Subtype-specific dimerization of alpha 1-adrenoceptors: effects on receptor expression and pharmacological properties

The potential role of dimerization in controlling the expression and pharmacological properties of alpha1-adrenoceptor subtypes was examined using coimmunoprecipitation of epitope-tagged receptors. Human alpha1-adrenoceptor subtypes (alpha1A, alpha1B, alpha1D) were tagged at their amino-termini with Flag or hemagglutinin epitopes and transfected into human embryonic kidney 293 cells. Homodimerization of all three subtypes was observed by coimmunoprecipitation of receptors with different tags and was not altered by norepinephrine treatment. Heterodimer formation between hemagglutinin-tagged alpha1B-adrenoceptors and Flag-tagged alpha1A- or alpha1D-adrenoceptors was also observed. However, no alpha1A/alpha1D-adrenoceptor heterodimers were observed, suggesting that dimerization is subtype-specific. The extent of heterodimerization was also unaltered by norepinephrine treatment. alpha1-Adrenoceptor truncation mutants lacking carboxyl or amino-terminal sequences formed homo- and heterodimers similarly to full-length receptors, suggesting that these domains play little or no role in dimerization. Biotinylation with a membrane-impermeable agent showed that monomers and homo- and hetero-oligomers of all three subtypes are expressed on the cell surface. Radioligand binding studies showed that heterodimerization did not alter the affinity of alpha1-adrenoceptors for norepinephrine, prazosin, or subtype-selective antagonists, suggesting that dimerization does not result in pharmacologically distinct subtypes. However, coexpression of alpha1B-adrenoceptors significantly increased both binding site density and protein expression of alpha1A- and alpha1D-adrenoceptors, and increased cell surface expression of alpha1D-adrenoceptors, suggesting a functional role for heterodimerization. Conversely, coexpression of alpha1A-with alpha1D-adrenoceptors, which did not heterodimerize, had no effect on receptor density or protein. These studies demonstrate subtype-selective heterodimerization of alpha1-adrenoceptors, which does not change their pharmacological properties but seems to have functional consequences in regulating receptor expression and trafficking.     

Nandrolone treatment decreases the alpha1B-adrenoceptor mRNA level in rat kidney, but not the density of alpha1B-adrenoceptors in cultured MDCK-D1 kidney cells

We have previously shown that treatment of rats with the anabolic androgen steroid nandrolone decreased the density of alpha1B-adrenoceptors in the rat kidney [Uhlén, S., Lindblom, J., Kindlundh, A., Muhisha, P., Nyberg, F., (2003). Nandrolone treatment decreases the level of rat kidney alpha1B-adrenoceptors. Naunyn-Schmiedeberg's Arch. Pharmacol. 368, 91-98]. This effect may have been caused either by decreased de novo synthesis of alpha1B-adrenoceptors or by increased degradation of alpha1B-adrenoceptors [corrected] In the present study, we show that treatment of rats with nandrolone decreases the level of mRNA for the alpha1B-adrenoceptor in the kidneys, implying decreased synthesis of alpha1B-adrenoceptors. On the other hand, nandrolone did not decrease the density of alpha1B-adrenoceptors in Madin-Darby Canine Kidney (MDCK) cells, even though the sub-cell line tested, MDCK-D1, expresses both the androgen receptor and the alpha1B-adrenoceptor. It is concluded that the regulation of alpha1B-adrenoceptor expression by anabolic androgenic steroids is intricate and cell-type specific.     

Antipsychotics lack alpha 1A/B adrenoceptor subtype selectivity in the rat.

The alpha1A- and alpha1B-adrenoceptor affinity of the typical (chlorpromazine, haloperidol, pimozide, thioridazine and trifluoperazine) and atypical (clozapine, olanzapine, quetiapine, risperidone and sertindole) antipsychotics was determined by competition binding at alpha1A- and alpha1B-adrenoceptors in rat submaxillary gland and liver. Although all antipsychotics bound to both subtypes with relatively high affinity (K(i)s<74 nM), none were selective (>10-fold). Comparison with published dopamine D2 receptor affinities suggests that antipsychotic blockade of alpha1A- and/or alpha1B-adrenoceptors may contribute to the antipsychotic activity of all the atypical and several of the typical antipsychotics examined.     

