalamus of similarly treated ovariectomized females. Serum LH rebounded to 157% of control levels at 15 h. The results indicate that the acute feedback suppression of LH by exposure to estrogens correlates with binding to ER and nuclear translocation. Replenishment and/or retention of cytoplasmic ER in hypothalamus appears to be required for full resumption of LH secretion, following acute suppression.     

Acute Administration of Non-Classical Estrogen Receptor Agonists Attenuates Ischemia-Induced Hippocampal Neuron Loss in Middle-Aged Female Rats

Background

Pretreatment with 17β-estradiol (E2) is profoundly neuroprotective in young animals subjected to focal and global ischemia. However, whether E2 retains its neuroprotective efficacy in aging animals, especially when administered after brain insult, is largely unknown.

Methodology/Principal Findings

We examined the neuroprotective effects of E2 and two agonists that bind to non-classical estrogen receptors, G1 and STX, when administered after ischemia in middle-aged rats after prolonged ovarian hormone withdrawal. Eight weeks after ovariectomy, middle-aged female rats underwent 10 minutes of global ischemia by four vessel occlusion. Immediately after reperfusion, animals received a single infusion of either E2 (2.25 µg), G1 (50 µg) or STX (50 µg) into the lateral ventricle (ICV) or a single systemic injection of E2 (100 µg/kg). Surviving pyramidal neurons in the hippocampal CA1 were quantified 1 week later. E2 and both agonists that target non-classical estrogen receptors (G1 and STX) administered ICV at the time of reperfusion provided significant levels of neuroprotection, with 55–60% of CA1 neurons surviving vs 15% survival in controls. A single systemic injection of a pharmacological dose of E2 also rescued approximately 50% of CA1 pyramidal neurons destined to die. To determine if E2 and G1 have similar mechanisms of action in hippocampal neurons, we compared the ability of E2 and G1 to modify CA1 pyramidal neuron responses to excitatory inputs from the Schaffer collaterals recorded in hippocampal slices derived from female rats not subjected to global ischemia. E2 and G1 (10 nM) significantly potentiated pyramidal neuron responses to excitatory inputs when applied to hippocampal slices.

Conclusions/Significance

These findings suggest (1) that middle-aged female rats retain their responsiveness to E2 even after a long period of hormone withdrawal, (2) that non-classical estrogen receptors may mediate the neuroprotective actions of E2 when given after ischemia, and (3) that the neuroprotective efficacy of estrogens may be related to their modulation of synaptic activity in hippocampal slices.     

An Exchange Assay for the Measurement of Cell Nuclear Estrogen Receptors in Microdissected Brain Regions

An exchange assay is described for the measurement of nuclear estrogen receptors (ERn) in microdissected brain regions. The distribution of ERn in the hypothalamus and amygdala of the rat 1 h after an injection of estradiol (E) is presented. Combining the exchange assay with a previously described method for measurement of cytosol estrogen receptors (ERc) in microdissected brain samples, gonadectomized male and female rats were compared for ERc and ERn. While ERc concentrations tended to be higher in females than in males in all regions of the hypothalamus, with a significant sex difference in the arcuate-median eminence, no sex difference in ERn concentrations was observed after E injection. These results suggest that ERc measurements alone are not sufficient to establish the capacity of the E receptor system: ERn measurements are also necessary to establish the relationship between receptor levels and physiologic estrogen responsiveness.     

Diabetes modulates differentially creatine kinase-specific activity responsiveness to estradiol-17beta and to raloxifene in rat organs

Diabetes mellitus increases the risk for CVD in women. While there is considerable evidence suggesting beneficial effects of estrogen on decreasing lipid peroxidation, atherosclerotic processes, and cardiovascular diseases, diabetes negates most estrogen protective effects as well as the skeletal protective effects of estrogens, which are not discernable in diabetic women. In the present study, we examined the in vivo effects of estradiol-17beta (E2), on creatine kinase (CK)-specific activity, in estrogen-responsive organs from healthy and diabetic rats. Healthy or diabetic (streptozotocin-induced) female rats were injected with either E2 (10-50 microg/rat) or raloxifene (Ral; 500-1,000 microg/rat). Twenty-four hours following the injection, animals were sacrificed; their organs removed and assayed for CK-specific activity. CK-specific activity in different organs [Left ventricle of heart (Lv), uterus (Ut), aorta (Ao), para uterine adipose tissue (Ad), epiphyseal cartilage (Ep), and diaphyseal bone (Di)] from healthy animals, was stimulated with increased doses of E2, with maximum at 20 microg/rat. Age-matched diabetic female rats exhibited a remarkable decreased response to E2 in all organs except Ut. In contrast, the response to Ral was not altered in diabetic rats. Similar results were observed in organs from ovariectomized female rats (Ovx), healthy or diabetic. These results support our previous in vitro findings, demonstrating that hyperglycemia decreases CK response to E2 but not to Ral in cultured human vascular and bone cells. In summary, diabetes mellitus decreases CK response to E2 but not that of Ral in skeletal and vascular tissues. The decreased response to E2 detected in organs derived from diabetic rats might be due to changes in nuclear and/or membrane estrogen receptors and/or other genomic and non-genomic pathways, as was shown in in vitro cellular models.     

Regulation of cytosol and nuclear progesterone receptors in rabbit uterus by estrogen, antiestrogen and progesterone administration.

A synthetic progestin, 16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione (ORG 2058), was utilized to measure progesterone receptors from the rabbit uterus. This steroid has a high affinity for both cytosol and nuclear receptors, with KD values of 1.2 nM (at 0--4 degrees C) and 2.3 nM (at 15 degrees C), respectively. Administration of estradiol-17 beta or a non-steroidal antiestrogen, tamoxifen, for 5 days to estrous rabbits led to a progressive rise in the cytosol receptor levels: from 34,000 to 120,000 (estradiol-17 beta) and 80,000 (tamoxifen) receptors/cell, without any major influence on the nuclear receptor content. A single intravenous injection of progesterone (5 mg/kg) elicited a 3-fold increase in the mean nuclear receptor content at 30 min after injection (from 18,000 to 48,000 receptors/nucleus). Nuclear receptor accumulation was short-lived and returned to control levels within 4 h after treatment. A second dose of progesterone given 24 h later doubled the nuclear receptor level (from 18,000 to 35,000 receptors/nucleus). The concomitant decline in the cytosol receptor content was twice that accounted for by the nuclear receptor accumulation (70,000 vs. 30,000, and 40,000 vs. 17,000 receptors/cell, after the first and second progesterone injection, respectively). Following progesterone administration, the cytosol receptor level reached a nadir by 30 min, exhibited minimal replenishment within the ensuing 24 h, and remained at approx. 50% of the pretreatment values. After a single dose or two consecutive doses of progesterone, total uterine progesterone receptor content declined to about 60% of the level prior to each dose, a nadir being reached at 2 h after treatment.