Receptor-estrogen complex in the nuclear fraction of rat uterine cells during the estrous cycle.

A quantitative method was used to determine the concentration of receptor-estrogen complex in the nuclear fraction of rat uterine cells throughout the estrous cycle. The concentrations of nuclear receptor-estrogen complex were: metestrus, 0.22; diestrus, 0.75; proestrus, 1.29; and estrus, 0.31 picomoles per milligram of DNA. This cyclic fluctuation in the nuclear complex closely parallels the secretion of ovarian estrogen during the estrous cycle, an indication that the accumulation of receptor-estrogen complex by the nuclear fraction of uterine cells may be of physiological significance, and under the control of endogenous estrogen.

Effect of ionic strength on charcoal adsorption assays of receptor-estradiol complexes.

The effect of increasing ionic strength on the efficacy of the charcoal adsorption assay for estrogen receptors has been examined. Cytosol prepared from immature rat uteri was exposed to variable concentrations of KCl and the saturable or "specific" binding of [3H]estradiol was measured by charcoal and hydroxylapatite adsorption, gel filtration, and/or density gradient centrifugation using excess diethylstilbestrol to correct for non-saturable or "non-specific" binding. As the concentration of KCl was increased, the number of estradiol binding sites as measured via charcoal adsorption decreased in proportion to the conversion of receptor from a low salt (8S) to a high salt (4S) complex. This "stripping" of [3H]estradiol from the 4S receptor species was both time- and charcoal concentration-dependent. It is concluded that the charcoal adsorption procedure quantitatively assesses the binding of [3H]estradiol to low salt, 8S receptor species but is not the assay of choice for salt-extracted or salt-treated receptors in view of the potential for artifactually low estimates of receptor number. Low values for receptor number under high salt conditions may result from exposure of the binding site of the estrogen receptor to charcoal.

Nuclear binding and retention of the receptor estrogen complex: relation to the agonistic and antagonistic properties of estriol.

The relationship between nuclear retention or residency or receptor estrogen complexes and the agonistic and antagonistic properties of estriol was examined. Cytoplasmic estrogen receptor (Rc) and uterine weight were measured 24 and 48 h after treatment with estradiol (E2), estriol (E3), or or both hormones (E2 and E3). Levels of Rc were elevated in all groups at either time. Since levels of Rc were above control in each case, Rc does not appear to be the limiting factor in the antagonistic effect of estriol. Therefore, the effect of these steroids on translocation and retention of nuclear receptor estrogen complexes (RnE) was examined. Rats were injectd as above and RnE measured by 3[H] estradiol exchange 1, 3, and 6 h after injection. All treatments cause equal translocation of receptor of the nuclear compartment. However, by 6 h the quantity of Rn present in E2 treated animals was significantly above controls while that in E2 + E3 animals was intermediate. E3 alone failed to cause significant nuclear retention of Rn. These patterns of retention correlate with the weight and cytoplasmic receptor responses above and suggest that the short nuclear residency time of RnE3 complexes relates to the antagonism of E3. To test this, these estrogens were administered via paraffin pellets to maintain blood levels of each hormone. Levels of Rn were elevated 24 and 48 h after implant with no significant differences between treatment groups. Likewise, there were no differences in the growth response. Uterine weights were highly stimulated in all three cases (300% above control). These results indicate that E3 acts as an estrogen antagonist when injected as a bolus because of the short nuclear retention time of RnE3 complexes. However, when E3 is present continuously and RnE3 is elevated and maintained, E3 is a potent estrogen without antagonistic properties.

Estrogen-induced uterine responses and growth: relationship to receptor estrogen binding by uterine nuclei.

