nM Of Estradiol Research Articles

Affinity for Estrogen Receptor α (ERα) in a <i>trans</i>-Stilbene Derivative Containing a Pyridoxine Fragment

Molecular targets for a promising antitumor agent based on trans-stilbene containing a pyridoxine fragment were identified. The lead compound, (E)-6-(3,4-dimethoxystyryl)-2,2,5,8-tetramethyl-4H-[1,3] dioxino[4,5-c]pyridine, was found to selectively induce apoptosis in MCF-7 breast adenocarcinoma cells overexpressing estrogen receptor, but not in MDA-MB-231 cells negative for estrogen receptor. The mechanism by which the novel trans-stilbene derivative acts as a selective estrogen receptor modulator was analyzed, and the affinity for human estrogen receptor α (ERα) was assessed by fluorescence polarization. Unlike its structural analogs—tamoxifen and raloxifene, the lead compound showed no affinity for ERα and did not form complexes with it. Therefore, it was concluded that the selective action of the pyridoxine-containing derivative of trans-stilbene on estrogen-positive breast cancer cells occurs through an alternative mechanism. The EC 50 values for the displacement of the fluorescent ligand from the ERα active site were 22, 120, and 595 nM for estradiol, raloxifene, and tamoxifen, respectively.

150P Association of hormone-sensitive miRNAs expression with main clinicopathologic characteristics of ER/PR-positive breast cancer

Difficulties in breast cancer (BC) treatment are associated with the occurrence of metastases at early stages of disease. It is known that ER and PR status may change during metastatic tumor progression. Recent studies have shown that changes in androgen receptor (AR) expression are associated with mammary gland carcinogenesis. Therefore a study of pathways involving these receptors might help understanding mechanisms of BC progression, as the expression of their targets, including their miRNA-targets, can also change. The search for miRNAs whose putative promoter regions contain AR, PR or ER binding sites was performed using Biostrings (R Bioconductor package). The level of miRNAs was evaluated by RT-PCR in MCF-7 cells treated for 6, 24, 48 hours with 10 nM or 100 nM of estradiol (E2), progesterone (P4) or testosterone (T). The expression of the miRNAs most sensitive to the action of hormones was analyzed in BC samples (n=94). 15 miRNAs with high expression in the mammary gland were selected to analyze their level in the MCF-7 cells. 5 miRNAs changed 1.3-2 times under the influence of hormones: miRNA-27a (decreased after incubation with T), -190a (increased after incubation with T, decreased after incubation with P4), -190b (increased after incubation with E2), -21 (increased after incubation with T), -193b (increased after incubation with T and E2), -324 (increased after incubation with P4). MiRNA-190a, -190b (lower level in T2-T3 tumors of luminal A), -324, (lower level in T2-T3 tumors of luminal B HER2-positive subtype) were found to be associated with tumor size. The levels of miRNA-193b, -190a decreased in the tissues of patients with lymph node metastases in luminal A. In luminal B HER2-positive BC, the levels of miRNA-190a, -190b, -193b, -21 were significantly lower in the tumor tissues of patients with lymph node metastases. Association with the level of ER expression in luminal B HER2-negative BC was found for miRNA-190b (increase at a higher level of ER). The study revealed an association between the expression of hormone-sensitive miRNAs (-190a, -190b, -193b, -21, -324) and T stage, N stage, the level of ER expression in some BC subtype.

Structural transition of lactoferrin upon interaction with estradiol as revealed by spectroscopic techniques: a molten globule state investigation

