Mary L. Thomas

Estrogen Receptor-α Detected on the Plasma Membrane of Aldehyde-Fixed GH3/B6/F10 Rat Pituitary Tumor Cells by Enzyme-Linked Immunocytochemistry1

A population of estrogen receptor-α (ERα) proteins, located at the plasma membrane, is postulated to mediate the rapid, nongenomic responses of GH3/B6/F10 pituitary cells to estrogen. To demonstrate the presence of ERα at the plasma membrane and to distinguish this receptor population from that in the nucleus, GH3/B6/F10 cells were first prepared in 2% paraformaldehyde/0.1% glutaraldehyde in PBS (P/G) without detergent, then exposed to one of several antibodies (Abs) raised against nuclear ERα. Ab binding was visualized as a fluorescent/chromagenic reaction product catalyzed by avidin-biotin-complexed alkaline phosphatase. With P/G fixation, Abs could only access antigens at the cell surface, as evidenced by the inability of 70K mol wt dextrans to permeate cells and the absence of intracellular staining by Abs to cytoplasmic or nuclear antigens. ERα Abs generated membrane, but not nuclear, staining in P/G-fixed cells; nuclear receptor labeling could only be detected in detergent-treated cells. Specificity...

Perimembrane Localization of the Estrogen Receptor α Protein in Neuronal Processes of Cultured Hippocampal Neurons

There is clear evidence of rapid, nongenomic responses to estrogen in a variety of neuronal model systems. To address the question of whether some of these rapid estrogen signals might be transduced by the classical estrogen receptor (ER) α or a closely related protein in nontransformed neurons, we undertook the present study using isolated fetal rat hippocampal neurons. Several antibodies developed to detect ERα were tested in this system and showed positive membrane staining in nonpermeabilized neurons. MC-20, an affinity purified anti-ERα, rabbit polyclonal IgG antibody which does not recognize ERβ was selected to carry out the majority of the experiments. When permeabilized, the hippocampal neurons exhibited low levels of nuclear staining for ERα, but abundant labeling for ERα throughout the entire cell including the neurites. In addition to traditional immunocytochemistry controls, incubation of neurons for 24 h in the presence of 10 µM antisense oligonucleotide directed against the translation start site of ERα reduced ERα immunoreactivity throughout the neurons providing further evidence that the immunostaining was specific for ERα. Confocal and conventional microscopy demonstrated that the antigen was predominately extranuclear and localization of ERα in the neurites suggests that the receptor is in close proximity to the plasma membrane. This localization is consistent with a role for ERα as a transducer of rapid, nongenomic estrogen responses in hippocampal neurons.

Estrogen regulation of gene expression in the brain: a possible mechanism altering the response to psychostimulants in female rats

Acute behavioral responses to cocaine are more pronounced in female than in male rats. We have shown that 3 weeks of treatment with 17beta-estradiol (E(2)) implants significantly enhanced the hyperactivity induced by cocaine in ovariectomized (OVX) rats. The ligand-bound estrogen receptor (ER) functions as a transcription factor to regulate the expression of E-responsive genes. Thus, we hypothesized that estrogen (E) modulates the behavioral response to cocaine via regulation of expression of components of dopamine (DA) and serotonin (5-HT) systems in mesolimbic nuclei important in the response to cocaine as well as the hypothalamus, a brain area known to be E-responsive. Adult female Sprague-Dawley rats were OVX; half of them then received E(2) implant (OVX+E) (n=8/group, two groups). Twenty-seven days later, brain tissue was collected to study E(2) effects on mRNA expression for DA D(1)-like (D(1)) and D(2)-like (D(2S), D(2L), D(3)) receptors, DA transporter (DAT), 5-HT(1A), 5-HT(1B), 5-HT(2A), 5-HT(2C) receptors, and 5-HT transporter (SERT) as well as ERalpha and ERbeta in amygdala, hypothalamus, nucleus accumbens, midbrain, and ventral tegmental area (VTA). We found that E(2) implants in OVX rats increased mRNA levels for D(1) receptor in hypothalamus, D(2L) receptor in midbrain, and D(3) receptor in VTA, and decreased D(3) receptor mRNA levels in midbrain relative to OVX controls. E(2) also increased 5-HT(2C) receptor mRNA levels in midbrain and hypothalamus. In addition, E(2) decreased mRNA levels for ERalpha in amygdala and hypothalamus and ERbeta in amygdala. The present study demonstrates that E can regulate mRNA expression for specific DA and 5-HT receptors in a region-specific manner and suggests that such modifications may contribute to the behavioral response to cocaine.