The relationship between estrogen receptor(R) binding by uterine nuclei and uterotrophic responses was examined. Immature rats received a single injection of estradiol (E2) or estriol (E3) and the following parameters were measured: accumulation and retention of the estrogen receptor by the nuceus of uterine cells; incorporation of 14C-glucose into CO2 lipid, protein and RNA; RNA polymerase activity; water imbibition and increased dry weight. E2 and E3 were of equal potency with regard to the rapid accumulation of R by the nucleus but differed with respect to long term retention of R. The concentrations of nuclear RE2 and RE3 complexes were equivalent between 1 and 3 hr after estrogen injection; however, by 6 hr RE2 remained significantly elevated while RE3 levels had fallen to control values. E2 and E3 were also of equal potency with respect to the stimulation of enhanced glucose utilization, water imbibition, the incorporation of 14C-glucose into lipid, protein and RNA 3 hours following an injection of the hormone. Likewise the activity of RNA polymerase was equally stimulated by E2 and E3 3 hr after injection. Thus all early uterotropic responses (0-3 hrs) that were measured were equally stimulated by E2 and E3. However, E3 failed to stimulate true uterine growth (increase dry weight 24 hr after injection), whereas E2 produced a significant stimulation of true uterine growth. These data suggest that the RE complex is capable of stimulating early uterotrohic events regardless of which estrogen is present; however, in order to produce true uterine growth the RE complex must be retained in the nucleus for long periods of time. This proposal was tested by the administration of repetitive injections of E3. This treatment resulted in an increase in dry weight that was equivalent to the growth that was produced by repetitive injections of E2. These results demonstrate that E2 and E3 elicit early uterotrophic responses with equal facility following a single injection but that only E2 causes true uterine growth. The ability of E2 to stimulate true uterine growth appears to be related to the time of residence of the RE complex in the nucleus.

Control of uterine estrogen receptor levels by progesterone.

The mechanism by which progesterone antagonizes estrogenic stimulation of uterine growth was examined in the immature rat. Rats received daily injections of 2.5 mug estradiol (E) for 2 days and on day 3 either 2.5 mug E or 2.5 mug E plus 2.5 mg of progesterone (P). The quantity of nuclear and cytoplasmic estrogen receptor was determined by [3H]estradiol exchange at various intervals after injection of E or E + P. In both groups, nuclear receptor estrogen complex (RnE) increased dramtically one hour after injection and showed a gradual decline from 4 to 24 h after injection. The quantity of cytoplasmic receptor, Rc, decreased to low levels by one hour and began a gradual increase from 4 to 8 h in both groups. However, between 8 and 24 h after injection, the level of Rc continued to increase in the E treatment group (2.39 +/- 0.21 pmol/uterus at 24 h) but remained at the 8 h level in the E + P group (1.09 +/- 0.04 pmol/uterus at 24 h). This observation suggests that two seperate processes are involved in the replenishment of Rc and that progesterone inhibits the second phase of replenishment. The binding affinity and specificity of Rc for estrogens following E + P pretreatment were identical to those of the E pretreatment group. Therefore, P does not alter the binding properties but rather the intrauterine level of Rc. Treatment with E on day 4, when Rc levels differ between E and E + P groups, stimulated uterine weight and protein content on day 5 in the E pretreatment group. However, minimal stimulation was observed in the E + P pretreatment group. The quantity of RnE and the time of nuclear retention of RnE following E injection on day 4 was greater in the E group than in the E + P group. The effect of progesterone on Rc replenishment was dose-dependent (range, 0.1-2.5 mg; 1/2 maximal, 0.5 mg). Injection of testosterone propionate (1.0 mg), a weak estrogen antagonist, with E on day 3 resulted in slightly reduced levels of Rc on day 4. This reduction also correlated with a reduced sensitivity to treatment with E on day 4. These data, together with previous studies from our laboratory, suggest that progesterone and other estrogen antagonists such as nafoxidine and testosterone propionate inhibit estrogen action by interfering with the replenishment of Rc, thereby reducing the number of receptor estrogen complexes that are translocated and retained by uterine nuclei.

Human anti-estrogen receptor antibodies: assay, characterization, and age- and sex-related differences.