Proteins have numerous physiological functions in the body. Their most important property is serving as depot carriers and as target molecules for many drugs. Three-dimensional fluorescence spectroscopy, zeta potential analysis, resonance light scattering (RLS), thermodynamic analysis, circular dichroism and fluorescence resonance energy transfer (FRET) measurements were performed to study the interactions between estradiol and human lactoferrin (Hlf). According to the three-dimensional fluorescence spectra, the binding of the drug to Hlf induced a slight destabilization of the protein. Circular dichroism results provided new insight into the course of structural transitions of Hlf after addition of estradiol, and revealed that a molten globule state was intermediate between the native and unfolded states. This is a compact state with a significant amount of native-like secondary structure, but a largely disordered tertiary structure. The experimental results further indicated that the fluorescence quenching mechanism between Hlf and estradiol corresponded to a static quenching procedure. The binding constant and the number of binding sites were obtained from modified Stern–Volmer diagrams. The distance r between donor and acceptor was obtained by FRET and found to be 1.96 nm for estradiol and Hlf. The critical induced aggregation concentrations (CCIAC) of estradiol on Hlf were determined by a non-linear relationship between the enhancement of the RLS intensities and drug concentrations. The zeta potential results were used to verify the existence of complexes and confirmed the CCIAC values obtained by the RLS technique. In addition, molecular modeling showed that estradiol was bound within the domain II of the C-lobe and N-lobe of the Hlf via hydrophobic interactions and hydrogen bonding.

Rapid activity-directed screening of estrogens by parallel coupling of liquid chromatography with a functional gene reporter assay and mass spectrometry

In this study we developed a new LC nanofractionation platform that combines a human cell (BG1.Luc) gene reporter assay with a high resolution mass spectrometer for the detection and identification of estrogenic and anti-estrogenic compounds in environmental waters. The selection of this assay was based on its high sensitivity and selectivity, which is required for environmental trace level detection. We modified an autosampler and controlled it with in-house developed software to collect fractions in the low second range in microtiter plates. This ensured that chromatographic separation was maintained and allowed straightforward hyphenation with the bioassay. After bioassay testing, bioassay chromatograms were reconstructed and directly correlated with MS chromatograms that were obtained in parallel. This enabled to pinpoint bioactives in the MS chromatogram within a single fractionation cycle and results in a significant increase in throughput compared to traditional EDA studies. The sensitivity of the platform was low enough for environmental waters (80nM for bisphenol A and 320pM and 3.2nM for estradiol and estriol, respectively). In addition, the ability of the platform to detect anti-estrogens was successfully demonstrated as well. Finally, real samples were analysed.

1459 RATIONALLY DESIGNED LXXLL PEPTIDOMIMETIC BLOCKS NON-ANDROGENIC ACTIVATION OF THE ANDROGEN RECEPTOR IN PROSTATE TUMOR

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 20111459 RATIONALLY DESIGNED LXXLL PEPTIDOMIMETIC BLOCKS NON-ANDROGENIC ACTIVATION OF THE ANDROGEN RECEPTOR IN PROSTATE TUMOR Lin Yang, Preethi Ravindranath, Jung-Mo Ahn, and Ganesh Raj Lin YangLin Yang Dallas, TX More articles by this author , Preethi RavindranathPreethi Ravindranath Dallas, TX More articles by this author , Jung-Mo AhnJung-Mo Ahn Dallas, TX More articles by this author , and Ganesh RajGanesh Raj Dallas, TX More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1372AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In our previous study, we have shown that Estrogen Receptor beta (ERβ), PELP1 and Androgen Receptor (AR) can form a heterotrimeric protein complex in prostate cancer (PCa) cells. This complex plays a central role in the non-androgenic activation of AR in PCa. We had also previously rationally designed an LXXLL peptidomimetic, D2, block the formation of the heterotrimeric complex between AR, PELP1 and ERβ. In this study, we examined if this peptidomimetic D2 could inhibit the non-androgenic activation of AR in PCa cells. METHODS PCa cells were pretreated with either the active peptidomimetic D2, control peptidomimetic D1, or vehicle (DMSO) for 2 hours, and then treated with 10 nM of estradiol for 24 hours. PCa cells were harvested and immunoprecipitation was done to check if D2 blocked the formation of the complex; MTT assay, luciferase assay and Western Blotting were done to check if D2 blocked estrogen-induced AR activation; PELP1 was then transfected into C4-2 cells to check if overexpression of PELP1 could rescue the D2 blocked estrogen-induced AR activation. RESULTS D2 can block the formation of ERβ, PELP1 and AR complex in PCa cells. Upon treatment with E2, this complex is induced, but pretreatment with D2 prevents induction of the heterotrimeric protein complex. D2 but not D1 can block estradiol induced PCa cell proliferation. D2 can downregulate estradiol induced PSA gene expression, and transcription from androgen responsive elements (ARE). Finally, D2 can block Estradiol-induced AR nuclear translocation. Overexpression of PELP1 in C4-2 cells can rescue the PCa cell proliferation, PSA gene expression, ARE luciferase activity and AR nuclear translocation from the suppressive effects of D2. CONCLUSIONS These data suggest that ERβ, PELP1 and AR form a heterotrimeric protein complex. The rationally signed LXXLL analogue D2 blocked the formation of this complex. Blockade of this protein complex with D2 blocks the non-androgenic activation of PCa cells, including PCa cell proliferation, ARE-driven gene expression and AR nuclear translocation. Over-expression of PELP1 in PCa cells can rescue the cells from the effect of D2, by restoring a functional concentration of PELP1 above the direct competitive inhibition of D2. These data indicate that the rationally designed peptidomimetic D2 can block both the androgenic and non-androgenic activation of AR in PCa cells. This feature makes the peptidomimetic D2 very attractive for further development. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e585 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Lin Yang Dallas, TX More articles by this author Preethi Ravindranath Dallas, TX More articles by this author Jung-Mo Ahn Dallas, TX More articles by this author Ganesh Raj Dallas, TX More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Membrane Estrogen Receptor-α Interacts with Metabotropic Glutamate Receptor Type 1a to Mobilize Intracellular Calcium in Hypothalamic Astrocytes

