Unexpected presence of estrogens in culture medium supplements: subsequent metabolism by the yeast Sacchromyces cerevisiae

We have previously shown the presence of 17 beta-estradiol in extracts of commercially prepared Saccharomyces cerevisiae ss well as the production of estradiol by yeast grown in the laboratory. In our current study, yeast grown in a chemically defined medium synthesized estradiol in only small amounts, (less than 500 pg/liter). We have analyzed a variety of media commonly used for growing yeast and found that substantial estradiol production (greater than 5 ng/liter) was obtained when yeast were grown in medium supplemented with Bacto-peptone. The peptone was shown to contain significant amounts of estrone, and the results of the experiments establish a precursor-product relationship where estrone from the medium is metabolized to estradiol by S. cerevisiae. Studies with added [3H]estrone demonstrated rapid conversion into [3H]estradiol and a 3H-labeled nonpolar estrogen derivative. The commercially obtained yeast used previously had been grown in a molasses medium. We demonstrate here that the molasses medium contains substantial amounts of estrone and estradiol. We conclude that the conversion of estrone in a culture medium to estradiol in laboratory grown yeast and estrone and estradiol present in the commercially grown yeast medium account for the majority of estradiol found in yeast.     

Estrogen receptors in cultured rat uterine cells: induction of progesterone receptors in the absence of estrogen receptor processing

When cultured rat uterine cells were treated for up to 6 h with 5 nM 17 beta-estradiol, no decrease in the [3H] estradiol-binding capacity of the cells was observed (i.e. no processing). This was true whether the cells were treated directly with 5 nM [3H]estradiol or with 5 nM unlabeled 17 beta-estradiol followed by homogenization and exchange with [3H]estradiol in vitro. In additional experiments, intact cells were treated with medium containing 5 nM [3H]estradiol for 30 min, and then that medium was removed and replaced with medium containing 5 nM unlabeled 17 beta-estradiol. Receptor-bound estradiol in intact cells was totally exchangeable with estradiol in the culture medium (t1/2, approximately 90 min). Six-hour treatment of cells with 5 nM 17 beta-estradiol led to a 50% increase in the [3H]progesterone-binding capacity of the cells, while no loss of estrogen-binding capacity occurred. These results indicate that progesterone receptors can be induced by estrogen in the rat uterus in the absence of estrogen receptor processing.     

Ginsenoside-Rb1 from Panax ginseng C.A. Meyer activates estrogen receptor-alpha and -beta, independent of ligand binding

We studied the estrogenic activity of a component of Panax ginseng, ginsenoside-Rb1. The activity of ginsenoside-Rb1 was characterized in a transient transfection system, using estrogen receptor isoforms and estrogen-responsive luciferase plasmids, in COS monkey kidney cells. Ginsenoside-Rb1 activated both alpha and beta estrogen receptors in a dose-dependent manner with maximal activity observed at 100 microm, the highest concentration examined. Activation was inhibited by the estrogen receptor antagonist ICI 182,780, indicating that the effects were mediated through the estrogen receptor. Treatment with 17beta-estradiol or ginsenoside-Rb1 increased expression of the progesterone receptor, pS2, and estrogen receptor in MCF-7 cells and of AP-1-driven luciferase genes in COS cells. Although these data suggest that it is functionally very similar to 17beta-estradiol, ginsenoside-Rb1 failed to displace specific binding of [(3)H]17beta-estradiol from estrogen receptors in MCF-7 whole-cell ligand binding assays. Our results indicate that the estrogen-like activity of ginsenoside-Rb1 is independent of direct estrogen receptor association.     