The effects of SB 216469, an antagonist which discriminates between the alpha 1A-adrenoceptor and the human prostatic alpha 1-adrenoceptor.

1. The affinity of the alpha 1-adrenoceptor antagonist SB 216469 (also known as REC 15/2739) has been determined at native and cloned alpha 1-adrenoceptor subtypes by radioligand binding and at functional alpha 1-adrenoceptor subtypes in isolated tissues. 2. In radioligand binding studies with [3H]-prazosin, SB 216469 had a high affinity at the alpha 1A-adrenoceptors of the rat cerebral cortex and kidney (9.5-9.8) but a lower affinity at the alpha 1B-adrenoceptors of the rat spleen and liver (7.7-8.2). 3. At cloned rat alpha 1-adrenoceptor subtypes transiently expressed in COS-1 cells and also at cloned human alpha 1-adrenoceptor subtypes stably transfected in Rat-1 cells, SB 216469 exhibited a high affinity at the alpha 1a-adrenoceptors (9.6-10.4) with a significantly lower affinity at the alpha 1b-adrenoceptor (8.0-8.4) and an intermediate affinity at the alpha 1d-adrenoceptor (8.7-9.2). 4. At functional alpha 1-adrenoceptors, SB 216469 had a similar pharmacological profile, with a high affinity at the alpha 1A-adrenoceptors of the rat vas deferens and anococcygeus muscle (pA2 = 9.5-10.0), a low affinity at the alpha 1B-adrenoceptors of the rat spleen (6.7) and guinea-pig aorta (8.0), and an intermediate affinity at the alpha 1D-adrenoceptors of the rat aorta (8.8). 5. Several recent studies have concluded that the alpha 1-adrenoceptor present in the human prostate has the pharmacological characteristics of the alpha 1A-adrenoceptor subtype. However, the affinity of SB 216469 at human prostatic alpha 1-adrenoceptors (pA2 = 8.1) determined in isolated tissue strips, was significantly lower than the values obtained at either the cloned alpha 1a-adrenoceptors (human, rat, bovine) or the native alpha 1A-adrenoceptors in radioligand binding and functional studies in the rat. 6. Our results with SB 216469, therefore, suggest that the alpha 1-adrenoceptor mediating contractile responses of the human prostate has properties which distinguish it from the cloned alpha 1a-adrenoceptor or native alpha 1A-adrenoceptor. Since it has previously been shown that the receptor is not the alpha 1B- or alpha 1D-adrenoceptor, the functional alpha 1-adrenoceptor of the human prostate may represent a novel receptor with properties which differ from any of the alpha 1-adrenoceptors currently defined by pharmacological means.     

Zearalenone exposure modulates the expression of ABC transporters and nuclear receptors in pregnant rats and fetal liver

The mycotoxin zearalenone (ZEN) is produced by a variety of Fusarium fungi and contaminates numerous cereals, fruits and vegetables. Interacting with the estrogen receptors, ZEN and reduced metabolites zearalenols cause hormonal effects in animals. Few data are available on the effects of repeated exposure to ZEN, particularly during pregnancy. The aim of our work was to assess the impact of this toxin on the expression of ABC transporters and nuclear receptors in fetal liver and pregnant rats that were exposed daily (gestation day 7-20) to 1 mg/kg ZEN. Significant variations were observed, depending on the tissue type, the tissue origin (maternal or fetal), and the time of analysis after the last exposure to ZEN (4 h or 24 h). The modulations of expression were independent of the magnitude of tissue impregnation by ZEN and its metabolites. The maternal uterus was the most sensitive tissue: Abcb1a, Abcb1b and Abcg2 mRNA and protein expressions were induced at both times, while Abcc1, Abcc3 and Esr1 mRNA and protein expressions were inhibited then induced 4 h and 24 h after exposure, respectively. In the fetal liver, Abcb1a and Esr1 protein expression was inhibited at both times, while mRNA expression was induced 24 h after the last exposure to ZEN. These results suggested that ZEN exposure could impact maternal and fetal exposure to ABC transporters substrates, and influence fetus development through nuclear receptor modulation.     