Estradiol, acting on a membrane-associated estrogen receptor-alpha (mERalpha), induces an increase in free cytoplasmic calcium concentration ([Ca(2+)](i)) needed for progesterone synthesis in hypothalamic astrocytes. To determine whether rapid estradiol signaling involves an interaction of mERalpha with metabotropic glutamate receptor type 1a (mGluR1a), changes in [Ca(2+)](i) were monitored with the calcium indicator, Fluo-4 AM, in primary cultures of female postpubertal hypothalamic astrocytes. 17beta-Estradiol over a range of 1 nm to 100 nm induced a maximal increase in [Ca(2+)](i) flux measured as a change in relative fluorescence [DeltaF Ca(2+) = 615 +/- 36 to 641 +/- 47 relative fluorescent units (RFU)], whereas 0.1 nm of estradiol stimulated a moderate [Ca(2+)](i) increase (275 +/- 16 RFU). The rapid estradiol-induced [Ca(2+)](i) flux was blocked with 1 microm of the estrogen receptor antagonist ICI 182,780 (635 +/- 24 vs. 102 +/- 11 RFU, P < 0.001) and 20 nmof the mGluR1a antagonist LY 367385 (617 +/- 35 vs. 133 +/- 20 RFU, P < 0.001). Whereas the mGluR1a receptor agonist (RS)-3,5-dihydroxyphenyl-glycine (50 microm) also stimulated a robust [Ca(2+)](i) flux (626 +/- 23 RFU), combined treatment of estradiol (1 nm) plus (RS)-3,5-dihydroxyphenyl-glycine (50 microm) augmented the [Ca(2+)](i) response (762 +/- 17 RFU) compared with either compound alone (P < 0.001). Coimmunoprecipitation demonstrated a direct physical interaction between mERalpha and mGluR1a in the plasma membrane of hypothalamic astrocytes. These results indicate that mERalpha acts through mGluR1a, and mGluR1a activation facilitates the estradiol response, suggesting that neural activity can modify estradiol-induced membrane signaling in astrocytes.