Clinically used estrogens differentially inhibit human aortic smooth muscle cell growth and mitogen-activated protein kinase activity

Some estrogenic compounds modify vascular smooth muscle cell (SMC) biology; however, whether such effects are mediated in part by estrogen receptors is unknown. The purpose of this study was to evaluate whether the actions of clinically used estrogens on human aortic SMC biology are mediated by estrogen receptors. We examined the effects of various clinically used estrogens in the presence and absence of ICI 182,780, an estrogen receptor antagonist, on cultured human aortic SMC DNA synthesis ([(3)H]thymidine incorporation), cellular proliferation (cell counting), cell migration (modified Boyden chamber), collagen synthesis ([(3)H]proline incorporation), and mitogen-activated protein kinase activity. FCS-induced DNA synthesis, cell proliferation, collagen synthesis, platelet-derived growth factor-induced SMC migration, and mitogen-activated protein kinase activity were significantly inhibited by physiological (10(-9) mol/L) concentrations of 17beta-estradiol and low concentrations (10(-8) to 10(-7) mol/L) of 17beta-estradiol, estradiol valerate, estradiol cypionate, and estradiol benzoate but not by estrone, estriol, 17alpha-estradiol, or estrone sulfate. The inhibitory effects of 17beta-estradiol and other inhibitory estrogens were completely reversed by 100 micromol/L ICI 182,780, and the rank-order potency of various estrogens to inhibit SMC biology matched their rank-order affinity for estrogen receptors. The inhibitory effects of estrogens on SMC biology are in part receptor-mediated. Because the cardioprotective effects of hormone replacement therapy are most likely mediated by modification of SMC biology, whether hormone replacement therapy protects a given postmenopausal woman against cardiovascular disease will depend partially on the affinity of the estrogen for estrogen receptors in vascular SMCs.     

Specificity of steroid binding in New World primate B95-8 cells with a vitamin D-resistant phenotype.

We recently described the existence of a competitive binding component in vitamin D-resistant New World primate cells that has a relatively low affinity (Kd, approximately 10(-8) M) but high capacity for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] compared to that possessed by the vitamin D receptor (VDR). Here we show that this binding component is capable of binding a vitamin D3 metabolite other than 1,25-(OH)2D3 as well as steroid hormones structurally disparate from vitamin D3 sterols. We studied the binding of [3H]1,25-(OH)2D3 and [3H]25-hydroxyvitamin D3 ([3H]25OHD3) in extracts of the vitamin D-resistant marmoset lymphoblastic cell line B95-8 in the presence and absence of potential competitive ligands, including 25OHD3, 1,25-(OH)2D3, 17 beta-estradiol, testosterone, and progesterone, at concentrations ranging from 1-100 nM. Compared to extracts containing the authentic VDR, extracts of B95-8 cells bound 180% more 1,25-(OH)2D3 and 12-fold more 25OHD3 on a weight basis. The affinity of this binder for 25OHD3 was 2.2 times as great as its affinity for 1,25-(OH)2D3. Further, at concentrations approaching the Kd of this binder for 1,25-(OH)2D3, 25OHD3 was 3 times more effective than 1,25-(OH)2D3 in competing with [3H]1,25-(OH)2D3 for binding. This binder eluted from a Sephadex G-100 column with an apparent mol wt of 58 kilodaltons, and pooled elution fractions from the column encompassing this mol wt range were capable of inhibiting binding of 1,25-(OH)2D3 to the VDR by 65%. Competitive steroid binding analyses showed estradiol to be at least as effective as 1,25-(OH)2D3 in inhibition of [3H]1,25-(OH)2D3 binding; homologous binding studies with 17 beta-estradiol as labeled and competitive ligand demonstrated that concentrations of the gonadal steroid that successfully displaced [3H]1,25-(OH)2D3 also displaced 17 beta-[3H] estradiol. Using [3H]25OHD3 as the labeled ligand and a more extensive array of competitive ligands, the rank order of steroid binding was 25OHD3 > 1,25-(OH)2D3 > or = estradiol = progesterone = testosterone. These results suggest that the phenotype of steroid hormone resistance in New World primates may result from the overexpression of an intracellular 58-kilodalton protein(s) that interferes with the steroid-receptor interaction by competing for ligand binding.     

