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Sample records for estrogen receptor-mediated mechanisms

  1. Androgen and estrogen receptor mediated mechanisms of testosterone action in male rat pelvic autonomic ganglia

    PubMed Central

    Purves-Tyson, T.D.; Arshi, M.S.; Handelsman, D. J.; Cheng, Y.; Keast, J. R.

    2007-01-01

    Although male reproductive function is primarily androgen dependent, many studies suggest that estrogens have direct actions on the male reproductive organs. Pelvic autonomic neurons provide the motor control of the internal reproductive organs and the penis and various properties of these neurons are affected by endogenous androgens. However, the possible role of estrogens at this site has not been examined. Here we have investigated the significance of estrogens produced by aromatisation of testosterone in the physiological actions of androgens on adult male rat pelvic ganglion neurons. RT-PCR studies showed that aromatase and both estrogen receptors (ERα and ERβ) are expressed in these ganglia. Western blotting also showed that aromatase is expressed in male pelvic ganglia. Using immunohistochemical visualisation, ERα was predominantly expressed by nitric oxide synthase (NOS)-positive parasympathetic pelvic ganglion neurons. In vivo studies showed that the decrease in pelvic ganglion soma size caused by gonadectomy could be prevented by administration of testosterone (T) or dihydrotestosterone (DHT), but not 17β-estradiol (E2), showing that this maintenance action of testosterone is mediated entirely by androgenic mechanisms. However, in vitro studies of cultured pelvic ganglion neurons revealed that T, DHT and E each stimulated the growth of longer and more complex neurites in both noradrenergic and cholinergic NOS-expressing neurons. The effects of T were attenuated by either androgen or estrogen receptor antagonists, or by inhibition of aromatase. Together these studies demonstrate that estrogens are likely to be synthesised in the male pelvic ganglia, produced from testosterone by local aromatase. The effects of androgens on axonal growth are likely to be at least partly mediated by estrogenic mechanisms, which may be important for understanding disease-, aging- and injury-induced plasticity in this part of the nervous system. PMID:17629410

  2. Mechanisms of G protein-coupled estrogen receptor-mediated spinal nociception

    PubMed Central

    Deliu, Elena; Brailoiu, G. Cristina; Arterburn, Jeffrey B.; Oprea, Tudor I.; Benamar, Khalid; Dun, Nae J.; Brailoiu, Eugen

    2012-01-01

    Human and animal studies suggest estrogens are involved in the processing of nociceptive sensory information and analgesic responses in the central nervous system. Rapid pro-nociceptive estrogenic effects have been reported, some of which likely involve G protein-coupled estrogen receptor (GPER) activation. Membrane depolarization, increases in cytosolic calcium and reactive oxygen species (ROS) levels are markers of neuronal activation, underlying pain sensitization in the spinal cord. Using behavioral, electrophysiological and fluorescent imaging studies, we evaluated GPER involvement in spinal nociceptive processing. Intrathecal challenging of mice with the GPER agonist G-1 results in pain-related behaviors. GPER antagonism with G15 reduces the G-1 induced response. Electrophysiological recordings from superficial dorsal horn neurons indicate neuronal membrane depolarization upon G-1 application, which is G15 sensitive. In cultured spinal sensory neurons G-1 increases intracellular calcium concentration and induces mitochondrial and cytosolic ROS accumulation. In the presence of G15, G-1 does not elicit the calcium and ROS responses, confirming specific GPER involvement in this process. Following G-1 intracellular microinjections, cytosolic calcium concentration elevates faster and with higher amplitude compared to extracellular exposure, suggesting subcellular GPER functionality. Thus, GPER activation results in spinal nociception, and the downstream mechanisms involve cytosolic calcium increase, ROS accumulation and neuronal membrane depolarization. Perspective Our results suggest that GPER modulates pain processing in spinal sensory neurons via cytosolic calcium increase and ROS accumulation. These findings extend the current knowledge on GPER involvement in physiology and disease, providing the first evidence of its pro-nociceptive effects at central levels and characterizing some of the underlying mechanisms. PMID:22858342

  3. Activation of membrane estrogen receptors attenuates opioid receptor-like1 receptor-mediated antinociception via an ERK-dependent non-genomic mechanism.

    PubMed

    Small, K M; Nag, S; Mokha, S S

    2013-01-01

    To our knowledge, the present data are the first to demonstrate that activation of membrane estrogen receptors (mERs) abolishes opioid receptor-like 1 (ORL1) receptor-mediated analgesia via extracellular signal-regulated kinase (ERK)-dependent non-genomic mechanisms. Estrogen was shown previously to both attenuate ORL1-mediated antinociception and down-regulate the ORL1 gene expression. The present study investigated whether non-genomic mechanisms contribute to estrogen-induced attenuation of ORL1-mediated antinociception by the mERs GPR30, Gq-coupled mER, ERα, and ERβ. E2BSA [β-estradiol-6-(O-carboxymethyl)oxime: bovine serum albumin] (0.5mM), a membrane impermeant analog of estradiol, injected intrathecally immediately prior to orphanin FQ (OFQ;10 nmol), the endogenous ligand for the ORL1 receptor, abolished OFQ's antinociceptive effect in both male and ovariectomized (OVX) female rats, assessed using the heat-induced tail-flick assay. This effect was not altered by protein synthesis inhibitor, anisomycin (125 μg), given intrathecally 15 min prior to E2BSA and OFQ. Intrathecal application of selective receptor agonists permitted the relative contributions of various estrogen receptors in mediating this blockade of the antinociceptive response of OFQ. Activation of GPR30, Gq-mER, ERα, but not ERβ abolished ORL1-mediated antinociception in males and OVX females. E2BSA produced a parallel and significant increase in the phosphorylation of ERK 2 only in OVX females, and pre-treatment with MEK/ERK 1/2 inhibitor, U0126 (10 μg), blocked the mER-mediated abolition of ORL1-mediated antinociception in OVX females. Taken together, the data are consistent with the interpretations that mER activation attenuates ORL1-mediated antinociception through a non-genomic, ERK 2-dependent mechanism in females. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  4. Age-related reduction in estrogen receptor-mediated mechanisms of vascular relaxation in female spontaneously hypertensive rats.

    PubMed

    Wynne, Fanisha L; Payne, Jason A; Cain, Ashley E; Reckelhoff, Jane F; Khalil, Raouf A

    2004-02-01

    Hypertension increases with aging, and changes in vascular estrogen receptors (ERs) may play a role in age-related hypertension in women. We tested whether age-related increases in blood pressure in female spontaneously hypertensive rats (SHRs) are associated with reduction in amount and/or vascular relaxation effects of estrogen and ER. Arterial pressure and plasma estradiol were measured in adult (12 weeks) and aging (16 months) female SHRs, and thoracic aorta was isolated for measurement of active stress, 45Ca2+ influx, and ERs. Arterial pressure was greater and plasma estradiol was less in aging females than in adult females. In aorta of adult females, Western blots revealed alpha- and beta-ERs that were slightly reduced in aging rats. In endothelium-intact vascular strips, phenylephrine (Phe; 10(-5) mol/L) caused greater active stress in aging rats (9.3+/-0.2) than in adult rats (6.2+/-0.3x10(4) N/m2). 17beta-estradiol (E2) caused relaxation of Phe contraction and stimulation of vascular nitrite/nitrate production, which was reduced in aging rats. In endothelium-denuded strips, E2 still caused relaxation of Phe contraction, which was smaller in aging rats than adult rats. KCl (51 mmol/L), which stimulates Ca2+ influx, produced greater active stress in aging rats (9.1+/-0.3) than in adult rats (5.9+/-0.2x10(4) N/m2). E2 caused relaxation of KCl contraction and inhibition of Phe- and KCl-induced 45Ca2+ influx, which were reduced in aging rats. Thus, aging in female SHR is associated with reduction in ER-mediated NO production from endothelial cells and decrease in inhibitory effects of estrogen on Ca2+ entry mechanisms of smooth muscle contraction. The age-related decrease in ER-mediated vascular relaxation may explain the increased vascular contraction and arterial pressure associated with aging in females.

  5. Chronic Exposure to Anabolic Androgenic Steroids Alters Neuronal Function in the Mammalian Forebrain via Androgen Receptor- and Estrogen Receptor-Mediated Mechanisms

    PubMed Central

    Penatti, Carlos A A; Porter, Donna M; Henderson, Leslie P

    2009-01-01

    Anabolic androgenic steroids (AAS) can promote detrimental effects on social behaviors for which γ-aminobutyric acid type A (GABAA) receptor-mediated circuits in the forebrain play a critical role. While all AAS bind to androgen receptors (AR), they may also be aromatized to estrogens and thus potentially impart effects via estrogen receptors (ER). Chronic exposure of wild type male mice to a combination of chemically distinct AAS increased action potential (AP) frequency, selective GABAA receptor subunit mRNAs, and GABAergic synaptic current decay in the medial preoptic area (mPOA). Experiments performed with pharmacological agents and in AR-deficient Tfm mutant mice suggest that the AAS-dependent enhancement of GABAergic transmission in wild type mice is AR-mediated. In AR-deficient mice, the AAS elicited dramatically different effects, decreasing AP frequency, sIPSC amplitude and frequency and the expression of selective GABAA receptor subunit mRNAs. Surprisingly, in the absence of AR signaling, the data indicate that the AAS do not act as ER agonists, but rather suggest a novel in vivo action in which the AAS inhibit aromatase and impair endogenous ER signaling. These results show that the AAS have the capacity to alter neuronal function in the forebrain via multiple steroid signaling mechanisms and suggest that effects of these steroids in the brain will depend not only on the balance of AR- vs. ER-mediated regulation for different target genes, but also on the ability of these drugs to alter steroid metabolism and thus the endogenous steroid milieu. PMID:19812324

  6. Chronic exposure to anabolic androgenic steroids alters neuronal function in the mammalian forebrain via androgen receptor- and estrogen receptor-mediated mechanisms.

    PubMed

    Penatti, Carlos A A; Porter, Donna M; Henderson, Leslie P

    2009-10-07

    Anabolic androgenic steroids (AAS) can promote detrimental effects on social behaviors for which GABA type A (GABA(A)) receptor-mediated circuits in the forebrain play a critical role. While all AAS bind to androgen receptors (AR), they may also be aromatized to estrogens and thus potentially impart effects via estrogen receptors (ER). Chronic exposure of wild-type male mice to a combination of chemically distinct AAS increased action potential (AP) frequency, selective GABA(A) receptor subunit mRNAs, and GABAergic synaptic current decay in the medial preoptic area (mPOA). Experiments performed with pharmacological agents and in AR-deficient Tfm mutant mice suggest that the AAS-dependent enhancement of GABAergic transmission in wild-type mice is AR-mediated. In AR-deficient mice, the AAS elicited dramatically different effects, decreasing AP frequency, spontaneous IPSC amplitude and frequency and the expression of selective GABA(A) receptor subunit mRNAs. Surprisingly, in the absence of AR signaling, the data indicate that the AAS do not act as ER agonists, but rather suggest a novel in vivo action in which the AAS inhibit aromatase and impair endogenous ER signaling. These results show that the AAS have the capacity to alter neuronal function in the forebrain via multiple steroid signaling mechanisms and suggest that effects of these steroids in the brain will depend not only on the balance of AR- versus ER-mediated regulation for different target genes, but also on the ability of these drugs to alter steroid metabolism and thus the endogenous steroid milieu.

  7. Direct Visualization of Estrogen Receptor-Mediated Transcription in Living Cells

    DTIC Science & Technology

    2007-10-01

    Receptor-Mediated Transcription in Living Cells PRINCIPAL INVESTIGATOR: Paul M. Yen, M.D...reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of...Sep 2007 4. TITLE AND SUBTITLE Direct Visualization of Estrogen Receptor-Mediated Transcription in Living Cells 5a. CONTRACT NUMBER 5b. GRANT

  8. Genomic variation in the MMP-1 promoter influences estrogen receptor mediated activity in a mechanically activated environment: potential implications for microgravity risk assessment

    NASA Astrophysics Data System (ADS)

    Thaler, John; Myers, Ken; Lu, Ting; Hart, David

    Background: Mechanotransduction, the conversion of mechanical forces (tensile, compression, shear etc.) into cellular signals is a significant response mechanism in bone that contributes to the balance between formation and resorption and helps maintain bone density. In microgravity the lack of mechanical signals can lead to a loss of bone density, however the signaling pathways responsible are not well understood. For women, sex-specific hormones are also important in maintaining bone density since estrogen deficiency is a major factor in the etiology of osteoporosis in postmenopausal women. Estrogen Receptors (ERs) are present in human connective tissue cells such as osteoblasts and may play a role in mechanotransduction responses. The two ER isoforms, alpha (ER-α) and beta (ER-β) differentially regulate expression of matrixmetalloproteinases (MMPs) which degrade extracellular matrix components found in connective tissues. Mechanical stimulation is known to affect the expression of MMP-1, a collagenase involved in the bone resorption process. The MMP-1 promoter region contains a single nucleotide polymorphism of an additional guanine (G) at position -1607 bp which creates a binding site for a member of the Ets family of mammalian transcription factors. The 2G allele is known to be present in 45-70% of healthy populations and has been associated with higher MMP-1 expression. The 2G allele has been linked to higher risk of several types of cancer but a link to osteoporosis or microgravity induced bone loss has not been explored. The purpose of the present study was to conduct a case-study to determine whether small genetic variations can influence cellular and tissue responses to mechanical loading. Specifically we examined the potential of the 1G/2G -1607 MMP-1 promoter SNP to alter the interplay between mechanical shear stress and estrogen receptors in controlling MMP-1 expression. Methods: Rabbit synovial cells (HIG-82) were used as an in vitro model system to

  9. Induction of aryl hydrocarbon receptor-mediated and estrogen receptor-mediated activities, and modulation of cell proliferation by dinaphthofurans.

    PubMed

    Vondrácek, Jan; Chramostová, Katerina; Plísková, Martina; Bláha, Ludek; Brack, Werner; Kozubík, Alois; Machala, Miroslav

    2004-09-01

    A group of heterocyclic aromatic compounds, dinaphthofurans (DNFs), recently have been identified as potentially significant contaminants in freshwater sediments. In the present study, a battery of in vitro assays was used for detection of toxic effects of DNFs that are potentially associated with endocrine disruption and tumor promotion. Dinaphthofurans were found to act as relatively potent inducers of aryl hydrocarbon receptor (AhR)-mediated activity in the chemical-activated luciferase reporter gene expression DR-CALUX assay. The relative AhR-inducing potencies of DNFs were similar or even higher than relative potencies of unsubstituted polycyclic aromatic hydrocarbons (PAHs), with dinaphtho[1,2-b;2'3'-d]furan being the most potent AhR agonist. Two compounds, dinaphtho[2,1-b;2'3'-d]furan and dinaphtho[1,2-b;1'2'-d]furan, induced estrogen receptor (ER)-mediated activity in the estrogen receptor-mediated CALUX (the ER-CALUX) assay. Two types of potential tumor-promoting effects of DNFs were investigated, using in vitro bioassays for detection of inhibition of gap-junctional intercellular communication and detection of a release from contact inhibition. Although the acute inhibition of gap-junctional intercellular communication was not observed, all six tested DNFs were able to release rat liver epithelial WB-F344 cells from contact inhibition at concentrations as low as 100 nM. In summary, the present study indicated that DNFs can exert multiple biological effects in vitro, including induction of the AhR-mediated activity, release of cells from contact inhibition, and induction of ER-mediated activity.

  10. Mechanics of receptor-mediated endocytosis

    NASA Astrophysics Data System (ADS)

    Gao, Huajian; Shi, Wendong; Freund, Lambert B.

    2005-07-01

    Most viruses and bioparticles endocytosed by cells have characteristic sizes in the range of tens to hundreds of nanometers. The process of viruses entering and leaving animal cells is mediated by the binding interaction between ligand molecules on the viral capid and their receptor molecules on the cell membrane. How does the size of a bioparticle affect receptor-mediated endocytosis? Here, we study how a cell membrane containing diffusive mobile receptors wraps around a ligand-coated cylindrical or spherical particle. It is shown that particles in the size range of tens to hundreds of nanometers can enter or exit cells via wrapping even in the absence of clathrin or caveolin coats, and an optimal particles size exists for the smallest wrapping time. This model can also be extended to include the effect of clathrin coat. The results seem to show broad agreement with experimental observations. Author contributions: H.G. and L.B.F. designed research; H.G., W.S., and L.B.F. performed research; and H.G., W.S., and L.B.F. wrote the paper.Abbreviations: CNT, carbon nanotube; SWNT, single-walled nanotube.

  11. Dietary Estrogens Act through Estrogen Receptor-Mediated Processes and Show No Antiestrogenicity in Cultured Breast Cancer Cells.

    PubMed Central

    Makela, S; Davis, VL; Tally, WC; Korkman, J; Salo, L; Vihko, R; Santti, R; Korach, KS

    1994-01-01

    Dietary estrogens are believed to exert their estrogenic or antiestrogenic (chemopreventive) action in estrogen responsive cells by interacting with the estrogen receptor (ER). The present study was undertaken to evaluate a direct role of ER in estrogenic or antiestrogenic activities of three dietary estrogens (coumestrol, genistein and zearalenone). HeLa cells were transiently co-transfected with an expression vector for ER and an estrogen-responsive reporter gene construct. Coumestrol, genistein, and zearalenone all increased the activity of the reporter gene, only in the presence of the ER, and the activation was blocked with the ER antagonist ICI 164,384, demonstrating an ER-specific, agonist response. In addition, in MCF-7 cells, coumestrol and zearalenone increased the expression of the estrogen-responsive pS2 gene. Coumestrol and genistein inhibited the purified estrogen-specific 17ß-hydroxysteroid oxidoreductase enzyme and the conversion of estrone to 17ß-estradiol in T-47D cells, which contain this enzyme. However, they did not inhibit the estrone-induced proliferation of T-47D cells. In conclusion, coumestrol, genistein, and zearalenone are all potent estrogens in vitro, and they act through ER mediated mechanism. Our findings give no evidence to support the idea that these compounds act as antiestrogens through competition for the binding sites of ER or by inhibition of the conversion of estrone to 17ß-estradiol in breast cancer cells, since this effect was nullified by their agonist action on cell proliferation. Therefore, their suggested chemopreventive action in estrogen-related cancers must be mediated through other mechanisms. Images Figure 2. A Figure 2. B Figure 2. C Figure 2. D Figure 2. E Figure 3. A Figure 3. B Figure 4. A Figure 4. B Figure 4. C Figure 4. D Figure 4. E Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. A Figure 9. B Figure 9. C PMID:9679118

  12. Sex-specificity and estrogen-dependence of kappa opioid receptor-mediated antinociception and antihyperalgesia

    PubMed Central

    Lawson, Kera P.; Nag, Subodh; Thompson, Analisa D.; Mokha, Sukhbir S.

    2010-01-01

    This investigation determined whether activation of the kappa opioid receptor (KOR) in the spinal cord produces estrogen-dependent, sex-specific modulation of acute and inflammation-induced persistent nociception. We demonstrate for the first time that KOR antinociception and gene expression are enhanced by exogenous or endogenous estrogen in the female. The lack of KOR antinociception and KOR gene expression are not altered by hormonal status (testosterone or estrogen) in males. Cannulae were implanted intrathecally in male, gonadectomized male (GDX), intact and ovariectomized female (OVX) Sprague-Dawley rats. Estradiol was injected subcutaneously, 48 h before testing (GDX+E and OVX+E). Intrathecal injection of U50, 488H, a selective KOR agonist, dose dependently increased heat-evoked tail flick latencies (TFLs) in proestrous and OVX+E groups, but not in male, GDX, GDX+E, OVX, and diestrous groups. Further, estrogen dose-dependently enhanced the effect of U50,488H in OVX rats. KOR selective antagonist, nor-binaltorphimine (Nor-BNI), blocked the antinociceptive effect of U50,488H. U50,488H reversed the carrageenan-induced thermal hyperalgesia in OVX+E rats, but not in male or OVX rats. However, U50,488H treatment did not alter mechanical thresholds in any group, with or without inflammation. KOR gene expression was enhanced in proestrous and OVX+E groups as compared to any other group. We conclude that selective activation of KOR in the spinal cord produces sex-specific, stimulus- and estrogen-dependent attenuation of acute and inflammatory pain in the rat via estrogen-induced upregulation of the KOR gene expression in the spinal cord. These findings may further implicate estrogen dependence of KOR effects in learning, epilepsy, stress response, addiction etc. Selective activation of the kappa opioid receptor by intrathecal U50,488H produces antinociception and antihyperalgesia which are sex-specific, stimulus dependent and require the presence of estrogen. PMID

  13. Estrogen receptor α can selectively repress dioxin receptor-mediated gene expression by targeting DNA methylation.

    PubMed

    Marques, Maud; Laflamme, Liette; Gaudreau, Luc

    2013-09-01

    Selective inhibitory crosstalk has been known to occur within the signaling pathways of the dioxin (AhR) and estrogen (ERα) receptors. More specifically, ERα represses a cytochrome P450-encoding gene (CYP1A1) that converts cellular estradiol into a metabolite that inhibits the cell cycle, while it has no effect on a P450-encoding gene (CYP1B1) that converts estrodiol into a genotoxic product. Here we show that ERα represses CYP1A1 by targeting the Dnmt3B DNA methyltransferase and concomitant DNA methylation of the promoter. We also find that histone H2A.Z can positively contribute to CYP1A1 gene expression, and its presence at that gene is inversely correlated with DNA methylation. Taken together, our results provide a framework for how ERα can repress transcription, and how that impinges on the production of an enzyme that generates genotoxic estradiol metabolites, and potential breast cancer progression. Finally, our results reveal a new mechanism for how H2A.Z can positively influence gene expression, which is by potentially competing with DNA methylation events in breast cancer cells.

  14. Estrogenic status modulates aryl hydrocarbon receptor - mediated hepatic gene expression and carcinogenicity

    USDA-ARS?s Scientific Manuscript database

    Estrogenic status is thought to influence the cancer risk in women and has been reported to affect toxicity of carcinogenic polycyclic aromatic hydrocarbons (PAHs) in animals. The objective of this study was to examine the influence of estradiol (E2) on hepatic gene expression changes mediated by 7,...

  15. Puerarin exerted anti-osteoporotic action independent of estrogen receptor-mediated pathway.

    PubMed

    Michihara, Seiwa; Tanaka, Teruyoshi; Uzawa, Yuki; Moriyama, Tatsuya; Kawamura, Yukio

    2012-01-01

    Puerarin, a daidzein-8-C glucoside, is the major isoflavonoid in Kudzu (Pueraria lobata), and is unique in that it contains C-C conjugated glucose at position 8 of the isoflavonoid structure. A puerarin diet at a dose of 5 mg/kg b.w./d to fed ovariectomized mice for 2 mo diminished the urinary deoxypyridinoline, a typical bone-degradation product. Since the bone absorption marker, serum tartarate-resistant acid phosphatase (TRAP) activity of puerarin-fed mice decreased but the bone formation marker, osteocalcin level, did not alter, the puerarin diet was proved to specifically depress the bone absorption, but not the overall bone metabolism. In accordance with that results, the femur structure of puerarin-fed mice was restored compared with that of puerarin-free diet mice. The atrophied uterine due to low estrogen (E2) level after ovariectomy was not restored by the puerarin diet, suggesting that puerarin exerted the anti-osteoporotic action through a non estrogen receptor (ER) mediated-pathway, in vivo. The growth of an ER-positive human breast cancer cell, MCF-70, was not enhanced by puerarin, suggesting that puerarin did not show estrogen-like action on MCF-7 cells, even at a ten thousand times higher concentration than that of E2. Furthermore, ICI182,780 (ICI), an estrogen antagonist, suppressed the enhanced growth of MCF-7 cells by E2, but not that by puerarin. In an ER-binding assay, puerarin was proved not to bind to ERα or β, or if all, extremely weakly, although daidzein, an aglycon of puerarin, showed a little stronger binding compared with puerarin. All these results strongly indicate that puerarin exerts its anti-osteoprotic action independently of the ER-mediated pathway.

  16. Androgen Receptor-Mediated Escape Mechanisms from Androgen Ablation Therapy

    DTIC Science & Technology

    2006-10-01

    well as mechanisms associated with prostate cancer growth and expansion, we may be able to develop therapies that prolong lives. Understanding the...and G. A. Coetzee. 2004. Androgen receptor signaling: mechanism of interleukin-6 inhibition. Cancer Res. 64:2619–2626. 19. Jia, L., and G. A. Coetzee...the prostate specific antigen locus: steroidal and non-steroidal mechanisms . Mol. Cancer Res. 1:385–392. 21. Johnstone, R. W. 2002. Histone

  17. Food-associated estrogenic compounds induce estrogen receptor-mediated luciferase gene expression in transgenic male mice.

    PubMed

    Ter Veld, Marcel G R; Zawadzka, E; van den Berg, J H J; van der Saag, Paul T; Rietjens, Ivonne M C M; Murk, Albertinka J

    2008-07-30

    The present paper aims at clarifying to what extent seven food-associated compounds, shown before to be estrogenic in vitro, can induce estrogenic effects in male mice with an estrogen receptor (ER)-mediated luciferase (luc) reporter gene system. The luc induction was determined in different tissues 8h after dosing the ER-luc male mice intraperitoneally (IP) or 14h after oral dosing. Estradiol-propionate (EP) was used as a positive control at 0.3 and 1mg/kg bodyweight (bw), DMSO as solvent control. The food-associated estrogenic compounds tested at non-toxic doses were bisphenol A (BPA) and nonylphenol (NP) (both at 10 and 50mg/kgbw), dichlorodiphenyldichloroethylene (p,p'-DDE; at 5 and 25mg/kgbw), quercetin (at 1.66 and 16.6mg/kgbw), di-isoheptyl phthalate (DIHP), di-(2-ethylhexyl) phthalate (DEHP) and di-(2-ethylhexyl) adipate (DEHA) all at 30 and 100mg/kgbw. In general IP dosing resulted in higher luc inductions than oral dosing. EP induced luc activity in the liver in a statistically significant dose-related way with the highest induction of all compounds tested which was 20,000 times higher than the induction by the DMSO-control. NP, DDE, DEHA and DIHP did not induce luc activity in any of the tissues tested. BPA induced luc in the liver up to 420 times via both exposure routes. BPA, DEHP and quercetin induced luc activity in the liver after oral exposure. BPA (50mg/kgbw IP) also induced luc activity in the testis, kidneys and tibia. The current study reveals that biomarker-responses in ER-luc male mice occur after a single oral exposure to food-associated estrogenic model compounds at exposure levels 10 to 10(4) times higher than the established TDI's for some of these compounds. Given the facts that (i) the present study did not include chronic exposure and that (ii) simultaneous exposure to multiple estrogenic compounds may be a realistic exposure scenario, it remains to be seen whether this margin is sufficiently high.

  18. Requirement of Histone Methyltransferase SMYD3 for Estrogen Receptor-mediated Transcription*

    PubMed Central

    Kim, Hyunjung; Heo, Kyu; Kim, Jeong Hoon; Kim, Kyunghwan; Choi, Jongkyu; An, Woojin

    2009-01-01

    SMYD3 is a SET domain-containing protein with histone methyltransferase activity on histone H3–K4. Recent studies showed that SMYD3 is frequently overexpressed in different types of cancer cells, but how SMYD3 regulates the development and progression of these malignancies remains unknown. Here, we report the previously unrecognized role of SMYD3 in estrogen receptor (ER)-mediated transcription via its histone methyltransferase activity. We demonstrate that SMYD3 functions as a coactivator of ERα and potentiates ERα activity in response to ligand. SMYD3 directly interacts with the ligand binding domain of ER and is recruited to the proximal promoter regions of ER target genes upon gene induction. Importantly, our chromatin immunoprecipitation analyses provide compelling evidence that SMYD3 is responsible for the accumulation of di- and trimethylation of H3–K4 at the induced ER target genes. Furthermore, RNA interference-directed down-regulation of SMYD3 reveals that SMYD3 is required for ER-regulated gene transcription in estrogen signaling pathway. Thus, our results identify SMYD3 as a new coactivator for ER-mediated transcription, providing a possible link between SMYD3 overexpression and breast cancer. PMID:19509295

  19. The Wedelolactone Derivative Inhibits Estrogen Receptor-Mediated Breast, Endometrial, and Ovarian Cancer Cells Growth

    PubMed Central

    Xu, Defeng; Lin, Tzu-Hua; Cheng, Max A.; Chen, Lu-Min; Chang, Chawnshang; Yeh, Shuyuan

    2014-01-01

    Estrogen and estrogen receptor (ER)-mediated signaling pathways play important roles in the etiology and progression of human breast, endometrial, and ovarian cancers. Attenuating ER activities by natural products and their derivatives is a relatively practical strategy to control and reduce breast, endometrial, and ovarian cancer risk. Here, we found 3-butoxy-1,8,9-trihydroxy-6H-benzofuro[3,2-c]benzopyran-6-one (BTB), a new derivative of wedelolactone, could effectively inhibit the 17-estradiol (E2)-induced ER transactivation and suppress the growth of breast cancer as well as endometrial and ovarian cancer cells. Our results indicate that 2.5 μM BTB effectively suppresses ER-positive, but not ER-negative, breast, endometrial, and ovarian cancer cells. Furthermore, our data indicate that BTB can modulate ER transactivation and suppress the expression of E2-mediated ER target genes (Cyclin D1, E2F1, and TERT) in the ER-positive MCF-7, Ishikawa, and SKOV-3 cells. Importantly, this BTB mediated inhibition of ER activity is selective since BTB does not suppress the activities of other nuclear receptors, including glucocorticoid receptor and progesterone receptor, suggesting that BTB functions as a selective ER signaling inhibitor with the potential to treat breast, endometrial, and ovarian cancers. PMID:25221777

  20. Androgen Receptor-Mediated Escape Mechanism from Androgen Ablation Therapy

    DTIC Science & Technology

    2008-10-31

    is a fundamental and an increasingly central aspect of transcriptional control in higher eukaryotic cells , and we therefore hypothesized that (i) the...regions (ARORs) in PCa cells . Our work suggests that only some ARORs function under the given physiological conditions, utilizing diverse mechanisms. This...exemplify the AR-dependence of ablation-resistant PCa cells . First, disruption of AR expression by a specific antibody or ribozyme inhibited

  1. Nanoscale imaging and mechanical analysis of Fc receptor-mediated macrophage phagocytosis against cancer cells.

    PubMed

    Li, Mi; Liu, Lianqing; Xi, Ning; Wang, Yuechao; Xiao, Xiubin; Zhang, Weijing

    2014-02-18

    Fc receptor-mediated macrophage phagocytosis against cancer cells is an important mechanism in the immune therapy of cancers. Traditional research about macrophage phagocytosis was based on optical microscopy, which cannot reveal detailed information because of the 200-nm-resolution limit. Quantitatively investigating the macrophage phagocytosis at micro- and nanoscale levels is still scarce. The advent of atomic force microscopy (AFM) offers an excellent analytical instrument for quantitatively investigating the biological processes at single-cell and single-molecule levels under native conditions. In this work, we combined AFM and fluorescence microscopy to visualize and quantify the detailed changes in cell morphology and mechanical properties during the process of Fc receptor-mediated macrophage phagocytosis against cancer cells. Lymphoma cells were discernible by fluorescence staining. Then, the dynamic process of phagocytosis was observed by time-lapse optical microscopy. Next, AFM was applied to investigate the detailed cellular behaviors during macrophage phagocytosis under the guidance of fluorescence recognition. AFM imaging revealed the distinct features in cellular ultramicrostructures for the different steps of macrophage phagocytosis. AFM cell mechanical property measurements indicated that the binding of cancer cells to macrophages could make macrophages become stiffer. The experimental results provide novel insights in understanding the Fc-receptor-mediated macrophage phagocytosis.

  2. Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line.

    PubMed

    Legler, J; van den Brink, C E; Brouwer, A; Murk, A J; van der Saag, P T; Vethaak, A D; van der Burg, B

    1999-03-01

    Development of an estrogen receptor-mediated, chemical-activated luciferase reporter gene-expression (ER-CALUX) assay was attempted by stable transfection of luciferase reporter genes in a number of cell lines. Stable transfection of the chimeric Gal4 estrogen receptor and luciferase gene constructs in MCF-7 breast cancer and Hepa.1c1c7 mouse hepatoma cell lines, as well as transfection of a newly constructed luciferase reporter gene pEREtata-Luc in the ECC-1 human endometrial cell line, resulted in constitutive, non-estradiol-inducible clones. Stable transfection of pEREtata-Luc in the T47D breast cancer cell line, however, resulted in an extremely sensitive, highly responsive cell line. Following a 24-h exposure to estradiol (E2), stably transfected T47D.Luc cells demonstrated a detection limit of 0.5 pM, an EC50 of 6 pM, and a maximum induction of 100-fold relative to solvent controls. No clear reduction in responsiveness has been found over extended culture periods (50 passages). Anti-estrogens ICI 182,780, TCDD, and tamoxifen inhibited the estradiol-mediated luciferase induction. Genistein, nonylphenol, and o,p'DDT were the most potent (pseudo-)estrogens tested in this system (EC50 100, 260, and 660 nM, respectively). Determination of interactive effects of the (pseudo-)estrogens nonylphenol, o,p'DDT, chlordane, endosulfan, dieldrin, and methoxychlor revealed that, in combination with 3 pM E2, (pseudo-)estrogens were additive. Slightly more than additive effects (less than 2-fold) were found for combinations of dieldrin and endosulfan tested in the range of 3 to 6 microM. At these concentrations, the combination of endosulfan and chlordane demonstrated additive interaction. The ER-CALUX assay with T47D cells can provide a sensitive, responsive, and rapid in vitro system to detect and measure substances with potential (anti-)estrogenic activity.

  3. beta-estradiol influences differentiation of hippocampal neurons in vitro through an estrogen receptor-mediated process.

    PubMed

    Audesirk, T; Cabell, L; Kern, M; Audesirk, G

    2003-01-01

    We utilized morphometric analysis of 3 day cultures of hippocampal neurons to determine the effects of both estradiol and the synthetic estrogen receptor modulator raloxifene on several parameters of neuronal growth and differentiation. These measurements included survival, neurite production, dendrite number, and axon and dendrite length and branching. 17 beta-Estradiol (10 nM) selectively stimulated dendrite branching; this effect was neither mimicked by alpha-estradiol, nor blocked by the estrogen receptor antagonist ICI 182780. The selective estrogen receptor modulator raloxifene (100 nM) neither mimicked nor reversed the effects of estradiol on dendritic branching. Western immunoblotting for the alpha and beta subtypes of estrogen receptor revealed the presence of alpha, but not beta, estrogen receptors in our hippocampal cultures. There is growing recognition of the effects of 17 beta-estradiol on neuronal development and physiology, with implications for brain sexual dimorphism, plasticity, cognition, and the maintenance of cognitive function during aging. The role of estradiol in hippocampal neuronal differentiation and function has particular implications for learning and memory. These data support the hypothesis that 17 beta-estradiol is acting via alpha estrogen receptors in influencing hippocampal development in vitro. Raloxifene, prescribed to combat osteoporosis in post-menopausal women, is a selective estrogen receptor modulator with tissue-specific agonist/antagonist properties. Because raloxifene had no effect on dendritic branching, we hypothesize that it does not interact with the alpha estrogen receptor in this experimental paradigm.

  4. Aryl hydrocarbon receptor-mediated and estrogenic activities of oxygenated polycyclic aromatic hydrocarbons and azaarenes originally identified in extracts of river sediments.

    PubMed

    Machala, M; Ciganek, M; Bláha, L; Minksová, K; Vondráck, J

    2001-12-01

    Reproductive dysfunction in wildlife populations can be a result of environmental contaminants binding to aryl hydrocarbon receptor (AhR) or estrogenic receptors. Signaling by both types of receptors can be affected by polycyclic aromatic hydrocarbons (PAHs), which are potential endocrine disruptors. However, our knowledge regarding the effects of oxygenated (oxy)-PAHs and azaarenes on AhR-mediated and estrogenic activities is incomplete. In the present study, we have identified 9-fluorenone, anthrone, anthraquinone, benzanthrone, benz[a]anthracene-7,12-dione, benz[c]acridine, and dibenz[a,h]acridine as prevalent oxy-PAHs and azaarenes found in river sediments. Their concentrations in sediment samples ranged from 2.1 to 165.2 ng g(-1) for oxy-PAHs and up to 27.3 ng g(-1) for azaarenes. Their relative AhR-inducing and estrogenic potencies were quantified in vitro using two cell lines that were stably transfected with a luciferase reporter gene system and expressed as induction equivalency factors (IEFs). The only oxy-PAHs with detectable levels of in vitro AhR-mediated activity were benzanthrone and benz[a]anthracene-7,12-dione. However, their IEFs were approximately three to four orders of magnitude lower than those of benzo[a]pyrene. On the other hand, azaarenes showed a strong AhR-mediated activity, with dibenzo[a,h]acridine being a far more potent inducer of activity than benzo[a]pyrene. Benzanthrone, benz[a]anthracene-7,12-dione, anthraquinone, and benz[a]acridine were weak inducers of in vitro estrogenic activity, with IEFs similar to that of benzo[a]pyrene. Based on concentrations and relative potencies, our results suggest that dibenzo[a,h]acridine can significantly contribute to the overall AhR-mediated activity in river sediments, whereas the remaining compounds do not. No studied compound was found to contribute significantly to estrogen receptor-mediated activity in vitro.

  5. Estrogen receptor-mediated neuroprotection: The role of the Alzheimer’s disease-related gene seladin-1

    PubMed Central

    Peri, Alessandro; Serio, Mario

    2008-01-01

    Experimental evidence supports a protective role of estrogen in the brain. According to the fact that Alzheimer’s disease (AD) is more common in postmenopausal women, estrogen treatment has been proposed. However, there is no general consensus on the beneficial effect of estrogen or selective estrogen receptor modulators in preventing or treating AD. It has to be said that several factors may markedly affect the efficacy of the treatment. A few years ago, the seladin-1 gene (for selective Alzheimer’s disease indicator-1) has been isolated and found to be down-regulated in brain regions affected by AD. Seladin-1 has been found to be identical to the gene encoding the enzyme 3-beta-hydroxysterol delta-24-reductase, involved in the cholesterol biosynthetic pathway, which confers protection against β-amyloid-mediated toxicity and from oxidative stress, and is an effective inhibitor of caspase-3 activity, a key mediator of apoptosis. Interestingly, we found earlier that the expression of this gene is up-regulated by estrogen. Furthermore, our very recent data support the hypothesis that seladin-1 is a mediator of the neuroprotective effects of estrogen. This review will summarize the current knowledge regarding the neuroprotective effects of seladin-1 and the relationship between this protein and estrogen. PMID:19043524

  6. Internalisation of the bleomycin molecules responsible for bleomycin toxicity: a receptor-mediated endocytosis mechanism.

    PubMed

    Pron, G; Mahrour, N; Orlowski, S; Tounekti, O; Poddevin, B; Belehradek, J; Mir, L M

    1999-01-01

    Bleomycin (BLM) does not diffuse through the plasma membrane but nevertheless displays cytotoxic activity due to DNA break generation. The aim of the study was to describe the mechanism of BLM internalisation. We previously provided evidence for the existence of BLM-binding sites at the surface of DC-3F Chinese hamster fibroblasts, as well as of their involvement in BLM cytotoxicity on DC-3F cells and related BLM-resistant sublines. Here we report that A253 human cells and their BLM-resistant subline C-10E also possessed a membrane protein of ca. 250 kDa specifically binding BLM. Part of this C-10E cell resistance could be explained by a decrease in the number of BLM-binding sites exposed at the cell surface with respect to A253 cells. The comparison between A253 and DC-3F cells exposing a similar number of BLM-binding sites revealed that the faster the fluid phase endocytosis, the greater the cell sensitivity to BLM. Moreover, the experimental modification of endocytotic vesicle size showed that BLM cytotoxicity was directly correlated with the flux of plasma membrane area engulfed during endocytosis rather than with the fluid phase volume incorporated. Thus, BLM would be internalised by a receptor-mediated endocytosis mechanism which would first require BLM binding to its membrane receptor and then the transfer of the complex into intracellular endocytotic vesicles, followed by BLM entry into the cytosol, probably from a nonacidic compartment.