Bisphenol A and Risk of Metabolic Disorders

IN THIS ISSUE OF JAMA, LANG AND COLLEAGUES 1 REPORT the results of the first major epidemiologic study to examine the health effects associated with the ubiquitous estrogenic chemical bisphenol A (BPA). This compound is the base chemical (monomer) used to make polycarbonate plastic food and beverage containers, the resin lining of cans, and dental sealants; it also is found in “carbonless” paper used for receipts as well as a wide range of other common household products. Based on their analysis of data from the National Health and Nutrition Examination Survey 2003-2004, Lang et al report a significant relationship between urine concentrations of BPA and cardiovascular disease, type 2 diabetes, and liver-enzyme abnormalities in a representative sample of the adult US population. This report, suggesting links between BPA and some of the most significant and economically burdensome human diseases, is based on a cross-sectional study and therefore cannot establish causality; follow-up longitudinal studies should thus be a high priority. Yet many peer-reviewed published studies report on related adverse effects of BPA in experimental animals, and cell culture studies identify the molecular mechanisms mediating these responses. These experimental findings add biological plausibility to the results reported by Lang et al. Based on this background information, the study by Lang et al, while preliminary with regard to these diseases in humans, should spur US regulatory agencies to follow the recent action taken by Canadian regulatory agencies, which have declared BPA a “toxic chemical” requiring aggressive action to limit human and environmental exposures. Alternatively, Congressional action could follow the precedent set with the recent passage of federal legislation designed to limit exposures to another family of compounds, phthalates, also used in plastic. Like BPA, phthalates are detectable in virtually everyone in the United States. This bill moves US policy closer to the European model, in which industry must provide data on the safety of a chemical before it can be used in products. Subsequent to an unexpected observation in 1997, numerous laboratory animal studies have identified lowdose drug-like effects of BPA at levels less than the dose used by the US Food and Drug Administration (FDA) and the Environmental Protection Agency to estimate the current human acceptable daily intake dose (ADI) deemed safe for humans. These studies have shown adverse effects of BPA on the brain, reproductive system, and—most relevant to the findings of Lang et al—metabolic processes, including alterations in insulin homeostasis and liver enzymes. However, no prior studies examining BPA for effects on cardiovascular function have been conducted in laboratory animals or humans. Epidemiologists are informed by animal studies that identify potential human health hazards when the animal models and exposure levels are relevant and effects are mediated via response mechanisms present in humans. For example, when adult rats were fed a 0.2-μg/kg per day dose of BPA for 1 month (a dose 250 times lower than the current ADI), BPA significantly decreased the activities of antioxidant enzymes and increased lipid peroxidation, thereby increasing oxidative stress. When adult mice were administered a 10-μg/kg dose of BPA once a day for 2 days (a dose 5 times lower than the ADI), BPA stimulated pancreatic cells to release insulin. After administration of 100 μg/kg per day of BPA via injection or feeding for 4 days, mice developed insulin resistance and postprandial hyperinsulinemia. Follow-up studies showed that stimulation of mouse -cell insulin production and secretion by between 0.1 to 1 nM of estradiol or BPA (23-230 pg/mL of BPA) is mediated by activation of the extracellular signal-related protein kinase 1/2 pathway by binding of BPA to estrogen receptor and that via this nonclassical estrogen-response mechanism, BPA and estradiol have equal potency and efficacy. BPA and estradiol are also equipotent at inhibiting adiponectin release from human adipocytes at 1 nM, further implicating BPA at current human exposure levels in insulin resistance and the metabolic syndrome.

Effect of High Concentration of Estradiol on Thyroid Specific Genes Expression and Cell Growth

Background: Since various thyroid diseases have dominant prevalence in women, it has been suggested that female sex hormone have important role on thyroid cell physiology. Interestingly, many thyroid disorders are newly diagnosed or changed their course around the period of high estrogen status, such as pregnancy. In this study, we questioned whether high concentration of estrogen could modulate thyroid cell function. Methods: We treated normal rat thyroid FRTL-5 cell line with different time and concentration of estradiol. Using cell count, FACscan, and Northern blot analysis, we compared the changes of cell growth, cell cycle progression and thyroid specific genes expression. To evaluate the influence of thyroid stimulating hormone (TSH), all experiment was designed as two different sets, with (6H) or without TSH (5H). Results: The concentration of 10-1000 nM estradiol had definite stimulatory function on thyroid cell growth in 5H condition as concentration dependent manner. FACscan revealed the increased cell growths were related to G1/S progression. The Pax-8, TTF-1 and NIS gene expressions were dramatically increased in 10-1000 nM of estradiol, too. With TSH (6H), however, we could not find any cell growth stimulating effects with 10-1000 nM of estradiol. Conclusion: High concentration of estradiol is one of important control factor for thyroid growth and thyroid specific genes expression, especially in 5H condition. It indicate that exposure to high concentration of female sex hormone, such as pregnancy, can be a direct stimulating factor to various thyroid function and related to autoimmune or nodular thyroid diseases around the period of pregnancy (J Kor Soc Endocrinol 21:32~39, 2006).