Triphenylethylene antiestrogen-binding sites in cockerel liver nuclei: evidence for an endogenous ligand

Salt extracts of purified nuclei from cockerel liver contain a limited number of sites that bind triphenylethylene nonsteroidal antiestrogens with high affinity and specificity. The assay of the [3H]tamoxifen (3H-labeled 1-[4-(2-dimethylaminoethyoxy)phenyl] 1,2-diphenylbut-1-(Z)ene)-binding sites is optimally achieved by preincubation of the salt extracts with charcoal-dextran suspension; a 4- to 8-fold increase in activity over that obtained with nontreated extracts is found. This suggests that the binding sites are occupied in vivo by an unknown endogenous ligand. The equilibrium dissociation constant for [3H]tamoxifen binding is 4.76 +/- 1.8 nM, and the binding site concentration is 1.7 +/- 0.7 fmol/microgram DNA. The concentration of high affinity estrogen-binding sites in the same extracts is almost 30-fold less (0.06 +/- 0.01 fmol/micrograms DNA). The relative binding affinities of various antiestrogens for the nuclear antiestrogen-binding sites (with tamoxifen arbitrarily set at 100%) are as follows: nafoxidine (1-[2-(p-[3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl]phenoxy)ethyl] pyrrolidine hydrochloride); 126%) greater than tamoxifen (100%) greater than N-des-methyltamoxifen (16%) greater than CI-628 (alpha-[p-[2-(1-pyrrolidine)ethyoxy]phenyl] 4-methoxy-alpha'-nitrostilbene; 14%) greater than 4-hydroxytamoxifen (7%). Estrogens (17 beta-estradiol, estriol, estrone, and diethylstilbestrol) and several other steroids (cholesterol, dihydrotestosterone, pregnenolone, progesterone, and hydrocortisone) show little or no affinity for binding to the nuclear sites (relative binding affinity, less than 0.5%). However, ether extracts of cockerel serum or liver nuclei contain a substance(s) that competitively inhibits [3H]tamoxifen binding to the nuclear antiestrogen-binding sites. The ether-soluble material does not compete for [3H]estradiol binding to the salt-soluble nuclear estrogen receptor. These studies suggest that cockerel serum and liver nuclei contain a natural compound which competes with the triphenylethylenes at the antiestrogen-binding site and may occupy the nuclear binding sites in vivo.     

Estrogen-binding proteins in the oviduct of the turtle, Chrysemys picta: evidence for a receptor species.

Estradiol-binding proteins in the reproductive tract of the turtle, Chrysemys picta, were characterized. Cytosol was prepared from the oviducts of mature female turtles, and estradiol binding was measured using charcoal adsorption and glycerol density gradient centrifugation. A sex steroid-binding protein (SBP) similar to that found in turtle plasma was demonstrated in oviduct cytosol. The characteristics of this SBP-like binding were as follows: Ka = 10(8) M-1; capacity, 10(-12) mol/mg protein; and sedimentation coefficient, 6--7S in low salt gradients. The SBP-like protein binds testosterone and progesterone as well as 17 beta-estradiol but does not bind diethylstilbestrol. No receptor-like binding activity could be demonstrated using these techniques. Explant culture and DNA cellulose affinity chromatography were used to remove the SBP-like material before assay of [3H]estradiol binding. Using these techniques, a high affinity (Ka = 10(9) M-1), low capacity (n = 10(-14) mol/mg cytosol protein) estradiol receptor was demonstrated. The putative turtle receptor exhibits steroid specificity and sedimentation profiles (6S and 8S in low salt, 4S and 5S in high salt) comparable to those of estrogen receptors in mammalian species. These results suggest a certain degree of physiochemical similarity between putative estrogen receptors in mammalian and turtle reproductive tracts.     