Gene expression and estrogen sensitivity in rat uterus after developmental exposure to the polybrominated diphenylether PBDE 99 and PCB

Considering the presence of polybrominated diphenylethers (PBDEs) in human milk and cord blood, and the estrogenic activity of some congeners, it is conceivable that PBDEs may interact with developing neuroendocrine systems. We investigated effects of 2,2′,4,4′,5-pentabromo-DE (PBDE 99), a major congener in human milk, on development of brain and reproductive organs, with focus on estrogen target gene expression. Time-pregnant Long Evans rats were subcutaneously injected with PBDE 99 (1 or 10 mg/kg/day), the PCB mixture Aroclor 1254 (10 mg/kg/day), known to interfere with sexual development, or vehicle, from gestational day (GD) 10 to GD 18. In female offspring, anogenital distance was unaffected by PBDE 99 but increased by Aroclor; puberty (vaginal opening) was not significantly changed. Adult PBDE 99-exposed offspring exhibited unchanged uterine weight but increased ovarian weight. Uterine mRNA levels of estrogen target genes were determined by real-time PCR. Progesterone receptor (PR) mRNA was down-regulated at both PBDE 99 doses, estrogen receptor alpha (ER alpha), ER beta and insulin-like growth factor-I (IGF-I) were up-regulated at the lower dose. Aroclor induced different effect patterns. In order to investigate possible changes in sensitivity of target genes to estrogen, some offspring were ovariectomized at 10 weeks of age, s.c. injected with estradiol-17β (E2, 10 μg/kg) or vehicle at 12 weeks, and sacrificed 6 h later. PBDE 99 dose-dependently reduced the magnitude of IGF-I mRNA induction by E2, and increased the magnitude of ER beta repression. PBDE 99 also influenced baseline levels of PR, IGF-I and ER beta mRNAs in ovariectomized, vehicle-injected controls. These data indicate that developmental exposure to PBDE 99 at doses devoid of general toxicity, affects the regulation of estrogen target genes in uterus. Since PBDE 99 was detected in blood and adipose tissue of adult offspring, these effects may result from interactions with developmental processes, adult functions, or a combination of both.     

淫羊藿苷对去卵巢大鼠骨和下丘脑不同核团ERβ mRNA表达的影响

目的观察淫羊藿苷对去卵巢大鼠骨和下丘脑不同核团ERβmRNA表达的影响,探讨其治疗绝经后骨质疏松的作用机制。方法将10～11月龄SD♀大鼠60只,随机分为假手术组、去卵巢模型组、淫羊藿苷小、中、大剂量组(每组12只)。以双侧卵巢切除法建立大鼠骨质疏松模型。分别用淫羊藿苷小、中、大剂量给药4个月,采用RT-PCR法,观察各组大鼠骨和下丘脑室旁核、视上核、弓状核ERβmRNA表达的变化。结果大鼠卵巢切除后,血清E2水平、椎骨骨密度、子宫湿重、胫骨和下丘脑弓状核ERβmRNA表达明显降低(P<0.01),下丘脑室旁核、视上核ERβmRNA表达均明显升高(P<0.01)。与去卵巢模型组比较,淫羊藿苷50.0和100.0 mg.kg-1组治疗后,血清E2水平、椎骨骨密度、胫骨和下丘脑弓状核ERβmRNA表达明显升高(P<0.01),下丘脑室旁核、视上核ERβmRNA表达均明显降低(P<0.01),子宫湿重无明显改变(P>0.05)。结论淫羊藿苷可以改善切除卵巢所致的骨质疏松大鼠的骨密度,对子宫无不良反应。其机制可能与其选择性调节去卵巢骨质疏松大鼠下丘脑不同核团ERβmRNA表达水平有关,调节下丘脑功能活动是其途径之一。     

Differential expression patterns of Wnt and beta-catenin/TCF target genes in the uterus of immature female rats exposed to 17alpha-ethynyl estradiol.