  7. Cellular mechanisms of the 5-HT7 receptor-mediated signaling

    PubMed Central

    Guseva, Daria; Wirth, Alexander; Ponimaskin, Evgeni

    2014-01-01

    Serotonin (5-hydroxytryptamine or 5-HT) is an important neurotransmitter regulating a wide range of physiological and pathological functions via activation of heterogeneously expressed 5-HT receptors. The 5-HT7 receptor is one of the most recently described members of the 5-HT receptor family. Functionally, 5-HT7 receptor is associated with a number of physiological and pathological responses, including serotonin-induced phase shifting of the circadian rhythm, control of memory as well as locomotor and exploratory activity. A large body of evidence indicates involvement of the 5-HT7 receptor in anxiety and depression, and recent studies suggest that 5-HT7 receptor can be highly relevant for the treatment of major depressive disorders. The 5-HT7 receptor is coupled to the stimulatory Gs-protein, and receptor stimulation results in activation of adenylyl cyclase (AC) leading to a rise of cAMP concentration. In addition, this receptor is coupled to the G12-protein to activate small GTPases of the Rho family. This review focuses on molecular mechanisms responsible for the 5-HT7 receptor-mediated signaling. We provide detailed overview of signaling cascades controlled and regulated by the 5-HT7 receptor and discuss the functional impact of 5-HT7 receptor for the regulation of different cellular and subcellular processes. PMID:25324743

  8. Receptor-mediated transcytosis: a mechanism for active extravascular transport of nanoparticles in solid tumors.

    PubMed

    Lu, Wei; Xiong, Chiyi; Zhang, Rui; Shi, Lifang; Huang, Miao; Zhang, Guodong; Song, Shaoli; Huang, Qian; Liu, Gang-Yu; Li, Chun

    2012-08-10

    Targeted nanoparticle-based delivery systems have been used extensively to develop effective cancer theranostics. However, how targeting ligands affect extravascular transport of nanoparticles in solid tumors remains unclear. Here, we show, using B16/F10 melanoma cells expressing melanocortin type-1 receptor (MC1R), that the nature of targeting ligands, i.e., whether they are agonists or antagonists, directs tumor uptake and intratumoral distribution after extravasation of nanoparticles from tumor vessels into the extravascular fluid space. Pegylated hollow gold nanospheres (HAuNS, diameter=40 nm) coated with MC1R agonist are internalized upon ligand-receptor binding, whereas MC1R antagonist-conjugated HAuNS remain attached on the cell surface. Transcellular transport of agonist-conjugated HAuNS was confirmed by a multilayer tumor cell model and by transmission electron microscopy. MC1R agonist- but not MC1R antagonist-conjugated nanoparticles exhibit significantly higher tumor uptake than nontargeted HAuNS and are quickly dispersed from tumor vessels via receptor-mediated endocytosis and subsequent transcytosis. These results confirm an active transport mechanism that can be used to overcome one of the major biological barriers for efficient nanoparticle delivery to solid tumors. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Cellular mechanisms of the 5-HT7 receptor-mediated signaling.

    PubMed

    Guseva, Daria; Wirth, Alexander; Ponimaskin, Evgeni

    2014-01-01

    Serotonin (5-hydroxytryptamine or 5-HT) is an important neurotransmitter regulating a wide range of physiological and pathological functions via activation of heterogeneously expressed 5-HT receptors. The 5-HT7 receptor is one of the most recently described members of the 5-HT receptor family. Functionally, 5-HT7 receptor is associated with a number of physiological and pathological responses, including serotonin-induced phase shifting of the circadian rhythm, control of memory as well as locomotor and exploratory activity. A large body of evidence indicates involvement of the 5-HT7 receptor in anxiety and depression, and recent studies suggest that 5-HT7 receptor can be highly relevant for the treatment of major depressive disorders. The 5-HT7 receptor is coupled to the stimulatory Gs-protein, and receptor stimulation results in activation of adenylyl cyclase (AC) leading to a rise of cAMP concentration. In addition, this receptor is coupled to the G12-protein to activate small GTPases of the Rho family. This review focuses on molecular mechanisms responsible for the 5-HT7 receptor-mediated signaling. We provide detailed overview of signaling cascades controlled and regulated by the 5-HT7 receptor and discuss the functional impact of 5-HT7 receptor for the regulation of different cellular and subcellular processes.

  10. Isoflavones made simple - genistein's agonist activity for the beta-type estrogen receptor mediates their health benefits.

    PubMed

    McCarty, Mark Frederick

    2006-01-01

    Soy isoflavones, the focus of much research and controversy, are often referred to as "weak estrogens". In fact, genistein is a relatively potent agonist for the recently characterized beta isoform of the estrogen receptor (ERbeta). The low nanomolar serum concentrations of unconjugated free genistein achieved with high-nutritional intakes of soy isoflavones are near the binding affinity of genistein for this receptor, but are about an order of magnitude lower than genistein's affinity for the "classical" alpha isoform of the estrogen receptor (ERalpha). Moreover, these concentrations are far too low to inhibit tyrosine kinases or topoisomerase II, in vitro activities of genistein often cited as potential mediators of its physiological effects. The thesis that these physiological effects are in fact mediated by ERbeta activation provides a satisfying rationale for genistein's clinical activities. Hepatocytes do not express ERbeta; this explains why soy isoflavones, unlike oral estrogen, neither modify serum lipids nor provoke the prothrombotic effects associated with increased risk for thromboembolic disorders. The lack of uterotrophic activity of soy isoflavones reflects the fact that ERalpha is the exclusive mediator of estrogen's impact in this regard. Vascular endothelium expresses both ERalpha and ERbeta, each of which has the potential to induce and activate nitric oxide synthase; this may account for the favorable influence of soy isoflavones on endothelial function in postmenopausal women and ovariectomized rats. The ERbeta expressed in osteoblasts may mediate the reported beneficial impact of soy isoflavones on bone metabolism. Suggestive evidence that soy-rich diets decrease prostate cancer risk, accords well with the observation that ERbeta appears to play an antiproliferative role in healthy prostate. In the breast, ERalpha promotes epithelial proliferation, whereas ERbeta has a restraining influence in this regard - consistent with the emerging view

  11. Assessment of Cellular Estrogenic Activity Based on Estrogen Receptor-Mediated Reduction of Soluble-Form Catechol-O-Methyltransferase (COMT) Expression in an ELISA-Based System

    PubMed Central

    Ho, Philip Wing-Lok; Tse, Zero Ho-Man; Liu, Hui-Fang; Lu, Song; Ho, Jessica Wing-Man; Kung, Michelle Hiu-Wai; Ramsden, David Boyer; Ho, Shu-Leong

    2013-01-01

    Xenoestrogens are either natural or synthetic compounds that mimic the effects of endogenous estrogen. These compounds, such as bisphenol-A (BPA), and phthalates, are commonly found in plastic wares. Exposure to these compounds poses major risk to human health because of the potential to cause endocrine disruption. There is huge demand for a wide range of chemicals to be assessed for such potential for the sake of public health. Classical in vivo assays for endocrine disruption are comprehensive but time-consuming and require sacrifice of experimental animals. Simple preliminary in vitro screening assays can reduce the time and expense involved. We previously demonstrated that catechol-O-methyltransferase (COMT) is transcriptionally regulated by estrogen via estrogen receptor (ER). Therefore, detecting corresponding changes of COMT expression in estrogen-responsive cells may be a useful method to estimate estrogenic effects of various compounds. We developed a novel cell-based ELISA to evaluate cellular response to estrogenicity by reduction of soluble-COMT expression in ER-positive MCF-7 cells exposed to estrogenic compounds. In contrast to various existing methods that only detect bioactivity, this method elucidates direct physiological effect in a living cell in response to a compound. We validated our assay using three well-characterized estrogenic plasticizers - BPA, benzyl butyl phthalate (BBP), and di-n-butyl phthalate (DBP). Cells were exposed to either these plasticizers or 17β-estradiol (E2) in estrogen-depleted medium with or without an ER-antagonist, ICI 182,780, and COMT expression assayed. Exposure to each of these plasticizers (10-9-10-7M) dose-dependently reduced COMT expression (p<0.05), which was blocked by ICI 182,780. Reduction of COMT expression was readily detectable in cells exposed to picomolar level of E2, comparable to other in vitro assays of similar sensitivity. To satisfy the demand for in vitro assays targeting different cellular

  12. Assessment of cellular estrogenic activity based on estrogen receptor-mediated reduction of soluble-form catechol-O-methyltransferase (COMT) expression in an ELISA-based system.

    PubMed

    Ho, Philip Wing-Lok; Tse, Zero Ho-Man; Liu, Hui-Fang; Lu, Song; Ho, Jessica Wing-Man; Kung, Michelle Hiu-Wai; Ramsden, David Boyer; Ho, Shu-Leong

    2013-01-01

    Xenoestrogens are either natural or synthetic compounds that mimic the effects of endogenous estrogen. These compounds, such as bisphenol-A (BPA), and phthalates, are commonly found in plastic wares. Exposure to these compounds poses major risk to human health because of the potential to cause endocrine disruption. There is huge demand for a wide range of chemicals to be assessed for such potential for the sake of public health. Classical in vivo assays for endocrine disruption are comprehensive but time-consuming and require sacrifice of experimental animals. Simple preliminary in vitro screening assays can reduce the time and expense involved. We previously demonstrated that catechol-O-methyltransferase (COMT) is transcriptionally regulated by estrogen via estrogen receptor (ER). Therefore, detecting corresponding changes of COMT expression in estrogen-responsive cells may be a useful method to estimate estrogenic effects of various compounds. We developed a novel cell-based ELISA to evaluate cellular response to estrogenicity by reduction of soluble-COMT expression in ER-positive MCF-7 cells exposed to estrogenic compounds. In contrast to various existing methods that only detect bioactivity, this method elucidates direct physiological effect in a living cell in response to a compound. We validated our assay using three well-characterized estrogenic plasticizers - BPA, benzyl butyl phthalate (BBP), and di-n-butyl phthalate (DBP). Cells were exposed to either these plasticizers or 17β-estradiol (E2) in estrogen-depleted medium with or without an ER-antagonist, ICI 182,780, and COMT expression assayed. Exposure to each of these plasticizers (10(-9)-10(-7)M) dose-dependently reduced COMT expression (p<0.05), which was blocked by ICI 182,780. Reduction of COMT expression was readily detectable in cells exposed to picomolar level of E2, comparable to other in vitro assays of similar sensitivity. To satisfy the demand for in vitro assays targeting different cellular

  13. EGCG functions through estrogen receptor-mediated activation of ADAM10 in the promotion of non-amyloidogenic processing of APP

    PubMed Central

    Fernandez, Jamie Winderbaum; Rezai-Zadeh, Kavon; Obregon, Demian; Tan, Jun

    2010-01-01

    Estrogen depletion following menopause has been correlated with an increased risk of developing Alzheimer’s disease (AD). We previously explored the beneficial effect of (-)-epigallocatechin-3-gallate (EGCG) on AD mice and found increased non-amyloidogenic processing of amyloid precursor protein (APP) through the α-secretase a-disintegrin-and-metallopeptidase-domain 10 (ADAM10). Our results in this study suggest that EGCG-mediated enhancement of non-amyloidogenic processing of APP is mediated by the maturation of ADAM10 via an estrogen receptor-α (ERα)/PI3K/Akt dependent mechanism, independent of furin-mediated ADAM10 activation. These data support prior assertions that central selective estrogen receptor modulation could be a therapeutic target for AD and support the use of EGCG as a well-tolerated alternative to estrogen therapy in the prophylaxis and treatment of this disease. PMID:20849853

  14. Dopamine receptor-mediated mechanisms involved in the expression of learned activity of primate striatal neurons.

    PubMed

    Watanabe, K; Kimura, M

    1998-05-01

    background discharge rates nor the responses of TANs were significantly influenced. Background discharge rate of TANs was also not affected by D1- or D2-class antagonists applied by either micropressure injection or iontophoresis. It was concluded that the nigrostriatal DA system enables TANs to express learned activity primarily through D2-class and partly through D1-class receptor-mediated mechanisms in the striatum.

  15. Octyl gallate markedly promotes anti-amyloidogenic processing of APP through estrogen receptor-mediated ADAM10 activation.

    PubMed

    Zhang, She-Qing; Sawmiller, Darrell; Li, Song; Rezai-Zadeh, Kavon; Hou, Huayan; Zhou, Shufeng; Shytle, Douglas; Giunta, Brian; Fernandez, Frank; Mori, Takashi; Tan, Jun

    2013-01-01

    Our previous studies showed that the green tea-derived polyphenolic compound (-)-epigallocatechin-3 gallate (EGCG) reduces amyloid-β (Aβ) production in both neuronal and mouse Alzheimer's disease (AD) models in concert with activation of estrogen receptor-α/phosphatidylinositide 3-kinase/protein kinase B (ERα/PI3K/Akt) signaling and anti-amyloidogenic amyloid precursor protein (APP) α-secretase (a disintegrin and metallopeptidase domain-10, ADAM10) processing. Since the gallate moiety in EGCG may correspond to the 7α position of estrogen, thereby facilitating ER binding, we extensively screened the effect of other gallate containing phenolic compounds on APP anti-amyloidogenic processing. Octyl gallate (OG; 10 µM), drastically decreased Aβ generation, in concert with increased APP α-proteolysis, in murine neuron-like cells transfected with human wild-type APP or "Swedish" mutant APP. OG markedly increased production of the neuroprotective amino-terminal APP cleavage product, soluble APP-α (sAPPα). In accord with our previous study, these cleavage events were associated with increased ADAM10 maturation and reduced by blockade of ERα/PI3k/Akt signaling. To validate these findings in vivo, we treated Aβ-overproducing Tg2576 mice with OG daily for one week by intracerebroventricular injection and found decreased Aβ levels associated with increased sAPPα. These data indicate that OG increases anti-amyloidogenic APP α-secretase processing by activation of ERα/PI3k/Akt signaling and ADAM10, suggesting that this compound may be an effective treatment for AD.

  16. IDENTIFICATION OF PUTATIVE ESTROGEN RECEPTOR-MEDIATED ENDOCRINE DISRUPTING CHEMICALS USING QSAR- AND STRUCTURE-BASED VIRTUAL SCREENING APPROACHES

    PubMed Central

    Zhang, Liying; Sedykh, Alexander; Tripathi, Ashutosh; Zhu, Hao; Afantitis, Antreas; Mouchlis, Varnavas D.; Melagraki, Georgia; Rusyn, Ivan; Tropsha, Alexander

    2013-01-01

    Identification of Endocrine Disrupting Chemicals is one of the important goals of environmental chemical hazard screening. We report on the development of validated in silico predictors of chemicals likely to cause Estrogen Receptor (ER)-mediated endocrine disruption to facilitate their prioritization for future screening. A database of relative binding affinity of a large number of ERα and/or ERβ ligands was assembled (546 for ERα and 137 for ERβ). Both single-task learning (STL) and multi-task learning (MTL) continuous Quantitative Structure-Activity Relationships (QSAR) models were developed for predicting ligand binding affinity to ERα or ERβ. High predictive accuracy was achieved for ERα binding affinity (MTL R2=0.71, STL R2=0.73). For ERβ binding affinity, MTL models were significantly more predictive (R2=0.53, p<0.05) than STL models. In addition, docking studies were performed on a set of ER agonists/antagonists (67 agonists and 39 antagonists for ERα, 48 agonists and 32 antagonists for ERβ, supplemented by putative decoys/non-binders) using the following ER structures (in complexes with respective ligands) retrieved from the Protein Data Bank: ERα agonist (PDB ID: 1L2I), ERα antagonist (PDB ID: 3DT3), ERβ agonist (PDB ID: 2NV7), ERβ antagonist (PDB ID: 1L2J). We found that all four ER conformations discriminated their corresponding ligands from presumed non-binders. Finally, both QSAR models and ER structures were employed in parallel to virtually screen several large libraries of environmental chemicals to derive a ligand- and structure-based prioritized list of putative estrogenic compounds to be used for in vitro and in vivo experimental validation. PMID:23707773

  17. Subtype-Specific Estrogen Receptor-Mediated Vasodilator Activity in the Cephalic, Thoracic and Abdominal Vasculature of Female Rat

    PubMed Central

    Reslan, Ossama M.; Yin, Zongzhi; do Nascimento, Graciliano R. A.; Khalil, Raouf A.

    2013-01-01

    Estrogen receptors (ERs) mediate genomic and nongenomic vasodilator effects, but estrogen therapy may not provide systemic vascular protection. To test whether this is due to regional differences in ER distribution or vasodilator activity, cephalic (carotid), thoracic (thoracic aorta, pulmonary) and abdominal arteries (abdominal aorta, mesenteric, renal) from female Sprague-Dawley rats were prepared to measure contraction to phenylephrine (Phe), and relaxation to acetylcholine (ACh) and the ER activators 17β-estradiol (E2) (all ERs), PPT (ERα), DPN (ERβ) and G1 (GPR30). Phe caused contraction that was enhanced in endothelium-denuded aorta, supporting endothelial release of vasodilators. In cephalic and thoracic arteries, ACh relaxation was abolished by the NOS inhibitor L-NAME, suggesting a role of NO. In mesenteric vessels, ACh-induced relaxation was partly inhibited by L-NAME+COX inhibitor indomethacin and blocked by the K+ channel blocker tetraethylammonium (TEA), suggesting a hyperpolarization pathway. E2 and PPT caused similar relaxation in all vessels. DPN and G1 caused smaller relaxation that was more prominent in abdominal vessels. RT-PCR revealed variable ERα mRNA expression, and increased ERβ in carotid artery and GPR30 in abdominal arteries. Western blots revealed greater amounts of ERα, ERβ and GPR30 in abdominal arteries. In thoracic aorta, E2, PPT and DPN-induced relaxation was blocked by L-NAME, and was associated with increased nitrite/nitrate production, suggesting a role of NO. In abdominal vessels, E2, PPT, DPN and G1-induced relaxation persisted in L-NAME+indomethacin+TEA-treated or endothelium-denuded arteries, suggesting direct effect on vascular smooth muscle (VSM). E2, PPT, DPN, and G1 caused greater relaxation of KCl-induced contraction in abdominal vessels, suggesting inhibitory effects on Ca2+ entry. Thus, E2 and ERα stimulation produce similar relaxation of the cephalic, thoracic and abdominal arteries. In the cephalic and

  18. Identification of putative estrogen receptor-mediated endocrine disrupting chemicals using QSAR- and structure-based virtual screening approaches

    SciTech Connect

    Zhang, Liying; Sedykh, Alexander; Tripathi, Ashutosh; Zhu, Hao; Afantitis, Antreas; Mouchlis, Varnavas D.; Melagraki, Georgia; Rusyn, Ivan; Tropsha, Alexander

    2013-10-01

    Identification of endocrine disrupting chemicals is one of the important goals of environmental chemical hazard screening. We report on the development of validated in silico predictors of chemicals likely to cause estrogen receptor (ER)-mediated endocrine disruption to facilitate their prioritization for future screening. A database of relative binding affinity of a large number of ERα and/or ERβ ligands was assembled (546 for ERα and 137 for ERβ). Both single-task learning (STL) and multi-task learning (MTL) continuous quantitative structure–activity relationship (QSAR) models were developed for predicting ligand binding affinity to ERα or ERβ. High predictive accuracy was achieved for ERα binding affinity (MTL R{sup 2} = 0.71, STL R{sup 2} = 0.73). For ERβ binding affinity, MTL models were significantly more predictive (R{sup 2} = 0.53, p < 0.05) than STL models. In addition, docking studies were performed on a set of ER agonists/antagonists (67 agonists and 39 antagonists for ERα, 48 agonists and 32 antagonists for ERβ, supplemented by putative decoys/non-binders) using the following ER structures (in complexes with respective ligands) retrieved from the Protein Data Bank: ERα agonist (PDB ID: 1L2I), ERα antagonist (PDB ID: 3DT3), ERβ agonist (PDB ID: 2NV7), and ERβ antagonist (PDB ID: 1L2J). We found that all four ER conformations discriminated their corresponding ligands from presumed non-binders. Finally, both QSAR models and ER structures were employed in parallel to virtually screen several large libraries of environmental chemicals to derive a ligand- and structure-based prioritized list of putative estrogenic compounds to be used for in vitro and in vivo experimental validation. - Highlights: • This is the largest curated dataset inclusive of ERα and β (the latter is unique). • New methodology that for the first time affords acceptable ERβ models. • A combination of QSAR and docking enables prediction of affinity and function.

  19. Estrogen-, androgen- and aryl hydrocarbon receptor mediated activities in passive and composite samples from municipal waste and surface waters.

    PubMed

    Jálová, V; Jarošová, B; Bláha, L; Giesy, J P; Ocelka, T; Grabic, R; Jurčíková, J; Vrana, B; Hilscherová, K

    2013-09-01

    Passive and composite sampling in combination with in vitro bioassays and identification and quantification of individual chemicals were applied to characterize pollution by compounds with several specific modes of action in urban area in the basin of two rivers, with 400,000 inhabitants and a variety of industrial activities. Two types of passive samplers, semipermeable membrane devices (SPMD) for hydrophobic contaminants and polar organic chemical integrative samplers (POCIS) for polar compounds such as pesticides and pharmaceuticals, were used to sample wastewater treatment plant (WWTP) influent and effluent as well as rivers upstream and downstream of the urban complex and the WWTP. Compounds with endocrine disruptive potency were detected in river water and WWTP influent and effluent. Year-round, monthly assessment of waste waters by bioassays documented estrogenic, androgenic and dioxin-like potency as well as cytotoxicity in influent waters of the WWTP and allowed characterization of seasonal variability of these biological potentials in waste waters. The WWTP effectively removed cytotoxic compounds, xenoestrogens and xenoandrogens. There was significant variability in treatment efficiency of dioxin-like potency. The study indicates that the WWTP, despite its up-to-date technology, can contribute endocrine disrupting compounds to the river. Riverine samples exhibited dioxin-like, antiestrogenic and antiandrogenic potencies. The study design enabled characterization of effects of the urban complex and the WWTP on the river. Concentrations of PAHs and contaminants and specific biological potencies sampled by POCIS decreased as a function of distance from the city.

  20. Wnt5a promotes cancer cell invasion and proliferation by receptor-mediated endocytosis-dependent and -independent mechanisms, respectively

    PubMed Central

    Shojima, Kensaku; Sato, Akira; Hanaki, Hideaki; Tsujimoto, Ikuko; Nakamura, Masahiro; Hattori, Kazunari; Sato, Yuji; Dohi, Keiji; Hirata, Michinari; Yamamoto, Hideki; Kikuchi, Akira

    2015-01-01

    Wnt5a activates the Wnt/β-catenin-independent pathway and its overexpression is associated with tumor aggressiveness enhancing invasive activity. For this action, Wnt5a-induced receptor endocytosis with clathrin is required. Wnt5a expression was previously believed to be associated with cancer cell motility but not proliferation. Recently, it was reported that Wnt5a is also implicated in cancer cell proliferation, but the mechanism was not clear. In this study, we generated a neutralizing anti-Wnt5a monoclonal antibody (mAb5A16) to investigate the mechanism by which Wnt5a regulates cancer cell proliferation. Wnt5a stimulated both invasion and proliferation of certain types of cancer cells, including HeLaS3 cervical cancer cells and A549 lung cancer cells although Wnt5a promoted invasion but not proliferation in other cancer cells such as KKLS gastric cancer cells. mAb5A16 did not affect the binding of Wnt5a to its receptor, but it suppressed Wnt5a-induced receptor-mediated endocytosis. mAb5A16 inhibited invasion but not proliferation of HeLaS3 and A549 cells. Wnt5a activated Src family kinases (SFKs) and Wnt5a-dependent cancer cell proliferation was dependent on SFKs, yet blockade of receptor-mediated endocytosis did not affect cancer cell proliferation and SFK activity. These results suggest that Wnt5a promotes invasion and proliferation of certain types of cancer cells through receptor-mediated endocytosis-dependent and -independent mechanisms, respectively. PMID:25622531

  1. The aryl hydrocarbon receptor mediates raloxifene-induced apoptosis in estrogen receptor-negative hepatoma and breast cancer cells.

    PubMed

    O'Donnell, E F; Koch, D C; Bisson, W H; Jang, H S; Kolluri, S K

    2014-01-30

    Identification of new molecular targets for the treatment of breast cancer is an important clinical goal, especially for triple-negative breast cancer, which is refractory to existing targeted treatments. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known primarily as the mediator of dioxin toxicity. However, the AhR can also inhibit cellular proliferation in a ligand-dependent manner and act as a tumor suppressor in mice, and thus may be a potential anticancer target. To investigate the AhR as an anticancer target, we conducted a small molecule screen to discover novel AhR ligands with anticancer properties. We identified raloxifene, a selective estrogen receptor (ER) modulator currently used in the clinic for prevention of ER-positive breast cancer and osteoporosis in post-menopausal women, as an AhR activator. Raloxifene directly bound the AhR and induced apoptosis in ER-negative mouse and human hepatoma cells in an AhR-dependent manner, indicating that the AhR is a molecular target of raloxifene and mediates raloxifene-induced apoptosis in the absence of ER. Raloxifene selectively induced apoptosis of triple-negative MDA-MB-231 breast cancer cells compared with non-transformed mammary epithelial cells via the AhR. Combined with recent data showing that raloxifene inhibits triple-negative breast cancer xenografts in vivo (Int J Oncol. 43(3):785-92, 2013), our results support the possibility of repurposing of raloxifene as an AhR-targeted therapeutic for triple-negative breast cancer patients. To this end, we also evaluated the role of AhR expression on survival of patients diagnosed with breast cancer. We found that higher expression of the AhR is significantly associated with increased overall survival and distant metastasis-free survival in both hormone-dependent (ER-positive) and hormone-independent (ER and progesterone receptor (PR)-negative) breast cancers. Together, our data strongly support the possibility of using the Ah

  2. The aryl hydrocarbon receptor mediates raloxifene-induced apoptosis in estrogen receptor-negative hepatoma and breast cancer cells

    PubMed Central

    O'Donnell, E F; Koch, D C; Bisson, W H; Jang, H S; Kolluri, S K

    2014-01-01

    Identification of new molecular targets for the treatment of breast cancer is an important clinical goal, especially for triple-negative breast cancer, which is refractory to existing targeted treatments. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known primarily as the mediator of dioxin toxicity. However, the AhR can also inhibit cellular proliferation in a ligand-dependent manner and act as a tumor suppressor in mice, and thus may be a potential anticancer target. To investigate the AhR as an anticancer target, we conducted a small molecule screen to discover novel AhR ligands with anticancer properties. We identified raloxifene, a selective estrogen receptor (ER) modulator currently used in the clinic for prevention of ER-positive breast cancer and osteoporosis in post-menopausal women, as an AhR activator. Raloxifene directly bound the AhR and induced apoptosis in ER-negative mouse and human hepatoma cells in an AhR-dependent manner, indicating that the AhR is a molecular target of raloxifene and mediates raloxifene-induced apoptosis in the absence of ER. Raloxifene selectively induced apoptosis of triple-negative MDA-MB-231 breast cancer cells compared with non-transformed mammary epithelial cells via the AhR. Combined with recent data showing that raloxifene inhibits triple-negative breast cancer xenografts in vivo (Int J Oncol. 43(3):785-92, 2013), our results support the possibility of repurposing of raloxifene as an AhR-targeted therapeutic for triple-negative breast cancer patients. To this end, we also evaluated the role of AhR expression on survival of patients diagnosed with breast cancer. We found that higher expression of the AhR is significantly associated with increased overall survival and distant metastasis-free survival in both hormone-dependent (ER-positive) and hormone-independent (ER and progesterone receptor (PR)-negative) breast cancers. Together, our data strongly support the possibility of using the Ah

  3. Down-regulation of cyclin-dependent kinase-4 and MAPK through estrogen receptor mediated cell cycle arrest in human breast cancer induced by gold nanoparticle tagged toxin protein NKCT1.

    PubMed

    Bhowmik, Tanmoy; Gomes, Antony

    2017-04-25

    The aim of this study was to determine whether gold nanoparticles conjugated cytotoxic protein NKCT1 (GNP-NKCT1) acted through the estrogen receptor mediated pathway in MCF-7 cells and to establish the MAPK and PI3k/Akt signal transduction pathway. Apoptosis was done by flow cytometry. BrdU incorporation and nuclear proliferating antigen was measured by flow cytometry. Wound healing assay along with matrigel chamber invasion and migration was done. Expression of MMP9 was checked by flow cytometry and also by gelatin zymography. To analyze the regulation of signaling protein, western blot was done. MTT assay was done to evaluate the ligand receptor pathway using the estrogen receptor negative cell line (MDA-MB-231) for inhibitor effects. Treatment of GNP-NKCT1 (3.9 μg/ml) exhibited 38.04% early apoptosis and 4.29% late apoptotic cell. GNP-NKCT1 significantly inhibited both cell migration and invasion with suppressed expression of MMP9. In addition, treatment of cultured human breast cancer MCF7 cells with GNP-NKCT1 reversely suppressed the incorporation of BrdU, with reduced expression of Ki-67. The western blot analysis showed that GNP-NKCT1 arrested cell cycle progression through upregulation of the kinase inhibitor protein p21 and inactivation of G1-cylin dependent kinase (CDK4). GNP-NKCT1 suppressed nuclear translocation of nuclear factor kappa B (NF-κB) and also abrogated the phosphorylation of p38 mitogen activated protein kinase (MAPK), phosphatidylinositide-3-kinase (PI3k), Akt and extracellular regulated kinase (ERK1/2). MTT assay indicated that GNP-NKCT1 reduced proliferation in the estrogen receptor induced ER negative breast cancer cell line (MDA-MB-231). Addition of, ER inhibitor (tamoxifen) and PI3K inhibitor (wortmannin) to cells resulted in reduced expression of Ki-67 and MMP-9. The data suggested that GNP-NKCT1 induced MCF7 cell inhibition may occur through estrogen receptor pathway via inactivation of CDK4 and inactivation of PI3K/Akt, ERK1

  4. Estrogen receptors mediate estradiol's effect on sensitization and CPP to cocaine in female rats: role of contextual cues.

    PubMed

    Segarra, Annabell C; Torres-Díaz, Yvonne M; Silva, Richard D; Puig-Ramos, Anabel; Menéndez-Delmestre, Raissa; Rivera-Bermúdez, José G; Amadeo, Waldo; Agosto-Rivera, José L

    2014-02-01

    Preclinical studies show that estradiol enhances sensitization to cocaine in females by mechanisms not fully understood. These studies consistently show that ovariectomized (OVX) rats exhibit little or no sensitization to cocaine compared to OVX rats administered estradiol. In this study we varied the dose of cocaine (10, 15, and 30mg/kg), the length of cocaine treatment (from 5 to 10days) and the context of cocaine injections to determine if these factors play a role on estradiol's effects on cocaine sensitization. Because OVX rats are hormonally compromised, they are not representative of the natural state of the animal, and thus the physiological context of these studies remains unclear. To address this issue, we blocked ERs in gonadally intact females by icv administration of the antiestrogen ICI-182,780. Varying the dose or length of exposure to cocaine does not alter estradiol's effect on cocaine sensitization. In contrast, a highly context-dependent sensitization protocol results in robust sensitization even in OVX rats. Interestingly, using this protocol, sensitization in OVX rats diminished with time, suggesting that estradiol is necessary for the maintenance of cocaine sensitization. Blocking brain ERs with ICI completely abolishes the development and expression of cocaine sensitization in gonadally intact female rats, even when tested in a highly context-dependent sensitization protocol. Given these findings, we propose that activation of brain ERs is required for the development and maintenance of sensitization and CPP. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Morphine inhibits an alpha9-acetylcholine nicotinic receptor-mediated response by a mechanism which does not involve opioid receptors.

    PubMed

    Lioudyno, M I; Verbitsky, M; Holt, J C; Elgoyhen, A B; Guth, P S

    2000-11-01

    Nicotinic acetylcholine (nACh) receptors are known to be targets for modulation by a number of substances, including the opiates. It is known that acetylcholine (ACh) coexists with opioid peptides in cochlear efferent neurons, and such a colocalization has been proposed for the vestibular system. In the present study we test the hypothesis that morphine, an opioid receptor agonist with a broad spectrum of selectivity, modulates alpha9nACh receptor-mediated responses in frog vestibular hair cells. Morphine dose-dependently and reversibly inhibited ACh-induced currents as recorded by the perforated patch-clamp method. In the presence of morphine the ACh dose-response curve was shifted to the right in a parallel fashion, suggesting a competitive interaction. However, naloxone did not antagonize the inhibition produced by morphine. To test the hypothesis that morphine could interact with the alpha9nACh receptor without the involvement of opioid receptors, experiments were performed using Xenopus laevis oocytes injected with the alpha9nACh receptor cRNA. The currents activated by ACh in Xenopus oocytes, a system that lacks opioid receptors, were also dose-dependently inhibited by morphine. We conclude that morphine inhibits the alpha9nACh receptor-mediated response in hair cells and Xenopus oocytes through a mechanism which does not involve opioid receptors but may be a direct block of the alpha9nACh receptor.

  6. Mechanism-Based Tumor-Targeting Drug Delivery System. Validation of Efficient Vitamin Receptor-Mediated Endocytosis and Drug Release

    SciTech Connect

    Chen, S.; Wong, S.; Zhao, X.; Chen, J.; Chen, J.; Kuznetsova, L.; Ojima, I.

    2010-05-01

    An efficient mechanism-based tumor-targeting drug delivery system, based on tumor-specific vitamin-receptor mediated endocytosis, has been developed. The tumor-targeting drug delivery system is a conjugate of a tumor-targeting molecule (biotin: vitamin H or vitamin B-7), a mechanism-based self-immolative linker and a second-generation taxoid (SB-T-1214) as the cytotoxic agent. This conjugate (1) is designed to be (i) specific to the vitamin receptors overexpressed on tumor cell surface and (ii) internalized efficiently through receptor-mediated endocytosis, followed by smooth drug release via glutathione-triggered self-immolation of the linker. In order to monitor and validate the sequence of events hypothesized, i.e., receptor-mediated endocytosis of the conjugate, drug release, and drug-binding to the target protein (microtubules), three fluorescent/fluorogenic molecular probes (2, 3, and 4) were designed and synthesized. The actual occurrence of these processes was unambiguously confirmed by means of confocal fluorescence microscopy (CFM) and flow cytometry using L1210FR leukemia cells, overexpressing biotin receptors. The molecular probe 4, bearing the taxoid linked to fluorescein, was also used to examine the cell specificity (i.e., efficacy of receptor-based cell targeting) for three cell lines, L1210FR (biotin receptors overexpressed), L1210 (biotin receptors not overexpressed), and WI38 (normal human lung fibroblast, biotin receptor negative). As anticipated, the molecular probe 4 exhibited high specificity only to L1210FR. To confirm the direct correlation between the cell-specific drug delivery and anticancer activity of the probe 4, its cytotoxicity against these three cell lines was also examined. The results clearly showed a good correlation between the two methods. In the same manner, excellent cell-specific cytotoxicity of the conjugate 1 (without fluorescein attachment to the taxoid) against the same three cell lines was confirmed. This mechanism

  7. Cell receptors: definition, mechanisms and regulation of receptor-mediated endocytosis.

    PubMed

    Féger, J; Gil-Falgon, S; Lamaze, C

    1994-12-01

    Receptors allow the cells to recognize specific ligands and to receive extracellular messages. They can be classified into five families: 1) receptors for lipidic or lipophilic ligands; 2) the seven transmembrane receptors which mediate their messages by transduction through the activation of G-proteins, effectors and second messengers to amplify the response; 3) receptors which present an enzymatic activity on their transmembrane domains; 4) channel-receptors, transmembrane oligomeric molecules which let ions flow into the cell and 5) receptors which role is to internalize ligands, whatever their various functions. In parallel a concept of membrane plasticity was developed: vesicles are constantly formed from the plasma membrane, addressing complexes of ligand-receptors to specific intracellular compartments. This receptor-mediated endocytosis of ligand plays a critical role in regulating the number of a given receptor at the plasma membrane and in the cellular uptake of nutrients, growth factors and hormones. Many pathways exist for these transports but little is known about the signals which select the ligands or the receptors and direct them to their appropriate intracellular destination.

  8. The influence of receptor-mediated interactions on reaction-diffusion mechanisms of cellular self-organisation.

    PubMed

    Klika, Václav; Baker, Ruth E; Headon, Denis; Gaffney, Eamonn A

    2012-04-01

    Understanding the mechanisms governing and regulating self-organisation in the developing embryo is a key challenge that has puzzled and fascinated scientists for decades. Since its conception in 1952 the Turing model has been a paradigm for pattern formation, motivating numerous theoretical and experimental studies, though its verification at the molecular level in biological systems has remained elusive. In this work, we consider the influence of receptor-mediated dynamics within the framework of Turing models, showing how non-diffusing species impact the conditions for the emergence of self-organisation. We illustrate our results within the framework of hair follicle pre-patterning, showing how receptor interaction structures can be constrained by the requirement for patterning, without the need for detailed knowledge of the network dynamics. Finally, in the light of our results, we discuss the ability of such systems to pattern outside the classical limits of the Turing model, and the inherent dangers involved in model reduction.

  9. Bisphenol a exposure promotes the migration of NCM460 cells via estrogen receptor-mediated integrin β1/MMP-9 pathway.

    PubMed

    Shi, Tonglin; Zhao, Chao; Li, Zhuoyu; Zhang, Quanbin; Jin, Xiaoting

    2016-07-01

    Bisphenol A (BPA) is a widely used industrial chemical and also an environmental endocrine disruptor (EED), which serves as a monomer in the manufacture of polycarbonate plastics. BPA enters human body mainly through oral intake, and has been reported as being linked to oncogenesis in many tissues. However, the association of BPA intake with gastrointestinal cancer, such as colon cancer, has received less attention. The present study was conducted to investigate the effects of BPA on the migration of normal colon epithelial cells (NCM460 cells) and further elucidate the underlying mechanisms. Our data showed that 1 × 10(-8) M (equivalent to environmental concentration) of BPA potently promoted the migration of NCM460 cells. Interestingly, BPA treatment induced an increase of integrin β1 expression, and the functional blocking of integrin β1 abolished the migration-promoting effects of BPA. Moreover, the results showed that it was estrogen receptor β but not estrogen receptor α that was involved in this migration promotion. In addition, cellular exposure of BPA stimulated the expression and activity of MMP-9, a well-known factor of cell migration. Taken together, these results indicate that environmental concentration of BPA exposure promotes cell migration through activating ERβ-mediated integrin β1/MMP-9 pathway, suggesting exposure to BPA in the colon may present a potential cancer risk. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 799-807, 2016.