Estrogen mimics bind with similar affinity and specificity to the hepatic estrogen receptor in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss)

A variety of organic chemicals have been documented to bind to and activate the estrogen receptor (ER) and consequently induce estrogenic effects in different animals. Although the function of the ER seems phylogenetically conserved in vertebrates, a considerable interspecies variation in the structure of the ER has been demonstrated. In this study, the hepatic ER from Atlantic salmon (Salmo salar) and rainbow trout (Oncorhunchus mykiss) were partly characterized, and the ligand-binding preference for a range of endogenous steroids and environmental estrogens (estrogen mimics) was determined by receptor-radio ligand studies. The results show that both Atlantic salmon and rainbow trout livers contain ERs that bind 1,2,4,6,7-[(3)H]estradiol ([(3)H]-E2) with high affinity and low capacity (K(d) = 2.5-4.4 nM and B(max) = 27-97 fmol/mg protein). The Atlantic salmon ER (asER) and rainbow trout ER (rtER) exhibit similar [(3)H]-E2 binding characteristics, although livers from female fish contained a two to three times higher amounts of ER than the males in the two species. In competition studies with [(3)H]-E2, the asER and rtER were found to bind both native steroids (E2 > estrone > 17 beta-estradiol 17-glucuronide > testosterone and 11-ketotestosterone) and putative estrogen mimics (diethylstilbestrol, 4-hydroxytamoxifen, ethynylestradiol > genistein, zearalenone > 4-t-octylphenol, 4-n-nonylphenol, and o,p'-DDT). The pesticides toxaphen and dieldrin, which are proposed to bind to and activate the human ER, did not display significant binding affinity for the fish ER, however. In general, the asER and rtER were found to bind both native steroids and estrogen mimics with similar affinity and specificity. The present results suggest that closely related species such as Atlantic salmon and rainbow trout display similar ER ligand-binding requirements, although interspecies differences in ER affinity and specificity between divergent species such as fish and humans may exist.     

Characterization of an estrogen-binding protein in the yeast Candida albicans

An estrogen-binding protein (EBP) has been identified and characterized in the cytosol of the pathogenic yeast Candida albicans. Binding of [3H]estradiol was found to be optimal at pH 7.4 in the presence of 0.3 M KCl and was linearly related to protein concentration. Binding was very rapid, reaching maximal levels in about 30 min, and was reversible with a dissociation rate constant of 13.2 +/- 1.7 x 10(-4) sec-1. EBP binding was destroyed by treatment with proteolytic enzymes and by high temperatures. Scatchard analysis of the [3H]estradiol equilibrium binding data of C. albicans (strain stn-1) yielded an apparent dissociation constant of 12.3 +/- 2.1 nM and a maximal binding capacity of 753 +/- 145 fmol/mg protein. Binding competition experiments showed very high specificity and stereoselectivity of EBP, demonstrating the following order of potency in displacing [3H]estradiol: 17 beta-estradiol greater than estrone greater than estriol greater than 17 alpha-estradiol. Negligible competitive potency was found for other mammalian steroid hormones, diethylstilbestrol, tamoxifen, or fungal hormones. The abundance of EBP was 4- to 10-fold higher during the early logarithmic growth phase of yeast cells than during the stationary phase. The molecular size of EBP, measured by Sephacryl S-200 gel exclusion chromatography, yielded a Stokes radius of approximately 29 A. Sucrose density gradient sedimentation showed a sedimentation coefficient (S2020,W) of 4, with no ionic dependent aggregation of the [3H]estradiol-EBP complex. The apparent mol wt of the EBP is approximately 46,000, with an axial ratio of 1, indicating the symmetrical shape of the molecule. In summary, in addition to the previously described corticosterone-binding protein, a separate high affinity, stereospecific, estrogen-selective binder has been demonstrated in the cytosol of C. albicans.     

The cyclic relationship of estrogen sulfurylation to the nuclear receptor level in human endometrial curettings.

Human endometrial curettings were selected principally from patients undergoing tubal ligations for elective sterilization. Specimens were analyzed for the metabolism of 17beta-[6,7-3H]estradiol and Na2 35SO4; assayed for the nuclear receptor content with 17beta-[6,7-3H]estradiol; and examined for the cytosol receptor content with 17beta-[2,4,6,7-3H]estradiol. The results of these experiments demonstrated that a) estrogen sulfotransferase activity is greatly stimulated during the secretory phase; b) although estrogen dehydrogenase is active throughout the menstrual cycle, the formation of estrone is elevated in concert with sulfurylation; and c) this increased metabolism of 17beta-estradiol is accompanied by a decreased nuclear uptake of the estrogen receptor complex. The importance of this endometrial estrogen metabolism in the maintenance of a secretory tissue is discussed.     