Human ADA3 Binds to Estrogen Receptor (ER) and Functions As a Coactivator for ER-mediated Transactivation

We have recently identified the hADA3 protein, the human homologue of yeast transcriptional coactivator yADA3, as a novel HPV16 E6 target. Using ectopic expression approaches, we further demonstrated that hADA3 directly binds to the 9-cis retinoic acid receptors alpha and beta, and functions as a coactivator for retinoid receptor-mediated transcriptional activation. Here, we examined the role of endogenous hADA3 as a coactivator for estrogen receptor (ER), an important member of the nuclear hormone receptor superfamily. We show that ADA3 directly interacts with ER alpha and ER beta. Using the chromatin immunoprecipitation assay, we also show that hADA3 is a component of the activator complexes bound to the native ER response element within the promoter of the estrogen-responsive gene pS2. Furthermore, using an ER response element-luciferase reporter, we show that overexpression of ADA3 enhances the ER alpha- and ER beta-mediated sequence-specific transactivation. Reverse transcription-PCR analysis showed an ADA3-mediated increase in estrogen-induced expression of the endogenous pS2 gene. More importantly, using RNA interference against hADA3, we demonstrate that inhibition of endogenous hADA3 inhibited ER-mediated transactivation and the estrogen-induced increase in the expression of pS2, cathepsin D, and progesterone receptor, three widely known ER-responsive genes. The HPV E6 protein, by targeting hADA3 for degradation, inhibited the ER alpha-mediated transactivation and the protein expression of ER target genes. Thus, our results demonstrate that ADA3 directly binds to human estrogen receptor and enhances the transcription of ER-responsive genes, suggesting a broader role of mammalian hADA3 as a coactivator of nuclear hormone receptors and the potential role of these pathways in HPV oncogenesis.

Differential p53 Protein Expression Level in Human Cancer-Derived Cell Lines After Estradiol Treatment

Background p53 has a remarkable number of biological activities, including a central role in cell cycle checkpoints, apoptosis, senescence, and maintenance of genomic integrity. Its expression is modified by estradiol in some epithelial cancer-derived cell lines from the reproductive tract. The aim of this study was to evaluate the effect of low and high doses of estradiol in p53 gene expression in epithelial cancer-derived cell lines from the reproductive tract. Methods p53 gene expression was assessed by Northern and Western blot methods in three human epithelial cancer-derived cell lines after estradiol treatment. Results These indicated that no changes in p53 mRNA content occurred after estradiol treatment at both low (10 nM) and high (1 μM) doses of estradiol in HeLa, CaLo, and C-33 cell lines. p53 protein content was nearly constant in HeLa and C-33 cell lines at administration of 10 nM of estradiol. However, when estradiol was administered at a higher dose (1 μM), an increase in p53 protein was observed over time in HeLa and CaLo cell lines. In contrast, estradiol was without variations in C-33. Conclusions Overall results indicate that estradiol induces variations of p53 protein levels in epithelial cancer-derived cell lines from the reproductive tract in vitro and that this effect may be related with status p53 and/or presence of E6/E7 from human papillomavirus.

An estradiol-Porphyrin conjugate selectively localizes into estrogen receptor-Positive breast cancer cells

A conjugate of a C 11-β-derivative of estradiol and an asymmetric tetraphenylporphyrin was synthesized to study its potential selective uptake by breast cancer cells naturally over-expressing the nuclear receptor for estrogen (ER). Competitive radioligand binding assays of this conjugate with recombinant ER showed that the conjugate bound to ER in a dose-dependent manner with an EC 50 of 274 nM, compared with 1 nM for estradiol, the natural ligand. Cellular uptake studies with ER-positive MCF-7 and ER-negative HS578t human breast cancer cells revealed that, the conjugate was taken up by MCF-7 cells in a dose-dependent manner, which was obliterated by co-incubation with a large excess of estradiol. On the other hand there was very little uptake of the un-conjugated porphyrin by MCF-7 and Hs578t cells. HS578t cells also showed insignificant uptake of the conjugate under the conditions of our experiment. These results strongly suggested that specific interaction between the endogenous ER in MCF-7 cells and the estrogen part of the conjugate enabled these cells to selectively internalize the conjugate over the un-conjugated porphyrin. Therefore, ER-binding conjugates of estradiol and porphyrins could potentially be used for ER-targeted photodynamic therapy of hormone-sensitive cancers of breast, ovary, gonads etc.