To characterize the effects of an estrogen receptor (ER) agonist on the gene expressions in the uterus, immature female rats were administered once orally with 17alpha-ethynyl estradiol (EE, 3 mug/kg), a potent ER agonist. We focused on four categories of sex steroid hormone receptor genes: well-known estrogen target genes, Wnt genes, and beta-catenin/T-cell factor (TCF) target genes. ERalpha, ERbeta, progesterone receptor, and androgen receptor mRNAs were all downregulated at 24 and/or 48 h after EE administration. Complement C3 and insulin-like growth factor 1 mRNAs were markedly induced after EE administration. Although the time courses of Wnt4, Wnt5a, and Wnt7a mRNA status varied until 12 h after EE administration, all of them were simultaneously downregulated at 24 and 48 h. The remarkable downregulation of Wnt7a mRNA in response to EE was considered to be important to understand the various uterine phenomena affected by ER agonists. In the beta-catenin/TCF target genes, the downregulation of anti-Mullerian hormone type 2 receptor and bone morphogenetic protein 4 mRNA after EE administration appeared to be closely related to the downregulation of Wnt7a. The upregulation of cyclin D1 and follistatin mRNA at the early phase after EE administration was considered to have been affected by the upregulation of Wnt4. These results indicate that an ER agonist influences not only the mRNA expression of sex steroid hormone receptor genes and well-known estrogen target genes but also Wnt genes (Wnt4, Wnt5a, Wnt7a) and beta-catenin/TCF target genes in the uterus of immature rats, indicating that their molecules are the potential players affected by estrogenic stimuli.     

Differential antagonism by conotoxin rho-TIA of contractions mediated by distinct alpha1-adrenoceptor subtypes in rat vas deferens, spleen and aorta

The ability of the conotoxin rho-TIA, a 19-amino acid peptide isolated from the marine snail Conus tulipa, to antagonize contractions induced by noradrenaline through activation of alpha1A-adrenoceptors in rat vas deferens, alpha1B-adrenoceptors in rat spleen and alpha1D-adrenoceptors in rat aorta, and to inhibit the binding of [125I]HEAT (2-[[beta-(4-hydroxyphenyl)ethyl]aminomethyl]-1-tetralone) to membranes of human embryonic kidney (HEK) 293 cells expressing each of the recombinant rat alpha1-adrenoceptors was investigated. rho-TIA (100 nM to 1 microM) antagonized the contractions of vas deferens and aorta in response to noradrenaline without affecting maximal effects and with similar potencies (pA2 approximately 7.2, n=4). This suggests that rho-TIA is a competitive antagonist of alpha1A- and alpha1D-adrenoceptors with no selectivity between these subtypes. Incubation of rho-TIA (30 to 300 nM) with rat spleen caused a significant reduction of the maximal response to noradrenaline, suggesting that rho-TIA is a non-competitive antagonist at alpha1B-adrenoceptors. After receptor inactivation with phenoxybenzamine, the potency of rho-TIA in inhibiting contractions was examined with similar occupancies (approximately 25%) at each subtype. Its potency (pIC50) was 12 times higher in spleen (8.3+/-0.1, n=4) than in vas deferens (7.2+/-0.1, n=4) or aorta (7.2+/-0.1, n=4). In radioligand binding assays, rho-TIA decreased the number of binding sites (B(max)) in membranes from HEK293 cells expressing the rat alpha1B-adrenoceptors without affecting affinity (K(D)). In contrast, in HEK293 cells expressing rat alpha1A- or alpha1D-adrenoceptors, rho-TIA decreased the K(D) without affecting the B(max). It is concluded that rho-TIA will be useful for distinguishing the role of particular alpha1-adrenoceptor subtypes in native tissues.     