  10. Presynaptic 5-HT3 receptor-mediated modulation of synaptic GABA release in the mechanically dissociated rat amygdala neurons

    PubMed Central

    Koyama, Susumu; Matsumoto, Nozomu; Kubo, Chiharu; Akaike, Norio

    2000-01-01

    Nystatin-perforated patch recordings were made from mechanically dissociated basolateral amygdala neurons with preserved intact native presynaptic nerve terminals to study the mechanism of 5-HT3 receptor-mediated serotonergic modulation of GABAergic inhibition. The specific 5-HT3 agonist mCPBG (1 μM) rapidly facilitated the frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) and this facilitation desensitized within 1 min. Tropisetron (30 nM), a specific 5-HT3 antagonist, blocked the mCPBG effect. mCPBG augmented mIPSC amplitude. However, no direct postsynaptic serotonergic currents were evoked by mCPBG. Neither GABA-evoked current amplitude nor the kinetics of individual GABAergic mIPSCs were affected by mCPBG. Therefore, the augmentation is unlikely to be due to postsynaptic effects evoked by mCPBG. At higher concentrations mCPBG produced shorter-duration facilitation of miniature events. While mCPBG increased the mIPSC frequency in calcium-containing solution with Cd2+, this increase was absent in Ca2+-free external solution. It appears that the Ca2+ influx through voltage-dependent calcium channels was not as crucial as that through 5-HT3 receptors for synaptic GABA release. When two pulses of mCPBG (each 1 μM, 1 min) were given, the response to the second pulse elicited full recovery when the interval between pulses was at least 9 min. Protein kinase A (PKA) activation by 8-Br-cAMP (300 μM) shortened and PKA inhibition by Rp-cAMP (100 μM) prolonged the recovery time. PKA activity did not affect the time course of fast desensitization. Our results suggest that a 5-HT3-specific agonist acts on presynaptic nerve terminals facilitating synaptic GABA release without postsynaptic effects. The facilitation requires calcium influx through presynaptic 5-HT3 receptors. PKA modulates the recovery process from desensitization of presynaptic 5-HT3 receptor-mediated regulation of synaptic GABA release. PMID:11101647

  11. Centrally administered neuromedin U elevates plasma adrenaline by brain prostanoid TP receptor-mediated mechanisms in rats.

    PubMed

    Sasaki, Tsuyoshi; Shimizu, Takahiro; Wakiguchi, Hiroshi; Yokotani, Kunihiko

    2008-09-11

    Neuromedin U is a hypothalamic peptide involved in energy homeostasis and stress responses. The peptide, when administered intracerebroventricularly (i.c.v.), decreases food intake and body weight while increasing body temperature and heat production. We examined the effect of i.c.v. administered neuromedin U on plasma catecholamines with regard to the brain prostanoid using anesthetized rats. Neuromedin U (0.1, 0.5 and 1 nmol/animal, i.c.v.) effectively elevated plasma adrenaline (a maximal response was obtained at 0.5 nmol/animal), but had little effect on plasma noradrenaline. However, intravenously administered neuromedin U (0.5 nmol/animal) had no effect on plasma catecholamines. Neuromedin U (0.5 nmol/animal, i.c.v.)-induced elevation of plasma adrenaline was effectively reduced by intracerebroventricular pretreatments with indomethacin (an inhibitor of cyclooxygenase) (0.6 and 1.2 micromol/animal), furegrelate (an inhibitor of thromboxane A2 synthase) (0.9 and 1.8 micromol/animal) and (+)-S-145 (a blocker of prostanoid TP receptors) (250 and 625 nmol/animal), respectively. The neuromedin U-induced adrenaline response was also abolished by acute bilateral adrenalectomy. These results suggest that centrally administered neuromedin U evokes the secretion of adrenaline from the adrenal medulla by brain prostanoid TP receptor-mediated mechanisms in rats.

  12. STAT1 Regulates the Homeostatic Component of Visual Cortical Plasticity via an AMPA Receptor-Mediated Mechanism

    PubMed Central

    Van Wart, Audra; Petravicz, Jeremy; Tropea, Daniela

    2014-01-01

    Accumulating evidence points to a role for Janus kinase/signal transducers and activators of transcription (STAT) immune signaling in neuronal function; however, its role in experience-dependent plasticity is unknown. Here we show that one of its components, STAT1, negatively regulates the homeostatic component of ocular dominance plasticity in visual cortex. After brief monocular deprivation (MD), STAT1 knock-out (KO) mice show an accelerated increase of open-eye responses, to a level comparable with open-eye responses after a longer duration of MD in wild-type (WT) mice. Therefore, this component of plasticity is abnormally enhanced in KO mice. Conversely, increasing STAT1 signaling by IFNγ treatment in WT mice reduces the homeostatic component of plasticity by impairing open-eye responses. Enhanced plasticity in KO mice is accompanied by sustained surface levels of GluA1 AMPA receptors and increased amplitude and frequency of AMPA receptor-mediated mEPSCs, which resemble changes in WT mice after a longer duration of MD. These results demonstrate a unique role for STAT1 during visual cortical plasticity in vivo through a mechanism that includes AMPA receptors. PMID:25080587

  13. Viral infection transiently reverses activation receptor-mediated NK cell hyporesponsiveness in an MHC class I-independent mechanism.

    PubMed

    Mazumdar, Budhaditya; Bolanos, Fred D; Tripathy, Sandeep K

    2013-05-01

    Continuous engagement of the Ly49H activating receptor with its ligand (m157) in a transgenic mouse expressing m157 (m157-Tg) results in hyporesponsiveness of Ly49H(+) NK cells. The same interaction, during murine cytomegalovirus (MCMV) infection, leads to activation of Ly49H(+) NK cells. MCMV infection results in decreased MHC class I (MHC-I) expression on the infected cell as well as inflammatory responses, both of which do not take place in the uninfected m157-Tg mouse, potentially allowing for activation of NK cells in the context of MCMV infection. In this study, we demonstrated that viral infection transiently reverses activation receptor-mediated NK cell hyporesponsiveness in an MHC-I-independent mechanism. Furthermore, Ly49H(+) NK cells in an MHC-I-deficient environment remained hyporesponsive in the context of m157 expression, even when mature WT splenocytes were transferred into m157-Tg mice in an MHC-I-deficient environment. However, the administration of cytokines TNF-α, IL-12, and IFN-β resulted in a partial recovery from activation receptor-induced hyporesponsiveness. Thus, the release of the aforementioned cytokines during MCMV infection and not the downregulation of MHC-I expression appears to be responsible for partial resolution of Ly49H receptor-induced NK cell hyporesponsiveness.

  14. Metabotropic glutamate receptor-mediated long-term depression: molecular mechanisms.

    PubMed

    Gladding, Clare M; Fitzjohn, Stephen M; Molnár, Elek

    2009-12-01

    The ability to modify synaptic transmission between neurons is a fundamental process of the nervous system that is involved in development, learning, and disease. Thus, synaptic plasticity is the ability to bidirectionally modify transmission, where long-term potentiation and long-term depression (LTD) represent the best characterized forms of plasticity. In the hippocampus, two main forms of LTD coexist that are mediated by activation of either N-methyl-d-aspartic acid receptors (NMDARs) or metabotropic glutamate receptors (mGluRs). Compared with NMDAR-LTD, mGluR-LTD is less well understood, but recent advances have started to delineate the underlying mechanisms. mGluR-LTD at CA3:CA1 synapses in the hippocampus can be induced either by synaptic stimulation or by bath application of the group I selective agonist (R,S)-3,5-dihydroxyphenylglycine. Multiple signaling mechanisms have been implicated in mGluR-LTD, illustrating the complexity of this form of plasticity. This review provides an overview of recent studies investigating the molecular mechanisms underlying hippocampal mGluR-LTD. It highlights the role of key molecular components and signaling pathways that are involved in the induction and expression of mGluR-LTD and considers how the different signaling pathways may work together to elicit a persistent reduction in synaptic transmission.

  15. Fc-receptor-mediated phagocytosis is regulated by mechanical properties of the target

    NASA Technical Reports Server (NTRS)

    Beningo, Karen A.; Wang, Yu-li

    2002-01-01

    Phagocytosis is an actin-based process used by macrophages to clear particles greater than 0.5 microm in diameter. In addition to its role in immunological responses, phagocytosis is also necessary for tissue remodeling and repair. To prevent catastrophic autoimmune reactions, phagocytosis must be tightly regulated. It is commonly assumed that the recognition/selection of phagocytic targets is based solely upon receptor-ligand binding. Here we report an important new criterion, that mechanical parameters of the target can dramatically affect the efficiency of phagocytosis. When presented with particles of identical chemical properties but different rigidity, macrophages showed a strong preference to engulf rigid objects. Furthermore, phagocytosis of soft particles can be stimulated with the microinjection of constitutively active Rac1 but not RhoA, and with lysophosphatidic acid, an agent known to activate the small GTP-binding proteins of the Rho family. These data suggest a Rac1-dependent mechanosensory mechanism for phagocytosis, which probably plays an important role in a number of physiological and pathological processes from embryonic development to autoimmune diseases.

  16. Fc-receptor-mediated phagocytosis is regulated by mechanical properties of the target

    NASA Technical Reports Server (NTRS)

    Beningo, Karen A.; Wang, Yu-li

    2002-01-01

    Phagocytosis is an actin-based process used by macrophages to clear particles greater than 0.5 microm in diameter. In addition to its role in immunological responses, phagocytosis is also necessary for tissue remodeling and repair. To prevent catastrophic autoimmune reactions, phagocytosis must be tightly regulated. It is commonly assumed that the recognition/selection of phagocytic targets is based solely upon receptor-ligand binding. Here we report an important new criterion, that mechanical parameters of the target can dramatically affect the efficiency of phagocytosis. When presented with particles of identical chemical properties but different rigidity, macrophages showed a strong preference to engulf rigid objects. Furthermore, phagocytosis of soft particles can be stimulated with the microinjection of constitutively active Rac1 but not RhoA, and with lysophosphatidic acid, an agent known to activate the small GTP-binding proteins of the Rho family. These data suggest a Rac1-dependent mechanosensory mechanism for phagocytosis, which probably plays an important role in a number of physiological and pathological processes from embryonic development to autoimmune diseases.

  17. Neurobiology of microglial action in CNS injuries: receptor-mediated signaling mechanisms and functional roles

    PubMed Central

    Hu, Xiaoming; Liou, Anthony K.F.; Leak, Rehana K.; Xu, Mingyue; An, Chengrui; Suenaga, Jun; Shi, Yejie; Gao, Yanqin; Zheng, Ping; Chen, Jun

    2014-01-01

    Microglia are the first line of immune defense against central nervous system (CNS) injuries and disorders. These highly plastic cells play dualistic roles in neuronal injury and recovery and are known for their ability to assume diverse phenotypes. A broad range of surface receptors are expressed on microglia and mediate microglial ‘On’ or ‘Off’ responses to signals from other host cells as well as invading microorganisms. The integrated actions of these receptors result in tightly regulated biological functions, including cell mobility, phagocytosis, the induction of acquired immunity, and trophic factor/inflammatory mediator release. Over the last few years, significant advances have been made towards deciphering the signaling mechanisms related to these receptors and their specific cellular functions. In this review, we describe the current state of knowledge of the surface receptors involved in microglial activation, with an emphasis on their engagement of distinct functional programs and their roles in CNS injuries. It will become evident from this review that microglial homeostasis is carefully maintained by multiple counterbalanced strategies, including, but not limited to, ‘On’ and ‘Off’ receptor signaling. Specific regulation of theses microglial receptors may be a promising therapeutic strategy against CNS injuries. PMID:24923657

  18. Ligand-Specific Transcriptional Mechanisms Underlie Aryl Hydrocarbon Receptor-Mediated Developmental Toxicity of Oxygenated PAHs

    PubMed Central

    Goodale, B. C.; La Du, J.; Tilton, S. C.; Sullivan, C. M.; Bisson, W. H.; Waters, K. M.; Tanguay, R. L.

    2015-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are priority environmental contaminants that exhibit mutagenic, carcinogenic, proinflammatory, and teratogenic properties. Oxygen-substituted PAHs (OPAHs) are formed during combustion processes and via phototoxidation and biological degradation of parent (unsubstituted) PAHs. Despite their prevalence both in contaminated industrial sites and in urban air, OPAH mechanisms of action in biological systems are relatively understudied. Like parent PAHs, OPAHs exert structure-dependent mutagenic activities and activation of the aryl hydrocarbon receptor (AHR) and cytochrome p450 metabolic pathway. Four-ring OPAHs 1,9-benz-10-anthrone (BEZO) and benz(a)anthracene-7,12-dione (7,12-B[a]AQ) cause morphological aberrations and induce markers of oxidative stress in developing zebrafish with similar potency, but only 7,12-B[a]AQ induces robust Cyp1a protein expression. We investigated the role of the AHR in mediating the toxicity of BEZO and 7,12-B[a]AQ, and found that knockdown of AHR2 rescued developmental effects caused by both compounds. Using RNA-seq and molecular docking, we identified transcriptional responses that precede developmental toxicity induced via differential interaction with AHR2. Redox-homeostasis genes were affected similarly by these OPAHs, while 7,12-B[a]AQ preferentially activated phase 1 metabolism and BEZO uniquely decreased visual system genes. Analysis of biological functions and upstream regulators suggests that BEZO is a weak AHR agonist, but interacts with other transcriptional regulators to cause developmental toxicity in an AHR-dependent manner. Identifying ligand-dependent AHR interactions and signaling pathways is essential for understanding toxicity of this class of environmentally relevant compounds. PMID:26141390

  19. Ligand-specific transcriptional mechanisms underlie aryl hydrocarbon receptor-mediated developmental toxicity of oxygenated PAHs

    DOE PAGES

    Goodale, B. C.; Geisel School of Medicine at Dartmouth, Hanover, NH; La Du, J.; ...

    2015-07-03

    Polycyclic aromatic hydrocarbons (PAHs) are priority environmental contaminants that exhibit mutagenic, carcinogenic, proinflammatory, and teratogenic properties. Oxygen-substituted PAHs (OPAHs) are formed during combustion processes and via phototoxidation and biological degradation of parent (unsubstituted) PAHs. Despite their prevalence both in contaminated industrial sites and in urban air, OPAH mechanisms of action in biological systems are relatively understudied. Like parent PAHs, OPAHs exert structure-dependent mutagenic activities and activation of the aryl hydrocarbon receptor (AHR) and cytochrome p450 metabolic pathway. Four-ring OPAHs 1,9-benz-10-anthrone (BEZO) and benz(a)anthracene-7,12-dione (7,12-B[a]AQ) cause morphological aberrations and induce markers of oxidative stress in developing zebrafish with similar potency, butmore » only 7,12-B[a]AQ induces robust Cyp1a protein expression. We investigated the role of the AHR in mediating the toxicity of BEZO and 7,12-B[a]AQ, and found that knockdown of AHR2 rescued developmental effects caused by both compounds. Using RNA-seq and molecular docking, we identified transcriptional responses that precede developmental toxicity induced via differential interaction with AHR2. Redox-homeostasis genes were affected similarly by these OPAHs, while 7,12-B[a]AQ preferentially activated phase 1 metabolism and BEZO uniquely decreased visual system genes. Analysis of biological functions and upstream regulators suggests that BEZO is a weak AHR agonist, but interacts with other transcriptional regulators to cause developmental toxicity in an AHR-dependent manner. Furthermore, identifying ligand-dependent AHR interactions and signaling pathways is essential for understanding toxicity of this class of environmentally relevant compounds.« less

  20. Ligand-specific transcriptional mechanisms underlie aryl hydrocarbon receptor-mediated developmental toxicity of oxygenated PAHs

    SciTech Connect

    Goodale, B. C.; La Du, J.; Tilton, S. C.; Sullivan, C. M.; Bisson, W. H.; Waters, K. M.; Tanguay, R. L.

    2015-07-03

    Polycyclic aromatic hydrocarbons (PAHs) are priority environmental contaminants that exhibit mutagenic, carcinogenic, proinflammatory, and teratogenic properties. Oxygen-substituted PAHs (OPAHs) are formed during combustion processes and via phototoxidation and biological degradation of parent (unsubstituted) PAHs. Despite their prevalence both in contaminated industrial sites and in urban air, OPAH mechanisms of action in biological systems are relatively understudied. Like parent PAHs, OPAHs exert structure-dependent mutagenic activities and activation of the aryl hydrocarbon receptor (AHR) and cytochrome p450 metabolic pathway. Four-ring OPAHs 1,9-benz-10-anthrone (BEZO) and benz(a)anthracene-7,12-dione (7,12-B[a]AQ) cause morphological aberrations and induce markers of oxidative stress in developing zebrafish with similar potency, but only 7,12-B[a]AQ induces robust Cyp1a protein expression. We investigated the role of the AHR in mediating the toxicity of BEZO and 7,12-B[a]AQ, and found that knockdown of AHR2 rescued developmental effects caused by both compounds. Using RNA-seq and molecular docking, we identified transcriptional responses that precede developmental toxicity induced via differential interaction with AHR2. Redox-homeostasis genes were affected similarly by these OPAHs, while 7,12-B[a]AQ preferentially activated phase 1 metabolism and BEZO uniquely decreased visual system genes. Analysis of biological functions and upstream regulators suggests that BEZO is a weak AHR agonist, but interacts with other transcriptional regulators to cause developmental toxicity in an AHR-dependent manner. Furthermore, identifying ligand-dependent AHR interactions and signaling pathways is essential for understanding toxicity of this class of environmentally relevant compounds.

  1. Tetrahydroisoquinoline alkaloids mimic direct but not receptor-mediated inhibitory effects of estrogens and phytoestrogens on testicular endocrine function. Possible significance for Leydig cell insufficiency in alcohol addiction

    SciTech Connect

    Stammel, W.; Thomas, H. ); Staib, W.; Kuehn-Velten, W.K. )

    1991-01-01

    Possible effects of various tetrahydroisoquinolines (TIQs) on rat testicular endocrine function were tested in vitro in order to prove whether these compounds may be mediators of the development of Leydig cell insufficiency. TIQ effects on different levels of regulation of testis function were compared in vitro with estrogen effects, since both classes of compounds have structural similarities. Gonadotropin-stimulated testosterone production by testicular Leydig cells was inhibited by tetrahydropapaveroline and isosalsoline, the IC{sub 50} values being comparable to those of estradiol, 2-hydroxyestradiol, and the phytoestrogens, coumestrol and genistein; salsolinol and salsoline were less effective, and salsolidine was ineffective. None of these TIQs interacted significantly with testicular estrogen receptor as analyzed by estradiol displacement. However, tetrahydropapaveroline, isosalsoline and salsolinol competitively inhibited substrate binding to cytochrome P45OXVII, with similar efficiency as the estrogens did; salsoline and salsolidine were again much less effective.

  2. Large effects from small exposures. I. Mechanisms for endocrine-disrupting chemicals with estrogenic activity.

    PubMed Central

    Welshons, Wade V; Thayer, Kristina A; Judy, Barbara M; Taylor, Julia A; Curran, Edward M; vom Saal, Frederick S

    2003-01-01

    Information concerning the fundamental mechanisms of action of both natural and environmental hormones, combined with information concerning endogenous hormone concentrations, reveals how endocrine-disrupting chemicals with estrogenic activity (EEDCs) can be active at concentrations far below those currently being tested in toxicological studies. Using only very high doses in toxicological studies of EEDCs thus can dramatically underestimate bioactivity. Specifically: a) The hormonal action mechanisms and the physiology of delivery of EEDCs predict with accuracy the low-dose ranges of biological activity, which have been missed by traditional toxicological testing. b) Toxicology assumes that it is valid to extrapolate linearly from high doses over a very wide dose range to predict responses at doses within the physiological range of receptor occupancy for an EEDC; however, because receptor-mediated responses saturate, this assumption is invalid. c) Furthermore, receptor-mediated responses can first increase and then decrease as dose increases, contradicting the assumption that dose-response relationships are monotonic. d) Exogenous estrogens modulate a system that is physiologically active and thus is already above threshold, contradicting the traditional toxicological assumption of thresholds for endocrine responses to EEDCs. These four fundamental issues are problematic for risk assessment methods used by regulatory agencies, because they challenge the traditional use of extrapolation from high-dose testing to predict responses at the much lower environmentally relevant doses. These doses are within the range of current exposures to numerous chemicals in wildlife and humans. These problems are exacerbated by the fact that the type of positive and negative controls appropriate to the study of endocrine responses are not part of traditional toxicological testing and are frequently omitted, or when present, have been misinterpreted. PMID:12826473

  3. Bisphenol-A rapidly promotes dynamic changes in hippocampal dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDA receptor subunit NR2B

    SciTech Connect

    Xu Xiaohong Ye Yinping; Li Tao; Chen Lei; Tian Dong; Luo Qingqing; Lu Mei

    2010-12-01

    Bisphenol-A (BPA) is known to be a potent endocrine disrupter. Evidence is emerging that estrogen exerts a rapid influence on hippocampal synaptic plasticity and the dendritic spine density, which requires activation of NMDA receptors. In the present study, we investigated the effects of BPA (ranging from 1 to 1000 nM), focusing on the rapid dynamic changes in dendritic filopodia and the expressions of estrogen receptor (ER) {beta} and NMDA receptor, as well as the phosphorylation of NMDA receptor subunit NR2B in the cultured hippocampal neurons. A specific ER antagonist ICI 182,780 was used to examine the potential involvement of ERs. The results demonstrated that exposure to BPA (ranging from 10 to 1000 nM) for 30 min rapidly enhanced the motility and the density of dendritic filopodia in the cultured hippocampal neurons, as well as the phosphorylation of NR2B (pNR2B), though the expressions of NMDA receptor subunits NR1, NR2B, and ER{beta} were not changed. The antagonist of ERs completely inhibited the BPA-induced increases in the filopodial motility and the number of filopodia extending from dendrites. The increased pNR2B induced by BPA (100 nM) was also completely eliminated. Furthermore, BPA attenuated the effects of 17{beta}-estradiol (17{beta}-E{sub 2}) on the dendritic filopodia outgrowth and the expression of pNR2B when BPA was co-treated with 17{beta}-E{sub 2}. The present results suggest that BPA, like 17{beta}-E{sub 2}, rapidly results in the enhanced motility and density of dendritic filopodia in the cultured hippocampal neurons with the concomitant activation of NMDA receptor subunit NR2B via an ER-mediated signaling pathway. Meanwhile, BPA suppressed the enhancement effects of 17{beta}-E{sub 2} when it coexists with 17{beta}-E{sub 2}. These results provided important evidence suggesting the neurotoxicity of the low levels of BPA during the early postnatal development of the brain.

  4. Uncovering the Mechanisms of Estrogen Effects on Hippocampal Function

    PubMed Central

    Spencer, Joanna L.; Waters, Elizabeth M.; Romeo, Russell D.; Wood, Gwendolyn E.; Milner, Teresa A.; McEwen, Bruce S.

    2008-01-01

    Estrogens have direct effects on the brain areas controlling cognition. One of the most studied of these regions is the dorsal hippocampal formation, which governs the formation of spatial and episodic memory formation. In laboratory animals, most investigators report that estrogen enhances synaptic plasticity and improves performance on hippocampal-dependent cognitive behaviors. This review summarizes work conducted in our laboratory and others toward identifying estrogen’s actions in the hippocampal formation, and the mechanisms for these actions. Physiologic and pharmacologic estrogen affects cognitive behavior in mammals, which may be applicable to human health and disease. The effects of estrogen in the hippocampal formation that lead to modulation of hippocampal function include effects on cell morphology, synapse formation, signaling, and excitability that have been studied in laboratory mice, rats, and primates. Finally, estrogen may signal through both nuclear and extranuclear hippocampal estrogen receptors to achieve its downstream effects. PMID:18078984

  5. Estrogens and the cognitive symptoms of schizophrenia: Possible neuroprotective mechanisms.

    PubMed

    McGregor, Claire; Riordan, Alexander; Thornton, Janice

    2017-06-30

    Schizophrenia is a complex neuropsychiatric illness with marked sex differences. Women have later onset and lesser symptoms, which has led to the hypothesis that estrogens are protective in schizophrenia. Cognitive dysfunction is a hallmark of the disease and the symptom most correlated with functional outcome. Here we describe a number of mechanisms by which estrogens may be therapeutic in schizophrenia, with a focus on cognitive symptoms. We review the relationship between estrogens and brain derived neurotrophic factor, neuroinflammation, NMDA receptors, GABA receptors, and luteinizing hormone. Exploring these pathways may enable novel treatments for schizophrenia and a greater understanding of this devastating disease. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Estrogen

    MedlinePlus

    ... life', the end of monthly menstrual periods). Some brands of estrogen are also used to treat vaginal ... prevent osteoporosis should consider a different treatment. Some brands of estrogen are also to relieve symptoms of ...

  7. R(+)-methanandamide induces cyclooxygenase-2 expression in human neuroglioma cells via a non-cannabinoid receptor-mediated mechanism.

    PubMed

    Ramer, R; Brune, K; Pahl, A; Hinz, B

    2001-09-07

    Cannabinoids affect prostaglandin (PG) formation in the central nervous system through as yet unidentified mechanisms. Using H4 human neuroglioma cells, the present study investigates the effect of R(+)-methanandamide (metabolically stable analogue of the endocannabinoid anandamide) on the expression of the cyclooxygenase-2 (COX-2) enzyme. Incubation of cells with R(+)-methanandamide was accompanied by concentration-dependent increases in COX-2 mRNA, COX-2 protein, and COX-2-dependent PGE(2) synthesis. Moreover, treatment of cells with R(+)-methanandamide in the presence of interleukin-1beta led to an overadditive induction of COX-2 expression. The stimulatory effect of R(+)-methanandamide on COX-2 expression was mimicked by the structurally unrelated cannabinoid Delta(9)-tetrahydrocannabinol. Stimulation of both COX-2 mRNA expression and subsequent PGE(2) synthesis by R(+)-methanandamide was not affected by the selective CB(1) receptor antagonist AM-251 or the G(i/o) protein inactivator pertussis toxin. Enhancement of COX-2 expression by R(+)-methanandamide was paralleled by time-dependent phosphorylations of p38 mitogen-activated protein kinase (MAPK) and p42/44 MAPK. Consistent with the activation of both kinases, R(+)-methanandamide-induced COX-2 mRNA expression and PGE(2) formation were abrogated in the presence of specific inhibitors of p38 MAPK (SB203580) and p42/44 MAPK activation (PD98059). Together, our results demonstrate that R(+)-methanandamide induces COX-2 expression in human neuroglioma cells via a cannabinoid receptor-independent mechanism involving activation of the MAPK pathway. In conclusion, induction of COX-2 expression may represent a novel mechanism by which cannabinoids mediate PG-dependent effects within the central nervous system.

  8. Molecular mechanisms of group I metabotropic glutamate receptor mediated LTP and LTD in basolateral amygdala in vitro.

    PubMed

    Chen, A; Hu, W W; Jiang, X L; Potegal, M; Li, H

    2017-02-01

    The roles of group I metabotropic glutamate receptors, metabotropic glutamate receptor 1 (mGluR1) and mGluR5, in regulating synaptic plasticity and metaplasticity in the basolateral amygdala (BLA) remain unclear. The present study examined mGluR1- and mGluR5-mediated synaptic plasticity in the BLA and their respective signaling mechanisms. Bath application of the group I mGluR agonist, 3,5-dihydroxyphenylglycine (DHPG) (20 μM), directly suppressed basal fEPSPs (84.5 ± 6.3% of the baseline). The suppressive effect persisted for at least 30 min after washout; it was abolished by the mGluR1 antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) but was unaffected by the mGluR5 antagonist 2-methyl-6- (phenylethynyl)-pyridine (MPEP). Interestingly, application of DHPG (at both 2 and 20 μM), regardless of the presence of CPCCOEt, could transform single theta burst stimulation (TBS)-induced short-term synaptic potentiation into a long-term potentiation (LTP). Such a facilitating effect could be blocked by the mGluR5 antagonist MPEP. Blockade of phospholipase C (PLC), the downstream enzyme of group I mGluR, with U73122, prevented both mGluR1- and mGluR5-mediated effects on synaptic plasticity. Nevertheless, blockade of protein kinase C (PKC), the downstream enzyme of PLC, with chelerythrine (5 μM) only prevented the transforming effect of DHPG on TBS-induced LTP and did not affect DHPG-induced long-term depression (LTD). These results suggest that mGluR1 activation induced LTD via a PLC-dependent and PKC-independent mechanism, while the priming action of mGluR5 receptor on the BLA LTP is both PLC and PKC dependent. The BLA metaplasticity mediated by mGluR1 and mGluR5 may provide signal switching mechanisms mediating learning and memory with emotional significance.

  9. Corticosterone suppresses vasotocin-enhanced clasping behavior in male rough-skinned newts by novel mechanisms interfering with V1a receptor availability and receptor-mediated endocytosis.

    PubMed

    Davis, Audrey; Abraham, Emily; McEvoy, Erin; Sonnenfeld, Sarah; Lewis, Christine; Hubbard, Catherine S; Dolence, E Kurt; Rose, James D; Coddington, Emma

    2015-03-01

    In rough-skinned newts, Taricha granulosa, exposure to an acute stressor results in the rapid release of corticosterone (CORT), which suppresses the ability of vasotocin (VT) to enhance clasping behavior. CORT also suppresses VT-induced spontaneous activity and sensory responsiveness of clasp-controlling neurons in the rostromedial reticular formation (Rf). The cellular mechanisms underlying this interaction remain unclear. We hypothesized that CORT blocks VT-enhanced clasping by interfering with V1a receptor availability and/or VT-induced endocytosis. We administered a physiologically active fluorescent VT conjugated to Oregon Green (VT-OG) to the fourth ventricle 9 min after an intraperitoneal injection of CORT (0, 10, 40 μg/0.1mL amphibian Ringers). The brains were collected 30 min post-VT-OG, fixed, and imaged with confocal microscopy. CORT diminished the number of endocytosed vesicles, percent area containing VT-OG, sum intensity of VT-OG, and the amount of VT-V1a within each vesicle; indicating that CORT was interfering with V1a receptor availability and VT-V1a receptor-mediated endocytosis. CORT actions were brain location-specific and season-dependent in a manner that is consistent with the natural and context-dependent expression of clasping behavior. Furthermore, the sensitivity of the Rf to CORT was much higher in animals during the breeding season, arguing for ethologically appropriate seasonal variation in CORT's ability to prevent VT-induced endocytosis. Our data are consistent with the time course and interaction effects of CORT and VT on clasping behavior and neurophysiology. CORT interference with VT-induced endocytosis may be a common mechanism employed by hormones across taxa for mediating rapid context- and season-specific behavioral responses.

  10. CaV3.2 calcium channels control NMDA receptor-mediated transmission: a new mechanism for absence epilepsy.

    PubMed

    Wang, Guangfu; Bochorishvili, Genrieta; Chen, Yucai; Salvati, Kathryn A; Zhang, Peng; Dubel, Steve J; Perez-Reyes, Edward; Snutch, Terrance P; Stornetta, Ruth L; Deisseroth, Karl; Erisir, Alev; Todorovic, Slobodan M; Luo, Jian-Hong; Kapur, Jaideep; Beenhakker, Mark P; Zhu, J Julius

    2015-07-15

    CaV3.2 T-type calcium channels, encoded by CACNA1H, are expressed throughout the brain, yet their general function remains unclear. We discovered that CaV3.2 channels control NMDA-sensitive glutamatergic receptor (NMDA-R)-mediated transmission and subsequent NMDA-R-dependent plasticity of AMPA-R-mediated transmission at rat central synapses. Interestingly, functional CaV3.2 channels primarily incorporate into synapses, replace existing CaV3.2 channels, and can induce local calcium influx to control NMDA transmission strength in an activity-dependent manner. Moreover, human childhood absence epilepsy (CAE)-linked hCaV3.2(C456S) mutant channels have a higher channel open probability, induce more calcium influx, and enhance glutamatergic transmission. Remarkably, cortical expression of hCaV3.2(C456S) channels in rats induces 2- to 4-Hz spike and wave discharges and absence-like epilepsy characteristic of CAE patients, which can be suppressed by AMPA-R and NMDA-R antagonists but not T-type calcium channel antagonists. These results reveal an unexpected role of CaV3.2 channels in regulating NMDA-R-mediated transmission and a novel epileptogenic mechanism for human CAE.

  11. CaV3.2 calcium channels control NMDA receptor-mediated transmission: a new mechanism for absence epilepsy

    PubMed Central

    Wang, Guangfu; Bochorishvili, Genrieta; Chen, Yucai; Salvati, Kathryn A.; Zhang, Peng; Dubel, Steve J.; Perez-Reyes, Edward; Snutch, Terrance P.; Stornetta, Ruth L.; Deisseroth, Karl; Erisir, Alev; Todorovic, Slobodan M.; Luo, Jian-Hong; Kapur, Jaideep; Beenhakker, Mark P.; Zhu, J. Julius

    2015-01-01

    CaV3.2 T-type calcium channels, encoded by CACNA1H, are expressed throughout the brain, yet their general function remains unclear. We discovered that CaV3.2 channels control NMDA-sensitive glutamatergic receptor (NMDA-R)-mediated transmission and subsequent NMDA-R-dependent plasticity of AMPA-R-mediated transmission at rat central synapses. Interestingly, functional CaV3.2 channels primarily incorporate into synapses, replace existing CaV3.2 channels, and can induce local calcium influx to control NMDA transmission strength in an activity-dependent manner. Moreover, human childhood absence epilepsy (CAE)-linked hCaV3.2(C456S) mutant channels have a higher channel open probability, induce more calcium influx, and enhance glutamatergic transmission. Remarkably, cortical expression of hCaV3.2(C456S) channels in rats induces 2- to 4-Hz spike and wave discharges and absence-like epilepsy characteristic of CAE patients, which can be suppressed by AMPA-R and NMDA-R antagonists but not T-type calcium channel antagonists. These results reveal an unexpected role of CaV3.2 channels in regulating NMDA-R-mediated transmission and a novel epileptogenic mechanism for human CAE. PMID:26220996

  12. CB1 cannabinoid receptor-mediated anandamide signaling mechanisms of the inferior colliculus modulate the haloperidol-induced catalepsy.

    PubMed

    Medeiros, P; de Freitas, R L; Silva, M O; Coimbra, N C; Melo-Thomas, L

    2016-11-19

    The inferior colliculus (IC), a midbrain structure that processes acoustic information of aversive nature, is distinguished from other auditory nuclei in the brainstem by its connections with structures of the motor system. Previous evidence relating the IC to motor behavior shows that glutamatergic and GABAergic mechanisms in the IC exert influence on systemic haloperidol-induced catalepsy. There is substantial evidence supporting a role played by the endocannabinoid system as a modulator of the glutamatergic neurotransmission, as well as the dopaminergic activity in the basal nuclei and therefore it may be considered as a potential pharmacological target for the treatment of movement disorders. The present study evaluated if the endocannabinoid system in the IC plays a role in the elaboration of systemic haloperidol-induced catalepsy. Male Wistar rats received intracollicular microinjection of either the endogenous cannabinoid anandamide (AEA) at different concentrations (5, 50 or 100pmol/0.2μl), the CB1 cannabinoid receptor antagonist AM251 at 50, 100 or 200pmol/0.2μl or vehicle, followed by intraperitoneal (IP) administration of either haloperidol at 0.5 or 1mg/kg or physiological saline. Systemic injection of haloperidol at both doses (0.5 or 1mg/kg, IP) produced a cataleptic state, compared to vehicle/physiological saline-treated group, lasting 30 and 50min after systemic administration of the dopaminergic receptors non-selective antagonist. The midbrain microinjection of AEA at 50pmol/0.2μl increased the latency for stepping down from the horizontal bar after systemic administration of haloperidol. Moreover, the intracollicular administration of AEA at 50pmol/0.2μl was able to increase the duration of catalepsy as compared to AEA at 100pmol/0.2-μl-treated group. Intracollicular pretreatment with AM251 at the intermediate concentration (100pmol/0.2μl) was able to decrease the duration of catalepsy after systemic administration of haloperidol. However

  13. Receptor-mediated signaling in Aspergillus fumigatus

    PubMed Central

    Grice, C. M.; Bertuzzi, M.; Bignell, E. M.

    2013-01-01

    Aspergillus fumigatus is the most pathogenic species among the Aspergilli, and the major fungal agent of human pulmonary infection. To prosper in diverse ecological niches, Aspergilli have evolved numerous mechanisms for adaptive gene regulation, some of which are also crucial for mammalian infection. Among the molecules which govern such responses, integral membrane receptors are thought to be the most amenable to therapeutic modulation. This is due to the localization of these molecular sensors at the periphery of the fungal cell, and to the prevalence of small molecules and licensed drugs which target receptor-mediated signaling in higher eukaryotic cells. In this review we highlight the progress made in characterizing receptor-mediated environmental adaptation in A. fumigatus and its relevance for pathogenicity in mammals. By presenting a first genomic survey of integral membrane proteins in this organism, we highlight an abundance of putative seven transmembrane domain (7TMD) receptors, the majority of which remain uncharacterized. Given the dependency of A. fumigatus upon stress adaptation for colonization and infection of mammalian hosts, and the merits of targeting receptor-mediated signaling as an antifungal strategy, a closer scrutiny of sensory perception and signal transduction in this organism is warranted. PMID:23430083

  14. Mechanism of the estrogen receptor interaction with 4-hydroxytamoxifen

    SciTech Connect

    Sasson, S.; Notides, A.C.

    1988-04-01

    The binding mechanism of the estrogen receptor with 4-(/sup 3/H)hydroxytamoxifen was investigated. The equilibrium binding analysis with 4-(/sup 3/H)hydroxytamoxifen indicated a positive cooperative interaction: the Scatchard plot was convex and the Hill coefficient was 1.4-1.5. This binding appears similar to the positively cooperative interaction of the estrogen receptor with (/sup 3/H)estradiol. However, a competitive binding assay with a saturating concentration of (/sup 3/H) estradiol and variable concentrations of 4-hydroxytamoxifen produced nonparallel displacement curves indicating that the binding mechanism of the receptor with these two ligands is different. The competitive binding assay with (/sup 3/H)estradiol and 4-hydroxytamoxifen at constant molar ratios demonstrated that the receptor's affinity for estradiol was reduced and the receptor preferentially bound 4-hydroxytamoxifen. These data suggest that 4-hydroxytamoxifen interacts with the receptor differently than estradiol; it antagonizes the binding of estradiol when these two ligands are simultaneously present.

  15. Receptor-Mediated Tobacco Toxicity

    PubMed Central

    Arredondo, Juan; Chernyavsky, Alexander I.; Marubio, Lisa M.; Beaudet, Arthur L.; Jolkovsky, David L.; Pinkerton, Kent E.; Grando, Sergei A.