Alterations of DNA methylation by glutathione depletion

One of the most consistent findings in cancer cells is an overall decrease of 5-methylcytosine content in DNA. The causes that lead to this alteration are not known. We have shown in a recent study that the methyl-donor, methionine (Met), can easily be depleted and that O- and S-methylation can be impaired in response to glutathione (GSH) depletion. This is because mammalian cells are capable of resynthesizing GSH after GSH is depleted, and GSH turnover occurs at the expense of Met. An extensive utilization of Met for the resynthesis of GSH causes Met depletion and impairment in methylation. In the present study we now demonstrate that GSH depletion has a significant impact on DNA methylation. An i.p. dose of a model GSH-depleting hepatotoxin, bromobenzene (BB), caused a progressive impairment in genomic DNA methylation in the Syrian hamster. The administration of a single i.p. dose of Met labeled with [14CH3]Met to BB-treated hamsters at either 1, 3, 5.5 or 9 h after BB resulted in an increase of methyl-group incorporation into liver genomic DNA at 24 h after BB. With respect to the time points chosen for Met administration, methyl-group incorporation found in the BB + Met groups were 1-, 2-, 4- and 12-fold of the controls that received only Met. We further employed an in vitro methylation assay using specific bacterial SssI CpG methylase as the catalyzing enzyme to demonstrate that BB caused a progressive increase of unmethylated CpG sites in genomic DNA. Interestingly, the time response curve of global DNA methylation in vitro showed an identical pattern to that observed in the in vivo experiment. The results provide strong evidence that GSH-depleting agents significantly impair cytosine methylation. Thus, alterations in gene expression could result from a high dose and/or prolonged exposure to GSH-depleting agents, e.g. medications, chemotherapeutic agents and environmental toxins.

Estrogen receptor-mediated direct stimulation of colon cancer cell growth in vitro.

In vivo and epidemiological data suggest a mitogenic role for estrogens (E) in colon cancer. The presence of estrogen receptor (ER) and ER mRNA in colonic epithelium and colon cancer cells, make it necessary to explore the possible direct effects of E on colon cancer growth. In this study, a 15-mer oligodoxynucleotide (oligo) antisense to the region of the translation start codon of estrogen receptor mRNA inhibited ER expression in a mouse colon cancer cell line (MC-26), as determined by receptor binding assay. Antisense oligo also decreased ER mRNA levels in MC-26 cells. The growth-stimulatory effect of E was abolished by antisense oligo treatment, demonstrating that the ER is directly involved in the regulation of colon cancer cell growth.