Effects of tamoxifen and raloxifene on normal human endometrial cells in an organotypic in vitro model

The selective estrogen receptor modulator tamoxifen is widely used in breast cancer therapy though its use is associated with an elevated risk of endometrial carcinoma. An organotypic culture model was employed here to examine the effects of tamoxifen and raloxifene, a related compound with no known adverse uterine effects, on epithelial cells of the premenopausal human endometrium. Changes in the expression levels of the proliferation marker Ki67, and estrogen and progesterone receptors were evaluated. No change in the Ki67 index compared to untreated controls was detected in cultures exposed to tamoxifen or tamoxifen+estradiol. In response to tamoxifen, the level of progesterone receptor-expressing organoids was shown to vary markedly between individual samples, whereas no change in estrogen receptor expression could be demonstrated. A significant decrease in Ki67 expression was observed in raloxifene-exposed cultures. Raloxifene or raloxifene+estradiol had no effect on progesterone receptor expression. The expression of estrogen receptor was markedly inhibited in response to raloxifene or raloxifene+estradiol in all but two samples displaying an intense estrogen receptor labelling. The present observations add to current clinical data on the respective estrogen receptor agonist and antagonist activities of tamoxifen and raloxifene on the human uterus by providing novel insights into the interindividual variation in cellular responses. Our organotypic model may have uses as an alternative to animal experimentation in preclinical screening of the endometrial effects of selective estrogen receptor modulators and may serve as a tool in personalized medicine by identifying patients with an increased risk of developing endometrial pathologies.     

Changes in mRNA expression induced by sustained noradrenaline stimulation are different for alpha1-adrenoceptor subtypes in HEK293 cells

1. The aim of the present study was to investigate noradrenaline (NA)-induced regulation of alpha1-adrenoceptor (AR) mRNA expression in human embryonic kidney (HEK) 293 cells stably expressing cloned alpha1-AR subtypes with similar receptor densities. Stable transfection was performed by calcium phosphate precipitation. Receptor expression was detected by radioligand binding assay. The mRNA expression was measured by RNase protection assay. 2. alpha1-Adrenoceptor subtype mRNA respond in distinct ways following prolonged exposure to NA. The mRNA level of the alpha 1A-AR subtype was unchanged, the mRNA level of the alpha 1B-AR subtype was increased and the mRNA level of the alpha 1D-AR subtype declined time dependently. The protein kinase C (PKC) inhibitor calphostin C or RO 31-8220 abolished the NA-induced downregulation of alpha 1D-AR mRNA. Phorbol myristate acetate (PMA), a PKC activator, similarly repressed the effects of NA on alpha 1D-AR. However, calphostin C, RO 31-8220 or PMA had no effect on the induction of alpha 1B-AR mRNA by NA. The Ca2+-ATPase inhibitor thapsigargin or the calcium chelator 1,2-bis-(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA/AM) had no effect on the repression of alpha 1D-AR mRNA, but did inhibit the induction of alpha 1B-AR mRNA by NA. Noradrenaline significantly decelerated the degradation of alpha 1B-AR mRNA, but had no effect on the degradation of alpha 1D-AR mRNA. 3. Thus, the mRNA expression of three alpha1-AR subtypes in HEK293 cells is differentially regulated through distinct signal transduction pathways under sustained NA stimulation. The upregulation of alpha 1B-AR mRNA is via the Ca2+ pathway, whereas the downregulation of alpha 1D-AR mRNA is via the PKC pathway.     

In situ hybridization and immunocytochemistry of alpha1-adrenoceptors in human peripheral blood lymphocytes