    2005-01-01

    Tobacco is a known cause of oral disease but the mechanism remains elusive. Nicotine (Nic) is a likely culprit of pathobiological effects because it displaces the local cytotransmitter acetylcholine from the nicotinic receptors (nAChRs) expressed by oral keratinocytes (KCs). To gain a mechanistic insight into tobacco-induced morbidity in the oral cavity, we studied effects of exposures to environmental tobacco smoke (ETS) versus equivalent concentration of pure Nic on human and murine KCs. Both ETS and Nic up-regulated expression of cell cycle and apoptosis regulators, differentiation marker filaggrin, and signal transduction factors at both the mRNA and protein levels. These changes could be abolished in cultured human oral KCs transfected with anti-α3 small interfering RNA or treated with the α3β2-preferring antagonist α-conotoxin MII. Functional inactivation of α3-mediated signaling in α3−/− mutant KCs prevented most of the ETS/Nic-dependent changes in gene expression. To determine relevance of the in vitro findings to the in vivo situation, we studied gene expression in oral mucosa of neonatal α3+/+ and α3−/− littermates delivered by heterozygous mice soon after their exposures to ETS or equivalent concentration of pure Nic in drinking water. In addition to reverse transcriptase-polymerase chain reaction and Western blot, the ETS/Nic-dependent alterations in gene expression were also detected by semiquantitative immunofluorescence assay directly in KCs comprising murine oral mucosa. Only wild-type mice consistently developed significant (P < 0.05) changes in the gene expression. These results identified α3β2 nAChR as a major receptor mediating effects of tobacco products on KC gene expression. Real-time polymerase chain reaction demonstrated that in all three model systems the common genes targeted by α3β2-mediated ETS/Nic toxicity were p21, Bcl-2, NF-κB, and STAT-1. The expression of the nAChR subunits α5 and β2 and the muscarinic

  16. Nongenomic mechanisms of physiological estrogen-mediated dopamine efflux.

    PubMed

    Alyea, Rebecca A; Watson, Cheryl S

    2009-06-16

    Neurological diseases and neuropsychiatric disorders that vary depending on female life stages suggest that sex hormones may influence the function of neurotransmitter regulatory machinery such as the dopamine transporter (DAT). In this study we tested the rapid nongenomic effects of several physiological estrogens [estradiol (E2), estrone (E1), and estriol (E3)] on dopamine efflux via the DAT in a non-transfected, NGF-differentiated, rat pheochromocytoma (PC12) cell model that expresses membrane estrogen receptors (ERs) alpha, beta, and GPR30. We examined kinase, ionic, and physical interaction mechanisms involved in estrogenic regulation of the DAT function. E2-mediated dopamine efflux is DAT-specific and not dependent on extracellular Ca2+-mediated exocytotic release from vesicular monoamine transporter vesicles (VMATs). Using kinase inhibitors we also showed that E2-mediated dopamine efflux is dependent on protein kinase C and MEK activation, but not on PI3K or protein kinase A. In plasma membrane there are ligand-independent associations of ERalpha and ERbeta (but not GPR30) with DAT. Conditions which cause efflux (a 9 min 10(-9) M E2 treatment) cause trafficking of ERalpha (stimulatory) to the plasma membrane and trafficking of ERbeta (inhibitory) away from the plasma membrane. In contrast, E1 and E3 can inhibit efflux with a nonmonotonic dose pattern, and cause DAT to leave the plasma membrane. Such mechanisms explain how gender biases in some DAT-dependent diseases can occur.

  17. Estrogen replacement and skeletal muscle: mechanisms and population health

    PubMed Central

    Lowe, Dawn A.; Brown, Marybeth

    2013-01-01

    There is a growing body of information supporting the beneficial effects of estrogen and estrogen-based hormone therapy (HT) on maintenance and enhancement of muscle mass, strength, and connective tissue. These effects are also evident in enhanced recovery from muscle atrophy or damage and have significant implications particularly for the muscular health of postmenopausal women. Evidence suggests that HT will also help maintain or increase muscle mass, improve postatrophy muscle recovery, and enhance muscle strength in aged females. This is important because this population, in particular, is at risk for a rapid onset of frailty. The potential benefits of estrogen and HT relative to skeletal muscle function and composition combined with other health-related enhancements associated with reduced risk of cardiovascular events, overall mortality, and metabolic dysfunction, as well as enhanced cognition and bone health cumulate in a strong argument for more widespread and prolonged consideration of HT if started proximal to menopausal onset in most women. Earlier reports of increased health risks with HT use in postmenopausal women has led to a decline in HT use. However, recent reevaluation regarding the health effects of HT indicates a general lack of risks and a number of significant health benefits of HT use when initiated at the onset of menopause. Although further research is still needed to fully delineate its mechanisms of action, the general use of HT by postmenopausal women, to enhance muscle mass and strength, as well as overall health, with initiation soon after the onset of menopause should be considered. PMID:23869062

  18. The Orphan Nuclear Receptor ERRγ Regulates Hepatic CB1 Receptor-Mediated Fibroblast Growth Factor 21 Gene Expression.

    PubMed

    Jung, Yoon Seok; Lee, Ji-Min; Kim, Don-Kyu; Lee, Yong-Soo; Kim, Ki-Sun; Kim, Yong-Hoon; Kim, Jina; Lee, Myung-Shik; Lee, In-Kyu; Kim, Seong Heon; Cho, Sung Jin; Jeong, Won-Il; Lee, Chul-Ho; Harris, Robert A; Choi, Hueng-Sik

    2016-01-01

    Fibroblast growth factor 21 (FGF21), a stress inducible hepatokine, is synthesized in the liver and plays important roles in glucose and lipid metabolism. However, the mechanism of hepatic cannabinoid type 1 (CB1) receptor-mediated induction of FGF21 gene expression is largely unknown. Activation of the hepatic CB1 receptor by arachidonyl-2'-chloroethylamide (ACEA), a CB1 receptor selective agonist, significantly increased FGF21 gene expression. Overexpression of estrogen-related receptor (ERR) γ increased FGF21 gene expression and secretion both in hepatocytes and mice, whereas knockdown of ERRγ decreased ACEA-mediated FGF21 gene expression and secretion. Moreover, ERRγ, but not ERRα and ERRβ, induced FGF21 gene promoter activity. In addition, deletion and mutation analysis of the FGF21 promoter identified a putative ERRγ-binding motif (AGGTGC, a near-consensus response element). A chromatin immunoprecipitation assay revealed direct binding of ERRγ to the FGF21 gene promoter. Finally, GSK5182, an ERRγ inverse agonist, significantly inhibited hepatic CB1 receptor-mediated FGF21 gene expression and secretion. Based on our data, we conclude that ERRγ plays a key role in hepatic CB1 receptor-mediated induction of FGF21 gene expression and secretion.

  19. The Orphan Nuclear Receptor ERRγ Regulates Hepatic CB1 Receptor-Mediated Fibroblast Growth Factor 21 Gene Expression

    PubMed Central

    Jung, Yoon Seok; Lee, Ji-Min; Kim, Don-Kyu; Lee, Yong-Soo; Kim, Ki-Sun; Kim, Yong-Hoon; Kim, Jina; Lee, Myung-Shik; Lee, In-Kyu; Kim, Seong Heon; Cho, Sung Jin; Jeong, Won-Il; Lee, Chul-Ho; Harris, Robert A.; Choi, Hueng-Sik

    2016-01-01

    Background Fibroblast growth factor 21 (FGF21), a stress inducible hepatokine, is synthesized in the liver and plays important roles in glucose and lipid metabolism. However, the mechanism of hepatic cannabinoid type 1 (CB1) receptor-mediated induction of FGF21 gene expression is largely unknown. Results Activation of the hepatic CB1 receptor by arachidonyl-2’-chloroethylamide (ACEA), a CB1 receptor selective agonist, significantly increased FGF21 gene expression. Overexpression of estrogen-related receptor (ERR) γ increased FGF21 gene expression and secretion both in hepatocytes and mice, whereas knockdown of ERRγ decreased ACEA-mediated FGF21 gene expression and secretion. Moreover, ERRγ, but not ERRα and ERRβ, induced FGF21 gene promoter activity. In addition, deletion and mutation analysis of the FGF21 promoter identified a putative ERRγ-binding motif (AGGTGC, a near-consensus response element). A chromatin immunoprecipitation assay revealed direct binding of ERRγ to the FGF21 gene promoter. Finally, GSK5182, an ERRγ inverse agonist, significantly inhibited hepatic CB1 receptor-mediated FGF21 gene expression and secretion. Conclusion Based on our data, we conclude that ERRγ plays a key role in hepatic CB1 receptor-mediated induction of FGF21 gene expression and secretion. PMID:27455076

  20. Chronic restraint stress causes a delayed increase in responding for palatable food cues during forced abstinence via a dopamine D1-like receptor-mediated mechanism.

    PubMed

    Ball, Kevin T; Best, Olivia; Luo, Jonathan; Miller, Leah R

    2017-02-15

    Relapse to unhealthy eating habits in dieters is often triggered by stress. Animal models, moreover, have confirmed a causal role for acute stress in relapse. The role of chronic stress in relapse vulnerability, however, has received relatively little attention. Therefore, in the present study, we used an abstinence-based relapse model in rats to test the hypothesis that exposure to chronic stress increases subsequent relapse vulnerability. Rats were trained to press a lever for highly palatable food reinforcers in daily 3-h sessions and then tested for food seeking (i.e., responding for food associated cues) both before and after an acute or chronic restraint stress procedure (3h/day×1day or 10days, respectively) or control procedure (unstressed). The second food seeking test was conducted either 1day or 7days after the last restraint. Because chronic stress causes dopamine D1-like receptor-mediated alterations in prefrontal cortex (a relapse node), we also assessed dopaminergic involvement by administering either SCH-23390 (10.0μg/kg; i.p.), a dopamine D1-like receptor antagonist, or vehicle prior to daily treatments. Results showed that chronically, but not acutely, stressed rats displayed increased food seeking 7days, but not 1day, after the last restraint. Importantly, SCH-23390 combined with chronic stress reversed this effect. These results suggest that drugs targeting D1-like receptors during chronic stress may help to prevent future relapse in dieters.

  1. Mechanisms of estrogens' dose-dependent neuroprotective and neurodamaging effects in experimental models of cerebral ischemia.

    PubMed

    Strom, Jakob O; Theodorsson, Annette; Theodorsson, Elvar

    2011-01-01

    Ever since the hypothesis was put forward that estrogens could protect against cerebral ischemia, numerous studies have investigated the mechanisms of their effects. Despite initial studies showing ameliorating effects, later trials in both humans and animals have yielded contrasting results regarding the fundamental issue of whether estrogens are neuroprotective or neurodamaging. Therefore, investigations of the possible mechanisms of estrogen actions in brain ischemia have been difficult to assess. A recently published systematic review from our laboratory indicates that the dichotomy in experimental rat studies may be caused by the use of insufficiently validated estrogen administration methods resulting in serum hormone concentrations far from those intended, and that physiological estrogen concentrations are neuroprotective while supraphysiological concentrations augment the damage from cerebral ischemia. This evidence offers a new perspective on the mechanisms of estrogens' actions in cerebral ischemia, and also has a direct bearing on the hormone replacement therapy debate. Estrogens affect their target organs by several different pathways and receptors, and the mechanisms proposed for their effects on stroke probably prevail in different concentration ranges. In the current article, previously suggested neuroprotective and neurodamaging mechanisms are reviewed in a hormone concentration perspective in an effort to provide a mechanistic framework for the dose-dependent paradoxical effects of estrogens in stroke. It is concluded that five protective mechanisms, namely decreased apoptosis, growth factor regulation, vascular modulation, indirect antioxidant properties and decreased inflammation, and the proposed damaging mechanism of increased inflammation, are currently supported by experiments performed in optimal biological settings.

  2. Mechanism of inhibition of estrogen biosynthesis by azole fungicides.

    PubMed

    Egbuta, Chinaza; Lo, Jessica; Ghosh, Debashis

    2014-12-01

    Biosynthesis of estrogens from androgens is catalyzed by cytochrome P450 aromatase. Aromatase inhibition by the triazole compounds letrozole (LTZ) and anastrozole is a prevalent therapy for estrogen-dependent postmenopausal breast cancer. Azoles are widely used as agricultural fungicides and antimycotic drugs that target 14α-demethylase. Some were previously shown to inhibit aromatase, thereby raising the possibility of endocrine disruptive effects. However, mechanistic analysis of their inhibition has never been undertaken. We have evaluated the inhibitory effects of 3 common fungicides, bifonazole, imazalil, and flusilazole, in human aromatase purified from placenta and compared them with LTZ, the most potent inhibitor of aromatase. Bifonazole exhibits strong inhibitory effects with an IC50 of 270nM and Ki (Michaeles-Menten inhibition constant) of 68nM, compared with 10nM and 13nM, respectively, for LTZ. The IC50 and Ki are 1100nM and 278nM for imazilil and 3200nM and 547nM for flusilazole, respectively. Analyses of inhibition kinetics suggest that the modes of inhibition by azole fungicides are mixed or competitive, whereas LTZ inhibition could be noncompetitive or mixed. We interpret the inhibition mechanism in the context of the x-ray structure of aromatase-androstenedione complex. Structural data show that aromatase has 3 binding pockets in relation to the heme. The substrate-binding cavity at the heme-distal site closely compliments the structures of the natural substrate, androstenedione, and steroidal aromatase inhibitors. Because the structures of LTZ and the azole fungicides are entirely dissimilar to the androstenedione backbone, the azoles possibly inhibit by binding to a structurally rearranged active site, the 2 other catalytically important sites, or both, in agreement with the kinetics data.

  3. Rapid effects of estrogens on behavior: environmental modulation and molecular mechanisms.

    PubMed

    Laredo, Sarah A; Villalon Landeros, Rosalina; Trainor, Brian C

    2014-10-01

    Estradiol can modulate neural activity and behavior via both genomic and nongenomic mechanisms. Environmental cues have a major impact on the relative importance of these signaling pathways with significant consequences for behavior. First we consider how photoperiod modulates nongenomic estrogen signaling on behavior. Intriguingly, short days permit rapid effects of estrogens on aggression in both rodents and song sparrows. This highlights the importance of considering photoperiod as a variable in laboratory research. Next we review evidence for rapid effects of estradiol on ecologically-relevant behaviors including aggression, copulation, communication, and learning. We also address the impact of endocrine disruptors on estrogen signaling, such as those found in corncob bedding used in rodent research. Finally, we examine the biochemical mechanisms that may mediate rapid estrogen action on behavior in males and females. A common theme across these topics is that the effects of estrogens on social behaviors vary across different environmental conditions.

  4. Rapid effects of estrogens on behavior: environmental modulation and molecular mechanisms

    PubMed Central

    Laredo, Sarah A.; Landeros, Rosalina Villalon; Trainor, Brian C.

    2014-01-01

    Estradiol can modulate neural activity and behavior via both genomic and nongenomic mechanisms. Environmental cues have a major impact on the relative importance of these signaling pathways with significant consequences for behavior. First we consider how photoperiod modulates nongenomic estrogen signaling on behavior. Intriguingly, short days permit rapid effects of estrogens on aggression in both rodents and song sparrows. This highlights the importance of considering photoperiod as a variable in laboratory research. Next we review evidence for rapid effects of estradiol on ecologically-relevant behaviors including aggression, copulation, communication, and learning. We also address the impact of endocrine disruptors on estrogen signaling, such as those found in corncob bedding used in rodent research. Finally, we examine the biochemical mechanisms that may mediate rapid estrogen action on behavior in males and females. A common theme across these topics is that the effects of estrogens on social behaviors vary across different environmental conditions. PMID:24685383

  5. Sources, mechanisms, and fate of steroid estrogens in wastewater treatment plants: a mini review.

    PubMed

    Ting, Yien Fang; Praveena, Sarva Mangala

    2017-04-01

    Steroid estrogens, such as estrone (E1), 17β-estradiol (E2), estriol (E3), and 17α-ethinylestradiol (EE2), are natural and synthetic hormones released into the environment through incomplete sewage discharge. This review focuses on the sources of steroid estrogens in wastewater treatment plants (WWTPs). The mechanisms and fate of steroid estrogens throughout the entire wastewater treatment system are also discussed, and relevant information on regulatory aspects is given. Municipal, pharmaceutical industry, and hospitals are the main sources of steroid estrogens that enter WWTPs. A typical WWTP comprises primary, secondary, and tertiary treatment units. Sorption and biodegradation are the main mechanisms for removal of steroid estrogens from WWTPs. The fate of steroid estrogens in WWTPs depends on the types of wastewater treatment systems. Steroid estrogens in the primary treatment unit are removed by sorption onto primary sludge, followed by sorption onto micro-flocs and biodegradation by microbes in the secondary treatment unit. Tertiary treatment employs nitrification, chlorination, or UV disinfection to improve the quality of the secondary effluent. Activated sludge treatment systems for steroid estrogens exhibit a removal efficiency of up to 100%, which is higher than that of the trickling filter treatment system (up to 75%). Moreover, the removal efficiency of advance treatment systems exceeds 90%. Regulatory aspects related to steroid estrogens are established, especially in the European Union. Japan is the only Asian country that implements a screening program and is actively involved in endocrine disruptor testing and assessment. This review improves our understanding of steroid estrogens in WWTPs, proposes main areas to be improved, and provides current knowledge on steroid estrogens in WWTPs for sustainable development.

  6. A novel mechanism of non-feminizing estrogens in neuroprotection.

    PubMed

    Engler-Chiurazzi, Elizabeth B; Covey, Douglas F; Simpkins, James W

    2016-11-03

    Estrogens are potent and efficacious neuroprotectants both in vitro and in vivo in a variety of models of neurotoxicity. We determined the structural requirements for neuroprotection in an in vitro assay using a panel of >70 novel estratrienes, synthesized to reduce or eliminate estrogen receptor (ER) binding. We observed that neuroprotection could be enhanced by as much as 200-fold through modifications that positioned a large bulky group at the C2 or C4 position of the phenolic A ring of the estratriene. Further, substitutions on the B, C or D rings either reduced or did not markedly change neuroprotection. Collectively, there was a negative correlation between binding to ERs and neuroprotection with the more potent compounds showing no ER binding. In an in vivo model for neuroprotection, transient cerebral ischemia, efficacious compounds were active in protection of brain tissue from this pro-oxidant insult. We demonstrated that these non-feminizing estrogens engage in a redox cycle with glutathione, using the hexose monophosphate shunt to apply cytosolic reducing potential to cellular membranes. Together, these results demonstrate that non-feminizing estrogens are neuroprotective and protect brain from the induction of ischemic- and Alzheimer's disease (AD)-like neuropathology in an animal model. These features of non-feminizing estrogens make them attractive compounds for assessment of efficacy in AD and stroke, as they are not expected to show the side effects of chronic estrogen therapy that are mediated by ER actions in the liver, uterus and breast.

  7. Spinal 5-HT1A, not the 5-HT1B or 5-HT3 receptors, mediates descending serotonergic inhibition for late-phase mechanical allodynia of carrageenan-induced peripheral inflammation.

    PubMed

    Kim, Joung Min; Jeong, Seong Wook; Yang, Jihoon; Lee, Seong Heon; Kim, Woon Mo; Jeong, Seongtae; Bae, Hong Beom; Yoon, Myung Ha; Choi, Jeong Il

    2015-07-23

    Previous electrophysiological studies demonstrated a limited role of 5-hydroxytryptamine 3 receptor (5-HT3R), but facilitatory role of 5-HT1AR and 5-HT1BR in spinal nociceptive processing of carrageenan-induced inflammatory pain. The release of spinal 5-HT was shown to peak in early-phase and return to baseline in late-phase of carrageenan inflammation. We examined the role of the descending serotonergic projections involving 5-HT1AR, 5-HT1BR, and 5-HT3R in mechanical allodynia of early- (first 4h) and late-phase (24h after) carrageenan-induced inflammation. Intrathecal administration of 5-HT produced a significant anti-allodynic effect in late-phase, but not in early-phase. Similarly, intrathecal 5-HT1AR agonist (8-OH-DPAT) attenuated the intensity of late-phase allodynia in a dose dependent fashion which was antagonized by 5-HT1AR antagonist (WAY-100635), but produced no effect on the early-phase allodynia. However, other agonists or antagonists of 5-HT1BR (CP-93129, SB-224289) and 5-HT3R (m-CPBG, ondansetron) did not produce any anti- or pro-allodynic effect in both early- and late- phase allodynia. These results suggest that spinal 5-HT1A, but not 5-HT1B or 5-HT3 receptors mediate descending serotonergic inhibition on nociceptive processing of late-phase mechanical allodynia in carrageenan-induced inflammation.

  8. High-density lipoprotein inhibits mechanical stress-induced cardiomyocyte autophagy and cardiac hypertrophy through angiotensin II type 1 receptor-mediated PI3K/Akt pathway.

    PubMed

    Lin, Li; Liu, Xuebo; Xu, Jianfeng; Weng, Liqing; Ren, Jun; Ge, Junbo; Zou, Yunzeng

    2015-08-01

    Mechanical stress triggers cardiac hypertrophy and autophagy through an angiotensin II (Ang II) type 1 (AT1) receptor-dependent mechanism. Low level of high density lipoprotein (HDL) is an independent risk factor for cardiac hypertrophy. This study was designed to evaluate the effect of HDL on mechanical stress-induced cardiac hypertrophy and autophagy. A 48-hr mechanical stretch and a 4-week transverse aortic constriction were employed to induce cardiomyocyte hypertrophy in vitro and in vivo, respectively, prior to the assessment of myocardial autophagy using LC3b-II and beclin-1. Our results indicated that HDL significantly reduced mechanical stretch-induced rise in autophagy as demonstrated by LC3b-II and beclin-1. In addition, mechanical stress up-regulated AT1 receptor expression in both cultured cardiomyocytes and in mouse hearts, whereas HDL significantly suppressed the AT1 receptor. Furthermore, the role of Akt phosphorylation in HDL-mediated action was assessed using MK-2206, a selective inhibitor for Akt phosphorylation. Our data further revealed that MK-2206 mitigated HDL-induced beneficial responses on cardiac remodelling and autophagy. Taken together, our data revealed that HDL inhibited mechanical stress-induced cardiac hypertrophy and autophagy through downregulation of AT1 receptor, and HDL ameliorated cardiac hypertrophy and autophagy via Akt-dependent mechanism. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  9. Estradiol-induced modulation of estrogen receptor-beta and GABA within the adult neocortex: a potential transsynaptic mechanism for estrogen modulation of BDNF.

    PubMed

    Blurton-Jones, Mathew; Tuszynski, Mark H

    2006-12-01

    Estrogen influences brain-derived neurotrophic factor (BDNF) expression in the neocortex. However, BDNF-producing cortical neurons do not express detectable levels of nuclear estrogen receptors; instead, the most abundant cortical nuclear estrogen receptor, ER-beta, is present in GABAergic neurons, prompting us to test the hypothesis that estrogen effects on BDNF are mediated via cortical inhibitory interneurons. Adult female ovariectomized rats were provided acute estrogen replacement and the number of cortical GABA, ER-beta, and ER-beta/GABA double-labeled neurons was examined. Within 48 hours of injection of 17-beta-estradiol, the number of perirhinal neurons double-labeled for ER-beta/GABA was reduced by 28% (P<0.01 compared to vehicle-treated ovariectomized controls), and all cells expressing detectable levels of GABA were reduced by 19% (P<0.01). To investigate potential relationships between estrogen receptors, GABAergic neurons, and BDNF-expressing cells, brain sections were double- or triple-labeled for ER-beta, GABAergic, and BDNF immunomarkers. The findings indicated that ER-beta-bearing inhibitory neurons project onto other GABAergic neurons that lack nuclear estrogen receptors; these inhibitory neurons in turn innervate BDNF-expressing excitatory cells. High estrogen states reduce cortical GABA levels, presumably releasing inhibition on BDNF-expressing neurons. This identifies a putative two-step transsynaptic mechanism whereby estrogen availability modulates expression of inhibitory transmitters, resulting in increased BDNF expression.

  10. Receptors mediating toxicity and their involvement in endocrine disruption.

    PubMed

    Rüegg, Joëlle; Penttinen-Damdimopoulou, Pauliina; Mäkelä, Sari; Pongratz, Ingemar; Gustafsson, Jan-Ake

    2009-01-01

    Many toxic compounds exert their harmful effects by activating of certain receptors, which in turn leads to dysregulation of transcription. Some of these receptors are so called xenosensors. They are activated by external chemicals and evoke a cascade of events that lead to the elimination of the chemical from the system. Other receptors that are modulated by toxic substances are hormone receptors, particularly the ones of the nuclear receptor family. Some environmental chemicals resemble endogenous hormones and can falsely activate these receptors, leading to undesired activity in the cell. Furthermore, excessive activation of the xenosensors can lead to disturbances of the integrity of the system as well. In this chapter, the concepts of receptor-mediated toxicity and hormone disruption are introduced. We start by describing environmental chemicals that can bind to xenosensors and nuclear hormone receptors. We then describe the receptors most commonly targeted by environmental chemicals. Finally, the mechanisms by which receptor-mediated events can disrupt the system are depicted.

  11. RDX binds to the GABA(A) receptor-convulsant site and blocks GABA(A) receptor-mediated currents in the amygdala: a mechanism for RDX-induced seizures.

    PubMed

    Williams, Larry R; Aroniadou-Anderjaska, Vassiliki; Qashu, Felicia; Finne, Huckelberry; Pidoplichko, Volodymyr; Bannon, Desmond I; Braga, Maria F M

    2011-03-01

    Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high-energy, trinitrated cyclic compound that has been used worldwide since World War II as an explosive in both military and civilian applications. RDX can be released in the environment by way of waste streams generated during the manufacture, use, and disposal of RDX-containing munitions and can leach into groundwater from unexploded munitions found on training ranges. For > 60 years, it has been known that exposure to high doses of RDX causes generalized seizures, but the mechanism has remained unknown. We investigated the mechanism by which RDX induces seizures. By screening the affinity of RDX for a number of neurotransmitter receptors, we found that RDX binds exclusively to the picrotoxin convulsant site of the γ-aminobutyric acid type A (GABA(A)) ionophore. Whole-cell in vitro recordings in the rat basolateral amygdala (BLA) showed that RDX reduces the frequency and amplitude of spontaneous GABA(A) receptor-mediated inhibitory postsynaptic currents and the amplitude of GABA-evoked postsynaptic currents. In extracellular field recordings from the BLA, RDX induced prolonged, seizure-like neuronal discharges. These results suggest that binding to the GABA(A) receptor convulsant site is the primary mechanism of seizure induction by RDX and that reduction of GABAergic inhibitory transmission in the amygdala is involved in the generation of RDX-induced seizures. Knowledge of the molecular site and the mechanism of RDX action with respect to seizure induction can guide therapeutic strategies, allow more accurate development of safe thresholds for exposures, and help prevent the development of new explosives or other munitions that could pose similar health risks.

  12. NMDA receptor mediated phosphorylation of GluR1 subunits contributes to the appearance of calcium-permeable AMPA receptors after mechanical stretch injury

    PubMed Central

    Spaethling, Jennifer; Le, Linda; Meaney, David F

    2016-01-01

    Alterations in neuronal cytosolic calcium is a key mediator of the traumatic brain injury (TBI) pathobiology, but less is known of the role and source of calcium in shaping early changes in synaptic receptors and neural circuits after TBI. In this study, we examined the calcium source and potential phosphorylation events leading to insertion of calcium-permeable AMPARs (CP-AMPARs) after in vitro traumatic brain injury, a receptor subtype that influences neural circuit dynamics for hours to days following injury. We found that both synaptic and NR2B-containing NMDARs contribute significantly to the calcium influx following stretch injury. Moreover, an early and sustained phosphorylation of the S-831 site of the GluR1 subunit appeared after mechanical injury, and this phosphorylation was blocked with the inhibition of either synaptic NMDARs or NR2B-containing NMDARs. In comparison, mechanical injury led to no significant change in the S-845 phosphorylation of the GluR1 subunit. Although no change in S-845 phosphorylation appeared in injured cultures, we observed that inhibition of NR2B-containing NMDARs significantly increased S-845 phosphorylation one hour after injury while blockade of synaptic NMDARs did not change S-845 phosphorylation at any time point following injury. These findings show that a broad class of NMDARs are activated in parallel and that targeting either subpopulation will reverse some of the consequences of mechanical injury, providing distinct paths to treat the effects of mechanical injury on neural circuits after TBI. PMID:22426393

  13. Importance of CRF receptor-mediated mechanisms of the bed nucleus of the stria terminalis in the processing of anxiety and pain.

    PubMed

    Tran, Lee; Schulkin, Jay; Greenwood-Van Meerveld, Beverley

    2014-10-01

    Corticotropin-releasing factor (CRF)-mediated mechanisms in the bed nucleus of the stria terminalis (BNST) have a pivotal role in stress-induced anxiety and hyperalgesia. Although CRF is known to activate two receptor subtypes, CRF1 and CRF2, attempts to delineate the specific role of each subtype in modulating anxiety and nociception have been inconsistent. Here we test the hypothesis that CRF1 and CRF2 receptor activation in the anteriolateral BNST (BNSTAL) facilitates divergent mechanisms modulating comorbid anxiety and hyperalgesia. Microinfusions of the specific antagonists CP376395 and Astressin2B into the BNSTAL were used to investigate CRF1 and CRF2 receptor functions, respectively. We found that CRF1 and CRF2 receptors in the BNSTAL had opposing effects on exploratory behavior in the elevated plus-maze, somatic mechanical threshold, and the autonomic and endocrine response to stress. However, CRF1 or CRF2 receptor antagonism in the BNSTAL revealed complementary roles in facilitating the acoustic startle and visceromotor reflexes. Our results suggest that the net effect of CRF1 and CRF2 receptor activation in the BNSTAL is pathway-dependent and provides important insight into the CRF receptor-associated circuitry that likely underpins stress-induced pathologies.

  14. ATP sensitivity of preBötzinger complex neurones in neonatal rat in vitro: mechanism underlying a P2 receptor-mediated increase in inspiratory frequency

    PubMed Central

    Lorier, A R; Lipski, J; Housley, G D; Greer, J J; Funk, G D

    2008-01-01

    P2 receptor (R) signalling plays an important role in the central ventilatory response to hypoxia. The frequency increase that results from activation of P2Y1Rs in the preBötzinger complex (preBötC; putative site of inspiratory rhythm generation) may contribute, but neither the cellular nor ionic mechanism(s) underlying these effects are known. We applied whole-cell recording to rhythmically-active medullary slices from neonatal rat to define, in preBötC neurones, the candidate cellular and ionic mechanisms through which ATP influences rhythm, and tested the hypothesis that putative rhythmogenic preBötC neurones are uniquely sensitive to ATP. ATP (1 mm) evoked inward currents in all non-respiratory neurones and the majority of respiratory neurons, which included inspiratory, expiratory and putative rhythmogenic inspiratory neurones identified by sensitivity to substance P (1 μm) and DAMGO (50 μm) or by voltage-dependent pacemaker-like activity. ATP current densities were similar in all classes of preBötC respiratory neurone. Reversal potentials and input resistance changes for ATP currents in respiratory neurones suggested they resulted from either inhibition of a K+ channel or activation of a mixed cationic conductance. The P2YR agonist 2MeSADP (1 mm) evoked only the latter type of current in inspiratory and pacemaker-like neurones. In summary, putative rhythmogenic preBötC neurones were sensitive to ATP. However, this sensitivity was not unique; ATP evoked similar currents in all types of preBötC respiratory neurone. The P2Y1R-mediated frequency increase is therefore more likely to reflect activation of a mixed cationic conductance in multiple types of preBötC neurone than excitation of one, highly sensitive group. PMID:18174215

  15. Gateways for the intracellular access of nanocarriers: a review of receptor-mediated endocytosis mechanisms and of strategies in receptor targeting.

    PubMed

    Zaki, Noha M; Tirelli, Nicola

    2010-08-01

    The last 10 years have seen a dramatic growth in understanding and controlling how complex, drug-loaded (nano)structures, as well as pathogens, or biopharmaceuticals can gather access to the cytoplasm, which is a key step to increasing the effectiveness of their action. The review offers an updated overview of the current knowledge of endocytic processes; furthermore, the cell surface receptors most commonly used in drug delivery are here discussed on the basis of their reported internalization mechanisms, with examples of their use as nanocarrier targets taken from the most recent scientific literature. Knowledge of molecular biology details is increasingly necessary for a rational design of drug delivery systems. Here, the aim is to provide the reader with an attempt to link a mechanistic knowledge of endocytic mechanisms with the identification of appropriate targets (internalization receptors) for nanocarriers. Much advance is still needed to create a complete and coherent biological picture of endocytosis, but current knowledge already allows individuation of a good number of targetable groups for a predetermined intracellular fate of nanocarriers.

  16. Signal Transduction Mechanism for Serotonin 5-HT2B Receptor-Mediated DNA Synthesis and Proliferation in Primary Cultures of Adult Rat Hepatocytes.

    PubMed

    Naito, Kota; Tanaka, Chizuru; Mitsuhashi, Manami; Moteki, Hajime; Kimura, Mitsutoshi; Natsume, Hideshi; Ogihara, Masahiko

    2016-01-01

    The involvement of serotonin (5-hydroxytryptamine; 5-HT) and the 5-HT2 receptor subtypes in the induction of DNA synthesis and proliferation was investigated in primary cultures of adult rat hepatocytes to elucidate the intracellular signal transduction mechanisms. Hepatocyte parenchymal cells maintained in a serum-free, defined medium, synthesized DNA and proliferated in the presence of 5-HT or a selective 5-HT2B receptor agonist, BW723C86, but not in the presence of 5-HT2A, or 5-HT2C receptor agonists (TCB-2 and CP809101, respectively), in a time- and dose-dependent manner. A selective 5-HT2B receptor antagonist, LY272015 (10(-7) M), and a specific phospholipase C (PLC) inhibitor, U-73122 (10(-6) M), as well as specific inhibitors of growth-related signal transducers-including AG1478, LY294002, PD98059, and rapamycin-completely inhibited 5-HT (10(-6) M)- or BW723C86 (10(-6) M)-induced hepatocyte DNA synthesis and proliferation. Both 5-HT and BW723C86 were shown to significantly stimulate the phosphorylation of epidermal growth factor (EGF)/transforming growth factor (TGF)-α receptor tyrosine kinase (p175 kDa) and extracellular signal-regulated kinase (ERK) 2 on Western blot analysis. These results suggest that the proliferative mechanism of activating 5-HT is mediated mainly through 5-HT2B receptor-stimulated Gq/PLC and EGF/TGF-α-receptor/phosphatidylinositol 3-kinase (PI3K)/ERK2/mammalian target of rapamycin (mTOR) signaling pathways in primary cultured hepatocytes.

  17. Distinct mechanisms of endocrine disruption of DDT-related pesticides toward estrogen receptor α and estrogen-related receptor γ.

    PubMed

    Zhuang, Shulin; Zhang, Jing; Wen, Yuezhong; Zhang, Chunlong; Liu, Weiping

    2012-11-01

    Dichlorodiphenyltrichloroethane (DDT) is ubiquitous in the environment, and the exposure to DDT and its related pesticides has long been linked to endocrine disruption. The mechanism of endocrine disruption toward targeted receptors, however, remains unclear. Probing the molecular recognition of DDT analogs by targeted receptors at the atomic level is critical for deciphering this mechanism. Molecular dynamics (MD) simulations were applied to probe the molecular recognition process of DDT and its five analogs, including dichlordiphenyldichloroethylene (DDE), dichlorodiphenyldichloroethane (DDD), methoxychlor (MXC), p,p'-hydroxy-DDT (HPTE), and dicofol by human estrogen receptor (ER) α and human ER-related receptor (ERR) γ. Van der Waals interactions mainly drive the interactions of DDT analogs with ERα ligand-binding domain (LBD) and ERRγ LBD. Minor structural changes of DDT analogs in the number and position of chlorine and phenolic hydroxyl moiety cause differences in binding modes through aromatic stacking and hydrogen bonding and thus affect differently conformational changes of ERα LBD and ERRγ LBD. The binding of DDT analogs affects the helix 12 orientation of ERα LBD but causes no rearrangement of helix 12 of ERRγ LBD. These results extend our understanding of how DDT analogs exert their estrogen-disrupting effects toward different receptors via multiple mechanisms.

  18. Fc-receptor-mediated intracellular delivery of Cu/Zn-superoxide dismutase (SOD1) protects against redox-induced apoptosis through a nitric oxide dependent mechanism.

    PubMed Central

    Vouldoukis, I.; Sivan, V.; Vozenin, M. C.; Kamaté, C.; Calenda, A.; Mazier, D.; Dugas, B.

    2000-01-01

    apoptosis and promoted the intracellular antioxidant armature. CONCLUSIONS: To define the pharmacologic mechanism of action of bovine SOD1, we attempted to identify the second messengers that are induced by SOD1 IC. In this work, we propose that Fc-mediated intracellular delivery of the SOD1 that reduced the production of superoxide anion and of peroxynitrite, promoted a NO-induced protective effect in inducing the antioxidant armature of the cells. Taken together, these data suggested that specific immune responses against antigenic SOD1 could promote the pharmacological properties of the antioxidant enzyme likely via a NO-dependent mechanism. PMID:11474120

  19. Mediator mechanisms involved in TRPV1, TRPA1 and P2X receptor-mediated sensory transduction of pulmonary ROS by vagal lung C-fibers in rats.

    PubMed

    Lin, Yu-Jung; Hsu, Hsao-Hsun; Ruan, Ting; Kou, Yu Ru

    2013-10-01

    We investigated the mediator mechanisms involved in the sensory transduction of pulmonary reactive oxygen species (ROS) by vagal lung C-fibers in anesthetized rats. Airway challenge of aerosolized H₂O₂ (0.4%) stimulated these afferent fibers. The H₂O₂-induced responses were reduced by a cyclooxygenase inhibitor or ATP scavengers and also attenuated by an antagonist of TRPV1, TRPA1 or P2X receptors. The suppressive effect of the cyclooxygenase inhibitor was not affected by a combined treatment with the TRPV1 or TRPA1 antagonist, but was amplified by a combined treatment with the P2X antagonists. The suppressive effect of ATP scavengers was not affected by a combined treatment with the P2X antagonist, but was amplified by a combined treatment with the TRPV1 or TRPA1 antagonist. Thus, the actions of cyclooxygenase metabolites are mediated through the functioning of the TRPV1 and TRPA1 receptors, whereas the action of ATP is mediated through the functioning of P2X receptors.

  20. P2X(7) receptor-mediated release of cathepsins from macrophages is a cytokine-independent mechanism potentially involved in joint diseases.

    PubMed

    Lopez-Castejon, Gloria; Theaker, Jill; Pelegrin, Pablo; Clifton, Andrew D; Braddock, Martin; Surprenant, Annmarie

    2010-08-15

    The ATP-gated P2X(7) receptor (P2X(7)R) is a promising therapeutic target in chronic inflammatory diseases with highly specific antagonists currently under clinical trials for rheumatoid arthritis. Anti-inflammatory actions of P2X(7)R antagonists are considered to result from inhibition of P2X(7)R-induced release of proinflammatory cytokines from activated macrophages. However, P2X(7)Rs are also expressed in resting macrophages, suggesting that P2X(7)R may also signal via cytokine-independent mechanisms involved in joint disease. In this study, we examined P2X(7)R function in resting human lung macrophages and mouse bone marrow-derived macrophages and found that ATP induced rapid release of the lysosomal cysteine proteases cathepsin B, K, L, and S and that was independent of the presence of the proinflammatory cytokines IL-1beta and IL-18. Cathepsins released into the medium were effective to degrade collagen extracellular matrix. ATP-induced cathepsin release was abolished by P2X(7)R antagonists, absent from P2X(7)R(-/-) mouse macrophages, and not associated with cell death. Our results suggest P2X(7)R activation may play a novel and direct role in tissue damage through release of cathepsins independently of its proinflammatory actions via IL-1 cytokines.