Effects of estrogen on muscarinic acetylcholine receptors in the rat hippocampus

The aim of the present study was to investigate whether different estrogen manipulations have effects on the expression of muscarinic acetylcholine receptors (mAChRs) in the adult female rat hippocampus. Hippocampus was obtained from rats in proestrus (control), ovariectomized for 2, 10 and 15 days, ovariectomized for 15 days and treated with 17beta-estradiol for 7 days, and treated with 17beta-estradiol immediately after ovariectomy for 21 days. Rats' estrogen status was monitored by measuring estradiol plasma levels and uterus relative weight. [3H]quinuclidinyl benzilate ([3H]QNB) binding studies indicated that ovariectomy time-dependently increases the number of mAChRs in hippocampus when compared to those obtained from control rats. Estradiol treatments for 21 days avoid the effect of ovariectomy. However, the estradiol treatments for 7 days after 15 days of ovariectomy slightly change the number of mAChRs. In conclusion, these results showed that ovariectomy time-dependently increases mAChRs number in the rat hippocampus. In addition, these data suggest that treatment with estradiol initiated within a specific period of time after the loss of ovarian function may be effective at preventing specific effects of hormone deprivation on hippocampus.     

Androgen and estrogen receptors in fetal rhesus monkey brain and anterior pituitary

In this study, we sought to identify and characterize cytosolic androgen and estrogen receptors in the brain and anterior pituitary gland (AP) of fetal rhesus monkeys using the technique of DNA-cellulose chromatography. Cytosolic extracts were prepared from fetal monkey (days 135-162 of gestation) tissues including hypothalamus-preoptic area/amygdala (HPOA/AMG), cerebral cortex, and AP. Extracts were incubated with [3H]testosterone, [3H]5 alpha-dihydrotestosterone, or [3H] 17 beta-estradiol and applied to DNA-cellulose columns. [3H]Androgen- and [3H]estrogen-binding activities from cytosolic extracts adhered to DNA-cellulose. After elution with a linear salt gradient (10-500 mM NaCl) [3H]androgen-binding activity exhibited elution maxima between 130-150 mM NaCl, while [3H] estrogen-binding activity exhibited elution maxima between 200-220 mM NaCl. These elution patterns were similar in every region examined and were characteristic of putative androgen and estrogen receptors found in other vertebrate species. Additional experiments established the high affinity-low capacity nature of both androgen- and estrogen-binding activities, as well as their inhibition by known competitors of receptor binding. Estimates of binding activity at ligand concentrations that approximated saturation suggested that the concentration (moles specific bound per mg cytosolic protein) of both androgen and estrogen receptor were highest in the AP, intermediate in the HPOA/AMG, and lowest in the cerebral cortex. Comparisons of androgen- and estrogen-binding activities revealed that in the AP, apparent concentrations of the estrogen receptor exceeded those of androgen. Androgen and estrogen receptor concentrations were roughly equivalent in the HPOA/AMG, whereas, in the cerebral cortex, androgen receptor concentration was greater than estrogen. Collectively, these data demonstrate that in the fetal primate brain and AP, distinct androgen and estrogen receptors are present which might mediate the action of gonadal steroids on sexual differentiation.     

A product of yeast RAS2 gene is a guanine nucleotide binding protein.