1 alpha1-Adrenoceptor subtypes were investigated in cytospin centrifuged preparations of human peripheral blood lymphocytes by in situ hybridization and immunocytochemistry. 2 In situ hybridization cytochemistry revealed alpha1A-, alpha1B-, and alpha1D-receptor mRNA in human peripheral blood lymphocytes. Lymphocytes hybridized for alpha1A receptor subtype represented approximately 30% of total lymphocytes, those hybridized for alpha1Beta- and alpha1D-receptor subtypes averaged 42 and 25% of total lymphocytes, respectively. 3 Cytospin centrifuged lymphocytes exposed to anti-alpha1A-, alpha1Beta- or alpha1D-receptor protein antibodies, developed specific immunostaining. Approximately 27% of total lymphocytes were immunoreactive for alpha1A-receptor protein, 40% displayed alpha1B-receptor protein immunoreactivity and 22% alpha1D-receptor protein immunoreactivity. Analysis of percentages as well as of lymphocyte morphology of in situ hybridized and immunolabelled lymphocytes suggests the co-expression of mRNA receptor signal and protein receptor immunostaining in the same lymphocyte. 4 The demonstration of both alpha1-adrenoceptor mRNA and receptor protein subtypes suggests that alpha1-adrenoceptors may have a role in regulating lymphocyte function. 5 The possibility of demonstrating receptor protein immunoreactivity in a small amount of blood, such as that required for preparing cytospin-centrifuged lymphocytes, may stimulate research to evaluate the role of these receptors in lymphocytes and to establish if assessment of lymphocyte alpha1-adrenoceptors may represent a marker of their status in health and disease.     

雌激素功能性调节人内皮细胞的生长调节致癌基因α表达

目的：研究雌激素在体外调节人脐带静脉内皮细胞（HUVEC）的生长调节致癌基因α（GROα）。方法：以体外培养的HUVEC为模型,Northern法检测CXC族趋化因子GROα mRNA;ELISA方法检测细胞表面的GROα蛋白表达;静态细胞粘附实验测定细胞表面的GROα蛋白的生理意义。结果：17β－雌二醇（0．05μmol/L）明显抑制HUVEC产生GROα mRNA和蛋白表达水平;而且17β－雌二醇抑制其蛋白表达水平显示剂量依赖性关系;雌激素受体α拮抗剂他莫昔芬（0．1μmol/L）单独使用不影响其蛋白表达,但可显著逆转17β－雌二醇抑制的GROα蛋白表达,显著逆转17β－雌二醇抑制单核细胞系细胞U937细胞粘附的HUVEC的作用达。结论：通过内皮细胞上雌激素受体α,雌激素可能功能性调节人内皮细胞的GROα的表达。     

Repeated imipramine and electroconvulsive shock increase alpha 1A-adrenoceptor mRNA level in rat prefrontal cortex

alpha(1)-Adrenoceptors have been implicated in the mechanism of action of antidepressants, but their action on specific receptor subtypes was rarely reported. We compared now the action of two prototypic antidepressant treatments: repeated imipramine and electroconvulsive shock, on the expression of the alpha(1A)- and alpha(1B)-adrenoceptor mRNAs and on the receptor density in rats. The mRNA expression was assessed by Northern blot in the prefrontal cortex and the hippocampus, the receptor density was measured by [3H]prazosin binding in the total cerebral cortex and hippocampus. In the cortex, both treatments elevated the alpha(1A)-adrenoceptor mRNA and the expression of receptor protein. The expression of alpha(1B)-adrenoceptor mRNA remained unaffected. In contrast, in the hippocampus, the antidepressant treatments augmented the density of alpha(1A)-adrenoceptor protein without changing the level of its mRNA expression there. The results suggest that the alpha(1A)-adrenoceptor subtype is specifically involved in the mechanism of action of classical antidepressant treatments.     

Uterine effects of the phytoestrogen 6-(1,1-dimethylallyl)naringenin in rats

Phytoestrogens are discussed as candidate substances to treat symptoms related to estrogen deficiency. In in vitro experiments, the naturally occurring flavonoid 6-(1,1-dimethylallyl)naringenin (6-DMAN) emerged as one of the most potent phytoestrogenic substances. 6-DMAN is not as well characterized as other flavonoids (8-prenylnaringenin) or isoflavones (genistein). We tested 6-DMAN for the first time in vivo, in a dose-dependent three-day uterotropic assay in ovariectomized Wistar rats, using 6-DMAN at three different concentrations (1.5 mg/kg; 7.5 mg/kg and 15 mg/kg BW/d). Estradiol (E2; 10 microg/kg BW/d) and the carrier castor oil were used as positive and negative controls. 6-DMAN did not have any effect on uterine wet weight, while the positive control E2 did. In contrast, 6-DMAN stimulated uterine mRNA expression of estrogen responsive genes in ovariectomized rats. Estrogen receptor alpha and beta mRNA were expressed in the uterus. They mediate the expression of genes with an estrogen responsive element in the promoter, e. g., complement C3 and the progesterone receptor. Therefore, we analyzed the expression of the above-mentioned genes in three different concentrations. 6-DMAN up-regulated progesterone receptor and particularly complement C3 mRNA expression however, less pronounced than E2. In conclusion, we demonstrated for the first time estrogenic activities of 6-DMAN in vivo. Surprisingly, although 6-DMAN regulated estrogen responsive gene expression, there was no uterine wet weight gain. These findings make 6-DMAN a very interesting candidate substance for further characterization, as it potentially represents a naturally occurring selective estrogen receptor modulator.     