  1. Drosophila Lipophorin Receptors Mediate the Uptake of Neutral Lipids in Oocytes and Imaginal Disc Cells by an Endocytosis-Independent Mechanism

    PubMed Central

    Parra-Peralbo, Esmeralda; Culi, Joaquim

    2011-01-01

    Lipids are constantly shuttled through the body to redistribute energy and metabolites between sites of absorption, storage, and catabolism in a complex homeostatic equilibrium. In Drosophila, lipids are transported through the hemolymph in the form of lipoprotein particles, known as lipophorins. The mechanisms by which cells interact with circulating lipophorins and acquire their lipidic cargo are poorly understood. We have found that lipophorin receptor 1 and 2 (lpr1 and lpr2), two partially redundant genes belonging to the Low Density Lipoprotein Receptor (LDLR) family, are essential for the efficient uptake and accumulation of neutral lipids by oocytes and cells of the imaginal discs. Females lacking the lpr2 gene lay eggs with low lipid content and have reduced fertility, revealing a central role for lpr2 in mediating Drosophila vitellogenesis. lpr1 and lpr2 are transcribed into multiple isoforms. Interestingly, only a subset of these isoforms containing a particular LDLR type A module mediate neutral lipid uptake. Expression of these isoforms induces the extracellular stabilization of lipophorins. Furthermore, our data indicate that endocytosis of the lipophorin receptors is not required to mediate the uptake of neutral lipids. These findings suggest a model where lipophorin receptors promote the extracellular lipolysis of lipophorins. This model is reminiscent of the lipolytic processing of triglyceride-rich lipoproteins that occurs at the mammalian capillary endothelium, suggesting an ancient role for LDLR–like proteins in this process. PMID:21347279

  2. Nanomolar concentrations of nicotine and cotinine alter the development of cultured hippocampal neurons via non-acetylcholine receptor-mediated mechanisms.

    PubMed

    Audesirk, T; Cabell, L

    1999-08-01

    We investigated the effects of nicotine and its metabolic byproduct cotinine on survival, differentiation and intracellular Ca2+ levels of cultured E18 rat hippocampal neurons. We used a range of concentrations from 1 nM to 10 microM, most of which are within the likely range of human fetal exposure from maternal smoking. Nicotine did not influence neuron survival or neurite production. However, at all concentrations tested, nicotine significantly increased branching of both axons and dendrites, an effect which was not reversed by co-culturing with alpha-bungarotoxin, which blocks the nicotinic acetylcholine receptors that predominate in hippocampal cultures (Alkondon and Albuquerque, 1993; Barrantes et al., 1995b). Cotinine at 100 nM and 1 microM significantly reduced neuron survival and neurite production of surviving neurons, but did not significantly alter axon or dendrite branching. These membrane-permeable compounds may work synergistically in the developing embryo to impair the survival and differentiation of hippocampal neurons via intracellular mechanisms.

  3. Maslinic Acid, a Natural Triterpene, Induces a Death Receptor-Mediated Apoptotic Mechanism in Caco-2 p53-Deficient Colon Adenocarcinoma Cells

    PubMed Central

    Reyes-Zurita, Fernando J.; Rufino-Palomares, Eva E.; García-Salguero, Leticia; Peragón, Juan; Medina, Pedro P.; Parra, Andrés; Cascante, Marta; Lupiáñez, José A.

    2016-01-01

    Maslinic acid (MA) is a natural triterpene present in high concentrations in the waxy skin of olives. We have previously reported that MA induces apoptotic cell death via the mitochondrial apoptotic pathway in HT29 colon cancer cells. Here, we show that MA induces apoptosis in Caco-2 colon cancer cells via the extrinsic apoptotic pathway in a dose-dependent manner. MA triggered a series of effects associated with apoptosis, including the cleavage of caspases -8 and -3, and increased the levels of t-Bid within a few hours of its addition to the culture medium. MA had no effect on the expression of the Bax protein, release of cytochrome-c or on the mitochondrial membrane potential. This suggests that MA triggered the extrinsic apoptotic pathway in this cell type, as opposed to the intrinsic pathway found in the HT29 colon-cancer cell line. Our results suggest that the apoptotic mechanism induced in Caco-2 may be different from that found in HT29 colon-cancer cells, and that in Caco-2 cells MA seems to work independently of p53. Natural antitumoral agents capable of activating both the extrinsic and intrinsic apoptotic pathways could be of great use in treating colon-cancer of whatever origin. PMID:26751572

  4. Prostaglandin E2 Regulates Its Own Inactivating Enzyme, 15-PGDH, by EP2 Receptor-Mediated Cervical Cell-Specific Mechanisms

    PubMed Central

    Kishore, A. Hari; Owens, David

    2014-01-01

    Context: Prostaglandins play important roles in parturition and have been used to induce cervical ripening and labor. Prior to cervical ripening at term, 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is highly expressed in the cervix and metabolizes cyclooxygenase-2-mediated increases in active prostaglandin E2 (PGE2) to inactive 15-keto PGE2. At term, 15-PGDH gene expression decreases and PGE2 accumulates, leading to cervical ripening and labor. Previously, we found that the cervical isoform of microphthalmia-associated transcription factor (MiTF-CX) serves as a progestational transcription factor that represses IL-8 and hypoxia-mediated increases in cyclooxygenase-2. Objective: We tested the hypothesis that PGE2 regulates its own inactivation through MiTF-CX. Design: We used human cervical stromal cells to investigate the regulation of 15-PGDH. Setting: This was a laboratory-based study using cells from clinical tissue samples. Main Outcome Measures: We evaluated the mechanisms by which PGE2 regulates 15-PGDH in human cervical stromal cells. Results: PGE2 repressed MiTF-CX and 15-PGDH, whereas ectopic overexpression of MiTF-CX induced 15-PGDH expression levels. Stabilization of HIF-1α by deferoxamine resulted in concomitant down-regulation of MiTF-CX and 15-PGDH. Ectopic overexpression of MiTF-CX abrogated PGE2- and deferoxamine-mediated loss of MiTF-CX and 15-PGDH. PGE2-induced loss of MiTF-CX and 15-PGDH was mediated through prostaglandin E2 receptor (EP2) receptors (PTGER2), but not cAMP. Conclusions: The 15-PGDH gene is a MiTF-CX target gene in cervical stromal cells and is down-regulated by PGE2 through EP2 receptors. The findings suggest that EP2 receptor-specific antagonists may be used as an adjunct to present clinical management for the prevention of preterm cervical ripening and preterm labor. PMID:24471568

  5. Methamphetamine blunts Ca2+ currents and excitatory synaptic transmission through D1/5 receptor-mediated mechanisms in the mouse medial prefrontal cortex

    PubMed Central

    González, Betina; Rivero-Echeto, Celeste; Muñiz, Javier A.; Cadet, Jean Lud; García-Rill, Edgar; Urbano, Francisco J.; Bisagno, Veronica

    2015-01-01

    Psychostimulant addiction is associated with dysfunctions in frontal cortex. Previous data demonstrated that repeated exposure to methamphetamine (METH) can alter prefrontal cortex (PFC) dependent functions. Here, we show that withdrawal from repetitive non-contingent METH administration (7 days, 1mg/kg) depressed voltage-dependent calcium currents (ICa) and increased IH amplitude and the paired-pulse ratio of evoked EPSCs in deep-layer pyramidal mPFC neurons. Most of these effects were blocked by systemic co-administration of the D1/D5 receptor antagonist SCH23390 (0.5 and 0.05 mg/kg). In vitro METH (i.e bath-applied to slices from naïve-treated animals) was able to emulate its systemic effects on ICa and evoked EPSCs paired-pulse ratio. We also provide evidence of altered mRNA expression of i) voltage-gated calcium channels P/Q-type Cacna1a (Cav2.1), N-type Cacna1b (Cav2.2), T-type Cav3.1 Cacna1g, Cav3.2 Cacna1h, Cav3.3 Cacna1i and the auxiliary subunit Cacna2d1 (α2δ1), ii) hyperpolarization-activated cyclic nucleotide-gated channels Hcn1 and Hcn2 and iii) glutamate receptors subunits AMPA-type Gria1, NMDA-type Grin1 and metabotropic Grm1 in the mouse mPFC after repeated METH treatment. Moreover, we show that some of these changes in mRNA expression were sensitive D1/5 receptor blockade. Altogether these altered mechanisms affecting synaptic physiology and transcriptional regulation may underlie prefrontal cortex functional alterations that could lead to PFC impairments observed in METH-addicted individuals. PMID:25871318

  6. Sigma-1 receptor-mediated increase in spinal p38 MAPK phosphorylation leads to the induction of mechanical allodynia in mice and neuropathic rats.

    PubMed

    Moon, Ji-Young; Roh, Dae-Hyun; Yoon, Seo-Yeon; Kang, Suk-Yun; Choi, Sheu-Ran; Kwon, Soon-Gu; Choi, Hoon-Seong; Han, Ho-Jae; Beitz, Alvin J; Lee, Jang-Hern

    2013-09-01

    The direct activation of the spinal sigma-1 receptor (Sig-1R) produces mechanical allodynia (MA) and thermal hyperalgesia (TH) in mice. In addition, the blockade of the spinal Sig-1R prevents the induction of MA, but not TH in chronic constriction injury (CCI)-induced neuropathic rats. The present study was designed to investigate whether the increase in spinal p38 MAPK phosphorylation (p-p38 MAPK) mediates Sig-1R-induced MA or TH in mice and the induction of MA in neuropathic rats. MA and TH were evaluated using von Frey filaments and a hot-plate apparatus, respectively. Neuropathic pain was produced by CCI of the right sciatic nerve in rats. Western blot assay and immunohistochemistry were performed to determine the changes of p-p38 MAPK expression in the spinal cord. Intrathecal (i.t.) injection of PRE084, a selective Sig-1R agonist, into naïve mice time-dependently increased the expression of p-p38 MAPK, which was blocked by pretreatment with BD1047, a Sig-1R antagonist. I.t. pretreatment with SB203580, a p38 MAPK inhibitor also dose-dependently inhibited PRE084-induced MA, whereas TH induction was not affected. In CCI rats, i.t. injection of BD1047 during the induction phase (postoperative days 0 to 5) reduced the CCI-induced increase in p-p38 MAPK. In addition, i.t. SB203580 treatment during the induction phase also suppressed the development of CCI-induced MA, but not TH. Conversely, i.t. SB203580 treatment during the maintenance phase (postoperative days 15 to 20) had no effect on CCI-induced MA or TH. These results demonstrate that the increase in spinal p-p38 MAPK is closely associated with the induction of Sig-1R mediated MA, but not TH. Sigma-1 receptor modulation of p-p38 MAPK also plays an important role in the induction, but not the maintenance, of MA in neuropathic pain.

  7. P2X3 Receptors Mediate Visceral Hypersensitivity during Acute Chemically-Induced Colitis and in the Post-Inflammatory Phase via Different Mechanisms of Sensitization

    PubMed Central

    Deiteren, Annemie; van der Linden, Laura; de Wit, Anouk; Ceuleers, Hannah; Buckinx, Roeland; Timmermans, Jean-Pierre; Moreels, Tom G.; Pelckmans, Paul A.; De Man, Joris G.; De Winter, Benedicte Y.

    2015-01-01

    more so in the post-inflammatory phase, albeit via different mechanisms of sensitization, validating P2X3 receptors as potential new targets in the treatment of abdominal pain syndromes. PMID:25885345

  8. Estrogen and female reproductive tract innervation: cellular and molecular mechanisms of autonomic neuroplasticity

    PubMed Central

    Brauer, M. Mónica; Smith, Peter G.

    2014-01-01

    The female reproductive tract undergoes remarkable functional and structural changes associated with cycling, conception and pregnancy, and it is likely advantageous to both individual and species to alter relationships between reproductive tissues and innervation. For several decades, it has been appreciated that the mammalian uterus undergoes massive sympathetic axon depletion in late pregnancy, possibly representing an adaptation to promote smooth muscle quiescence and sustained blood flow. Innervation to other structures such as cervix and vagina also undergo pregnancy-related changes in innervation that may facilitate parturition. These tissues provide highly tractable models for examining cellular and molecular mechanisms underlying peripheral nervous system plasticity. Studies show that estrogen elicits rapid degeneration of sympathetic terminal axons in myometrium, which regenerate under low-estrogen conditions. Degeneration is mediated by the target tissue: under estrogen's influence, the myometrium produces proteins repulsive to sympathetic axons including BDNF, neurotrimin, semaphorins, and pro-NGF, and extracellular matrix components are remodeled. Interestingly, nerve depletion does not involve diminished levels of classical sympathetic neurotrophins that promote axon growth. Estrogen also affects sympathetic neuron neurotrophin receptor expression in ways that appear to favor pro-degenerative effects of the target tissue. In contrast to the uterus, estrogen depletes vaginal autonomic and nociceptive axons, with the latter driven in part by estrogen-induced suppression BMP4 synthesis. These findings illustrate that hormonally mediated physiological plasticity is a highly complex phenomenon involving multiple, predominantly repulsive target-derived factors acting in concert to achieve rapid and selective reductions in innervation. PMID:25530517

  9. The Immune System Is a Natural Target for Estrogen Action: Opposing Effects of Estrogen in Two Prototypical Autoimmune Diseases.

    PubMed

    Khan, Deena; Ansar Ahmed, S

    2015-01-01

    Analogous to other physiological systems, the immune system also demonstrates remarkable sex differences. Although the reasons for sex differences in immune responses are not precisely understood, it potentially involves differences in sex hormones (estrogens, androgens, and differential sex hormone receptor-mediated events), X-chromosomes, microbiome, epigenetics among others. Overall, females tend to have more responsive and robust immune system compared to their male counterparts. It is therefore not surprising that females respond more aggressively to self-antigens and are more susceptible to autoimmune diseases. Female hormone (estrogen or 17β-estradiol) can potentially act on all cellular subsets of the immune system through estrogen receptor-dependent and -independent mechanisms. This minireview highlights differential expression of estrogen receptors on immune cells, major estrogen-mediated signaling pathways, and their effect on immune cells. Since estrogen has varied effects in female-predominant autoimmune diseases such as multiple sclerosis and systemic lupus erythematosus, we will mechanistically postulate the potential differential role of estrogen in these chronic debilitating diseases.

  10. The Immune System Is a Natural Target for Estrogen Action: Opposing Effects of Estrogen in Two Prototypical Autoimmune Diseases

    PubMed Central

    Khan, Deena; Ansar Ahmed, S.

    2016-01-01

    Analogous to other physiological systems, the immune system also demonstrates remarkable sex differences. Although the reasons for sex differences in immune responses are not precisely understood, it potentially involves differences in sex hormones (estrogens, androgens, and differential sex hormone receptor-mediated events), X-chromosomes, microbiome, epigenetics among others. Overall, females tend to have more responsive and robust immune system compared to their male counterparts. It is therefore not surprising that females respond more aggressively to self-antigens and are more susceptible to autoimmune diseases. Female hormone (estrogen or 17β-estradiol) can potentially act on all cellular subsets of the immune system through estrogen receptor-dependent and -independent mechanisms. This minireview highlights differential expression of estrogen receptors on immune cells, major estrogen-mediated signaling pathways, and their effect on immune cells. Since estrogen has varied effects in female-predominant autoimmune diseases such as multiple sclerosis and systemic lupus erythematosus, we will mechanistically postulate the potential differential role of estrogen in these chronic debilitating diseases. PMID:26779182

  11. Diversity of mechanisms involved in aromatase regulation and estrogen action in the brain.

    PubMed

    Charlier, Thierry D; Cornil, Charlotte A; Ball, Gregory F; Balthazart, Jacques

    2010-10-01

    The mechanisms through which estrogens modulate neuronal physiology, brain morphology, and behavior in recent years have proven to be far more complex than previously thought. For example, a second nuclear estrogen receptor has been identified, a new family of coregulatory proteins regulating steroid-dependent gene transcriptions was discovered and, finally, it has become clear that estrogens have surprisingly rapid effects based on their actions on cell membranes, which in turn result in the modulation of intracellular signaling cascades. This paper presents a selective review of new findings in this area related to work in our laboratories, focusing on the role of estrogens in the activation of male sexual behavior. Two separate topics are considered. We first discuss functions of the steroid receptor coactivator-1 (SRC-1) that has emerged as a key limiting factor for behavioral effects of estradiol. Knocking-down its expression by antisense oligonucleotides drastically inhibits male-typical sexual behaviors. Secondly, we describe rapid regulations of brain estradiol production by calcium-dependent phosphorylations of the aromatase enzyme, themselves under the control of neurotransmitter activity. These rapid changes in estrogen bioavailability have clear behavioral consequences. Increases or decreases in estradiol concentrations respectively obtained by an acute injection of estradiol itself or of an aromatase inhibitor lead within 15-30 min to parallel changes in sexual behavior frequencies. These new controls of estrogen action offer a vast array of possibilities for discrete local controls of estrogen action. They also represent a formidable challenge for neuroendocrinologists trying to obtain an integrated view of brain function in relation to behavior. Copyright © 2010 Elsevier B.V. All rights reserved.

  12. Diversity of mechanisms involved in aromatase regulation and estrogen action in the brain

    PubMed Central

    Charlier, Thierry D.; Cornil, Charlotte A.; Ball, Gregory F.; Balthazart, Jacques

    2010-01-01

    The mechanisms through which estrogens modulate neuronal physiology, brain morphology, and behavior in recent years have proven to be far more complex than previously thought. For example, a second nuclear estrogen receptor has been identified, a new family of coregulatory proteins regulating steroid-dependent gene transcriptions was discovered and, finally, it has become clear that estrogens have surprisingly rapid effects based on their actions on cell membranes, which in turn results in the modulation of intracellular signaling cascades. This paper presents a selective review of new findings in this area related to work in our laboratories, focusing on the role of estrogens in the activation of male sexual behavior. Two separate topics are considered. We first discuss functions of the steroid receptor coactivator-1 (SRC-1) that has emerged as a key limiting factor for behavioral effects of estradiol. Knocking-down its expression by antisense oligonucleotides drastically inhibits male-typical sexual behaviors. Secondly, we describe rapid regulations of brain estradiol production by calcium-dependent phosphorylations of the aromatase enzyme, themselves under the control of neurotransmitter activity. These rapid changes in estrogen bioavailability have clear behavioral consequences. Increases or decreases in estradiol concentrations respectively obtained by an acute injection of estradiol itself or of an aromatase inhibitor lead within 15-30 min to parallel changes in sexual behavior frequencies. These new controls of estrogens action offer a vast array of possibilities for discrete local controls of estrogen action. They also represent a formidable challenge for neuroendocrinologists trying to obtain an integrated view of brain function in relation to behavior. PMID:20060879

  13. Estrogen-induced effects on the neuro-mechanics of hopping in humans.

    PubMed

    Bryant, Adam L; Crossley, Kay M; Bartold, Simon; Hohmann, Erik; Clark, Ross A

    2011-02-01

    Estrogen receptors in skeletal muscle suggest a tissue-based mechanism for influencing neuromuscular control. This has important physiological implications for both eumenorrheic women with fluctuating estrogen levels and those with constant and attenuated estrogen levels, i.e., women using the monophasic oral contraceptive pill (MOCP). This study examined the effects of endogenous plasma estrogen levels on leg stiffness (K (LEG)) and foot center of pressure (COP) during hopping. Nineteen females (Age = 28.0 ± 4.2 years, Ht = 1.67 ± 0.07 m, Mass = 61.6 ± 6.8 kg) who had been using the MOCP for at least 12 months together with 19 matched, female, non-MOCP users (Age = 31.9 ± 7.3 years, Ht = 1.63 ± 0.05 m, Mass = 62.5 ± 5.9 kg) participated. Non-MOCP users were tested at the time of lowest (menstruation) and highest (≈ ovulation) estrogen whilst MOCP users were tested at Day 1 and Day 14 of their cycle. At each test session, K (LEG) (N m(-1) kg(-1)) and foot COP path length (mm) and path velocity (mm s(-1)) were determined from ground reaction force data as participants hopped at 2.2 Hz on a force plate. Statistical analysis revealed no significant (p < 0.05) differences for K (LEG). In contrast, significantly higher COP path length (30%) and COP path velocity (25%) were identified at ≈ ovulation compared to menstruation in the non-MOCP users. Whilst there was no evidence of an estrogen-induced effect on K (LEG); significantly elevated estrogen at ≈ ovulation presumably increased extensibility of connective tissue and/or diminished neuromuscular control. Consistent lower limb dynamics of MOCP users demands less reliance on acutely modified neuromuscular control strategies during dynamic tasks and may explain the lower rate of lower limb musculoskeletal injuries in this population compared to non-MOCP users.

  14. Estrogen inhibits lysyl oxidase and decreases mechanical function in engineered ligaments.

    PubMed

    Lee, Cassandra A; Lee-Barthel, Ann; Marquino, Louise; Sandoval, Natalie; Marcotte, George R; Baar, Keith

    2015-05-15

    Women are more likely to suffer an anterior cruciate ligament (ACL) rupture than men, and the incidence of ACL rupture in women rises with increasing estrogen levels. We used an engineered ligament model to determine how an acute rise in estrogen decreases the mechanical properties of ligaments. Using fibroblasts isolated from human ACLs from male or female donors, we engineered ligaments and determined that ligaments made from female ACL cells had more collagen and were equal in strength to those made from male ACL cells. We then treated engineered ligaments for 14 days with low (5 pg/ml), medium (50 pg/ml), or high (500 pg/ml) estrogen, corresponding to the range of in vivo serum estrogen concentrations and found that collagen within the grafts increased without a commensurate increase in mechanical strength. Mimicking the menstrual cycle, with 12 days of low estrogen followed by 2 days of physiologically high estrogen, resulted in a decrease in engineered ligament mechanical function with no change in the amount of collagen in the graft. The decrease in mechanical stiffness corresponded with a 61.7 and 76.9% decrease in the activity of collagen cross-linker lysyl oxidase with 24 and 48 h of high estrogen, respectively. Similarly, grafts treated with the lysyl oxidase inhibitor β-aminoproprionitrile (BAPN) for 24 h showed a significant decrease in ligament mechanical strength [control (CON) = 1.58 ± 0.06 N; BAPN = 1.06 ± 0.13 N] and stiffness (CON = 7.7 ± 0.46 MPa; BAPN = 6.1 ± 0.71 MPa) without changing overall collagen levels (CON = 396 ± 11.5 μg; BAPN = 382 ± 11.6 μg). Together, these data suggest that the rise in estrogen during the follicular phase decreases lysyl oxidase activity in our engineered ligament model and if this occurs in vivo may decrease the stiffness of ligaments and contribute to the elevated rate of ACL rupture in women.

  15. The Natural Estrogenic Compound Diarylheptanoid (D3): In Vitro Mechanisms of Action and in Vivo Uterine Responses via Estrogen Receptor α

    PubMed Central

    Winuthayanon, Wipawee; Piyachaturawat, Pawinee; Suksamrarn, Apichart; Burns, Katherine A.; Arao, Yukitomo; Hewitt, Sylvia C.; Pedersen, Lars C.

    2013-01-01

    Background: Diarylheptanoid (D3) isolated from the medicinal plant, Curcuma comosa, has estrogenic activity. Objective: We aimed to elucidate the mechanism(s) of D3 action and compare it with that of 17β-estradiol (E2) using both in vitro and in vivo uterine models. Methods: We used human uterine (Ishikawa) cells to determine the estrogenic action of D3 on the activation and nuclear translocation of estrogen receptor α (ERα). In addition, we further characterized the uterine response to D3 treatment in vivo. Results: D3 activated an estrogen responsive element (ERE) luciferase reporter through ERα, and molecular modeling suggested that D3 could be accommodated in the ERα binding pocket. Using modified ERα to assay ligand-dependent nuclear translocation, we observed D3-dependent ERα interaction and translocation. In mouse uteri, early- and late-phase estrogen-regulated gene responses were increased in D3-treated ovariectomized wild-type animals, in a manner similar to that of E2; no response was seen in ERα knockout animals. We observed a divergence in estrogen responses after D3 treatment: D3 induced robust DNA synthesis in uterine epithelial cells, linked to an increase in cell-cycle–related genes; however, no increase in uterine weight was observed 24 hr after treatment. D3 also affected uterine progesterone receptor expression patterns similar to E2. When D3 and E2 were administered together, we observed no additive or antagonistic effects of D3 on E2. Our findings suggest that D3 is a weak estrogenic agonist compound. Conclusion: D3 is a weakly acting phytoestrogen that mimics the mitogenic responses produced by E2 in an ERα-dependent manner, but it is unable to increase uterine weight or enhance or antagonize the effects of estrogen. PMID:23552522

  16. From empirical to mechanism-based discovery of clinically useful Selective Estrogen Receptor Modulators (SERMs)

    PubMed Central

    Wardell, Suzanne E.; Nelson, Erik R.; McDonnell, Donald P.

    2014-01-01

    Our understanding of the molecular mechanisms underlying the pharmacological actions of estrogen receptor (ER) ligands has evolved considerably in recent years. Much of this knowledge has come from a detailed dissection of the mechanism(s) of action of the Selective Estrogen Receptor Modulators (SERMs) tamoxifen and raloxifene, drugs whose estrogen receptor (ER) agonist/antagonist properties are influenced by the cell context in which they operate. These studies have revealed that notwithstanding differences in drug pharmokinetics, the activity of an ER ligand is determined primarily by (a) the impact that a given ligand has on the receptor conformation and (b) the ability of structurally distinct ER-ligand complexes to interact with functionally distinct coregulators. Exploitation of the established relationships between ER structure and activity has led to the development of improved SERMs with more favorable therapeutic properties and of tissue-selective estrogen complexes, drugs in which a SERM and an ER agonist are combined to yield a blended activity that results in distinct clinical profiles. Remarkably, endogenous ligands that exhibit SERM activity have also been identified. One of these ligands, 27-hydroxycholesterol (27HC), has been shown to manifest ER-dependent pathological activities in the cardiovascular system, bone and mammary gland. Whereas the physiological activity of 27HC remains to be determined, its discovery highlights how cells have adopted mechanisms to allow the same receptor ligand complex to manifest different activities in different cells, and also how these processes can be exploited for new drug development. PMID:25084324

  17. Divergent mechanisms for trophic actions of estrogens in the brain and peripheral tissues

    PubMed Central

    Walf, Alicia A.; Paris, Jason J.; Rhodes, Madeline E.; Simpkins, James W.; Frye, Cheryl A.

    2011-01-01

    17β-estradiol (E2) can enhance reproductive, cognitive, and affective functions; however, the mechanisms by which E2 has these effects need to be better understood. Pleiotrophic effects of E2 can occur via traditional and novel actions at various forms of estrogen receptors (ERs). In the central nervous system, trophic effects of E2 may be related to beneficial effects of hormone replacement therapy (HRT). However, in peripheral reproductive tissues, E2's capacity to evoke growth can increase risk of cancers. This review focuses on investigations aimed at elucidating divergent mechanisms of steroids to promote trophic effects in the brain, independent of effects on peripheral reproductive tissues. First, actions of estrogens via ERα or ERβ for peripheral growth (carcinogen-induced tumors, uterine growth) and hippocampus-dependent behaviors (affect, cognition) are described. Second, factors that influence these effects of estrogens are described (e.g. experience, timing/critical windows, non-ER mechanisms). Third, effects of estrogens at ERβ related to actions of progestogens, such as 5α-pregnan-3α-ol-20-one (3α,5α-THP) are described. In summary, effects of E2 may occur via multiple mechanisms, which may underlie favorable effects in the brain with minimal peripheral trophic effects. PMID:21130078

  18. Endocrine disrupting chemicals targeting estrogen receptor signaling: Identification and mechanisms of action

    PubMed Central

    Shanle, Erin K.; Xu, Wei

    2011-01-01

    Many endocrine disrupting chemicals (EDCs) adversely impact estrogen signaling by interacting with two estrogen receptors (ERs): ERα and ERβ. Though the receptors have similar ligand binding and DNA binding domains, ERα and ERβ have some unique properties in terms of ligand selectivity and target gene regulation. EDCs that target ER signaling can modify genomic and non-genomic ER activity through direct interactions with ERs, indirectly through transcription factors like the aryl hydrocarbon receptor (AhR), or through modulation of metabolic enzymes that are critical for normal estrogen synthesis and metabolism. Many EDCs act through multiple mechanisms as exemplified by chemicals that bind both AhR and ER, such as 3-methylcholanthrene. Other EDCs that target ER signaling include phytoestrogens, bisphenolics, and organochlorine pesticides and many alter normal ER signaling through multiple mechanisms. EDCs can also display tissue-selective ER agonist and antagonist activities similar to selective estrogen receptor modulators (SERMs) designed for pharmaceutical use. Thus, biological effects of EDCs need to be carefully interpreted because EDCs can act through complex tissue-selective modulation of ERs and other signaling pathways in vivo. Current requirements by the U.S. Environmental Protection Agency require some in vitro and cell-based assays to identify EDCs that target ER signaling through direct and metabolic mechanisms. Additional assays may be useful screens for identifying EDCs that act through alternative mechanisms prior to further in vivo study. PMID:21053929

  19. Receptor-mediated DNA-targeted photoimmunotherapy.

    PubMed

    Karagiannis, Tom C; Lobachevsky, Pavel N; Leung, Brenda K Y; White, Jonathan M; Martin, Roger F

    2006-11-01

    We show the efficacy of a therapeutic strategy that combines the potency of a DNA-binding photosensitizer, UV(A)Sens, with the tumor-targeting potential of receptor-mediated endocytosis. The photosensitizer is an iodinated bibenzimidazole, which, when bound in the minor groove of DNA and excited by UV(A) irradiation, induces cytotoxic lesions attributed to a radical species resulting from photodehalogenation. Although reminiscent of photochemotherapy using psoralens and UV(A) irradiation, an established treatment modality in dermatology particularly for the treatment of psoriasis and cutaneous T-cell lymphoma, a critical difference is the extreme photopotency of the iodinated bibenzimidazole, approximately 1,000-fold that of psoralens. This feature prompted consideration of combination with the specificity of receptor-mediated targeting. Using two in vitro model systems, we show the UV(A) cytotoxicity of iodo ligand/protein conjugates, implying binding of the conjugate to cell receptors, internalization, and degradation of the conjugate-receptor complex, with release and translocation of the ligand to nuclear DNA. For ligand-transferrin conjugates, phototoxicity was inhibited by coincubation with excess native transferrin. Receptor-mediated UV(A)-induced cytotoxicity was also shown with the iodo ligand conjugate of an anti-human epidermal growth factor receptor monoclonal antibody, exemplifying the potential application of the strategy to other cancer-specific targets to thus improve the specificity of phototherapy of superficial lesions and for extracorporeal treatments.

  20. A Novel Mechanism for Adenylyl Cyclase Inhibition from the Crystal Structure of its Complex with Catechol Estrogen

    SciTech Connect

    Steegborn,C.; Litvin, T.; Hess, K.; Capper, A.; Taussig, R.; Buck, J.; Levin, L.; Wu, H.

    2005-01-01

    Catechol estrogens are steroid metabolites that elicit physiological responses through binding to a variety of cellular targets. We show here that catechol estrogens directly inhibit soluble adenylyl cyclases and the abundant trans-membrane adenylyl cyclases. Catechol estrogen inhibition is non-competitive with respect to the substrate ATP, and we solved the crystal structure of a catechol estrogen bound to a soluble adenylyl cyclase from Spirulina platensis in complex with a substrate analog. The catechol estrogen is bound to a newly identified, conserved hydrophobic patch near the active center but distinct from the ATP-binding cleft. Inhibitor binding leads to a chelating interaction between the catechol estrogen hydroxyl groups and the catalytic magnesium ion, distorting the active site and trapping the enzyme substrate complex in a non-productive conformation. This novel inhibition mechanism likely applies to other adenylyl cyclase inhibitors, and the identified ligand-binding site has important implications for the development of specific adenylyl cyclase inhibitors.

  1. Estrogen inhibits vascular calcification via vascular RANKL system: common mechanism of osteoporosis and vascular calcification.

    PubMed

    Osako, Mariana Kiomy; Nakagami, Hironori; Koibuchi, Nobutaka; Shimizu, Hideo; Nakagami, Futoshi; Koriyama, Hiroshi; Shimamura, Munehisa; Miyake, Takashi; Rakugi, Hiromi; Morishita, Ryuichi

    2010-08-20

    Arterial calcification and osteoporosis are associated in postmenopausal women. RANK (the receptor activator of nuclear factor kappaB), RANKL (RANK ligand), and osteoprotegerin are key proteins in bone metabolism and have been found at the site of aortic calcification. The role of these proteins in vasculature, as well as the contribution of estrogen to vascular calcification, is poorly understood. To clarify the mechanism of RANKL system to vascular calcification in the context of estrogen deficiency. RANKL induced the calcification inducer bone morphogenetic protein-2 by human aortic endothelial cells (HAECs) and decreased the calcification inhibitor matrix Gla protein (MGP) in human aortic smooth muscle cells (HASMCs), as quantified by real-time PCR and Western blot analysis. RANKL also induced bone-related gene mRNA expression and calcium deposition (Alizarin red staining) followed by the osteogenic differentiation of HASMCs. Estrogen inhibited RANKL signaling in HAECs and HASMCs mainly through estrogen receptor alpha. Apolipoprotein E-deficient mice fed with Western high-fat diet for 3 months presented atherosclerotic calcification (Oil red and Alizarin red staining) and osteoporosis (microcomputed tomographic analysis) after ovariectomy and increased expression of RANKL, RANK, and osteopontin in atherosclerotic lesion, as detected by in situ hybridization. Estrogen replacement inhibited osteoporosis and the bone morphogenetic protein osteogenic pathway in aorta by decreasing phosphorylation of smad-1/5/8 and increasing MGP mRNA expression. RANKL contributes to vascular calcification by regulating bone morphogenetic protein-2 and MGP expression, as well as bone-related proteins, and is counteracted by estrogen in a receptor-dependent manner.

  2. Estrogen receptor β in Alzheimer's disease: From mechanisms to therapeutics.

    PubMed

    Zhao, Liqin; Woody, Sarah K; Chhibber, Anindit

    2015-11-01

    Alzheimer's disease (AD) disproportionally affects women and men. The female susceptibility for AD has been largely associated with the loss of ovarian sex hormones during menopause. This review examines the current understanding of the role of estrogen receptor β (ERβ) in the regulation of neurological health and its implication in the development and intervention of AD. Since its discovery in 1996, research conducted over the last 15-20 years has documented a great deal of evidence indicating that ERβ plays a pivotal role in a broad spectrum of brain activities from development to aging. ERβ genetic polymorphisms have been associated with cognitive impairment and increased risk for AD predominantly in women. The role of ERβ in the intervention of AD has been demonstrated by the alteration of AD pathology in response to treatment with ERβ-selective modulators in transgenic models that display pronounced plaque and tangle histopathological presentations as well as learning and memory deficits. Future studies that explore the potential interactions between ERβ signaling and the genetic isoforms of human apolipoprotein E (APOE) in brain aging and development of AD-risk phenotype are critically needed. The current trend of lost-in-translation in AD drug development that has primarily been based on early-onset familial AD (FAD) models underscores the urgent need for novel models that recapitulate the etiology of late-onset sporadic AD (SAD), the most common form of AD representing more than 95% of the current human AD population. Combining the use of FAD-related models that generally have excellent face validity with SAD-related models that hold more reliable construct validity would together increase the predictive validity of preclinical findings for successful translation into humans.

  3. Estrogens facilitate memory processing through membrane mediated mechanisms and alterations in spine density

    PubMed Central

    Luine, Victoria N.; Frankfurt, Maya

    2012-01-01

    Estrogens exert sustained, genomically mediated effects on memory throughout the female life cycle, but here we review new studies documenting rapid effects of estradiol on memory, which are exerted through membrane-mediated mechanisms. Use of recognition memory tasks in rats, shows that estrogens enhance memory consolidation within one hour. 17α-estradiol is more potent than 17β-estradiol, and the dose response relationship between estrogens and memory is an inverted U shape. Use of specific estrogen receptor (ER) agonists suggests mediation by an ERβ-like membrane receptor. Enhanced memory is associated with increased spine density and altered noradrenergic activity in the medial prefrontal cortex and hippocampus within 30 min. of administration. The environmental chemical, bisphenol-A, rapidly antagonizes enhancements in memory in both sexes possibly through actions on spines. Thus, estradiol and related compounds exert rapid alterations in cognition through non-genomic mechanisms, a finding which may provide a basis for better understanding and treating memory impairments. PMID:22981654

  4. Anti-osteoporosis effect of Epimedium via an estrogen-like mechanism based on a system-level approach.

    PubMed

    Xu, Feifei; Ding, Yan; Guo, Yingying; Liu, Baoyue; Kou, Zinong; Xiao, Wei; Zhu, Jingbo

    2016-01-11

    Epimedium is a popular traditional herbal medicine worldwide that has long been used to relieve osteoporosis. The estrogenic properties of the herb are conferred by several phytoestrogens, such as flavonoids, lignans, and steroids. However, the poor understanding on the estrogen-like mechanism of Epimedium at the molecular and system levels limits the applications of this herb in osteoporosis treatment. In this study, systems pharmacology was established to investigate the relationship between Epimedium and estrogen against osteoporosis by integrating active component screening, drug-likeness evaluation, herb feature mapping, target prediction and validation, and network analysis. A total of 77 active components that possessed similar structural features to estrogen as determined using herb feature mapping were selected from Epimedium by oral bioavailability prediction and drug-likeness evaluation. Twenty three osteoporosis-related targets were obtained from the active components of Epimedium as potential targets, 11 of which were common targets with estrogen. All osteoporosis-related targets were further mapped to compound-target and target-pathway networks. Results displayed that Epimedium can exert anti-osteoporosis effects by directly regulating the 11 estrogen-related targets and a set of target proteins on five estrogen-related pathways. This study explained the estrogen-like mechanism of Epimedium in preventing and treating osteoporosis, and provided a new standpoint for exploring the traditional herbal medicine against osteoporosis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  5. DDT and its metabolites alter gene expression in human uterine cell lines through estrogen receptor-independent mechanisms.

    PubMed Central

    Frigo, Daniel E; Burow, Matthew E; Mitchell, Kamron A; Chiang, Tung-Chin; McLachlan, John A

    2002-01-01

    Endocrine-disrupting organochlorines, such as the pesticide dichlorodiphenyltrichloroethane (DDT), bind to and activate estrogen receptors (ERs), thereby eliciting estrogen-like effects. Although ERs function predominantly through activation of transcription via estrogen-responsive elements, both ERs, alpha and ss, can interact with various transcription factors such as activator protein-1 (AP-1). Additionally, estrogens may regulate early signaling events, suggesting that the biological effects of environmental estrogens may not be mediated through classic ER (alpha and ss) activity alone. We hypothesized that known environmental estrogens, such as DDT and its metabolites, activate AP-1-mediated gene transactivation through both ER-dependent and ER-independent means. Using two Ishikawa human endometrial adenocarcinoma cell line variants that we confirmed to be estrogen responsive [Ishikawa(+)] and estrogen unresponsive [Ishikawa(-)], we generated stably transfected AP-1 luciferase cell lines to identify the role of an estrogen-responsive mechanism in AP-1-mediated gene expression by various stimuli. Our results demonstrate that DDT and dichlorodiphenyldichloroethane (DDD) were the most potent activators of AP-1 activity; 2,2-bis(p-chlorophenyl) acetic acid failed to activate. Although stimulated in both Ishikawa(+) and Ishikawa(-) cells by DDT and its congeners, AP-1 activation was more pronounced in the estrogen-unresponsive Ishikawa(-) cells. In addition, DDT, DDD, and dichlorodiphenyldichloroethylene (DDE) could also stimulate AP-1 activity in the estrogen-unresponsive human embryonic kidney 293 cells using a different promoter context. Thus, our data demonstrate that DDT and its metabolites activate the AP-1 transcription factor independent of ER (alpha or ss) status. PMID:12460804

  6. Differential modulation of expression of nuclear receptor mediated genes by tris(2-butoxyethyl) phosphate (TBOEP) on early life stages of zebrafish (Danio rerio).