Selective Estrogen Receptor Modulator Effects in the Rat Brain

The effects in the brain of selective estrogen receptor modulators (SERMs) such as tamoxifen and raloxifene have not yet been fully elucidated. Based upon the hypothesis that serotonin (5-HT)-steroid hormone interactions are important in mood regulation, we have compared six SERMs (tamoxifen, raloxifene, levormeloxifene, NNC 45-0781, NNC 45-0320, NNC 45-1506) with 17β-estradiol (E2) in terms of their ability to regulate mRNA levels of estrogen receptor (ER)α, ERβ, 5-HT1A receptor, and 5-HT reuptake transporter (SERT) in the midbrain, amygdala, and hypothalamus of ovariectomized (OVX) rats. Female rats (n = 6/group, 8 groups total) were OVX and allowed to recover for 2 weeks. During the third post-OVX week, rats were injected subcutaneously with E2 (0.1 mg/kg) or one of the SERMs (5 mg/kg) once per day for 7 days. Twenty-four hours after the last injection, tissue was collected for the determination of mRNA levels by ribonuclease protection assay (RPA). E2 treatment significantly decreased mRNA levels for ERα, ERβ, and SERT in midbrain and ERα in hypothalamus. Tamoxifen increased ERβ mRNA levels in hypothalamus, while raloxifene increased ERβ mRNA levels in amygdala. NNC 45-0320 decreased ERα mRNA in hypothalamus and decreased ERβ mRNA in amygdala. These results suggest that while SERMs are not full estrogen receptor agonists in the brain, the agonist/antagonist profiles for individual SERMs may differ among brain areas. This raises the possibility of developing new SERMs for selective functions in specific brain areas.

Influence of estrous cycle and estradiol on behavioral sensitization to cocaine in female rats.

The hypotheses that the estrous cycle and estradiol modulate behavioral sensitization to cocaine in female rats were assessed. In an analysis of sensitization across the estrous cycle, female rats were administered saline or cocaine (15 mg/kg) twice daily for 5 days. Sensitization developed in the intact female rats as measured by the significant increase in stimulant behaviors seen between day 1 and day 5 of treatment. Rats were challenged with cocaine (5 mg/kg) at 3 days following discontinuation of drug treatment. The expression of sensitization as measured between cocaine and saline-treated rats was evident only in female rats in diestus at the time of the challenge test with cocaine. To explore the role of estradiol in sensitization, female rats were ovariectomized or ovariectomized and implanted with estradiol for two weeks prior to treatment with cocaine (15 mg/kg) twice daily for 5 days. Sensitization developed in both ovariectomized and ovariectomized+estradiol rats treated with cocaine as measured by the significant increase in stimulant-like behaviors seen between day 1 and day 5 of treatment. Rats were challenged with 5 mg/kg of cocaine at 3, 13 and 34 days following discontinuation of drug treatment. While neither hormone treatment group exposed to the cocaine regimen expressed sensitization at 3 days of withdrawal, both groups exhibited sensitization at 13 and 34 days following discontinuation of cocaine treatment. The estradiol-treated groups exhibited higher levels of activity relative to their untreated cohorts in both saline or cocaine treatment groups. These results suggest that detection of sensitization in female rats is not only influenced by injection regimen and length of abstinence but also by the presence of estrogens which effectively enhance the response to an acute cocaine challenge in the presence or absence of prior cocaine exposure.

Estradiol effects on the dopamine transporter – protein levels, subcellular location, and function

Background The effects of estrogens on dopamine (DA) transport may have important implications for the increased incidence of neurological disorders in women during life stages when hormonal fluctuations are prevalent, e.g. during menarche, reproductive cycling, pregnancy, and peri-menopause. Results The activity of the DA transporter (DAT) was measured by the specific uptake of 3H-DA. We found that low concentrations (10-14 to 10-8 M) of 17β-estradiol (E2) inhibit uptake via the DAT in PC12 cells over 30 minutes, with significant inhibition taking place due to E2 exposure during only the last five minutes of the uptake period. Such rapid action suggests a non-genomic, membrane-initiated estrogenic response mechanism. DAT and estrogen receptor-α (ERα) were elevated in cell extracts by a 20 ng/ml 2 day NGFβ treatment, while ERβ was not. DAT, ERα and ERβ were also detectable on the plasma membrane of unpermeabilized cells by immunocytochemical staining and by a fixed cell, quantitative antibody (Ab)-based plate assay. In addition, PC12 cells contained RNA coding for the alternative membrane ER GPR30; therefore, all 3 ER subtypes are candidates for mediating the rapid nongenomic actions of E2. At cell densities above 15,000 cells per well, the E2-induced inhibition of transport was reversed. Uptake activity oscillated with time after a 10 nM E2 treatment; in a slower room temperature assay, inhibition peaked at 9 min, while uptake activity increased at 3 and 20–30 min. Using an Ab recognizing the second extracellular loop of DAT (accessible only on the outside of unpermeabilized cells), our immunoassay measured membrane vs. intracellular/nonvesicular DAT; both were found to decline over a 5–60 min E2 treatment, though immunoblot analyses demonstrated no total cellular loss of protein. Conclusion Our results suggest that physiological levels of E2 may act to sequester DAT in intracellular compartments where the transporters second extramembrane loop is inaccessible (inside vesicles) and that rapid estrogenic actions on this differentiated neuronal cell type may be regulated via membrane ERs of several types.