Yeast Saccharomyces cerevisiae contains two genes, RAS1 and RAS2, which show remarkable homology to mammalian ras genes. To characterize these gene products, we have expressed the RAS2 gene in yeast using an inducible GAL10 promoter. After labeling with [35S]methionine and immunoprecipitating with a monoclonal antibody Y13-259, which reacts with p21 encoded by mammalian ras genes, a major band having an apparent molecular weight of 41,000 is detected. This band has also been identified in cell-free translation products of polyadenylated RNA extracted from yeast cells grown in the presence of galactose. Crude extracts of cells expressing the RAS2 gene exhibit guanine nucleotide binding activity. This is detected by incubation with [3H]GDP followed by immunoprecipitation with the antibody Y13-259. The binding of labeled GDP is inhibited by a 20-fold excess of GDP, GTP, and, to a lesser extent, by UTP, a characteristic similar to that possessed by the mammalian ras proteins. However, the activity of the yeast protein differs from that of the mammalian proteins in its strong dependence on temperature. The guanine nucleotide binding activity provides an assay to purify the yeast protein.     

Estrogen responsiveness of normal mouse mammary cells in primary cell culture: association of mammary fibroblasts with estrogenic regulation of progesterone receptors

Estrogens enhance proliferation of normal mouse mammary cells in vivo. However, when cultured alone, normal mouse mammary epithelial cells fail to exhibit a proliferative response to estrogen in vitro; the basis for this lack of in vitro responsiveness to estrogen is not known. The purpose of the present study is to determine if cultured normal mouse mammary cells possess estrogen receptors (ER) and/or progesterone receptors (PgR) and if the ER mechanism is functional, as measured by the ability of estrogens to regulate PgR. Recent findings that mammary fibroblasts can influence the behavior of mammary epithelial cells in vitro led us to investigate their effect on epithelial cell responsiveness to estrogen. In these studies, collagenase-dissociated mammary glands of midpregnant BALB/c mice were the source of mixed cultures (containing both epithelial cells and fibroblasts) and epithelial or fibroblast cultures. The purity of epithelial or fibroblast cultures was quantified immunocytochemically using antivimentin antibody as a fibroblast marker. Steroid hormone binding was quantified in intact cultured cells using [3H]R5020 and 17 beta-[3H]estradiol as the ligands. Specific high affinity binding sites for estrogen (Kd = 3.1 +/- 0.8 X 10(-10] and progestins (Kd = 3.3 +/- 1.2 X 10(-9) M) were detected in mixed cultures. To assess the possible role of mammary fibroblasts, we investigated cultures containing only fibroblasts which were derived by differential centrifugation. When 17 beta-estradiol was added to the culture medium, a significant (P less than 0.01) increase in PgR concentration was observed in mixed cultures. While mixed cultures maintain responsiveness to estrogen in vitro, as measured herein, the epithelial cultures, derived by differential centrifugation and Percoll gradient sedimentation, did not. However, estrogenic regulation of PgR appears to be specific to epithelial cells in mixed cultures, since fibroblast cultures neither contained PgR nor displayed estrogen-inducible PgR. The lack of responsiveness of epithelial cultures is not due to a loss or decrease in the ER concentration. Thus, the presence of mammary fibroblasts appears to be associated with epithelial cell responsiveness to estrogen in vitro.     

Binding of xenobiotics to hepatic estrogen receptor and plasma sex steroid binding protein in the teleost fish, the common carp (Cyprinus carpio)

Competitive receptor binding assays have been suggested as an in vitro screening tool for assessing the activity of alleged estrogenic substances. In this study, we determined the ability of steroidal and nonsteroidal substances to inhibit the binding of [(3)H]17 beta-estradiol (E2) to the hepatic estrogen receptor (ER) and the plasma sex steroid binding protein (SBP) of the teleost fish, the common carp (Cyprinus carpio). The objectives of the study were (1) to characterize ER binding in the liver cytosol of male and female carp, (2) to establish complete [(3)H]E2 displacement curves from carp ER for a range of natural and xenobiotic substances and to compare the ligand data of carp ER with published data from other vertebrate species to reveal possible species differences, and (3) to determine the interaction of natural and xenobiotic substances with the steroid binding site of SBP in carp plasma. The results indicate the presence of a single class of estrogen binding sites with high affinity and limited capacity in liver cytosol of carp. The various test agents showed partly quantitative differences in their binding affinities, with the xenobiotics generally showing limited ability to displace [(3)H]E2 from the hepatic ER or from plasma SBP of carp. However, we found no evidence that a compound is an ER ligand exclusively in one species. The findings of this study indicate that interspecies extrapolation of steroid receptor binding data is possible on a yes/no basis but not on a quantitative basis.     