    PubMed

    Ma, Zhiyuan; Yu, Yijun; Tang, Song; Liu, Hongling; Su, Guanyong; Xie, Yuwei; Giesy, John P; Hecker, Markus; Yu, Hongxia

    2015-12-01

    As one substitute for phased-out brominated flame retardants (BFRs), tris(2-butoxyethyl) phosphate (TBOEP) is frequently detected in aquatic organisms. However, knowledge about endocrine disrupting mechanisms associated with nuclear receptors caused by TBOEP remained restricted to results from in vitro studies with mammalian cells. In the study, results of which are presented here, embryos/larvae of zebrafish (Danio rerio) were exposed to 0.02, 0.1 or 0.5μM TBOEP to investigate expression of genes under control of several nuclear hormone receptors (estrogen receptors (ERs), androgen receptor (AR), thyroid hormone receptor alpha (TRα), mineralocorticoid receptor (MR), glucocorticoid receptor (GR), aryl hydrocarbon (AhR), peroxisome proliferator-activated receptor alpha (PPARα), and pregnane×receptor (P×R)) pathways at 120hpf. Exposure to 0.5μM TBOEP significantly (p<0.05, one-way analysis of variance) up-regulated expression of estrogen receptors (ERs, er1, er2a, and er2b) genes and ER-associated genes (vtg4, vtg5, pgr, ncor, and ncoa3), indicating TBOEP modulates the ER pathway. In contrast, expression of most genes (mr, 11βhsd, ube2i,and adrb2b) associated with the mineralocorticoid receptor (MR) pathway were significantly down-regulated. Furthermore, in vitro mammalian cell-based (MDA-kb2 and H4IIE-luc) receptor transactivation assays, were also conducted to investigate possible agonistic or antagonistic effects on AR- and AhR-mediated pathways. In mammalian cells, none of these pathways were affected by TBOEP at the concentrations studied. Receptor-mediated responses (in vivo) and mammalian cell lines receptor binding assay (in vitro) combined with published information suggest that TBOEP can modulate receptor-mediated, endocrine process (in vivo/in vitro), particularly ER and MR. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Estrogen binding, receptor mRNA, and biologic response in osteoblast-like osteosarcoma cells

    SciTech Connect

    Komm, B.S.; Terpening, C.M.; Benz, D.J.; Graeme, K.A.; Gallegos, A.; Korc, M.; Greene, G.L.; O'Malley, B.W.; Haussler, M.R.

    1988-07-01

    High specific activity estradiol labeled with iodine-125 was used to detect approximately 200 saturable, high-affinity (dissociation constant approximately equal to 1.0 nM) nuclear binding sites in rat (ROS 17/2.8) and human (HOS TE85) clonal osteoblast-like osteosarcoma cells. Of the steroids tested, only testosterone exhibited significant cross-reactivity with estrogen binding. RNA blot analysis with a complementary DNA probe to the human estrogen receptor revealed putative receptor transcripts of 6 to 6.2 kilobases in both rat and human osteosarcoma cells. Type I procollagen and transforming growth factor-beta messenger RNA levels were enhanced in cultured human osteoblast-like cells treated with 1 nM estradiol. Thus, estrogen can act directly on osteoblasts by a receptor-mediated mechanism and thereby modulate the extracellular matrix and other proteins involved in the maintenance of skeletal mineralization and remodeling.

  8. Long-term exposure to estrogen enhances chemotherapeutic efficacy potentially through epigenetic mechanism in human breast cancer cells

    PubMed Central

    Chang, Yu-Wei

    2017-01-01

    Chemotherapy is the most common clinical option for treatment of breast cancer. However, the efficacy of chemotherapy depends on the age of breast cancer patients. Breast tissues are estrogen responsive and the levels of ovarian estrogen vary among the breast cancer patients primarily between pre- and post-menopausal age. Whether this age-dependent variation in estrogen levels influences the chemotherapeutic efficacy in breast cancer patients is not known. Therefore, the objective of this study was to evaluate the effects of natural estrogen 17 beta-estradiol (E2) on the efficacy of chemotherapeutic drugs in breast cancer cells. Estrogen responsive MCF-7 and T47D breast cancer cells were long-term exposed to 100 pg/ml estrogen, and using these cells the efficacy of chemotherapeutic drugs doxorubicin and cisplatin were determined. The result of cell viability and cell cycle analysis revealed increased sensitivities of doxorubicin and cisplatin in estrogen-exposed MCF-7 and T47D cells as compared to their respective control cells. Gene expression analysis of cell cycle, anti-apoptosis, DNA repair, and drug transporter genes further confirmed the increased efficacy of chemotherapeutic drugs in estrogen-exposed cells at molecular level. To further understand the role of epigenetic mechanism in enhanced chemotherapeutic efficacy by estrogen, cells were pre-treated with epigenetic drugs, 5-aza-2-deoxycytidine and Trichostatin A prior to doxorubicin and cisplatin treatments. The 5-aza-2 deoxycytidine pre-treatment significantly decreased the estrogen-induced efficacy of doxorubicin and cisplatin, suggesting the role of estrogen-induced hypermethylation in enhanced sensitivity of these drugs in estrogen-exposed cells. In summary, the results of this study revealed that sensitivity to chemotherapy depends on the levels of estrogen in breast cancer cells. Findings of this study will have clinical implications in selecting the chemotherapy strategies for treatment of breast

  9. Postmenopausal osteoporosis in rheumatoid arthritis: The estrogen deficiency-immune mechanisms link.

    PubMed

    Sapir-Koren, Rony; Livshits, Gregory

    2017-10-01

    Rheumatoid arthritis (RA) is characterized, among other factors, by systemic bone loss, reaching ~50% prevalence of osteoporosis in postmenopausal women. This is roughly a doubled prevalence in comparison with age-matched non-RA women. Postmenopausal RA women are more likely to be sero-positive for the anti-citrullinated peptide antibody (ACPA). Our extensive review of recent scientific literature enabled us to propose several mechanisms as responsible for the accelerated bone loss in ACPA(+) RA postmenopausal women. Menopause-associated estrogen deficiency plays a major role in these pathological mechanisms, as follows. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Estrogen Is Renoprotective via a Nonreceptor-dependent Mechanism after Cardiac Arrest In Vivo

    PubMed Central

    Hutchens, Michael P.; Nakano, Takaaki; Kosaka, Yasuharu; Dunlap, Jennifer; Zhang, Wenri; Herson, Paco S.; Murphy, Stephanie J.; Anderson, Sharon; Hurn, Patricia D.

    2010-01-01

    Background Severe ischemia induces renal injury less frequently in women than men. In this study, cardiac arrest and cardiopulmonary resuscitation were used to assess whether estradiol is renoprotective via an estrogen receptor (ER)-dependent mechanism. Materials and Methods Male and female C57BL/6 and ER gene-deleted mice underwent 10 min of cardiac arrest followed by cardiopulmonary resuscitation. Serum chemistries and renal stereology were measured 24 h after arrest. Results Estrogen did not affect mean arterial pressure, regional renal cortical blood flow, and arterial blood gases. Hence, female kidneys were protected (mean ± SEM: blood urea nitrogen, 65 ± 21 vs.149 ± 27 mg/dl, P = 0.04; creatinine, 0.14 ± 0.05 vs. 0.73 ± 0.16 mg/dl, P = 0.01; volume of necrotic tubules, 7 ± 1% vs. 10 ± 0%, P = 0.04). Estrogen also reduced renal injury. In intact females (n = 5), ovariectomized/vehicle-treated (n = 8), and ovariectomized/estrogen-treated (n = 8) animals, blood urea nitrogen was 65 ± 21, 166 ± 28, and 50 ± 14 mg/dl (P = 0.002); creatinine was 0.14 ± 0.05, 0.74 ± 0.26, and 0.23 ± 0.27 mg/dl (P = 0.014); necrotic tubules were 2.5 ± 0.25%, 12.0 ± 1.9%, and 5.0 ± 1.6% (P = 0.004), respectively. In ER-α and ER-β gene-deleted mice and controls estradiol-reduced functional injury (blood urea nitrogen: estradiol 117 ± 71, vehicle 167 ± 56, P = 0.007; creatinine: estradiol 0.5 ± 0.5, vehicle 1.0 ± 0.4, P = 0.013), but the effect of estradiol was not different between ER-α or ER-β gene-deleted mice. Adding ICI 182,780 to estradiol did not alter injury. Conclusions In women, kidneys were protected from cardiac arrest through estrogen. Estradiol-mediated renoprotection was not affected by ER deletion or blockade. Estradiol is renoprotective after cardiac arrest. The results indicate that estradiol renoprotection is ER-α and ER-β independent. PMID:20068453

  11. The Role of Estrogen Related Receptor in Modulating Estrogen Receptor Mediated Transcription in Breast Cancer Cells

    DTIC Science & Technology

    2005-04-01

    receptors ) by demonstrating that mitochondrial biogenesis and fatty acid P- oxidation , processes ERRa is known to regulate , are robustly...gluconeogenesis, and fatty acid oxidation (Lin 2003; Puigserver 1998; Wu 1999; Yoon 2001). In addition to its activity on a number of nuclear receptors , this...in target cells. They were generated by replacing the receptor interaction domains in peroxisome proliferator activated receptor

  12. Estrogen and Osteoporosis.

    ERIC Educational Resources Information Center

    Lindsay, Robert

    1987-01-01

    This article reviews the use of estrogen in the prevention and treatment of osteoporosis. Dosage levels, interactions with other factors, side effects, and the mechanism of estrogen action are discussed. (Author/MT)

  13. Estrogen and Osteoporosis.

    ERIC Educational Resources Information Center

    Lindsay, Robert

    1987-01-01

    This article reviews the use of estrogen in the prevention and treatment of osteoporosis. Dosage levels, interactions with other factors, side effects, and the mechanism of estrogen action are discussed. (Author/MT)

  14. Quercetin-induced apoptotic cascade in cancer cells: antioxidant versus estrogen receptor alpha-dependent mechanisms.

    PubMed

    Galluzzo, Paola; Martini, Chiara; Bulzomi, Pamela; Leone, Stefano; Bolli, Alessandro; Pallottini, Valentina; Marino, Maria

    2009-06-01

    The flavonol quercetin, especially abundant in apple, wine, and onions, is reported to have anti-proliferative effects in many cancer cell lines. Antioxidant or pro-oxidant activities and kinase inhibition have been proposed as molecular mechanisms for these effects. In addition, an estrogenic activity has been observed but, at the present, it is poorly understood whether this latter activity plays a role in the quercetin-induced anti-proliferative effects. Here, we studied the molecular mechanisms of quercetin committed to the generation of an apoptotic cascade in cancer cells devoid or containing transfected estrogen receptor alpha (ERalpha; i.e., human cervix epitheloid carcinoma HeLa cells). Although none of tested quercetin concentrations increase reactive oxygen species (ROS) generation in HeLa cells, quercetin stimulation prevents the H(2)O(2)-induced ROS production both in the presence and in the absence of ERalpha. However, this flavonoid induces the activation of p38/MAPK, leading to the pro-apoptotic caspase-3 activation and to the poly(ADP-ribose) polymerase cleavage only in the presence of ERalpha. Notably, no down-regulation of survival kinases (i.e., AKT and ERK) was reported. Taken together, these findings suggest that quercetin results in HeLa cell death through an ERalpha-dependent mechanism involving caspase- and p38 kinase activation. These findings indicate new potential chemopreventive actions of flavonoids on cancer growth.

  15. Receptor-mediated signaling at plasmodesmata.

    PubMed

    Faulkner, Christine

    2013-01-01

    Plasmodesmata (PD) generate continuity between plant cells via the cytoplasm, endoplasmic reticulum (ER) and plasma membrane (PM), allowing movement of different classes of molecules between cells. Proteomic data indicates that the PD PM hosts many receptors and receptor kinases, as well as lipid raft and tetraspanin enriched microdomain associated proteins, suggesting the hypothesis that the PD PM is specialized with respect to both composition and function. PD-located receptor proteins and receptor kinases are responsible for perception of microbe associated molecular patterns at PD and initiate signaling that mediates changes to PD flux. In addition, developmentally relevant receptor kinases have different interactions dependent upon whether located at the PD PM or the cellular PM. The implications of these findings are that receptor-mediated signaling in PD membranes differs from that in the cellular PM and, in light the identification of PD-located proteins associated with membrane microdomains and the role of membrane microdomains in analogous signaling processes in animals, suggests that the PD PM contains specialized signaling platforms.

  16. Hops (Humulus lupulus) inhibits oxidative estrogen metabolism and estrogen-induced malignant transformation in human mammary epithelial cells (MCF-10A).

    PubMed

    Hemachandra, L P; Madhubhani, P; Chandrasena, R; Esala, P; Chen, Shao-Nong; Main, Matthew; Lankin, David C; Scism, Robert A; Dietz, Birgit M; Pauli, Guido F; Thatcher, Gregory R J; Bolton, Judy L

    2012-01-01

    Long-term exposure to estrogens including those in traditional hormone replacement therapy (HRT) increases the risk of developing hormone-dependent cancers. As a result, women are turning to over-the-counter (OTC) botanical dietary supplements, such as black cohosh (Cimicifuga racemosa) and hops (Humulus lupulus), as natural alternatives to HRT. The two major mechanisms which likely contribute to estrogen and/or HRT cancer risk are: the estrogen receptor-mediated hormonal pathway; and the chemical carcinogenesis pathway involving formation of estrogen quinones that damage DNA and proteins, hence initiating and promoting carcinogenesis. Because, OTC botanical HRT alternatives are in widespread use, they may have the potential for chemopreventive effects on estrogen carcinogenic pathways in vivo. Therefore, the effect of OTC botanicals on estrogen-induced malignant transformation of MCF-10A cells was studied. Cytochrome P450 catalyzed hydroxylation of estradiol at the 4-position leads to an o-quinone believed to act as the proximal carcinogen. Liquid chromatography/tandem mass spectrometry analysis of estradiol metabolites showed that 4-hydroxylation was inhibited by hops, whereas black cohosh was without effect. Estrogen-induced expression of CYP450 1B1 and CYP450 1A1 was attenuated by the hops extract. Two phenolic constituents of hops (xanthohumol, XH; 8-prenylnaringenin, 8-PN) were tested: 8-PN was a potent inhibitor, whereas XH had no effect. Finally, estrogen-induced malignant transformation of MCF-10A cells was observed to be significantly inhibited by hops (5 μg/mL) and 8-PN (50 nmol/L). These data suggest that hops extracts possess cancer chemopreventive activity through attenuation of estrogen metabolism mediated by 8-PN.

  17. Behavioral stress may increase the rewarding valence of cocaine-associated cues through a dynorphin/kappa-opioid receptor-mediated mechanism without affecting associative learning or memory retrieval mechanisms.

    PubMed

    Schindler, Abigail G; Li, Shuang; Chavkin, Charles

    2010-08-01

    Stress exposure increases the risk of addictive drug use in human and animal models of drug addiction by mechanisms that are not completely understood. Mice subjected to repeated forced swim stress (FSS) before cocaine develop significantly greater conditioned place preference (CPP) for the drug-paired chamber than unstressed mice. Analysis of the dose dependency showed that FSS increased both the maximal CPP response and sensitivity to cocaine. To determine whether FSS potentiated CPP by enhancing associative learning mechanisms, mice were conditioned with cocaine in the absence of stress, then challenged after association was complete with the kappa-opioid receptor (KOR) agonist U50,488 or repeated FSS, before preference testing. Mice challenged with U50,488 60 min before CPP preference testing expressed significantly greater cocaine-CPP than saline-challenged mice. Potentiation by U50,488 was dose and time dependent and blocked by the KOR antagonist norbinaltorphimine (norBNI). Similarly, mice subjected to repeated FSS before the final preference test expressed significantly greater cocaine-CPP than unstressed controls, and FSS-induced potentiation was blocked by norBNI. Novel object recognition (NOR) performance was not affected by U50,488 given 60 min before assay, but was impaired when given 15 min before NOR assay, suggesting that KOR activation did not potentiate CPP by facilitating memory retrieval or expression. The results from this study show that the potentiation of cocaine-CPP by KOR activation does not result from an enhancement of associative learning mechanisms and that stress may instead enhance the rewarding valence of cocaine-associated cues by a dynorphin-dependent mechanism.

  18. Estrogens and aging skin

    PubMed Central

    Thornton, M. Julie

    2013-01-01

    Estrogen deficiency following menopause results in atrophic skin changes and acceleration of skin aging. Estrogens significantly modulate skin physiology, targeting keratinocytes, fibroblasts, melanocytes, hair follicles and sebaceous glands, and improve angiogenesis, wound healing and immune responses. Estrogen insufficiency decreases defense against oxidative stress; skin becomes thinner with less collagen, decreased elasticity, increased wrinkling, increased dryness and reduced vascularity. Its protective function becomes compromised and aging is associated with impaired wound healing, hair loss, pigmentary changes and skin cancer.   Skin aging can be significantly delayed by the administration of estrogen. This paper reviews estrogen effects on human skin and the mechanisms by which estrogens can alleviate the changes due to aging. The relevance of estrogen replacement, selective estrogen receptor modulators (SERMs) and phytoestrogens as therapies for diminishing skin aging is highlighted. Understanding estrogen signaling in skin will provide a basis for interventions in aging pathologies. PMID:24194966

  19. Monoclonal antibody to the type I insulin-like growth factor (IGF-I) receptor blocks IGF-I receptor-mediated DNA synthesis: clarification of the mitogenic mechanisms of IGF-I and insulin in human skin fibroblasts

    SciTech Connect

    Flier, J.S.; Usher, P.; Moses, A.C.

    1986-02-01

    Insulin and insulin-like growth factor type I (IGF-I) stimulate an overlapping spectrum of biological responses in human skin fibroblasts. Although insulin and IGF-I are known to stimulate the incorporation of (/sup 3/H)thymidine into DNA in these cells, the identify of the receptor(s) that mediates this effect has not been fully clarified. The mouse anti-human IGF-I receptor antibody ..cap alpha..IR-3 binds with specificity to IGF-I but not to insulin receptors in human placental membranes; it also specifically inhibits the binding of /sup 125/I-labeled IGF-I but not /sup 125/I-labeled insulin to suspensions of human skin fibroblasts in a dose-dependent manner. ..cap alpha..IR-3 competitively inhibits IGF-I-mediated stimulation of (/sup 3/H)thymidine incorporation into DNA. This inhibition is dependent on the concentration of ..cap alpha..IR-3 and in the presence of a fixed antibody concentration can be partially overcome by high concentrations of IGF-I. In contrast, at concentrations of < 1 ..mu..g/ml, the effect of insulin to stimulate (/sup 3/H)thymidine incorporation is not inhibited by ..cap alpha..IR-3. However, the incremental effects of higher concentrations (> 1 ..mu..g/ml) of insulin on (/sup 3/H)thymidine incorporation are inhibited by ..cap alpha..IR-3. ..cap alpha..IR-3 is a highly specific antagonist of IGF-I receptor-mediated mitogenesis in human skin fibroblasts. By using this antibody, it is shown directly that insulin can act through the IGF-I receptor to stimulate DNA synthesis but can also activate this effect through the insulin receptor itself.

  20. Receptor subtypes and signal transduction mechanisms contributing to the estrogenic attenuation of cannabinoid-induced changes in energy homeostasis

    PubMed Central

    Washburn, Neal; Borgquist, Amanda; Wang, Kate; Jeffery, Garrett S.; Kelly, Martin J.; Wagner, Edward J.

    2013-01-01

    We examined the receptor subtypes and signal transduction mechanisms contributing to the estrogenic modulation of cannabinoid-induced changes in energy balance. Food intake and, in some cases, O2 consumption, CO2 production and the respiratory exchange ratio, were evaluated in ovariectomized female guinea pigs treated s.c. with the cannabinoid receptor agonist WIN 55,212-2 or its cremephor/ethanol/0.9% saline vehicle, and either with estradiol benzoate (EB), the estrogen receptor (ER)α agonist PPT, the ERβ agonist DPN, the Gq-coupled membrane ER agonist STX, the GPR30 agonist G-1 or their respective vehicles. Patch-clamp recordings were performed in hypothalamic slices. EB, STX, PPT and G-1 decreased daily food intake. Of these, EB, STX and PPT blocked the WIN 55,212-2-induced increase in food intake within 1-4 hr. The estrogenic diminution of cannabinoid-induced hyperphagia correlated with a rapid (within 15 min) attenuation of cannabinoid-mediated decreases in glutamatergic synaptic input onto arcuate neurons, which was completely blocked by inhibition of protein kinase C (PKC) and attenuated by inhibition of protein kinase A (PKA). STX, but not PPT, mimicked this rapid estrogenic effect. However, PPT abolished the cannabinoid-induced inhibition of glutamatergic neurotransmission in cells from animals treated 24 hr prior. The estrogenic antagonism of this presynaptic inhibition was observed in anorexigenic POMC neurons. These data reveal that estrogens negatively modulate cannabinoid-induced changes in energy balance via Gq-coupled membrane ER- and ERα-mediated mechanisms involving activation of PKC and PKA. As such, they further our understanding of the pathways through which estrogens act to temper cannabinoid sensitivity in regulating energy homeostasis in females. PMID:22538462

  1. Hemoglobin Uptake by Paracoccidioides spp. Is Receptor-Mediated

    PubMed Central

    Bailão, Elisa Flávia Luiz Cardoso; Parente, Juliana Alves; Pigosso, Laurine Lacerda; de Castro, Kelly Pacheco; Fonseca, Fernanda Lopes; Silva-Bailão, Mirelle Garcia; Báo, Sônia Nair; Bailão, Alexandre Melo; Rodrigues, Marcio L.; Hernandez, Orville; McEwen, Juan G.; Soares, Célia Maria de Almeida

    2014-01-01

    Iron is essential for the proliferation of fungal pathogens during infection. The availability of iron is limited due to its association with host proteins. Fungal pathogens have evolved different mechanisms to acquire iron from host; however, little is known regarding how Paracoccidioides species incorporate and metabolize this ion. In this work, host iron sources that are used by Paracoccidioides spp. were investigated. Robust fungal growth in the presence of the iron-containing molecules hemin and hemoglobin was observed. Paracoccidioides spp. present hemolytic activity and have the ability to internalize a protoporphyrin ring. Using real-time PCR and nanoUPLC-MSE proteomic approaches, fungal growth in the presence of hemoglobin was shown to result in the positive regulation of transcripts that encode putative hemoglobin receptors, in addition to the induction of proteins that are required for amino acid metabolism and vacuolar protein degradation. In fact, one hemoglobin receptor ortholog, Rbt5, was identified as a surface GPI-anchored protein that recognized hemin, protoporphyrin and hemoglobin in vitro. Antisense RNA technology and Agrobacterium tumefaciens-mediated transformation were used to generate mitotically stable Pbrbt5 mutants. The knockdown strain had a lower survival inside macrophages and in mouse spleen when compared with the parental strain, which suggested that Rbt5 could act as a virulence factor. In summary, our data indicate that Paracoccidioides spp. can use hemoglobin as an iron source most likely through receptor-mediated pathways that might be relevant for pathogenic mechanisms. PMID:24831516

  2. Hemoglobin uptake by Paracoccidioides spp. is receptor-mediated.

    PubMed

    Bailão, Elisa Flávia Luiz Cardoso; Parente, Juliana Alves; Pigosso, Laurine Lacerda; de Castro, Kelly Pacheco; Fonseca, Fernanda Lopes; Silva-Bailão, Mirelle Garcia; Báo, Sônia Nair; Bailão, Alexandre Melo; Rodrigues, Marcio L; Hernandez, Orville; McEwen, Juan G; Soares, Célia Maria de Almeida

    2014-05-01

    Iron is essential for the proliferation of fungal pathogens during infection. The availability of iron is limited due to its association with host proteins. Fungal pathogens have evolved different mechanisms to acquire iron from host; however, little is known regarding how Paracoccidioides species incorporate and metabolize this ion. In this work, host iron sources that are used by Paracoccidioides spp. were investigated. Robust fungal growth in the presence of the iron-containing molecules hemin and hemoglobin was observed. Paracoccidioides spp. present hemolytic activity and have the ability to internalize a protoporphyrin ring. Using real-time PCR and nanoUPLC-MSE proteomic approaches, fungal growth in the presence of hemoglobin was shown to result in the positive regulation of transcripts that encode putative hemoglobin receptors, in addition to the induction of proteins that are required for amino acid metabolism and vacuolar protein degradation. In fact, one hemoglobin receptor ortholog, Rbt5, was identified as a surface GPI-anchored protein that recognized hemin, protoporphyrin and hemoglobin in vitro. Antisense RNA technology and Agrobacterium tumefaciens-mediated transformation were used to generate mitotically stable Pbrbt5 mutants. The knockdown strain had a lower survival inside macrophages and in mouse spleen when compared with the parental strain, which suggested that Rbt5 could act as a virulence factor. In summary, our data indicate that Paracoccidioides spp. can use hemoglobin as an iron source most likely through receptor-mediated pathways that might be relevant for pathogenic mechanisms.

  3. The effects and mechanism of estrogen on rats with Parkinson’s disease in different age groups

    PubMed Central

    Li, Xue-Zhong; Sui, Chen-Yan; Chen, Qiang; Zhuang, Yuan-Su; Zhang, Hong; Zhou, Xiao-Ping

    2016-01-01

    Objective: In order to investigate the effect and mechanism of estrogen in rotenone-induced Parkinson’s disease (PD) rats in different age groups. Methods: we established rat models of PD by rotenone at different interventions. Then, behavioral tests, immunohistochemistry, western blot, high-performance liquid chromatography-electrochemical detector (HPLC-ECD) and electron microscopy were performed. Results: Results revealed the following: (1) Rotenone significantly reduced rotarod latencies in senile rats, prolonged their climbing pole time, and decreased TH positive cells, DA and its metabolite, DOPAC. Estrogen ameliorated this effect, in which weaker effects were observed in younger rats compared with older rats. (2) Rotenone increased the expression of LC3-II in older rats, but estrogen and tamoxifen did not show the same effect. (3) Rotenone increased the number of autophagosomes, but estrogen increased the proportion of autolysosomes/autophagosomes in the rotenone-treated group. (4) U0126 could reduce the number of autophagosomes in the rotenone-treated group, but this did not change the proportion of autolysosome/autophagosome in combining rotenone with the estrogen group. Rapamycin did not increase the number of autophagosomes in the rotenone-treated group, but combining rapamycin with estrogen and rotenone was able to further increase the proportion of autolysome/autophagosomes. Therefore, we speculate that the senile rat model of PD was more reliable than that in young rats. Conclusions: In addition, estrogen could promote autophagy maturation through the ERK pathway, and had an obvious therapeutic effect on the rat model of PD. PMID:27829998

  4. Effects of neuron-specific estrogen receptor (ER) α and ERβ deletion on the acute estrogen negative feedback mechanism in adult female mice.

    PubMed

    Cheong, Rachel Y; Porteous, Robert; Chambon, Pierre; Abrahám, István; Herbison, Allan E

    2014-04-01

    The negative feedback mechanism through which 17β-estradiol (E2) acts to suppress the activity of the GnRH neurons remains unclear. Using inducible and cell-specific genetic mouse models, we examined the estrogen receptor (ER) isoforms expressed by neurons that mediate acute estrogen negative feedback. Adult female mutant mice in which ERα was deleted from all neurons in the neonatal period failed to exhibit estrous cycles or negative feedback. Adult mutant female mice with neonatal neuronal ERβ deletion exhibited normal estrous cycles, but a failure of E2 to suppress LH secretion was seen in ovariectomized mice. Mutant mice with a GnRH neuron-selective deletion of ERβ exhibited normal cycles and negative feedback, suggesting no critical role for ERβ in GnRH neurons in acute negative feedback. To examine the adult roles of neurons expressing ERα, an inducible tamoxifen-based Cre-LoxP approach was used to ablate ERα from neurons that express calmodulin kinase IIα in adults. This resulted in mice with no estrous cycles, a normal increase in LH after ovariectomy, but an inability of E2 to suppress LH secretion. Finally, acute administration of ERα- and ERβ-selective agonists to adult ovariectomized wild-type mice revealed that activation of ERα suppressed LH secretion, whereas ERβ agonists had no effect. This study highlights the differences in adult reproductive phenotypes that result from neonatal vs adult ablation of ERα in the brain. Together, these experiments expand previous global knockout studies by demonstrating that neurons expressing ERα are essential and probably sufficient for the acute estrogen negative feedback mechanism in female mice.

  5. Estrogen regulates the rate of bone turnover but bone balance in ovariectomized rats is modulated by prevailing mechanical strain

    NASA Technical Reports Server (NTRS)

    Westerlind, K. C.; Wronski, T. J.; Ritman, E. L.; Luo, Z. P.; An, K. N.; Bell, N. H.; Turner, R. T.

    1997-01-01

    Estrogen deficiency induced bone loss is associated with increased bone turnover in rats and humans. The respective roles of increased bone turnover and altered balance between bone formation and bone resorption in mediating estrogen deficiency-induced cancellous bone loss was investigated in ovariectomized rats. Ovariectomy resulted in increased bone turnover in the distal femur. However, cancellous bone was preferentially lost in the metaphysis, a site that normally experiences low strain energy. No bone loss was observed in the epiphysis, a site experiencing higher strain energy. The role of mechanical strain in maintaining bone balance was investigated by altering the strain history. Mechanical strain was increased and decreased in long bones of ovariectomized rats by treadmill exercise and functional unloading, respectively. Functional unloading was achieved during orbital spaceflight and following unilateral sciatic neurotomy. Increasing mechanical loading reduced bone loss in the metaphysis. In contrast, decreasing loading accentuated bone loss in the metaphysis and resulted in bone loss in the epiphysis. Finally, administration of estrogen to ovariectomized rats reduced bone loss in the unloaded and prevented loss in the loaded limb following unilateral sciatic neurotomy in part by reducing indices of bone turnover. These results suggest that estrogen regulates the rate of bone turnover, but the overall balance between bone formation and bone resorption is influenced by prevailing levels of mechanical strain.

  6. DEHP exposure impairs mouse oocyte cyst breakdown and primordial follicle assembly through estrogen receptor-dependent and independent mechanisms.

    PubMed

    Mu, Xinyi; Liao, Xinggui; Chen, Xuemei; Li, Yanli; Wang, Meirong; Shen, Cha; Zhang, Xue; Wang, Yingxiong; Liu, Xueqing; He, Junlin

    2015-11-15

    Estrogen plays an essential role in the development of mammalian oocytes, and recent studies suggest that it also regulates primordial follicle assembly in the neonatal ovaries. During the last decade, potential exposure of humans and animals to estrogen-like endocrine disrupting chemicals has become a growing concern. In the present study, we focused on the effect of diethylhexyl phthalate (DEHP), a widespread plasticizer with estrogen-like activity, on germ-cell cyst breakdown and primordial follicle assembly in the early ovarian development of mouse. Neonatal mice injected with DEHP displayed impaired cyst breakdown. Using ovary organ cultures, we revealed that impairment was mediated through estrogen receptors (ERs), as ICI 182,780, an efficient antagonist of ER, reversed this DEHP-mediated effect. DEHP exposure reduced the expression of ERβ, progesterone receptor (PR), and Notch2 signaling components. Finally, DEHP reduced proliferation of pregranulosa precursor cells during the process of primordial folliculogenesis. Together, our results indicate that DEHP influences oocyte cyst breakdown and primordial follicle formation through several mechanisms. Therefore, exposure to estrogen-like chemicals during fetal or neonatal development may adversely influence early ovarian development. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Testosterone Represses Estrogen Signaling by Upregulating miR-22: A Mechanism for Imbalanced Steroid Hormone Production in Preeclampsia.

    PubMed

    Shao, Xuan; Liu, Yanlei; Liu, Ming; Wang, Yongqing; Yan, Liying; Wang, Hao; Ma, Liyang; Li, Yu-Xia; Zhao, Yangyu; Wang, Yan-Ling

    2017-04-01

    Preeclampsia, a multisystem syndrome occurring during mid- to late gestation in humans, is a leading cause of maternal and perinatal morbidity and mortality. Patients usually present with high circulating testosterone and reduced estradiol production, but the mechanisms remain unclear. Revealing the mechanism that modulating the imbalance of testosterone and estradiol in preeclampsia is of great value in understanding the cause of the disease. The placenta is the predominant source of steroid hormone production during gestation, and we observed markedly increased 17β-HSD3 (17β-hydroxysteroid dehydrogenase 3) levels and downregulated aromatase expression, the key enzymes responsible for synthesis of testosterone and estradiol, respectively, in preeclamptic placentas compared with controls. Furthermore, we found a significant upregulation of microRNA (miR)-22 in preeclamptic placentas. In a trophoblast cell line, JEG-3 cells, testosterone repressed the expression of aromatase and estrogen receptor α and the production of estradiol while promoting miR-22 expression. miR-22 directly targeted and inhibited estrogen receptor α expression while indirectly decreasing aromatase expression and estradiol production by interfering with estrogen receptor α signaling. Furthermore, inhibition of miR-22 expression significantly reversed the inhibitory effect of testosterone on de novo estradiol synthesis in human trophoblastic cells. The findings reveal a mechanism underlying the balanced production of androgen and estrogen modulated by miR-22 in the human placenta and provide new insights into the pathogenesis of preeclampsia from the aspect of endocrine regulation. © 2017 American Heart Association, Inc.

  8. Heterogeneous estrogen receptor expression in circulating tumor cells suggests diverse mechanisms of fulvestrant resistance.

    PubMed

    Paoletti, Costanza; Larios, Jose M; Muñiz, Maria C; Aung, Kimberly; Cannell, Emily M; Darga, Elizabeth P; Kidwell, Kelley M; Thomas, Dafydd G; Tokudome, Nahomi; Brown, Martha E; Connelly, Mark C; Chianese, David A; Schott, Anne F; Henry, N Lynn; Rae, James M; Hayes, Daniel F

    2016-08-01

    Fulvestrant is a dose dependent selective estrogen receptor (ER) down-regulator (SERD) used in ER-positive metastatic breast cancer (MBC). Nearly all patients develop resistance. We performed molecular analysis of circulating tumor cells (CTC) to gain insight into fulvestrant resistance. Preclinical studies were performed with cultured breast cancer cells spiked into human blood and analyzed on the CellSearch(®) system. Clinical data are limited to a subset of patients with ER-positive MBC from a previously reported pilot trial whose disease was progressing on fulvestrant (N = 7) or aromatase inhibitors (AIs) (N = 10). CTCs were enumerated and phenotyped for ER and B-cell lymphoma (BCL2) using the CellSearch(®) CXC kit. In preclinical modeling, tamoxifen and AIs resulted in stabilized ER expression, whereas fulvestrant eliminated it. Five of seven patients progressing on fulvestrant had ≥5CTC/7.5 ml WB. Two of these five, treated with 500 mg/month fulvestrant, had no detectable CTC-expression of ER and BCL2 (an ER regulated gene). Three patients had heterogeneous CTC-ER and BCL2 expression indicating incomplete degradation of the ER target by fulvestrant. Two of these patients received 250 mg/month whereas the third patient received 500 mg/month fulvestrant. Her cancer harbored a mutation (Y537S) in the estrogen receptor alpha gene (ESR1). All seven ER positive patients progressing on AIs had heterogeneous CTC-ER expression. These results suggest heterogeneous mechanisms of resistance to fulvestrant, including insufficient dosage, ESR1 mutation, or conversion to dependence on non-ER pathways. CTC enumeration, phenotyping, and genotyping might identify patients who would benefit from fulvestrant dose escalation versus switching to alternative therapies.

  9. A pp32-retinoblastoma protein complex modulates androgen receptor-mediated transcription and associates with components of the splicing machinery

    SciTech Connect

    Adegbola, Onikepe; Pasternack, Gary R. . E-mail: gpastern@jhmi.edu

    2005-08-26

    We have previously shown pp32 and the retinoblastoma protein interact. pp32 and the retinoblastoma protein are nuclear receptor transcriptional coregulators: the retinoblastoma protein is a coactivator for androgen receptor, the major regulator of prostate cancer growth, while pp32, which is highly expressed in prostate cancer, is a corepressor of the estrogen receptor. We now show pp32 increases androgen receptor-mediated transcription and the retinoblastoma protein modulates this activity. Using affinity purification and mass spectrometry, we identify members of the pp32-retinoblastoma protein complex as PSF and nonO/p54nrb, proteins implicated in coordinate regulation of nuclear receptor-mediated transcription and splicing. We show that the pp32-retinoblastoma protein complex is modulated during TPA-induced K562 differentiation. Present evidence suggests that nuclear receptors assemble multiprotein complexes to coordinately regulate transcription and mRNA processing. Our results suggest that pp32 and the retinoblastoma protein may be part of a multiprotein complex that coordinately regulates nuclear receptor-mediated transcription and mRNA processing.

  10. Receptor-Mediated Drug Delivery Systems Targeting to Glioma

    PubMed Central

    Wang, Shanshan; Meng, Ying; Li, Chengyi; Qian, Min; Huang, Rongqin

    2015-01-01

    Glioma has been considered to be the most frequent primary tumor within the central nervous system (CNS). The complexity of glioma, especially the existence of the blood-brain barrier (BBB), makes the survival and prognosis of glioma remain poor even after a standard treatment based on surgery, radiotherapy, and chemotherapy. This provides a rationale for the development of some novel therapeutic strategies. Among them, receptor-mediated drug delivery is a specific pattern taking advantage of differential expression of receptors between tumors and normal tissues. The strategy can actively transport drugs, such as small molecular drugs, gene medicines, and therapeutic proteins to glioma while minimizing adverse reactions. This review will summarize recent progress on receptor-mediated drug delivery systems targeting to glioma, and conclude the challenges and prospects of receptor-mediated glioma-targeted therapy for future applications.

  11. Mechanisms of action of estrogen in the brain: insights from human neuroimaging and psychopharmacologic studies.

    PubMed

    Maki, Pauline M; Dumas, Julie

    2009-05-01

    Use of estrogen therapy in the perimenopausal and postmenopausal periods has been shown in several clinical trials to help women maintain a premenopausal level of cognitive function. What is not yet fully understood is how the neurobiological effects of estrogen contribute to these cognitive effects. This review explores data from two related bodies of human literature that provide compelling evidence in support of the biological plausibility that estrogen treatment can benefit cognition. The first half of the literature review focuses on studies from the estrogen neuroimaging literature, and the second half focuses on pharmacologic challenge studies assessing estrogen-neurotransmitter interactions. We integrate these two bodies of literature by focusing on the neurophysiologic underpinnings of estrogen effects on cognition and linking these clinical studies to preclinical studies. The focus on verbal memory is important because it is a cognitive function that has been shown to change with estrogen treatment and predict Alzheimer's disease risk but is not addressed by preclinical studies. Overall, we conclude that estrogen interacts with cholinergic and serotonergic systems to affect hippocampal and frontal cortical brain areas and thereby enhance memory, particularly at the retrieval stage.

  12. Estrogens of multiple classes and their role in mental health disease mechanisms

    PubMed Central

    Watson, Cheryl S; Alyea, Rebecca A; Cunningham, Kathryn A; Jeng, Yow-Jiun

    2010-01-01

    Gender and sex hormones can influence a variety of mental health states, including mood, cognitive development and function, and vulnerability to neurodegenerative diseases and brain damage. Functions of neuronal cells may be altered by estrogens depending upon the availability of different physiological estrogenic ligands; these ligands and their effects vary with life stages, the genetic or postgenetic regulation of receptor levels in specific tissues, or the intercession of competing nonphysiological ligands (either intentional or unintentional, beneficial to health or not). Here we review evidence for how different estrogens (physiological and environmental/dietary), acting via different estrogen receptor subtypes residing in alternative subcellular locations, influence brain functions and behavior. We also discuss the families of receptors and transporters for monoamine neurotransmitters and how they may interact with the estrogenic signaling pathways. PMID:21072308

  13. High-throughput screening and mechanism-based evaluation of estrogenic botanical extracts

    PubMed Central

    Overk, Cassia R.; Yao, Ping; Chen, Shaonong; Deng, Shixing; Imai, Ayano; Main, Matthew; Schinkovitz, Andreas; Farnsworth, Norman R.; Pauli, Guido F.; Bolton, Judy L.