Simultaneous Determination of Estradiol and Medroxyprogesterone Acetate in Pharmaceutical Formulations by Second-Derivative Spectrophotometry

This paper reports a simple and fast method for the simultaneous determination of estradiol (ED) and medroxyprogesterone acetate (MP) in pharmaceutical formulations by second-derivative spectrophotometry. Methanol was used to extract the drugs from formulations, and subsequently the extracts were evaluated directly by derivative spectrophotometry. The drugs were determined simultaneously by using the graphic method at 297.4 nm for ED and the zero-crossing method at 273.4 nm for MP. If both compounds are present together in a sample, it is possible to quantitate one in the presence of the other. The best signal-to-noise ratio was found when the second derivative of the spectrum was used. The linear ranges for determination of the drugs were 4.7 x 10(-6) to 1.6 x 10(-4) and 7.2 x 10(-6) to 2.0 x 10(-4) mol/L for ED and MP, respectively. The ingredients commonly found in commercial pharmaceutical formulations do not interfere with the determination. Chemical and spectral variables were optimized for the determination of both analytes. Good levels of repeatability (relative standard deviation), 1.4 and 1.9%, were obtained for ED and MP, respectively. The proposed method was applied to the determination of these drugs in pharmaceutical formulations.

Manipulation of culture conditions for BHK cell growth inhibition by IRF-1 activation.

The activation of interferon-regulatory-factor-1 (IRF-1) hasbeen applied to regulate the cell growth of BHK cells. Theconstitutively expressed IRF-1-estrogen receptor fusion protein(IRF-1-hER) activated by the addition to the culture medium ofan estrogen analogue (estradiol), enabled IRF-1 to gain itstranscriptional activator function. By using a dicistronicstabilised self-selecting construct it was possible to controlcell proliferation. With the addition of 100 nM of estradiol at the beginning of the exponential phase, the IRF-1 activationled to a rapid cell growth inhibition. Two days after estradioladdition cell concentration was still maintained but a decreasein cell viability was observed. This cell response isindependent on clone (producer and non-producer) and culturesystem (static and stirred cultures). Specificrecombinant-protein productivity of the producer clone was notsignificantly altered. Control experiments confirmed that IRF-1activation effect was not due to the addition of estradiol per se, estradiol solvent or serum concentration. The extent ofcell growth inhibition is dependent on estradiol concentrationand estradiol addition time, although a decrease in cellviability was always observed. Reducing the time span ofestradiol exposure allowed the decrease in the cell viability tobe controlled and the stationary inhibited phase to be extended:when the time of contact between the cells and estradiol isreduced cell viability increases, archieving values similar tothose obtained if no estradiol is added. During this recoveryphase the cells passed two different phases: first a stationaryphase extension where cell growth was still inhibited, followedby an increase of cell concentration. The IRF-1 system isreversible. This pattern can be repeated for an extended period when estradiol addition and removal are repeated, showing acyclic response. Thus, it is possible to modulate the IRF-1effect by manipulating cycles of addition/removal of estradioland in this way the stationary phase can be maintained.

Sex-hormone binding globulin from sheep serum: purification and effects of pregnancy and treatment with exogenous estradiol

Sex-hormone binding globulin (SHBG) is a protein that binds sex steroids in the serum of many species. SHBG binds androgens and estrogens in humans and primates with high affinity, but behaves as an androgen binding protein in other species. Here we purified SHBG from ewe and ram sera to homogeneity, by a simple and rapid method. The K D of the purified protein was found to be 3.63 nM for testosterone and around 600 nM for estradiol. We also studied the effect of pregnancy on SHBG levels in ewes and the effect of exogenous estradiol administration either orally or parenterally on SHBG levels in rams. Basal levels of SHBG in sheep are not affected by pregnancy or exposure to exogenous estradiol. It is concluded that SHBG regulation of expression in ewes and rams differs from that in humans in that it is not affected by estrogen and possibly is species specific.