A binding assay was developed to measure the estrogen receptor (ER)-binding activity in serum. The method employed immunoglobulin G (IgG) adsorption with protein A-bearing Staphylococcus aureus cells before incubation with tritium-labeled ER. Specificity studies employing chromatographic and electrophoretic analyses suggested that an IgG antibody (Anti-ER) was responsible for the serum activity. Anti-ER was found in human, rat, and mouse serum and exhibited species cross-reactivity. The antibody recognized both the 8S and 5S forms of the ER. Anti-ER was measured in 262 individuals ranging in age from 1-85 yr. The antibody was detected in all serum samples examined, suggesting its natural occurrence. When the study group was divided into three arbitrary age groups (young, 1-13 yr; middle age, 13-51 yr; older, greater than 51 yr), significant differences in levels of antibody were found, with highest levels in the young, followed by the elderly, and lowest levels in the middle years. An examination of sex- as well as age-related differences in the population revealed a striking sex difference. Thus, the young male population had a lower level of antibody than the corresponding female population, and these levels in males declined throughout life to reach their lowest point in old age, whereas the high levels in young females declined in middle years and then increased significantly in the elderly. We postulate that the antibody is a subgroup of IgG of multifactorial etiology. The idiotypic network theory offers an explanation for the occurrence of these antibodies in normal serum, whereas an autoimmune mechanism could account for the secondary rise of anti-ER in the aging female population.

In vitro LHRH release: correlation with the LH surge and alteration by a mammary carcinogen.

The releasability of LHRH from neurosecretory terminals, as assessed in vitro by depolarization of hypothalamic synaptosomes with elevated K+, varies as a function of the time of day in parallel with the LH surge. Pretreatment of ovariectomized female rats with dimethylbenz(a)anthracene (DMBA) results in a blockade of the estrogen-induced LH surge and a blunting of LHRH release as measured in vitro. An examination of the LHRH release process revealed that K+-stimulated release of particulate LHRH was inhibited by DMBA pretreatment while basal release of particulate LHRH and both basal and K+-stimulated release of soluble LHRH were not altered. These results suggest that release of particulate LHRH is closely coupled with the estrogen-induced LH surge.

Estrogen and the subcellular distribution of luteinizing hormone releasing hormone: rate sedimentation studies.

Rate sedimentation of the 900 X G supernatants (S1) of hypothalamic homogenates from untreated male rats or ovariectomized rats with or without 5 micrograms estradiol benzoate (EB) revealed two populations of LHRH particles: a minor, slowly sedimenting one (peak 1) and a major, more rapidly sedimenting one (peak 2). Some LHRH-containing material also sedimented to the bottom of the gradient. The ovariectomized rats displayed more heterogeneity of particulate LHRH than did the male rats. Furthermore, the administration of EB to ovariectomized rats altered the relative sedimentation pattern of LHRH. In ovariectomized rats, hypotonic shock of S1 prior to rate sedimentation eliminated peak 2 and post-peak 2 LHRH and increased free LHRH at the top of the gradient. Peak 1 LHRH was still present and was elevated after EB treatment. Also, EB treatment lowered the free LHRH at the top of the gradient. These data demonstrate that the administration of EB to an ovariectomized rat alters the subcellular distribution of LHRH.

Kinetic and pharmacologic characterization of gamma-aminobutyric acid receptive sites from mammalian brain.