    2009-01-01

    Symptoms associated with menopause can greatly affect the quality of life for women. Botanical dietary supplements have been viewed by the public as safe and effective despite a lack of evidence indicating a urgent necessity to standardize these supplements chemically and biologically. Seventeen plants were evaluated for estrogenic biological activity using standard assays: competitive estrogen receptor (ER) binding assay for both alpha and beta subtypes, transient transfection of the estrogen response element luciferase plasmid into MCF-7 cells expressing either ER alpha or ER beta, and the Ishikawa alkaline phosphatase induction assay for both estrogenic and antiestrogenic activities. Based on the combination of data pooled from these assays, the following was determined: a) a high rate of false positive activity for the competitive binding assays, b) some extracts had estrogenic activity despite a lack of ability to bind the ER, c) one extract exhibited selective estrogen receptor modulator (SERM) activity, and d) several extracts show additive/synergistic activity. Taken together, these data indicate a need to reprioritize the order in which the bioassays are performed for maximal efficiency of programs involving bioassay-guided fractionation. In addition, possible explanations for the conflicts in the literature over the estrogenicity of Cimicifuga racemosa (black cohosh) are suggested. PMID:18473738

  14. Neonatal exposure to bisphenol A alters estrogen-dependent mechanisms governing sexual behavior in the adult female rat.

    PubMed

    Monje, L; Varayoud, J; Muñoz-de-Toro, M; Luque, E H; Ramos, J G

    2009-12-01

    This study examines the effects of neonatal exposure to the endocrine disruptor bisphenol A (BPA) on the hypothalamic circuitry controlling the female sexual behaviors of adult rats. From postnatal day 1 (PND1) to PND7, pups were injected with corn oil (control) or BPA (BPA20: 20mg/kg-d; BPA.05: 0.05 mg/kg-d) and at PND85 the rats were bilaterally ovariectomized (OVX). At PND100, OVX-rats received estradiol alone or estradiol and progesterone to evaluate estrogen-dependent gene expression in the hypothalamus and sexual behavior. In BPA-exposed females, estrogen receptor alpha (ER alpha) expression was down-regulated in both the medial preoptic (MPN) and ventromedial nucleus (VMHvl), while repressor of estrogen receptor activity (REA) expression was up-regulated in the VMHvl. Interestingly, BPA-exposed females displayed significantly lower levels of proceptive behavior. Our results show that BPA permanently alters the hypothalamic estrogen-dependent mechanisms that govern sexual behavior in the adult female rat.

  15. Evidence that somatostatin sst2 receptors mediate striatal dopamine release

    PubMed Central

    Hathway, G J; Humphrey, P P A; Kendrick, K M

    1999-01-01

    Somatostatin (SRIF) is a cyclic tetradecapeptide present in medium-sized aspiny interneurones in the rat striatum. We have previously shown that exogenous SRIF potently stimulates striatal dopamine (DA) release via a glutamate-dependent mechanism. We now report the ability of the selective sst2 receptor agonist, BIM-23027, to mimic this effect of SRIF.In vivo microdialysis studies were performed in anaesthetized male Wistar rats. In most experiments, compounds were administered by retrodialysis into the striatum for 15 min periods, 90 min and 225 min after sampling commenced, with levels of neurotransmitters being measured by HPLC with electrochemical and fluorescence detection.BIM-23027 (50 and 100 nM) stimulated DA release with extracellular levels increasing by up to 18 fold.Prior retrodialysis of BIM-23027 (50 nM) abolished the effects of subsequent administration of SRIF (100 nM).The agonist effects of both BIM-23027 and SRIF were abolished by the selective sst2 receptor antagonist, L-Tyr8-CYN-154806 (100 nM).The AMPA/kainate receptor antagonist, DNQX (100 μM), abolished the agonist effects of BIM-23027 as previously shown for SRIF.This study provides evidence that the sst2 receptor mediates the potent dopamine-releasing actions observed with SRIF in the rat striatum. Dopamine release evoked by both peptides appears to be mediated indirectly via a glutamatergic pathway. Other subtype-specific somatostatin receptor ligands were unable to elicit any effects and therefore we conclude that no other somatostatin receptor types are involved in mediating the dopamine-releasing actions of SRIF in the striatum. PMID:10578151

  16. Mechanisms of uterine estrogen signaling during early pregnancy in mice: an update

    PubMed Central

    Robertshaw, I; Bian, F; Das, S K

    2016-01-01

    Adherence of an embryo to the uterus represents the most critical step of the reproductive process. Implantation is a synchronized event between the blastocyst and uterine luminal epithelium leading to structural and functional changes for further embryonic growth and development. The milieu comprising the complex process of implantation is mediated by estrogen through diverse but interdependent signaling pathways. Mouse models have demonstrated the relevance of the expression of estrogen modulated paracrine factors to uterine receptivity and implantation window. More importantly, some factors seem to serve as molecular links between different estrogen pathways promoting cell growth, acting as molecular chaperones or amplifying estrogenic effects. Abnormal expression of these factors can lead to implantation failure and infertility. This review provides an overview of several well characterized signaling pathways that elucidates molecular cross-talk involved in the uterus during early pregnancy. PMID:26887389

  17. In Silico Elucidation of the Molecular Mechanism Defining the Adverse Effect of Selective Estrogen Receptor Modulators

    PubMed Central

    Xie, Lei; Wang, Jian; Bourne, Philip E

    2007-01-01

    Early identification of adverse effect of preclinical and commercial drugs is crucial in developing highly efficient therapeutics, since unexpected adverse drug effects account for one-third of all drug failures in drug development. To correlate protein–drug interactions at the molecule level with their clinical outcomes at the organism level, we have developed an integrated approach to studying protein–ligand interactions on a structural proteome-wide scale by combining protein functional site similarity search, small molecule screening, and protein–ligand binding affinity profile analysis. By applying this methodology, we have elucidated a possible molecular mechanism for the previously observed, but molecularly uncharacterized, side effect of selective estrogen receptor modulators (SERMs). The side effect involves the inhibition of the Sacroplasmic Reticulum Ca2+ ion channel ATPase protein (SERCA) transmembrane domain. The prediction provides molecular insight into reducing the adverse effect of SERMs and is supported by clinical and in vitro observations. The strategy used in this case study is being applied to discover off-targets for other commercially available pharmaceuticals. The process can be included in a drug discovery pipeline in an effort to optimize drug leads and reduce unwanted side effects. PMID:18052534

  18. Isoflavone genistein inhibits estrogen-induced chloride and bicarbonate secretory mechanisms in the uterus in rats.

    PubMed

    Chinigarzadeh, Asma; Karim, Kamarulzaman; Muniandy, Sekaran; Salleh, Naguib

    2017-04-01

    We hypothesized that genistein could affect the chloride (Cl(-) ) and bicarbonate (HCO3(-) ) secretory mechanisms in uterus. Ovariectomized female rats were given estradiol or estradiol plus progesterone with 25, 50, or 100 mg/kg/day genistein. Following completion of the treatment, uterine fluid Cl(-) and HCO3(-) concentrations were determined by in vivo uterine perfusion. Uteri were subjected for molecular biological analysis (Western blot, qPCR, and immunohistochemistry) to detect levels of expression of Cystic Fibrosis transmembrane regulator (CFTR), Cl(-) /HCO3(-) exchanger (SLC26a6), Na(+) /HCO3(-) cotransporter (SLC4a4), and estrogen receptor (ER)-α and β. Coadministration of genistein resulted in decrease in Cl(-) and HCO3(-) concentrations and expression of CFTR, SLC26a6, SLC4a4, and ER-α and ER-β in the uteri of estradiol-treated rats. In estradiol plus progesterone-treated rats, a significant increase in the above parameters were observed following high-dose genistein treatment except for the SLC24a4 level. In conclusion, genistein-induced changes in the uterus could affect the reproductive processes that might result in infertility. © 2016 Wiley Periodicals, Inc.

  19. Identifying a Mechanism for Crosstalk Between the Estrogen and Glucocorticoid Receptors | Center for Cancer Research

    Cancer.gov

    Estrogen has long been known to play important roles in the development and progression of breast cancer. Its receptor (ER), a member of the steroid receptor family, binds to estrogen response elements (EREs) in DNA and regulates gene transcription. More recently, another steroid receptor family member, the glucocorticoid receptor (GR), has been implicated in breast cancer progression, and ER/GR status is an important predictor of breast cancer outcome.

  20. Extracellular ATP and P2Y2 receptors mediate intercellular Ca2+ waves induced by mechanical stimulation in submandibular gland cells: role of mitochondrial regulation of store operated Ca2+ entry

    PubMed Central

    Ryu, Shin-Young; Peixoto, Pablo M.; Won, Jong-Hak; Yule, David I.; Kinnally, Kathleen W.

    2009-01-01

    SUMMARY Coordination of Ca2+ signaling among cells contributes to synchronization of salivary gland cell function. However, mechanisms that underlie this signaling remain elusive. Here, intercellular Ca2+ waves (ICW) in submandibular gland cells were investigated using fura-2 fluorescence imaging. Mechanical stimulation of single cells induced ICW propagation from the stimulated cells through ~7 layers of cells or ~120 μm. Our findings indicate that an extracellular ATP-dependent pathway is involved because the purinergic receptor antagonist suramin and the ATP hydrolyzing enzyme apyrase blocked ICW propagation. However, the gap junction uncoupler oleamide had no effect. ATP is released from mechanically stimulated cells possibly through opening of mechanosensitive maxi-anion channels, and does not appear to be directly linked to cytosolic Ca2+. The ICW is propagated by diffusing ATP, which activates purinergic receptors in neighboring cells. This purinergic signaling induces a Ca2+ transient that is dependent on Ca2+ release via IP3 receptors in the ER and store operated Ca2+ entry (SOCE). Finally, inhibition of mitochondrial Ca2+ uptake modified ICW indicating an important role of these organelles in this phenomenon. These studies increase our understanding of purinergic receptor signaling in salivary gland cells, and its role as a coordination mechanism of Ca2+ signals induced by mechanical stimulation. PMID:20022109

  1. NMDA Receptors Mediate Synaptic Competition in Culture

    PubMed Central

    She, Kevin; Craig, Ann Marie

    2011-01-01

    Background Activity through NMDA type glutamate receptors sculpts connectivity in the developing nervous system. This topic is typically studied in the visual system in vivo, where activity of inputs can be differentially regulated, but in which individual synapses are difficult to visualize and mechanisms governing synaptic competition can be difficult to ascertain. Here, we develop a model of NMDA-receptor dependent synaptic competition in dissociated cultured hippocampal neurons. Methodology/Principal Findings GluN1 -/- (KO) mouse hippocampal neurons lacking the essential NMDA receptor subunit were cultured alone or cultured in defined ratios with wild type (WT) neurons. The absence of functional NMDA receptors did not alter neuron survival. Synapse development was assessed by immunofluorescence for postsynaptic PSD-95 family scaffold and apposed presynaptic vesicular glutamate transporter VGlut1. Synapse density was specifically enhanced onto minority wild type neurons co-cultured with a majority of GluN1 -/- neighbour neurons, both relative to the GluN1 -/- neighbours and relative to sister pure wild type cultures. This form of synaptic competition was dependent on NMDA receptor activity and not conferred by the mere physical presence of GluN1. In contrast to these results in 10% WT and 90% KO co-cultures, synapse density did not differ by genotype in 50% WT and 50% KO co-cultures or in 90% WT and 10% KO co-cultures. Conclusions/Significance The enhanced synaptic density onto NMDA receptor-competent neurons in minority coculture with GluN1 -/- neurons represents a cell culture paradigm for studying synaptic competition. Mechanisms involved may include a retrograde ‘reward’ signal generated by WT neurons, although in this paradigm there was no ‘punishment’ signal against GluN1 -/- neurons. Cell culture assays involving such defined circuits may help uncover the rules and mechanisms of activity-dependent synaptic competition in the developing nervous

  2. Elsevier Trophoblast Research Award lecture: Molecular mechanisms underlying estrogen functions in trophoblastic cells--focus on leptin expression.

    PubMed

    Gambino, Y P; Maymó, J L; Pérez Pérez, A; Calvo, J C; Sánchez-Margalet, V; Varone, C L

    2012-02-01

    The steroid hormone 17β-estradiol is an estrogen that influences multiple aspects of placental function and fetal development in humans. During early pregnancy it plays a role in the regulation of blastocyst implantation, trophoblast differentiation and invasiveness, remodeling of uterine arteries, immunology and trophoblast production of hormones such as leptin. Estradiol exerts some effects through the action of classical estrogen receptors ERα and ERβ, which act as ligand-activated transcription factors and regulate gene expression. In addition, estradiol can elicit rapid responses from membrane-associated receptors, like activation of protein-kinase pathways. Thus, the cellular effects of estradiol will depend on the specific receptors expressed and the integration of their signaling events. Leptin, the 16,000MW protein product of the obese gene, was originally considered an adipocyte-derived signaling molecule for the central control of metabolism. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy. The leptin gene is expressed in placenta, where leptin promotes proliferation and survival of trophoblastic cells. Expression of leptin in placenta is highly regulated by key pregnancy molecules as hCG and estradiol. The aim of this paper is to review the molecular mechanisms underlying estrogen functions in trophoblastic cells; focusing on mechanisms involved in estradiol regulation of placental leptin expression. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Multiple direct and indirect mechanisms drive estrogen-induced tumor growth in high grade serous ovarian cancers.

    PubMed

    Ciucci, Alessandra; Zannoni, Gian Franco; Buttarelli, Marianna; Lisi, Lucia; Travaglia, Daniele; Martinelli, Enrica; Scambia, Giovanni; Gallo, Daniela

    2016-02-16

    The notion that menopausal estrogen replacement therapy increases ovarian cancer risk, but only for the two more common types (i.e. serous and endometrioid), while possibly decreasing risk for clear cell tumors, is strongly suggestive of causality. However, whether estradiol (E2) is tumorigenic or promotes development of occult preexisting disease is unknown. The present study investigated molecular and cellular mechanisms by which E2 modulates the growth of high grade serous ovarian cancer (HGSOC). Results showed that ERα expression was necessary and sufficient to induce the growth of HGSOC cells in in vitro models. Conversely, in vivo experimental studies demonstrated that increasing the levels of circulating estrogens resulted in a significant growth acceleration of ERα-negative HGSOC xenografts, as well. Tumors from E2-treated mice had significantly higher proliferation rate, angiogenesis, and density of tumor-associated macrophage (TAM) compared to ovariectomized females. Accordingly, immunohistochemical analysis of ERα-negative tissue specimens from HGSOC patients showed a significantly greater TAM infiltration in premenopausal compared to postmenopausal women. This study describes novel insights into the impact of E2 on tumor microenvironment, independently of its direct effect on tumor cell growth, thus supporting the idea that multiple direct and indirect mechanisms drive estrogen-induced tumor growth in HGSOC.

  4. CD3ζ-based chimeric antigen receptors mediate T cell activation via cis- and trans-signalling mechanisms: implications for optimization of receptor structure for adoptive cell therapy.

    PubMed

    Bridgeman, J S; Ladell, K; Sheard, V E; Miners, K; Hawkins, R E; Price, D A; Gilham, D E

    2014-02-01

    Chimeric antigen receptors (CARs) can mediate redirected lysis of tumour cells in a major histocompatibility complex (MHC)-independent manner, thereby enabling autologous adoptive T cell therapy for a variety of malignant neoplasms. Currently, most CARs incorporate the T cell receptor (TCR) CD3ζ signalling chain; however, the precise mechanisms responsible for CAR-mediated T cell activation are unclear. In this study, we used a series of immunoreceptor tyrosine-based activation motif (ITAM)-mutant and transmembrane-modified receptors to demonstrate that CARs activate T cells both directly via the antigen-ligated signalling chain and indirectly via associated chains within the TCR complex. These observations allowed us to generate new receptors capable of eliciting polyfunctional responses in primary human T cells. This work increases our understanding of CAR function and identifies new avenues for the optimization of CAR-based therapeutic interventions.

  5. Effects of estrogen on the mechanical behavior of the human Achilles tendon in vivo.

    PubMed

    Bryant, Adam L; Clark, Ross A; Bartold, Simon; Murphy, Aron; Bennell, Kim L; Hohmann, Erik; Marshall-Gradisnik, Sonya; Payne, Craig; Crossley, Kay M

    2008-10-01

    The purpose of this study was to elucidate the effect of normal fluctuating [non-monophasic oral contraceptive pill (MOCP) users] and low, consistent (MOCP users) endogenous plasma estrogen levels on the strain behavior of the Achilles tendon in vivo. Twenty women (age 28.0 +/- 4.2 yr, height 1.67 +/- 0.07 m, mass 61.6 +/- 6.8 kg) who had been using the MOCP for at least 12 mo together with 20 matched women who were non-MOCP users (age 31.9 +/- 7.3 yr, height 1.63 +/- 0.05 m, mass 62.5 +/- 5.9 kg) participated in this study. Non-MOCP users were tested at the time of lowest (menstruation) and highest (approximately same as ovulation) estrogen, whereas MOCP users, who exhibited constant and attenuated endogenous estrogen levels, were tested at day 1 and day 14 of their cycle. At each test session, maximal isometric plantarflexion efforts were performed on a calf-raise apparatus while synchronous real-time ultrasonography of the triceps surae aponeurosis was recorded. Achilles tendon strain (%) was calculated by dividing tendon displacement during plantarflexion by resting tendon length. Repeated-measures ANOVA revealed a significant (P < 0.05) main effect of subject group with significantly lower Achilles strain (25.5%) in the MOCP users compared with the non-MOCP users. In conclusion, acute fluctuations in plasma estrogen across the menstrual cycle in non-MOCP users did not alter the strain behavior of the Achilles tendon. Conversely, long-term exposure to attenuated estrogen in MOCP users resulted in a decrease in Achilles tendon strain, which is thought to be attributed to the effects of endogenous estrogen on collagen synthesis. These findings have a number of important functional and clinical implications.

  6. Ritonavir binds to and downregulates estrogen receptors: Molecular mechanism of promoting early atherosclerosis

    SciTech Connect

    Xiang, Jin; Wang, Ying; Su, Ke; Liu, Min; Hu, Peng-Chao; Ma, Tian; Li, Jia-Xi; Wei, Lei; Zheng, Zhongliang; Yang, Fang

    2014-10-01

    Estrogenic actions are closely related to cardiovascular disease. Ritonavir (RTV), a human immunodeficiency virus (HIV) protease inhibitor, induces atherosclerosis in an estrogen-related manner. However, how RTV induce pathological phenotypes through estrogen pathway remains unclear. In this study, we found that RTV increases thickness of coronary artery walls of Sprague Dawley rats and plasma free fatty acids (FFA) levels. In addition, RTV could induce foam cell formation, downregulate both estrogen receptor α (ERα) and ERβ expression, upregulate G protein-coupled estrogen receptor (GPER) expression, and all of them could be partially blocked by 17β-estradiol (E2), suggesting RTV acts as an antagonist for E2. Computational modeling shows a similar interaction with ERα between RTV and 2-aryl indoles, which are highly subtype-selective ligands for ERα. We also found that RTV directly bound to ERα and selectively inhibited the nuclear localization of ERα, and residue Leu536 in the hydrophobic core of ligand binding domain (LBD) was essential for the interaction with RTV. In addition, RTV did not change the secondary structure of ERα-LBD like E2, which explained how ERα lost the capacity of nuclear translocation under the treatment of RTV. All of the evidences suggest that ritonavir acts as an antagonist for 17β-estradiol in regulating α subtype estrogen receptor function and early events of atherosclerosis. - Graphical abstract: RTV directly binds to ERα and Leu536 in the hydrophobic core of ligand binding domain is essential for the interaction. - Highlights: • RTV increases the thickness of rat coronary artery wall and foam cell formation. • RTV downregulates the expression of ERα and ERβ. • RTV inhibits ERα promoter activity. • RTV directly binds to ERα and the key amino acid is Leu536. • RTV inhibits the nuclear translocation of ERα and GPER.

  7. Comparative analyses of lysophosphatidic acid receptor-mediated signaling.

    PubMed

    Fukushima, Nobuyuki; Ishii, Shoichi; Tsujiuchi, Toshifumi; Kagawa, Nao; Katoh, Kazutaka

    2015-06-01

    Lysophosphatidic acid (LPA) is a bioactive lipid mediator that activates G protein-coupled LPA receptors to exert fundamental cellular functions. Six LPA receptor genes have been identified in vertebrates and are classified into two subfamilies, the endothelial differentiation genes (edg) and the non-edg family. Studies using genetically engineered mice, frogs, and zebrafish have demonstrated that LPA receptor-mediated signaling has biological, developmental, and pathophysiological functions. Computational analyses have also identified several amino acids (aa) critical for LPA recognition by human LPA receptors. This review focuses on the evolutionary aspects of LPA receptor-mediated signaling by comparing the aa sequences of vertebrate LPA receptors and LPA-producing enzymes; it also summarizes the LPA receptor-dependent effects commonly observed in mouse, frog, and fish.

  8. Receptor-mediated Endocytosis in the Caenorhabditis elegans Oocyte

    PubMed Central

    Grant, Barth; Hirsh, David

    1999-01-01

    The Caenorhabditis elegans oocyte is a highly amenable system for forward and reverse genetic analysis of receptor-mediated endocytosis. We describe the use of transgenic strains expressing a vitellogenin::green fluorescent protein (YP170::GFP) fusion to monitor yolk endocytosis by the C. elegans oocyte in vivo. This YP170::GFP reporter was used to assay the functions of C. elegans predicted proteins homologous to vertebrate endocytosis factors using RNA-mediated interference. We show that the basic components and pathways of endocytic trafficking are conserved between C. elegans and vertebrates, and that this system can be used to test the endocytic functions of any new gene. We also used the YP170::GFP assay to identify rme (receptor-mediated endocytosis) mutants. We describe a new member of the low-density lipoprotein receptor superfamily, RME-2, identified in our screens for endocytosis defective mutants. We show that RME-2 is the C. elegans yolk receptor. PMID:10588660

  9. Multiscale Modeling of Virus Entry via Receptor-Mediated Endocytosis

    NASA Astrophysics Data System (ADS)

    Liu, Jin

    2012-11-01

    Virus infections are ubiquitous and remain major threats to human health worldwide. Viruses are intracellular parasites and must enter host cells to initiate infection. Receptor-mediated endocytosis is the most common entry pathway taken by viruses, the whole process is highly complex and dictated by various events, such as virus motions, membrane deformations, receptor diffusion and ligand-receptor reactions, occurring at multiple length and time scales. We develop a multiscale model for virus entry through receptor-mediated endocytosis. The binding of virus to cell surface is based on a mesoscale three dimensional stochastic adhesion model, the internalization (endocytosis) of virus and cellular membrane deformation is based on the discretization of Helfrich Hamiltonian in a curvilinear space using Monte Carlo method. The multiscale model is based on the combination of these two models. We will implement this model to study the herpes simplex virus entry into B78 cells and compare the model predictions with experimental measurements.

  10. Estrogen promotes the brain metastatic colonization of triple negative breast cancer cells via an astrocyte-mediated paracrine mechanism

    PubMed Central

    Sartorius, Carol A.; Hanna, Colton T.; Gril, Brunilde; Cruz, Hazel; Serkova, Natalie J.; Huber, Kendra M.; Kabos, Peter; Schedin, Troy B.; Borges, Virginia F.; Steeg, Patricia S.; Cittelly, Diana M.

    2015-01-01

    Brain metastases (BM) are a devastating consequence of breast cancer. BM occur more frequently in patients with estrogen receptor-negative (ER−) breast cancer subtypes; HER2 overexpressing (HER2+) tumors and triple-negative (TN) (ER−, progesterone receptor-negative (PR−) and normal HER2) tumors. Young age is an independent risk factor for development of BM, thus we speculated that higher circulating estrogens in young, pre-menopausal women could exert paracrine effects through the highly estrogen-responsive brain microenvironment. Using a TN experimental metastases model, we demonstrate that ovariectomy decreased the frequency of MRI detectable lesions by 56% as compared to estrogen supplementation, and that the combination of ovariectomy and letrozole further reduced the frequency of large lesions to 14.4% of the estrogen control. Human BM expressed 4.2-48.4% ER+ stromal area, particularly ER+ astrocytes. In vitro, E2-treated astrocytes increased proliferation, migration and invasion of 231BR-EGFP cells in an ER-dependent manner. E2 upregulated EGFR ligands Egf, Ereg, and Tgfa mRNA and protein levels in astrocytes, and activated EGFR in brain metastatic cells. Co-culture of 231BR-EGFP cells with E2-treated astrocytes led to upregulation of the metastatic mediator S100 Calcium-binding protein A4 (S100A4) (1.78-fold, P<0.05). Exogenous EGF increased S100A4 mRNA levels in 231BR-EGFP cells (1.40±0.02 fold, P<0.01 compared to vehicle-control) and an EGFR/HER2 inhibitor blocked this effect, suggesting that S100A4 is a downstream effector of EGFR activation. ShRNA-mediated S100A4 silencing in 231BR-EGFP cells decreased their migration and invasion in response to E2-CM, abolished their increased proliferation in co-cultures with E2-treated astrocytes, and decreased brain metastatic colonization. Thus, S100A4 is one effector of the paracrine action of E2 in brain metastatic cells. These studies provide a novel mechanism by which estrogens, acting through ER

  11. Estrogen Receptor α Mediates Proliferation of Osteoblastic Cells Stimulated by Estrogen and Mechanical Strain, but Their Acute Down-regulation of the Wnt Antagonist Sost Is Mediated by Estrogen Receptor β*

    PubMed Central

    Galea, Gabriel L.; Meakin, Lee B.; Sugiyama, Toshihiro; Zebda, Noureddine; Sunters, Andrew; Taipaleenmaki, Hanna; Stein, Gary S.; van Wijnen, Andre J.; Lanyon, Lance E.; Price, Joanna S.

    2013-01-01

    Mechanical strain and estrogens both stimulate osteoblast proliferation through estrogen receptor (ER)-mediated effects, and both down-regulate the Wnt antagonist Sost/sclerostin. Here, we investigate the differential effects of ERα and -β in these processes in mouse long bone-derived osteoblastic cells and human Saos-2 cells. Recruitment to the cell cycle following strain or 17β-estradiol occurs within 30 min, as determined by Ki-67 staining, and is prevented by the ERα antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride. ERβ inhibition with 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-β]pyrimidin-3-yl] phenol (PTHPP) increases basal proliferation similarly to strain or estradiol. Both strain and estradiol down-regulate Sost expression, as does in vitro inhibition or in vivo deletion of ERα. The ERβ agonists 2,3-bis(4-hydroxyphenyl)-propionitrile and ERB041 also down-regulated Sost expression in vitro, whereas the ERα agonist 4,4′,4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl]tris-phenol or the ERβ antagonist PTHPP has no effect. Tamoxifen, a nongenomic ERβ agonist, down-regulates Sost expression in vitro and in bones in vivo. Inhibition of both ERs with fulvestrant or selective antagonism of ERβ, but not ERα, prevents Sost down-regulation by strain or estradiol. Sost down-regulation by strain or ERβ activation is prevented by MEK/ERK blockade. Exogenous sclerostin has no effect on estradiol-induced proliferation but prevents that following strain. Thus, in osteoblastic cells the acute proliferative effects of both estradiol and strain are ERα-mediated. Basal Sost down-regulation follows decreased activity of ERα and increased activity of ERβ. Sost down-regulation by strain or increased estrogens is mediated by ERβ, not ERα. ER-targeting therapy may facilitate structurally appropriate bone formation by enhancing the distinct ligand-independent, strain-related contributions to proliferation

  12. Developmental changes in AMPA and kainate receptor-mediated quantal transmission at thalamocortical synapses in the barrel cortex.

    PubMed

    Bannister, Neil J; Benke, Timothy A; Mellor, Jack; Scott, Helen; Gürdal, Esra; Crabtree, John W; Isaac, John T R

    2005-05-25

    During the first week of life, there is a shift from kainate to AMPA receptor-mediated thalamocortical transmission in layer IV barrel cortex. However, the mechanisms underlying this change and the differential properties of AMPA and kainate receptor-mediated transmission remain essentially unexplored. To investigate this, we studied the quantal properties of AMPA and kainate receptor-mediated transmission using strontium-evoked miniature EPSCs. AMPA and kainate receptor-mediated transmission exhibited very different quantal properties but were never coactivated by a single quantum of transmitter, indicating complete segregation to different synapses within the thalamocortical input. Nonstationary fluctuation analysis showed that synaptic AMPA receptors exhibited a range of single-channel conductance (gamma) and a strong negative correlation between gamma and functional channel number, indicating that these two parameters are reciprocally regulated at thalamocortical synapses. We obtained the first estimate of gamma for synaptic kainate receptors (<2 pS), and this primarily accounted for the small quantal size of kainate receptor-mediated transmission. Developmentally, the quantal contribution to transmission of AMPA receptors increased and that of kainate receptors decreased. No changes in AMPA or kainate quantal amplitude or in AMPA receptor gamma were observed, demonstrating that the developmental change was attributable to a decrease in the number of kainate synapses and an increase in the number of AMPA synapses contributing to transmission. Therefore, we demonstrate fundamental differences in the quantal properties for these two types of synapse. Thus, the developmental switch in transmission will dramatically alter information transfer at thalamocortical inputs to layer IV.

  13. Estrogens and Androgens Inhibit Association of RANKL with the Pre-osteoblast Membrane through Post-translational Mechanisms.

    PubMed

    Martin, Anthony; Yu, Jiali; Xiong, Jian; Khalid, Aysha B; Katzenellenbogen, Benita; Kim, Sung Hoon; Katzenellenbogen, John A; Malaivijitnond, Suchinda; Gabet, Yankel; Krum, Susan A; Frenkel, Baruch

    2017-02-18

    We have recently demonstrated that RUNX2 promoted, and 17-β-Estradiol (E2) diminished, association of RANKL with the cell membrane in pre-osteoblast cultures. Here we show that, similar to E2, dihydrotestosterone (DHT) diminishes association of RANKL and transiently transfected GFP-RANKL with the pre-osteoblast membrane without decreasing total RANKL mRNA or protein levels. Diminution of membrane-associated RANKL was accompanied with marked suppression of osteoclast differentiation from co-cultured pre-osteoclasts, even though DHT increased, not decreased, RANKL concentrations in pre-osteoblast conditioned media. A marked decrease in membrane-associated RANKL was observed after 30 minutes of either E2 or DHT treatment, and near-complete inhibition was observed by 1 hour, suggesting that the diminution of RANKL membrane association was mediated through non-genomic mechanisms. Further indicating dispensability of nuclear action of estrogen receptor, E2-mediated inhibition of RANKL membrane association was mimicked by an estrogen dendrimer conjugate (EDC) that cannot enter the cell nucleus. Finally, the inhibitory effect of E2 and DHT on RANKL membrane association was counteracted by the MMP inhibitor NNGH, and the effect of E2 (and not DHT) was antagonized by the Src inhibitor SU6656. Taken together, these results suggest that estrogens and androgens inhibit osteoblast-driven osteoclastogenesis through non-genomic, MMP-mediated RANKL dissociation from the cell membrane. This article is protected by copyright. All rights reserved.

  14. Neuregulin-1-beta1 enters brain and spinal cord by receptor-mediated transport.

    PubMed

    Kastin, Abba J; Akerstrom, Victoria; Pan, Weihong

    2004-02-01

    Proteins of the neuregulin (NRG) family play important regulatory roles in neuronal survival and synaptic activity. NRG-1-beta1 has particular potential as a therapeutic agent because it enhances myelination of neurites in spinal cord explants. In this study, we determined the permeation of NRG-1-beta1 across the blood-brain and blood-spinal cord barriers (BBB and BSCB respectively). Intact radioactively labeled NRG-1-beta1 had a saturable and relatively rapid influx rate from blood to the CNS in mice. Capillary depletion studies showed that NRG-1-beta1 entered the parenchyma of the brain and spinal cord rather than being trapped in the capillaries that compose the BBB. The possible mechanism of receptor-mediated transport was shown by the ability of antibodies to erbB3 and erbB4 receptors to inhibit the influx. Lipophilicity, less important for such saturable transport mechanisms, was measured by the octanol : buffer partition coefficient and found to be low. The results indicate that NRG-1-beta1 enters spinal cord and brain by a saturable receptor-mediated mechanism, which provides the opportunity for possible therapeutic manipulation at the BBB level.

  15. Evaluation of estrogenic activities and mechanism of action of perfluorinated chemicals determined by vitellogenin induction in primary cultured tilapia hepatocytes.

    PubMed

    Liu, Chunsheng; Du, Yongbing; Zhou, Bingsheng

    2007-12-30

    Perfluorochemicals (PFCs) are emerging persistent organic pollutants (POPs) and are widely present in the environment, wildlife and humans. Recently, reports have suggested that PFCs may have endocrine-disrupting activities. In the present study, we have developed a non-competitive enzyme-linked immunosorbent assay (ELISA) method to investigate estrogenic activities of selected PFCs using vitellogenin (VTG) induction in primary cultured hepatocytes of freshwater male tilapia (Oreochromis niloticus). Cultured hepatocytes were exposed to various concentrations of perfluorooctanyl sulfonate (PFOS), pentadecafluorooctanoic acid (PFOA), 1H, 1H, 2H, 2H-nonafluoro-1-hexanol (4:2 FTOH), 1H, 1H, 2H, 2H-perfluorooctanol (6:2 FTOH) and 1H, 1H, 2H, 2H-perfluoro-1-decanol (8:2 FTOH) for 48 h, while 17beta-estradiol (E2) and 4-nonylphenol (4-NP) were used as positive controls. A dose-dependent induction of VTG was observed in E2-, 4-NP-, PFOS-, PFOA- and 6:2 FTOH-treated cells, whereas VTG levels remained unchanged in the 4:2 FTOH and 8:2 FTOH exposure groups at the concentrations tested. The estimated 48-h EC(50) values for E2, 4-NP, PFOS, PFOA and 6:2 FTOH were 4.7 x 10(-7), 7.1 x 10(-6), 1.5 x 10(-5), 2.9 x 10(-5) and 2.8 x 10(-5)M, respectively. In the time-course study, significant VTG induction took place at 24 h (E2), 6 h (4-NP), 48 h (PFOS), 48 h (PFOA), 72 h (4:2 FTOH), 12 h (6:2 FTOH), 72 h (8:2 FTOH), and increased further after 96 h of exposure. Co-exposure to binary mixtures of individual PFCs and E2 for 48 h significantly inhibited E2-induced hepatocellular VTG production in a dose-dependent manner except for 4:2 FTOH. The estimated 48-h IC(50) (concentration of a compound that elicits 50% inhibition of maximally E2-induced VTG) values for PFOS, PFOA, 6:2 FTOH and 8:2 FTOH were 3.1 x 10(-7), 5.1 x 10(-7), 1.1 x 10(-6) and 7.5 x 10(-7)M, respectively. In order to further investigate the estrogenic mechanism of PFCs, the hepatocytes were co-exposed to binary mixtures

  16. Tracking Progesterone Receptor-Mediated Actions in Breast Cancer

    PubMed Central

    Knutson, Todd P.; Lange, Carol A.

    2014-01-01

    Ovarian steroid hormones contribute to breast cancer initiation and progression primarily through the actions of their nuclear transcription factors, the estrogen receptor alpha (ERα) and progesterone receptors (PRs). These receptors are important drivers of the luminal A and B subtypes of breast cancer, where estrogen-blocking drugs have been effective endocrine therapies for patients with these tumors. However, many patients do not respond, or become resistant to treatment. When endocrine therapies fail, the luminal subtypes of breast cancer are more difficult to treat because these subtypes are among the most heterogeneous in terms of mutation diversity and gene expression profiles. Recent evidence suggests that progestin and PR actions may be important drivers of luminal breast cancers. Clinical trial data has demonstrated that hormone replacement therapy with progestins drives invasive breast cancer and results in greater mortality. PR transcriptional activity is dependent upon cross-talk with growth factor signaling pathways that alter PR phosphorylation, acetylation, or SUMOylation as mechanisms for regulating PR target gene selection required for increased cell proliferation and survival. Site-specific PR phosphorylation is the primary driver of gene-selective PR transcriptional activity. However, PR phosphorylation and heightened transcriptional activity is coupled to rapid PR protein degradation; the range of active PR detected in tumors is likely to be dynamic. Thus, PR target gene signatures may provide a more accurate means of tracking PR’s contribution to tumor progression rather than standard clinical protein-based (IHC) assays. Further development of antiprogestin therapies should be considered along side antiestrogens and aromatase inhibitors. PMID:24291072

  17. Androgen Receptor-Mediated Escape Mechanisms from Androgen Ablation Therapy

    DTIC Science & Technology

    2005-10-01

    03- catenin in lipogenesis can be assigned. In addition to being highly expressed in adipose tissue, both PPAR8 and PPARy are thought to serve...ProliferationPrlfato? a) Androgen Dependent PrCa b) Androgen Independent PrCa Figure 3 a) ER TR VDR VDR Wnt -*P-cat - *lo AR Wn Ic AR: RAR, RAR, RXR RXR* PPAR8 PPARy ...97) Tcf-4 PPARy Activation Increased PPAR7 detected in colonic cancer cells (85) Table 11. Nuclear Receptor Modulation of Wnt//J-catenin/Tcf

  18. Glucocorticoid hormone resistance during primate evolution: receptor-mediated mechanisms.

    PubMed Central

    Chrousos, G P; Renquist, D; Brandon, D; Eil, C; Pugeat, M; Vigersky, R; Cutler, G B; Loriaux, D L; Lipsett, M B

    1982-01-01

    The concentrations of total and protein-unbound plasma cortisol of New World monkeys are higher than those of Old World primates and prosimians. The urinary free-cortisol excretion also is increased markedly. However, there is no physiologic evidence of increased cortisol effect. These findings suggest end-organ resistance to glucocorticoids. This was confirmed by showing that the hypothalamic-pituitary adrenal axis is resistant to suppression by dexamethasone. To study this phenomenon, glucocorticoid receptors were examined in circulating mononuclear leukocytes and cultured skin fibroblasts from both New and Old World species. The receptor content is the same in all species, but the New World monkeys have a markedly decreased binding affinity for dexamethasone. Thus, the resistance of these species to the action of cortisol is due to the decreased binding affinity of the glucocorticoid receptor. This presumed mutation must have occurred after the bifurcation of Old and New World primates (approximately 60 x 10(6) yr ago) and before the diversion of the New World primates from each other (approximately 15 x 10(6) yr ago). Images PMID:6952251

  19. Glucocorticoid hormone resistance during primate evolution: receptor-mediated mechanisms.

    PubMed

    Chrousos, G P; Renquist, D; Brandon, D; Eil, C; Pugeat, M; Vigersky, R; Cutler, G B; Loriaux, D L; Lipsett, M B

    1982-03-01

    The concentrations of total and protein-unbound plasma cortisol of New World monkeys are higher than those of Old World primates and prosimians. The urinary free-cortisol excretion also is increased markedly. However, there is no physiologic evidence of increased cortisol effect. These findings suggest end-organ resistance to glucocorticoids. This was confirmed by showing that the hypothalamic-pituitary adrenal axis is resistant to suppression by dexamethasone. To study this phenomenon, glucocorticoid receptors were examined in circulating mononuclear leukocytes and cultured skin fibroblasts from both New and Old World species. The receptor content is the same in all species, but the New World monkeys have a markedly decreased binding affinity for dexamethasone. Thus, the resistance of these species to the action of cortisol is due to the decreased binding affinity of the glucocorticoid receptor. This presumed mutation must have occurred after the bifurcation of Old and New World primates (approximately 60 x 10(6) yr ago) and before the diversion of the New World primates from each other (approximately 15 x 10(6) yr ago).