Type II estrogen-binding sites and 17 beta-hydroxysteroid dehydrogenase activity in human peripheral blood mononuclear cells

Type II estrogen-binding sites (type II EBS) have been demonstrated in human peripheral blood mononuclear cells (PBMC) using a whole cell assay with [6,7-3H]estradiol [( 3H]E2) as tracer. During whole cell incubations for 60 min at 37 C for type II EBS quantification, we found that PBMC contain 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) activity, which led to errors in estimating type II EBS concentrations by diminishing, by about 70%, the amount of available labeled E2. On the other hand, after 150 min at 4 C only 16% of the tracer was converted to estrone. Thus, we measured the maximal steady state binding in PBMC by incubating the cells with [3H]E2 at 4 C for 150 min. Equilibrium binding analysis of PBMC yielded sigmoid saturation curves with a saturation point at a ligand concentration of about 40 nmol/L. Scatchard analysis of binding data yielded a concave plot, which together with a Hill coefficient of 2.13, suggests that the type II EBS may have multiple binding sites which display positive cooperativity. The apparent equilibrium dissociation constant (Kd), determined from the [3H]E2 concentration required for half-saturation, was about 22 nmol/L. The type II EBS were estrogen specific, as demonstrated by competition experiments. Only those steroids with estrogenic activity inhibited binding of [3H]E2; nonestrogenic steroids did not. The type II EBS were found to be 3S macromolecules based on analysis of postlabeled fractions prepared by sucrose density gradient centrifugation. The number of type II EBS in PBMC from normal women was highest during the late follicular-early luteal phase of the menstrual cycle. We conclude that human PBMC specifically take up, retain, and metabolize E2.     

Estrogens act in rat hippocampus and frontal cortex to produce rapid, receptor-mediated decreases in serotonin 5-HT(1A) receptor function

Previously our laboratory has shown that 17beta-estradiol in vivo rapidly decreases R(+)-8-OH-DPAT-stimulated [(35)S]GTPgammaS binding (a measure of the initial biochemical event in the intracellular signaling pathway associated with 5-HT(1A) receptors) in the hippocampus, frontal cortex and amygdala. Studies were designed to determine if 17beta-estradiol also acts in vitro on estrogen receptors in the hippocampus and frontal cortex to decrease 5-HT(1A) receptor function. Hippocampus and frontal cortex were dissected from ovariectomized rats and incubated for up to 3 h with various estrogens and antiestrogens; membrane homogenates were prepared for R(+)-8-OH-DPAT-stimulated [(35)S]GTPgammaS binding assays. 17beta-Estradiol (10(-6) M) decreased the maximal response in the R(+)-8-OH-DPAT-stimulated [(35)S]GTPgammaS binding assay in a time-dependent manner (observed at 30, 60 and 120 min) in both hippocampus and frontal cortex. The hormone, however, did not alter the EC(50) of R(+)-8-OH-DPAT. When hippocampus and frontal cortex were incubated in graded concentrations of 17beta-estradiol for 1 h, the calculated EC(50) was approximately 2.5 x 10(-8) M in both brain regions. The nonestradiol estrogen diethylstilbestrol also decreased 5-HT(1A) receptor function while the less potent estrogens 17alpha-estradiol and estriol were inactive at 5 x 10(-8) M. The estrogen receptor antagonist ICI 182,780 potently and completely blocked the effects of 17beta-estradiol on 5-HT(1A) receptor function with an apparent K(B) of approximately 10(-9) M. These data demonstrate clearly that estrogens can act on estrogen receptors located in hippocampus and frontal cortex of ovariectomized rats to produce rapid heterologous decreases in 5-HT(1A) receptor function.     