Estradiol-sertraline synergy in ovariectomized rats.

This study investigated estradiol (E(2)) modulation of the antidepressant effects of a selective serotonin (5-HT) reuptake inhibitor (SSRI; sertraline) and a tricyclic antidepressant (imipramine) as measured by the forced swim test (FST) followed by assessment of gene and protein expression for the 5-HT transporter (SERT) and multiple 5-HT receptors. Female Sprague-Dawley rats were ovariectomized (OVX) and two-thirds of the rats received E(2) implants (OVE). 4 weeks later, implants were withdrawn in half of the OVE rats (OVW) to capture a time point when E(2) levels were rapidly declining. Rats in each hormone group were treated with vehicle, sertraline (10 mg/kg) or imipramine (10 mg/kg), 24, 5 and 1h before the FST. Immediately after the FST, midbrain, hippocampus and prefrontal cortex tissue was removed and frozen for analysis of gene expression via quantitative real-time PCR (midbrain tissue) and protein expression via Western blot (prefrontal cortex and hippocampal tissue). In the FST, sertraline decreased immobility and increased swimming in OVE rats, as well as increased swimming in OVW rats. In contrast, no sertraline effect was observed in OVX rats. Rats treated with imipramine showed increased climbing but no changes in immobility or swimming. No changes in protein expression were detected in any treatment group. However, in vehicle-treated rats, E(2) increased midbrain SERT mRNA expression, with no effect on midbrain mRNA for the 5-HT receptors. In sertraline-treated rats, E(2) decreased 5-HT(2A) receptor mRNA, and E(2)-withdrawal increased 5-HT(1A), 5-HT(2A) and 5-HT(2C) receptor mRNA. In imipramine-treated rats, E(2) (and E(2)-withdrawal) did not affect mRNA expression for any of the target genes. Thus, E(2) synergized behaviorally and neurochemically with an SSRI but not a tricyclic antidepressant.

Intracellular signaling involved in estrogen regulation of serotonin reuptake.

17beta-estradiol (E2) regulates neuronal activity via genomic and rapid, non-genomic mechanisms. The rat serotonergic neuronal cell line (RN46A) was used to investigate the rapid effects of E2 on serotonin (5-HT) reuptake and on potential intracellular signaling pathways. RN46A cells express the serotonin transporter (SERT) and estrogen receptor (ER)beta, but not ERalpha. Fifteen minute E2 treatment (10(-9)M) decreased 5-HT uptake. Intracellular cAMP levels were not increased by 15 min E2 treatment; however, E2 caused an increase in intracellular Ca2+ levels, with a maximum response within the first minute. The response was E2 specific, since other steroids (17alpha-estradiol, testosterone, and progesterone) had no effect. The ER antagonist ICI 182,780 blocked the rapid E2 effects on intracellular Ca2+ levels as did the selective ER modulator tamoxifen. In summary, changes in intracellular Ca2+ levels caused by E2 and mediated through ERbeta may be responsible for observed rapid effects of E2 on SERT activity.