CAMP accumulation in opioid-sensitive SH-SY5Y neuroblastoma cells is modified by estradiol and progesterone

We have recently demonstrated that acute and chronic treatments with estradiol and progesterone induce changes in the responsiveness of endogenous opioid systems to painful stimulation. In the present study the neuroblastoma SH-SY5Y subclone known to contain predominantly mu opioid receptors was used as a model to characterize the gonadal steroid effect on this opioid receptor system. The function of opioid receptors was assessed by measuring prostaglandin E 1 (PGE 1)-induced cyclic AMP accumulation after various treatments with estradiol and progesterone. Differentiated SH-SY5Y cells respond to PGE 1 with a dramatic increase in cAMP level. Morphine (MOR) inhibits by about 75% the stimulatory effect of PGE 1 on cAMP. Pretreatment with 5 nM of estradiol for 6 days resulted in a significant increase of PGE 1-stimulated cAMP accumulation. Exposure of cells for 48 h to estradiol in doses of 5 nM or 50 nM did not affect cell sensitivity to the PGE 1 effect on cAMP. Moreover, neither dose of estradiol changed the inhibitory effect of morphine on PGE 1-induced cAMP response. There was a significant increase in PGE 1-stimulated cAMP accumulation after treatment with 100 nM progesterone for l h or 15 min and a marked elevation of cAMP levels was also measured after 15 min treatment with 10 nM progesterone. Exposure to either dose of progesterone for 8 h, 48 h or 6 days did not affect basal or PGE 1-induced cAMP in neuroblastoma cells. Progesterone-treated groups responded to MOR with 56–67% inhibition of PGE 1-stimulated cAMP accumulation. The potency of MOR-induced inhibition was comparable to the MOR effect in cells not treated with the steroid. Although estradiol and progesterone do not affect the magnitude of MOR-induced suppression of cAMP levels, the concentrations of cAMP remain significantly elevated in those MOR-treated cells in which steroid pretreatment produced a significant increase of PGE 1-induced cAMP accumulation. We conclude that under in vitro conditions short-term treatment with progesterone and chronic exposure to estradiol potentiate the stimulatory effect of PGE 1 on cAMP accumulation in SH-SY5Y neuroblastoma cells. Although MOR retains its ability to suppress cAMP levels in steroid-treated cells, the MOR-suppressed levels of the cyclic nucleotide remain higher than in non-steroid-treated cells. SH-SY5Y cells may serve as a suitable model for investigating the interaction of ovarian steroids with opioid receptors.

The immature rat ovary is innervated by vasoactive intestinal peptide (VIP)-containing fibers and responds to VIP with steroid secretion.

The nature and role of the peptidergic innervation of the ovary were examined by determining the location and function of vasoactive intestinal peptide (VIP)-containing nerve fibers in the immature rat ovary. Immunohistofluorescence analysis of prepubertal ovaries using a specific VIP antibody revealed sparse delicate VIP-immunoreactive fibers localized around veins and arterioles, in the interstitial tissue, and associated with the thecal layers of developing follicles. Radioimmunoassayable VIP content was found to be approximately 100 pg/ovary (3 nM). The VIP immunoreactivity coeluted with authentic VIP when subjected to Sephadex G-25 chromatography. VIP enhanced in vitro progesterone release from infantile (12 days old), juvenile (30 days old), and peripubertal ovaries and estradiol release during the two latter developmental periods. The maximal estradiol response to VIP occurred during the early and first proestrous phases of puberty. No response was observed during estrus or first diestrus. The progesterone response to VIP increased moderately between day 12 and first proestrus, and then strikingly at estrus and first diestrus. The stimulatory effect of VIP on ovarian steroid production was dose related, as determined in ovaries from PMSG-treated immature rats (ED50 = 215, 44, and 51 nM for estradiol, androgen, and progesterone, respectively). The specificity of the VIP effect was tested using five other gastrointestinal peptides (porcine peptide histidine isoleucine, gastric inhibitory polypeptide, secretin, motilin, and glucagon). Only peptide histidine isoleucine, which has the greatest sequence homology with VIP, enhanced ovarian steroid production at 50% of VIP effectiveness. VIPergic nerves thus appear to be involved in the developmental regulation of ovarian steroidogenesis.