Estrogen biphasic regulation of paracellular permeability of cultured human vaginal-cervical epithelia

The objective of the study was to understand effects of estrogen and aging on paracellular permeability of human vaginal-cervical epithelia. Assays included determinations of transepithelial electrical conductance across cultures of normal human ectocervical epithelial cells on filters. Baseline transepithelial electrical conductance across steroid-deprived cells from postmenopausal women was lower than across cells of premenopausal women. Short-term (24-48 h) treatment with 10 nM 17beta-estradiol increased transepithelial electrical conductance in both groups of cells. In cells of premenopausal women longer-term treatment with estrogen for up to 14 d had no additional effect on permeability, but in cells of postmenopausal women it caused a secondary increase in transepithelial electrical conductance that continued for the duration of the 2-wk treatment. Binding assays of 17beta-[(3)H]estradiol revealed saturable binding to high affinity (1.2-1.3 nM), low capacity sites (0.2-1.2 pmol/mg DNA) in cells of both premenopausal and postmenopausal women. In both types of cells treatment with 17beta-estradiol increased 17beta-[(3)H]estradiol binding activity in a time- and dose-related manner (EC(50) 1 nM; maximal effect within 9-12 h), and increased estrogen receptor-alpha and -beta mRNA. 8-Br-cGMP, a stable cell-permeant analog of cGMP, could mimic the estrogen first phase increase in transepithelial electrical conductance, but not the secondary increase. Treatment with estrogen augmented the increase in transepithelial electrical conductance that was induced by hydrostatic gradients (modulator of the resistance of the lateral intercellular space), and the effect was independent of woman's age or baseline transepithelial electrical conductance. In contrast, the effect of low extracellular calcium (modulator of the tight junctional resistance) was more potent in cells of premenopausal women than in cells of postmenopausal women and was independent of treatment with estrogen. These results suggest that changes in vaginal-cervical epithelial permeability after menopause are determined by aging-related increase in tight junctional resistance, and by low estrogen-dependent increase in lateral intercellular space that lead to net increase in total paracellular resistance and decreased permeability and result in reduced lubrication of the lower genital canal.     

Characterization of membrane estrogen binding proteins from rabbit uterus

Estrogens exert fast non-genomic actions in their target tissues which may involve the participation of receptors located at the cell membrane. Studies were performed to identify and characterize membrane-associated 17beta-estradiol binding proteins in rabbit uterus. Specific and saturable [3H]17beta-estradiol binding sites of high affinity (Kd = 0.36 nM) were detected in uterine microsomes at higher concentration than in cytosol (370 +/- 98 vs. 270 +/- 87 fmol/mg protein, respectively). Various other steroid hormones, the stereoisomer 17alpha-estradiol and the antiestrogen tamoxifen were significantly less effective than 17beta-estradiol to compete with the radioactive ligand for binding to the membranes. The microsome binding sites were trypsin-sensitive and could be extracted to a great extent (80-90%) with 0.4/0.6 M KCl. Assays of the marker enzyme glucose-6-P dehydrogenase excluded membrane contamination with cytosolic soluble components. Immunoblot analysis of particulate and soluble fractions using monoclonal antibodies against the transactivation, heat shock protein recognition, and steroid binding domains of the nuclear estrogen receptor (ER; 67 kDa), revealed lower concentrations of the ER in membranes and the presence of five additional immunoreactive proteins of 57, 50, 32, 28, and 11 kDa which were absent in cytosol. Moreover, the antibody against the steroid binding domain was as effective as an inhibitor for cytosolic and membrane specific radioligand binding. Extraction of microsomes with the nondenaturing detergent CHAPS allowed a 2-fold enrichment of ER-like binding proteins as shown by antibody labeling and [3H]17beta-estradiol binding analysis. The results of this work are consistent with the existence of novel 17beta-estradiol membrane binding proteins structurally related to the intracellular ER. Future studies should investigate whether any of these proteins are involved in the primary events (e.g. receptor function) mediating nongenomic estrogen effects.