Estrogenic activity and estrogen receptor beta binding of the UV filter 3-benzylidene camphor. Comparison with 4-methylbenzylidene camphor

UV filters represent new classes of estrogenic [Environ. Health Perspect. 109 (2001) 239] or antiandrogenic [Toxicol. Sci. 74 (2003) 43] chemicals. We tested 3-benzylidene camphor (3-BC), reported as estrogenic in fish [Pharmacol. Toxicol. 91 (2002) 204], and mammalian systems in comparison to 4-methylbenzylidene camphor (4-MBC), shown to be active in rats, and analyzed binding to estrogen receptor subtypes. 3-BC and 4-MBC stimulated MCF-7 cell proliferation (EC(50): 0.68 and 3.9 microM). The uterotrophic assay of 3-BC (oral gavage) in immature rats showed unexpected potency with ED50 45.3mg/kg per day; lowest effective dose 2mg/kg per day, and maximum effect with 70% of ethinylestradiol. After comparing with literature data, we found that the oral 3-BC was considerably more potent than oral bisphenol A and almost as active as subcutaneous genistein. 3-BC and 4-MBC displaced 16alpha 125I-estradiol from porcine uterine cytosolic receptors (IC(50): 14.5 and 112 microM), and from recombinant human estrogen receptor beta (hERbeta) (IC(50): 3-BC, 11.8 microM; 4-MBC, 35.3 microM), whereas no displacement was detected at human estrogen receptor alpha (hERalpha) up to 3mM. This subtype selectivity makes the two camphor derivatives interesting model compounds. Their activity on immature rat uterus is not easily explained by ERbeta activation. It cannot be excluded that active metabolites with possibly different receptor binding characteristics are formed in vivo.     

Molecular identification of potential selective estrogen receptor modulator (SERM) like properties of phytoestrogens in the human breast cancer cell line MCF-7

Numerous epidemiologic studies revealed that ethnic populations with higher dietary intake of phytoestrogens have the lowest incidence for breast cancer. The molecular mechanisms which may be responsible for this cancer protective action of phytoestrogens are so far only barely characterised. There are some hints that phytoestrogens may act like selective estrogen receptor modulators (SERMs) on the breast. For this reason we have investigated potential SERM-like properties of the phytoestrogens daidzein (Dai), coumestrol (Cou), and genistein (Gen) in the human breast cancer cell line MCF-7. Effects of these substances on progesterone (PR) and estrogen receptor alpha (ER) mRNA expression and estrogen receptor alpha protein levels were studied in comparison to estradiol (E2) and the synthetic SERMs raloxifene (Ral) and faslodex (ICI 182 780). PR mRNA expression was up-regulated after administration of Cou, whereas treatment with Dai and Gen induced only a faint increase. ER mRNA expression was down-regulated by Cou but not affected by Dai and Gen. The content of ER protein in the breast cancer cells was strongly decreased by Gen, only a faint reduction could be observed following administration of Cou, whereas administration of Dai slightly increases ER protein levels. In summary and in comparison to the effects observed after administration of E2, Ral, and ICI it turned out that Cou shows molecular properties which are very similar to an estrogen receptor agonist like E2, whereas the molecular properties of Gen are comparable to the SERMs ICI and Ral. These results clearly indicate that phytoestrogens differ significantly in regard to their molecular action on breast cancer cells and can be subdivided into distinct functional categories.