Sodium-dependent (+Na) and sodium-independent (-Na) receptive sites for gamma-aminobutyric acid (GABA) have been characterized using synaptic plasma membranes from bovine and rat brain. Synaptic plasma membranes were prepared from either rat cerebellar cortex or calf cerebral cortex by discontinuous sucrose gradient flotation centrifugation of crude mitochondrial pellets, and assayed using equilibrium ligand binding assays to obtain the maximum binding capacity (Bmax) and the thermodynamic constant (KD). Values for KD from equilibrium studies were subsequently confirmed by kinetic analyses of association and dissociation reactions. The KD for +Na GABA binding (5.0 +/- 0.2 micron) corresponds to the apparent Michaelis constant for neuronal GABA transport (3.8 +/- 0.1 micron)22, while the KD for -Na binding (0.17 +/- 0.04 micron) agrees with that determined by Enna and Snyder for the putative postsynaptic receptor. Maximal binding activities of about 5 and 55 pmole/mg protein were obtained for -Na and +Na binding respectively. The pharmacologic specificities of the two sites were determined using competition binding studies. Nipecotic acid and diaminobutyric acid inhibit both synaptosomal GABA uptake (Ki approximately 25 micron and 120 micron respectively) and +Na binding of GABA to synaptic plasma membrane (IC50 approximately 40 micron and 350 micron respectively) but do not inhibit -Na binding. Bicuculline inhibits -Na [3H]GABA binding at low concentrations (IC50 approximately 15 micron), while affecting the uptake and +Na binding of [3H]GABA only at high concentrations (IC50 approximately 520 micron and 300 micron respectively). beta-Alanine inhibits the -Na binding site (IC50 approximately 100 micron), but is ineffective at the +Na binding site and does not interfere with synaptosomal uptake of GABA. Finally, chlorpromazine and N-ethylmaleimide inhibit the +Na binding, albeit at high concentrations (IC50 approximately 600 micron and 5 mM respectively) but are ineffective at the -Na binding site. From these results the -Na binding site is tentatively identified as a postsynaptic receptor and the +Na binding site is identified as the neuronal uptake receptive site.

Biphasic response of hippocampal pyramidal neurons to GABA.

GABA released either iontophoretically or synaptically near pyramidal neurons in the CA1 region of the rat hippocampal slice could produce a biphasic response: a hyperpolarization followed by a depolarization. The depolarizing component elicited by either method was accompanied by an increased membrane conductance, and a reduction in neuronal discharge. The depolarization was reversed at a potential which was less negative than the resting membrane potential; it was blocked by antagonists of GABA action such as picrotoxin; it was sensitive to manipulation of extracellular chloride concentration; and it persisted in the presence of concentrations of cobalt or manganese which were sufficient to block evoked synaptic activity. Iontophoresis of GABA near the apical dendrites elicited an initial depolarization rather than an initial hyperpolarization, suggesting a dendritic origin for the depolarizing component. Together, these results suggest that GABA can produce, in the same neuron, both hyperpolarizing and depolarizing responses which depend at least in part upon changes in chloride conductances.

Pentobarbital and synaptic high-affinity receptive sites for gamma-aminobutyric acid.

Electrophysiological investigations of others show that pentobarbital enhances the inhibitory inflences of gamma-aminobutyric acid (GABA). Specifically, receptor activation is amplified and prolonged, suggesting the presence of an increased number of GABA molecules in the synaptic cleft. Either inactivation of high-affinity GABA transport or alteration of post-synaptic GABA receptors might account for these influences of pentobarbital. In this sudy the effect of pentobarbital on high-affinity uptake and binding of GABA to synaptic receptive sites has been examined. Using synaptosomes and subsynaptosomal fractions of cerebral cortex and hippocampus, it si shown that concentrations of pentobarbital, exceeding 1 mM have no appreciable effect on GABA uptake or binding. Thus the synaptic influence of pentobarbital, evident at 0.1 mM in electrophysiologic experiments, must originate from mechanisms other than the high-affinity uptake or binding of GABA. Possible sites of action include the presynaptic release of GABA and the ionophores coupled with postsynapitc sites.

The residual effect of chronic neuroleptic treatment on the neuroleptic binding assay in rats.

Chronic chlorpromazine administration to rats (25 mg/Kg/day) for 30 days followed by a washout period of 10 days resulted in an increase in both the measured maximum number of binding sites, Bmax, and the apparent dissociation constant, Kd, for the binding of 3H-spiroperidol to neural membranes of the brain. When membrane suspensions were progressively diluted before the binding assay, it was found that the apparent Bmax did not change with dilution, remaining higher in membranes of chlorpromazine-treated rats than in controls. The apparent increase in Kd, on the other hand, was found to be an artifact of the assay. Thus extrapolation of the measured or apparent Kd value to infinite dilution resulted in identical value for Kd regardless of the treatment.