Estrogen and glucocorticoid receptors in adult canine articular cartilage.

The cytoplasmic and crude nuclear fractions of adult mongrel dog articular cartilage contained estradiol- and dexamethasone-binding components which had properties of physiologic steroid receptors. The equilibrium dissociation constants averaged 0.37 nM for estradiol and 2.27 nM for dexamethasone. The concentrations estrogen receptors ranged from below 6 to 101 fmol/mg protein in the cytosols and from below 2.8 to 17.5 fmol/micrograms DNA in the nuclear fractions. Glucocorticoid receptors were detected in only 4 of 13 cytosols (range: 61.2-132 fmol/mg protein), whereas 10 of 13 nuclear fractions contained 0.8 to 46.8 femtomoles of the receptors for each microgram of DNA. There appeared to be no marked difference between the contents of either steroid receptor in female or male dog cartilage. No receptors were detected for androgen and progesterone.

Zearalenones: Characterization of the Estrogenic Potencies and Receptor Interactions of a Series of Fungal β-Resorcylic Acid Lactones*

This study investigates the direct estrogen receptor interactions and the character of the biological activities of three estrogenic resorcylic acid lactones in the immature rat uterus. These compounds are fungal metabolites (P-1492; zearalenone) or derivatives thereof (P-1496 and P-1560; epimeric zearalanols) that have been associated with estrogenizing syndromes in cattle fed mold-infected grain. The compounds compete with estradiol for binding to the cytoplasmic receptor (P- 1496, 13.6%; P-1492, 1.8%; P-1560, 0.8% that of estradiol), and they translocate estrogen receptor sites to the nucleus, with P- 1496 showing the most prolonged nuclear receptor interaction. The three compounds induce the synthesis of the uterine induced protein (P-1496 > P-1560 > P-1492) and increase uterine weight. Direct binding studies with the most potent compound P-1496, in tritium-labeled form indicates a Kd of 1.8 nM (compared to 0.12 nM for estradiol) for interaction with uterine cytoplasmic receptor. Cytoplasmic receptor ...

Effect of Cu 2+ ions on the binding of estrogen to the human myometrial estrogen binding protein

The binding of estrogens to the estrogen binding protein in human myometrial cytosol and the effect of Cu 2+ ions on the binding characteristics have been investigated. The dissociation constants at 20 °C for the estrogen receptor complex were 0.081, 0.21 and 0.24 nM for estradiol, estriol and estrone, respectively. Both the affinity and the number of binding sites were affected in the presence of Cu 2+ ions which indicated a mixed type inhibition of the binding of steroid to the receptor. The second order association rate constants decreased in the presence of Cu 2+ ions and the effect seemed to be reinforced by an increase in temperature. No change in the sedimentation coefficient of the estrogen receptor complex occurred in the presence of Cu 2+ ions although less steroid was bound. Cu 2+ ion concentrations above 10 μM were required before remarkable alterations in the binding characteristics were established. As the copper level in human uterine fluid of women using a Cu-IUD have been shown to be in the range of 20 μM, the interaction of Cu 2+ ions with the binding of estrogens to their specific receptors has to be considered as one factor responsible for the changes in the uterus in the presence of the device.

Spectrophotometric estimation of estradiol-17β, progesterone, and testosterone

An ultraviolet spectrophotometric method is presented for the quantitative estimation of steroids which are in concentrations of more than 1.25 μ/ml. Estradiol-17β, progesterone, and testosterone were used in these studies. Based on uv light absorption at 230 nm for estradiol and progesterone, and at 240 nm for testosterone, the relative concentration of each steroid could be estimated. This method is very simple and rapid. It is economical, requires no sophisticated instruments, and is very practical for estimating steroids in pharmaceutical preparations, chemicals, or biological specimens.