  20. Mechanisms of Estrogen Carcinogenesis: The Role of E2/E1- Quinone Metabolites Suggests New Approaches to Preventive Intervention – A Review

    PubMed Central

    Yager, James D.

    2014-01-01

    Studies in hamsters, mice and rats have demonstrated that estradiol (E2), its interconvertible metabolite estrone (E1) and their catechol metabolites, in particular 4-hydroxy E2/E1, are carcinogenic in the kidney, uterus and mammary gland. Observational studies and clinical trials consistently show that sustained exposure to E2/E1 is associated with the development of sporadic breast cancer. The weight of evidence supports the contribution of two complementary pathways in the initiation, promotion and progression of breast cancer. One pathway involves activation of nuclear and cytoplasmic signaling pathways through the binding of estrogen to nuclear and membrane-bound estrogen receptors leading to increased cell proliferation. The other pathway involves the oxidative metabolism of E2/E1 to catechols and then reactive quinones that can contribute to oxidative DNA damage and form specific, mutagenic depurinating adducts with adenine and guanine which then in turn can serve as biomarkers for the occurrence of these processes. Both pathways can serve as portals to preventive intervention. Antiestrogens are used clinically to block receptor-mediated signaling to block tumor growth. Various chemopreventive agents such as sulforaphane (SFN) and resveratrol have been shown in cell culture to block oxidative metabolism of E2/E1 and thus prevent DNA damage. Pretreatment of MCF-7 and MCF-10F cells with and inhibitor of catechol-O-methyltransferase (COMT) followed by treatment with E2 or 4-OH E2 caused increased oxidative DNA damage (8-oxo-dG) and depurinating DNA adducts showing the importance of E2-catechol O-methylation by COMT as a protective pathway. E2 Treatment of MCF-10A cells with E2 or 4-OH E2 caused an increase in E2-adenine and guanine adducts. Treatment with sulforaphane increased NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione-S-transferase A1 (GSTA1) expression without affecting expression of catechol-O-methyltransferase (COMT) or cytochrome P450 1B1

  1. Adaptation in sound localization: from GABA(B) receptor-mediated synaptic modulation to perception.

    PubMed

    Stange, Annette; Myoga, Michael H; Lingner, Andrea; Ford, Marc C; Alexandrova, Olga; Felmy, Felix; Pecka, Michael; Siveke, Ida; Grothe, Benedikt

    2013-12-01

    Across all sensory modalities, the effect of context-dependent neural adaptation can be observed at every level, from receptors to perception. Nonetheless, it has long been assumed that the processing of interaural time differences, which is the primary cue for sound localization, is nonadaptive, as its outputs are mapped directly onto a hard-wired representation of space. Here we present evidence derived from in vitro and in vivo experiments in gerbils indicating that the coincidence-detector neurons in the medial superior olive modulate their sensitivity to interaural time differences through a rapid, GABA(B) receptor-mediated feedback mechanism. We show that this mechanism provides a gain control in the form of output normalization, which influences the neuronal population code of auditory space. Furthermore, psychophysical tests showed that the paradigm used to evoke neuronal GABA(B) receptor-mediated adaptation causes the perceptual shift in sound localization in humans that was expected on the basis of our physiological results in gerbils.

  2. Estrogen and the cardiovascular system.

    PubMed

    Knowlton, A A; Lee, A R

    2012-07-01

    Estrogen is a potent steroid with pleiotropic effects, which have yet to be fully elucidated. Estrogen has both nuclear and non-nuclear effects. The rapid response to estrogen, which involves a membrane associated estrogen receptor(ER) and is protective, involves signaling through PI3K, Akt, and ERK 1/2. The nuclear response is much slower, as the ER-estrogen complex moves to the nucleus, where it functions as a transcription factor, both activating and repressing gene expression. Several different ERs regulate the specificity of response to estrogen, and appear to have specific effects in cardiac remodeling and the response to injury. However, much remains to be understood about the selectivity of these receptors and their specific effects on gene expression. Basic studies have demonstrated that estrogen treatment prevents apoptosis and necrosis of cardiac and endothelial cells. Estrogen also attenuates pathologic cardiac hypertrophy. Estrogen may have great benefit in aging as an anti-inflammatory agent. However, clinical investigations of estrogen have had mixed results, and not shown the clear-cut benefit of more basic investigations. This can be explained in part by differences in study design: in basic studies estrogen treatment was used immediately or shortly after ovariectomy, while in some key clinical trials, estrogen was given years after menopause. Further basic research into the underlying molecular mechanisms of estrogen's actions is essential to provide a better comprehension of the many properties of this powerful hormone.

  3. QSAR prediction of estrogen activity for a large set of diverse chemicals under the guidance of OECD principles.

    PubMed

    Liu, Huanxiang; Papa, Ester; Gramatica, Paola

    2006-11-01

    A large number of environmental chemicals, known as endocrine-disrupting chemicals, are suspected of disrupting endocrine functions by mimicking or antagonizing natural hormones, and such chemicals may pose a serious threat to the health of humans and wildlife. They are thought to act through a variety of mechanisms, mainly estrogen-receptor-mediated mechanisms of toxicity. However, it is practically impossible to perform thorough toxicological tests on all potential xenoestrogens, and thus, the quantitative structure--activity relationship (QSAR) provides a promising method for the estimation of a compound's estrogenic activity. Here, QSAR models of the estrogen receptor binding affinity of a large data set of heterogeneous chemicals have been built using theoretical molecular descriptors, giving full consideration to the new OECD principles in regulation for QSAR acceptability, during model construction and assessment. An unambiguous multiple linear regression (MLR) algorithm was used to build the models, and model predictive ability was validated by both internal and external validation. The applicability domain was checked by the leverage approach to verify prediction reliability. The results obtained using several validation paths indicate that the proposed QSAR model is robust and satisfactory, and can provide a feasible and practical tool for the rapid screening of the estrogen activity of organic compounds.

  4. Calcium regulates estrogen increase in permeability of cultured CaSki epithelium by eNOS-dependent mechanism.

    PubMed

    Gorodeski, G I

    2000-11-01

    Estrogen increases baseline transepithelial permeability across CaSki cultures and augments the increase in permeability in response to hypertonic gradients. In estrogen-treated cells, lowering cytosolic calcium abrogated the hypertonicity-induced augmented increase in permeability and decreased baseline permeability to a greater degree than in estrogen-deprived cells. Steady-state levels of cytosolic calcium in estrogen-deprived cells were higher than in estrogen-treated cells. Increases in extracellular calcium increased cytosolic calcium more in estrogen-deprived cells than in estrogen-treated cells. However, in estrogen-treated cells, increasing cytosolic calcium was associated with greater increases in permeability in response to hypertonic gradients than in estrogen-deprived cells. Lowering cytosolic calcium blocked the estrogen-induced increase in nitric oxide (NO) release and in the in vitro conversion of L-[(3)H]arginine to L-[(3)H]citrulline. Treatment with estrogen upregulated mRNA of the NO synthase isoform endothelial nitric oxide synthase (eNOS). These results indicate that cytosolic calcium mediates the responses to estrogen and suggest that the estrogen increase in permeability and the augmented increase in permeability in response to hypertonicity involve an increase in NO synthesis by upregulation of the calcium-dependent eNOS.

  5. The miR-199-dynamin regulatory axis controls receptor-mediated endocytosis.

    PubMed

    Aranda, Juan F; Canfrán-Duque, Alberto; Goedeke, Leigh; Suárez, Yajaira; Fernández-Hernando, Carlos

    2015-09-01

    Small non-coding RNAs (microRNAs) are important regulators of gene expression that modulate many physiological processes; however, their role in regulating intracellular transport remains largely unknown. Intriguingly, we found that the dynamin (DNM) genes, a GTPase family of proteins responsible for endocytosis in eukaryotic cells, encode the conserved miR-199a and miR-199b family of miRNAs within their intronic sequences. Here, we demonstrate that miR-199a and miR-199b regulate endocytic transport by controlling the expression of important mediators of endocytosis such as clathrin heavy chain (CLTC), Rab5A, low-density lipoprotein receptor (LDLR) and caveolin-1 (Cav-1). Importantly, miR-199a-5p and miR-199b-5p overexpression markedly inhibits CLTC, Rab5A, LDLR and Cav-1 expression, thus preventing receptor-mediated endocytosis in human cell lines (Huh7 and HeLa). Of note, miR-199a-5p inhibition increases target gene expression and receptor-mediated endocytosis. Taken together, our work identifies a new mechanism by which microRNAs regulate intracellular trafficking. In particular, we demonstrate that the DNM, miR-199a-5p and miR-199b-5p genes act as a bifunctional locus that regulates endocytosis, thus adding an unexpected layer of complexity in the regulation of intracellular trafficking.

  6. Target shape dependence in a simple model of receptor-mediated endocytosis and phagocytosis.

    PubMed

    Richards, David M; Endres, Robert G

    2016-05-31

    Phagocytosis and receptor-mediated endocytosis are vitally important particle uptake mechanisms in many cell types, ranging from single-cell organisms to immune cells. In both processes, engulfment by the cell depends critically on both particle shape and orientation. However, most previous theoretical work has focused only on spherical particles and hence disregards the wide-ranging particle shapes occurring in nature, such as those of bacteria. Here, by implementing a simple model in one and two dimensions, we compare and contrast receptor-mediated endocytosis and phagocytosis for a range of biologically relevant shapes, including spheres, ellipsoids, capped cylinders, and hourglasses. We find a whole range of different engulfment behaviors with some ellipsoids engulfing faster than spheres, and that phagocytosis is able to engulf a greater range of target shapes than other types of endocytosis. Further, the 2D model can explain why some nonspherical particles engulf fastest (not at all) when presented to the membrane tip-first (lying flat). Our work reveals how some bacteria may avoid being internalized simply because of their shape, and suggests shapes for optimal drug delivery.

  7. The applications of machine learning algorithms in the modeling of estrogen-like chemicals.

    PubMed

    Liu, Huanxiang; Yao, Xiaojun; Gramatica, Paola

    2009-06-01

    Increasing concern is being shown by the scientific community, government regulators, and the public about endocrine-disrupting chemicals that, in the environment, are adversely affecting human and wildlife health through a variety of mechanisms, mainly estrogen receptor-mediated mechanisms of toxicity. Because of the large number of such chemicals in the environment, there is a great need for an effective means of rapidly assessing endocrine-disrupting activity in the toxicology assessment process. When faced with the challenging task of screening large libraries of molecules for biological activity, the benefits of computational predictive models based on quantitative structure-activity relationships to identify possible estrogens become immediately obvious. Recently, in order to improve the accuracy of prediction, some machine learning techniques were introduced to build more effective predictive models. In this review we will focus our attention on some recent advances in the use of these methods in modeling estrogen-like chemicals. The advantages and disadvantages of the machine learning algorithms used in solving this problem, the importance of the validation and performance assessment of the built models as well as their applicability domains will be discussed.

  8. Dose-response approaches for nuclear receptor-mediated ...

    EPA Pesticide Factsheets

    A public workshop, organized by a Steering Committee of scientists from government, industry, universities, and research organizations, was held at the National Institute of Environmental Health Sciences (NIEHS) in September, 2010. The workshop explored the dose-response implications of toxicant modes of action (MOA) mediated by nuclear receptors. The dominant paradigm in human health risk assessment has been linear extrapolation without a threshold for cancer, and estimation of sub-threshold doses for non-cancer and (in appropriate cases) cancer endpoints. However, recent publications question the application of dose-response modeling approaches with a threshold. The growing body of molecular toxicology information and computational toxicology tools has allowed for exploration of the presence or absence of subthreshold doses for a number of receptor-mediated MOPs. The workshop explored the development of dose-response approaches for nuclear receptor-mediated liver cancer, within a MOA Human Relevance framework (HRF). Case studies addressed activation of the AHR; the CAR/PXR, and the PPARa. This paper describes the workshop process, key issues discussed, and conclusions. The value of an interactive workshop approach to apply current MOA/HRF frameworks was demonstrated. The results may help direct research on the MOA and dose-response of receptor-based toxicity, since there are commonalities for many receptors in the basic pathways involved for late steps in the

  9. Stronger Dopamine D1 Receptor-Mediated Neurotransmission in Dyskinesia.

    PubMed

    Farré, Daniel; Muñoz, Ana; Moreno, Estefanía; Reyes-Resina, Irene; Canet-Pons, Júlia; Dopeso-Reyes, Iria G; Rico, Alberto J; Lluís, Carme; Mallol, Josefa; Navarro, Gemma; Canela, Enric I; Cortés, Antonio; Labandeira-García, José L; Casadó, Vicent; Lanciego, José L; Franco, Rafael

    2015-12-01

    Radioligand binding assays to rat striatal dopamine D1 receptors showed that brain lateralization of the dopaminergic system were not due to changes in expression but in agonist affinity. D1 receptor-mediated striatal imbalance resulted from a significantly higher agonist affinity in the left striatum. D1 receptors heteromerize with dopamine D3 receptors, which are considered therapeutic targets for dyskinesia in parkinsonian patients. Expression of both D3 and D1-D3 receptor heteromers were increased in samples from 6-hydroxy-dopamine-hemilesioned rats rendered dyskinetic by treatment with 3, 4-dihydroxyphenyl-L-alanine (L-DOPA). Similar findings were obtained using striatal samples from primates. Radioligand binding studies in the presence of a D3 agonist led in dyskinetic, but not in lesioned or L-DOPA-treated rats, to a higher dopamine sensitivity. Upon D3-receptor activation, the affinity of agonists for binding to the right striatal D1 receptor increased. Excess dopamine coming from L-DOPA medication likely activates D3 receptors thus making right and left striatal D1 receptors equally responsive to dopamine. These results show that dyskinesia occurs concurrently with a right/left striatal balance in D1 receptor-mediated neurotransmission.

  10. Dose-response approaches for nuclear receptor-mediated ...

    EPA Pesticide Factsheets

    A public workshop, organized by a Steering Committee of scientists from government, industry, universities, and research organizations, was held at the National Institute of Environmental Health Sciences (NIEHS) in September, 2010. The workshop explored the dose-response implications of toxicant modes of action (MOA) mediated by nuclear receptors. The dominant paradigm in human health risk assessment has been linear extrapolation without a threshold for cancer, and estimation of sub-threshold doses for non-cancer and (in appropriate cases) cancer endpoints. However, recent publications question the application of dose-response modeling approaches with a threshold. The growing body of molecular toxicology information and computational toxicology tools has allowed for exploration of the presence or absence of subthreshold doses for a number of receptor-mediated MOPs. The workshop explored the development of dose-response approaches for nuclear receptor-mediated liver cancer, within a MOA Human Relevance framework (HRF). Case studies addressed activation of the AHR; the CAR/PXR, and the PPARa. This paper describes the workshop process, key issues discussed, and conclusions. The value of an interactive workshop approach to apply current MOA/HRF frameworks was demonstrated. The results may help direct research on the MOA and dose-response of receptor-based toxicity, since there are commonalities for many receptors in the basic pathways involved for late steps in the

  11. High dose 17 beta-estradiol and the alpha-estrogen agonist PPT trigger apoptosis in human adrenal carcinoma cells but the beta-estrogen agonist DPN does not.

    PubMed

    Prieto, L M; Brown, J W; Perez-Stable, C; Fishman, L M

    2008-05-01

    Previous studies have shown that high dose 17beta-estradiol (10 (-5) M) has a G2/M blocking effect in SW-13 human adrenal carcinoma cultures and strongly enhances apoptosis. To examine the differential effects of estrogen alpha and beta-receptors in this system, we incubated SW-13 cells with specific alpha- and beta-estrogen receptor agonists, PPT [4,4',4''-(propyl-[ (1)H]-pyrazole-1,3,5-triyl) trisphenol] and DPN [2,3-bis (4-hydroxyphenyl) propionitrile], respectively (each at 10 (-5) M). Flow cytometry was used to analyze the percentages of cells in various phases of the cell cycle [sub-G1 (apoptosis), G1, S, and G2/M] in each experimental condition. Exposure to 17 beta-estradiol for 48 hours increased apoptosis more than 5-fold (from 3.6+/-0.5 to 20+/-2.2% of cells; p<0.01). The alpha-estrogen agonist PPT had a similar effect, increasing apoptosis to 22+/-1.7% (p<0.01), but the beta-agonist DPN caused no change (3.6+/-0.5 vs. 3.9+/-0.8%). While estrogen and the alpha-estrogen agonist decrease apoptosis in this system, both of these compounds decreased the percentage of cells in G1 (from 59+/-1.4% for control to 34+/-2.3% for estrogen and 40+/-2.0% for PPT; p<0.01 for both agents relative to control); the beta-agonist again had no effect. Estrogen was also found to block the cell cycle in G2/M, increasing it from 15+/-0.4 to 21+/-1.0% of cells (p<0.01), but neither the alpha- nor beta-estrogen agonists had any effect at this point in the cell cycle, indicating that the influence of estrogen was not likely to be either alpha- or beta-receptor mediated. There was no apparent effect of any of these agents on DNA synthesis, as indicated by unchanged percentages of cells in S phase. These studies suggest that induction of apoptosis by estrogen in SW-13 human adrenal cortical carcinoma cultures is mediated by the alpha-receptor, but the G2/M blocking effect of estrogen is not likely to be related to either alpha or beta mechanisms.

  12. Tibolone Reduces Oxidative Damage and Inflammation in Microglia Stimulated with Palmitic Acid through Mechanisms Involving Estrogen Receptor Beta.

    PubMed

    Hidalgo-Lanussa, Oscar; Ávila-Rodriguez, Marco; Baez-Jurado, Eliana; Zamudio, Jairo; Echeverria, Valentina; Garcia-Segura, Luis Miguel; Barreto, George E

    2017-09-25

    High concentrations of palmitic acid in plasma increase both the inflammation associated with obesity and the susceptibility to develop a neurodegenerative event. In the brain, the inflammatory response is mediated by activated microglial cells, which undergo morphological and biochemical changes and can directly affect cell viability. Recent evidence shows that the use of estrogenic compounds can control microglia-induced inflammation with promising results. In this study, we explored the actions of the synthetic steroid tibolone on BV-2 microglia cells stimulated with palmitic acid. Our results demonstrated that tibolone increased cell viability and reduced nuclear fragmentation and the production of reactive oxygen species, as well as preserved mitochondrial membrane potential. These effects were accompanied by reduced nuclear translocation of NF-κB p65, upregulation of neuroglobin, and improved antioxidant defense. Furthermore, estrogen receptor beta (ERβ) inhibition partially dampened tibolone's protective actions in BV-2 cells stimulated with palmitic acid. In conclusion, tibolone protects BV-2 cells by a mechanism involving ERβ and upregulation of neuroglobin.

  13. Environmental Estrogens and Obesity

    PubMed Central

    Newbold, Retha R.; Padilla-Banks, Elizabeth; Jefferson, Wendy N.

    2009-01-01

    Many chemicals in the environment, in particular those with estrogenic activity, can disrupt the programming of endocrine signaling pathways that are established during development and result in adverse consequences that may not be apparent until much later in life. Most recently, obesity and diabetes join the growing list of adverse consequences that have been associated with developmental exposure to environmental estrogens during critical stages of differentiation. These diseases are quickly becoming significant public health issues and are fast reaching epidemic proportions worldwide. In this review, we summarize the literature from experimental animal studies documenting an association of environmental estrogens and the development of obesity, and further describe an animal model of exposure to diethylstilbestrol (DES) that has proven useful in studying mechanisms involved in abnormal programming of various differentiating estrogen- target tissues. Other examples of environmental estrogens including the phytoestrogen genistein and the environmental contaminant Bisphenol A are also discussed. Together, these data suggest new targets (i.e., adipocyte differentiation and molecular mechanisms involved in weight homeostasis) for abnormal programming by estrogenic chemicals, and provide evidence that support the scientific hypothesis termed “the developmental origins of adult disease”. The proposal of an association of environmental estrogens with obesity and diabetes expands the focus on the diseases from intervention/treatment to include prevention/avoidance of chemical modifiers especially during critical windows of development. PMID:19433252

  14. Tamoxifen prevents induction of hepatic neoplasia by zeranol, an estrogenic food contaminant.

    PubMed

    Coe, J E; Ishak, K G; Ward, J M; Ross, M J

    1992-02-01

    Zeranol (alpha-zearalanol) is a beta-resorcylic acid lactone (RAL) that has estrogen activity. It is synthesized by molds and is difficult to avoid in human food products. We tested the ability of this mycoestrogen to damage the liver of the Armenian hamster, a rodent that is especially sensitive to hepatotoxic effects of exogenous estrogens. Zeranol induced acute hepatotoxicity and, subsequently, hepatic carcinogenesis; both effects were blocked by tamoxifen, suggesting estrogen receptor mediation. Because zeranol is acting alone as a primary initiator of hepatic neoplasms, this model provides an unusual opportunity to study the pathogenesis of estrogen-initiated tumorigenesis.

  15. Xenoestrogens down-regulate aryl-hydrocarbon receptor nuclear translocator 2 mRNA expression in human breast cancer cells via an estrogen receptor alpha-dependent mechanism.

    PubMed

    Qin, Xian-Yang; Zaha, Hiroko; Nagano, Reiko; Yoshinaga, Jun; Yonemoto, Junzo; Sone, Hideko

    2011-10-10

    Environmental chemicals with estrogenic activity, known as xenoestrogens, may cause impaired reproductive development and endocrine-related cancers in humans by disrupting endocrine functions. Aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) is believed to play important roles in a variety of physiological processes, including estrogen signaling pathways, that may be involved in the pathogenesis and therapeutic responses of endocrine-related cancers. However, much of the underlying mechanism remains unknown. In this study, we investigated whether ARNT2 expression is regulated by a range of representative xenoestrogens in human cancer cell lines. Bisphenol A (BPA), benzyl butyl phthalate (BBP), and 1,1,1-trichloro-2,2-bis(2-chlorophenyl-4-chlorophenyl)ethane (o,p'-DDT) were found to be estrogenic toward BG1Luc4E2 cells by an E-CALUX bioassay. ARNT2 expression was downregulated by BPA, BBP, and o,p'-DDT in a dose-dependent manner in estrogen receptor 1 (ESR1)-positive MCF-7 and BG1Luc4E2 cells, but not in estrogen receptor-negative LNCaP cells. The reduction in ARNT2 expression in cells treated with the xenoestrogens was fully recovered by the addition of a specific ESR1 antagonist, MPP. In conclusion, we have shown for the first time that ARNT2 expression is modulated by xenoestrogens by an ESR1-dependent mechanism in MCF-7 breast cancer cells. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  16. Receptor-Mediated Transport of Insulin across Endothelial Cells

    NASA Astrophysics Data System (ADS)

    King, George L.; Johnson, Sandra M.

    1985-03-01

    Hormones such as insulin are transported from the interior to the exterior of blood vessels. Whether endothelial cells, which line the inner walls of blood vessels have a role in this transport of hormones is not clear, but it is known that endothelial cells can internalize and release insulin rapidly with little degradation. The transport of iodine-125-labeled insulin was measured directly through the use of dual chambers separated by a horizontal monolayer of cultured bovine aortic endothelial cells. In this setting, endothelial cells took up and released the labeled insulin, thereby transporting it across the cells. The transport of insulin across the endothelial cells was temperature sensitive and was inhibited by unlabeled insulin and by antibody to insulin receptor in proportion to the ability of these substances to inhibit insulin binding to its receptor. More than 80 percent of the transported insulin was intact. These data suggest that insulin is rapidly transported across endothelial cells by a receptor-mediated process.

  17. Receptor mediated cellular uptake of low molecular weight dendritic polyglycerols.

    PubMed

    Calderón, Marcelo; Reichert, Stephanie; Welker, Pia; Licha, Kai; Kratz, Felix; Haag, Rainer

    2014-01-01

    The development of effective polymer-based nanocarriers which are able to target diseased tissues still remains a great challenge in current research. Dendritic polyglycerols have emerged as novel polymeric scaffolds that have demonstrated a great potential for diverse biomedical applications. These architectures have already proven their usefulness in therapeutic approaches related to multivalency, given by the synergy between the nanosized dimensions combined with the high density of functional groups. However, a continuous effort is necessary to modify and tailor polyglycerol architectures to fit the future demands of biomedical applications. The present work deals with the development of a general synthetic strategy that allows the linkage of low molecular weight dendritic polyglycerols to fluorescent dyes and cell targeting ligands. The receptor mediated cellular uptake of the polyglycerol conjugates highlight their potential to acts as new targeted nanocarriers that should be able to decrease non-specific cellular uptake.

  18. Menthol inhibits 5-HT3 receptor-mediated currents.

    PubMed

    Ashoor, Abrar; Nordman, Jacob C; Veltri, Daniel; Yang, Keun-Hang Susan; Shuba, Yaroslav; Al Kury, Lina; Sadek, Bassem; Howarth, Frank C; Shehu, Amarda; Kabbani, Nadine; Oz, Murat

    2013-11-01

    The effects of alcohol monoterpene menthol, a major active ingredient of the peppermint plant, were tested on the function of human 5-hydroxytryptamine type 3 (5-HT3) receptors expressed in Xenopus laevis oocytes. 5-HT (1 μM)-evoked currents recorded by two-electrode voltage-clamp technique were reversibly inhibited by menthol in a concentration-dependent (IC50 = 163 μM) manner. The effects of menthol developed gradually, reaching a steady-state level within 10-15 minutes and did not involve G-proteins, since GTPγS activity remained unaltered and the effect of menthol was not sensitive to pertussis toxin pretreatment. The actions of menthol were not stereoselective as (-), (+), and racemic menthol inhibited 5-HT3 receptor-mediated currents to the same extent. Menthol inhibition was not altered by intracellular 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid injections and transmembrane potential changes. The maximum inhibition observed for menthol was not reversed by increasing concentrations of 5-HT. Furthermore, specific binding of the 5-HT3 antagonist [(3)H]GR65630 was not altered in the presence of menthol (up to 1 mM), indicating that menthol acts as a noncompetitive antagonist of the 5-HT3 receptor. Finally, 5-HT3 receptor-mediated currents in acutely dissociated nodose ganglion neurons were also inhibited by menthol (100 μM). These data demonstrate that menthol, at pharmacologically relevant concentrations, is an allosteric inhibitor of 5-HT3 receptors.

  19. Estrogen Enhances Linkage in the Vascular Endothelial Calmodulin Network via a Feedforward Mechanism at the G Protein-coupled Estrogen Receptor 1.

    PubMed

    Tran, Quang-Kim; Firkins, Rachel; Giles, Jennifer; Francis, Sarah; Matnishian, Vahe; Tran, Phuong; VerMeer, Mark; Jasurda, Jake; Burgard, Michelle Ann; Gebert-Oberle, Briana

    2016-05-13

    Estrogen exerts many effects on the vascular endothelium. Calmodulin (CaM) is the transducer of Ca(2+) signals and is a limiting factor in cardiovascular tissues. It is unknown whether and how estrogen modifies endothelial functions via the network of CaM-dependent proteins. Here we show that 17β-estradiol (E2) up-regulates total CaM level in endothelial cells. Concurrent measurement of Ca(2+) and Ca(2+)-CaM indicated that E2 also increases free Ca(2+)-CaM. Pharmacological studies, gene silencing, and receptor expression-specific cell studies indicated that the G protein-coupled estrogen receptor 1 (GPER/GPR30) mediates these effects via transactivation of EGFR and subsequent MAPK activation. The outcomes were then examined on four distinct members of the intracellular CaM target network, including GPER/GPR30 itself and estrogen receptor α, the plasma membrane Ca(2+)-ATPase (PMCA), and endothelial nitric-oxide synthase (eNOS). E2 substantially increases CaM binding to estrogen receptor α and GPER/GPR30. Mutations that reduced CaM binding to GPER/GPR30 in separate binding domains do not affect GPER/GPR30-Gβγ preassociation but decrease GPER/GPR30-mediated ERK1/2 phosphorylation. E2 increases CaM-PMCA association, but the expected stimulation of Ca(2+) efflux is reversed by E2-stimulated tyrosine phosphorylation of PMCA. These effects sustain Ca(2+) signals and promote Ca(2+)-dependent CaM interactions with other CaM targets. Consequently, E2 doubles CaM-eNOS interaction and also promotes dual phosphorylation of eNOS at Ser-617 and Ser-1179. Calculations using in-cell and in vitro data revealed substantial individual and combined contribution of these effects to total eNOS activity. Taken together, E2 generates a feedforward loop via GPER/GPR30, which enhances Ca(2+)/CaM signals and functional linkage in the endothelial CaM target network.

  20. D2-like dopamine receptors mediate the response to amphetamine in a mouse model of ADHD

    PubMed Central

    Fan, Xueliang; Hess, Ellen J.

    2007-01-01

    The mechanisms underlying the effects of psychostimulants in attention deficit hyperactivity disorder (ADHD) are not well understood, but indirect evidence implicates D2 dopamine receptors. Here we dissect the components of dopaminergic neurotransmission in the hyperactive mouse mutant coloboma to identify pre- and postsynaptic elements essential for the effects of amphetamine in these mice. Amphetamine treatment reduced locomotor activity in coloboma mice, but induced a robust increase in dopamine overflow suggesting that abnormal regulation of dopamine efflux does not account for the behavioral effect. However, the D2-like dopamine receptor antagonists haloperidol and raclopride, but not the D1-like dopamine receptor antagonist SCH23390, blocked the amphetamine-induced reduction in locomotor activity in coloboma mice, providing direct evidence that D2-like dopamine receptors mediate the effect of amphetamine in these mice. With the precedent established that it is possible to directly antagonize this response, this strategy should prove useful for identifying novel therapeutics in ADHD. PMID:17291774

  1. ESTROGENIC STATUS MODULATES DMBA-MEDIATED HEPATIC GENE EXPRESSION: MICROARRAY-BASED ANALYSIS

    USDA-ARS?s Scientific Manuscript database

    Estrogenic status in women influences the metabolism and toxicity of polycyclic aromatic hydrocarbons (PAH). The objective of this study was to examine the influence of estradiol (E2) on 7,12 dimethylbenz(a)anthracene (DMBA), a ligand for aryl hydrocarbon receptor, mediated changes on gene expressio...

  2. Cyclic AMP Modulation of Estrogen-Induced Effects: A Novel Mechanism for Hormonal Resistance in Breast Cancer

    DTIC Science & Technology

    1997-10-01

    Marden E, Martin G, MacKay H, Abbon- danza C, Brown M 1994 Estrogen receptor-associated proteins: possible mediators of hormone-induced tran...cells. Nucleic Ac- ids Res 19:6595-6602 42. Halachmi S, Marden E, Martin G, MacKay H, Abbon- danza C, Brown M 1994 Estrogen receptor-associated...the estrogen re- ceptor. EMBO J 14:3741-3751 26. Halachmi S, Marden E, Martin G, MacKay H, Abbon- danza C, Brown M 1994 Estrogen receptor-associated

  3. Estrogenic and progestagenic activities coexisting in steroidal drugs: quantitative evaluation by in vitro bioassays with human cells.

    PubMed

    Markiewicz, L; Gurpide, E

    1994-01-01

    The progestin-specific stimulation of alkaline phosphatase (AP) activity in cells of the T47D human breast cancer line was applied to the development of a sensitive microtiter plate bioassay for the quantitative evaluation of progestagenic and antiprogestagenic potencies of natural and synthetic compounds. Some of the steroids tested (viz. progesterone, medroxyprogesterone acetate, norethynodrel) behaved as full-agonists, capable of inducing AP activities to the same maximal levels (equal efficacy), while others (norethindrone, gestrinone, R5020, norgestrel, Org OD 14 and its 4-ene metabolite) behaved as partial agonists, eliciting lower maximal effects. Efficacy, EC50 values (concentrations at which they induce one-half of the maximal response) and "slope factors" serve to characterize agonistic effects. Relative progestagenic potencies among the full-agonists were evaluated by comparing EC50 concentrations. Several 19-nor synthetic progestins (norethynodrel, norethindrone, Org OD 14 and its 4-ene isomer, dl-norgestrel, levo-norgestrel, RU2323), but none of the tested progestins with the pregnane structure, showed intrinsic estrogenic activity, as evaluated by using a similar in vitro bioassay based on a previously reported estrogen-specific induction of AP in human endometrial adenocarcinoma cells of the Ishikawa Var-1 line. Maximal estrogenic effects of all the tested progestins with dual activity were as high as those of estradiol. However, these compounds widely varied in their EC50 values for estrogenic activity. Consequently, the in vitro bioassays can reveal differences in the ratio of progestagenic and estrogenic activities intrinsic to these compounds. The reduced capability of the partial agonists to exert progestagenic or estrogenic effects on AP expression may reflect an impeded, receptor-mediated action, a mechanism that would also account for their inhibitory effects on the induction of AP activity by full agonists. Partial progestagenic agonists

  4. [Antagonistic effects of estrogen on iron-induced bone resorption and its mechanism].

    PubMed

    Wang, Xiao; Liu, Lulin; Gao, Chao; Zhao, Guoyang; Shen, Guangsi; Zhang, Wen; Xie, Zhanli; Xu, Youjia

    2014-09-09

    To explore the antagonistic effect of estrogen on iron-induced bone resorption and the role of oxidative stress. In vivo, 8-week-old female imprinting control region mice were randomly divided into 3 groups of ferritin (F), ovariectomy (OVX) and F+OVX. Intervention was made by ferric ammonium citrate (FAC) and OVX. Serum levels of ferritin, malondialdehyde (MDA) and superoxide dismutase (SOD) were measured. The expression changes of TRAP, CTR, matrix metallopeptidase 9 (MMP9) and CTK derived from murine bilateral tibia were detected by reverse transcription-polymerase chain reaction (RT-PCR). A high-resolution micro-computed tomography was utilized for scanning distal femur. In vitro, RAW264.7 cells were used and intervened by FAC and estradiol. Tartrate resistant acid phosphatase (TRAP) staining was performed and wine-red TRAP positive cells were counted. ROS level was detected by 2', 7'-dichloro-dihydrofluorescein diacetate (DCFH-DA) with a multi-detection reader. The serum ferritin were heightened in F and F+OVX groups [(335.30 ± 44.10) vs (41.38 ± 5.56) µg/L, (324.80 ± 38.60) vs (41.38 ± 5.56) µg/L respectively, P < 0.01]. The trend of MDA level was F+OVX>OVX>F while SOD level was quite opposite. Body mass density of F+OVX group was lower than that of OVX group (0.114 ± 0.013 vs 0.187 ± 0.029 mg/mm³, P < 0.05) or F group (0.114 ± 0.013 vs 0.902 ± 0.064 mg/mm³, P < 0.05). TRAP and CTK gene expression of OVX group was lower than that of F+OVX group. However, TRAP, CTR and CTK gene expression of F+OVX group was higher than that of F group. TRAP staining: FAC increased the number of TRAP positive cells (41.7 ± 5.5 vs 20.0 ± 4.0, P < 0.05) while estradiol decreased it (14.8 ± 5.1 vs 41.7 ± 5.5, P < 0.05). DCFH-DA test show that reactive oxygen species was elevated by FAC (160% ± 8% vs 100% ± 9%, P < 0.05) and reduced by estradiol (53% ± 13% vs 160% ± 8%, P < 0.05). The antagonistic effect of estrogen on iron-induced bone resorption is probably

  5. Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients.

    PubMed

    Gorvin, Caroline M; Wilmer, Martijn J; Piret, Sian E; Harding, Brian; van den Heuvel, Lambertus P; Wrong, Oliver; Jat, Parmjit S; Lippiat, Jonathan D; Levtchenko, Elena N; Thakker, Rajesh V

    2013-04-23

    Receptor-mediated endocytosis, involving megalin and cubilin, mediates renal proximal-tubular reabsorption and is decreased in Dent disease because of mutations of the chloride/proton antiporter, chloride channel-5 (CLC-5), resulting in low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. To facilitate studies of receptor-mediated endocytosis and the role of CLC-5, we established conditionally immortalized proximal-tubular epithelial cell lines (ciPTECs) from three patients with CLC-5 mutations (30:insH, R637X, and del132-241) and a normal male. Confocal microscopy using the tight junction marker zona occludens-1 (ZO-1) and end-binding protein-1 (EB-1), which is specific for the plus end of microtubules demonstrated that the ciPTECs polarized. Receptor-mediated endocytic uptake of fluorescent albumin and transferrin in 30:insH and R637X ciPTECs was significantly decreased, compared with normal ciPTECs, and could be further reduced by competition with 10-fold excess of unlabeled albumin and transferrin, whereas in the del132-241 ciPTEC, receptor-mediated endocytic uptake was abolished. Investigation of endosomal acidification by live-cell imaging of pHluorin-VAMP2 (vesicle-associated membrane protein-2), a pH-sensitive-GFP construct, revealed that the endosomal pH in normal and 30:insH ciPTECs was similar, whereas in del132-241 and R637X ciPTECs, it was significantly more alkaline, indicating defective acidification in these ciPTECs. The addition of bafilomycin-A1, a V-ATPase inhibitor, raised the pH significantly in all ciPTECs, demonstrating that the differences in acidification were not due to alterations in the V-ATPase, but instead to abnormalities of CLC-5. Thus, our studies, which have established human Dent disease ciPTECs that will facilitate studies of mechanisms in renal reabsorption, demonstrate that Dent disease-causing CLC-5 mutations have differing effects on endosomal acidification and receptor-mediated endocytosis

  6. Pulsed Estrogen Therapy Prevents Post-OVX Porcine Dura Mater Microvascular Network Weakening via a PDGF-BB-Dependent Mechanism

    PubMed Central

    Glinskii, Olga V.; Huxley, Virginia H.; Glinskii, Vladimir V.; Rubin, Leona J.; Glinsky, Vladislav V.

    2013-01-01

    In postmenopausal women, estrogen (E2) deficiencies are frequently associated with higher risk of intracranial hemorrhage, increased incidence of stroke, cerebral aneurysm, and decline in cognitive abilities. In younger postpartum women and those using oral contraceptives, perturbations in E2 are associated with higher risk of cerebral venous thrombosis. A number of serious intracranial pathologic conditions linked to E2 deficiencies, such as dural sinus thrombosis, dural fistulae, non-parenchymal intracranial hemorrhages, migraines, and spontaneous cerebrospinal fluid leaks, involve the vessels not of the brain itself, but of the outer fibrous membrane of the brain, the dura mater (DM). The pathogenesis of these disorders remains mysterious and how estrogen regulates structural and functional integrity of DM vasculature is largely unknown. Here, we demonstrate that post ovariectomy (OVX) DM vascular remodeling is manifested by microvessel destabilization, capillary rarefaction, increased vascular permeability, and aberrant angio-architecture, and is the result of disrupted E2-regulated PDGF-BB signaling within dura microvasculature. These changes, associated with the reduction in systemic PDGF-BB levels, are not corrected by a flat-dose E2 hormone replacement therapy (HRT), but are largely prevented using HRT schedules mimicking physiological E2 fluctuations. We demonstrate that 1) E2 regulates PDGF-BB production by endothelial cells in a dose-dependent manner and 2) optimization of PDGF-BB levels and induction of robust PDGF-mediated endothelial cell-vascular pericyte interactions require high (estrous) E2 concentrations. We conclude that high (estrous) levels of E2 are important in controlling PDGF-mediated crosstalk between endothelial cells and pericytes, a fundamental mechanism governing microvessel stability and essential for preserving intracranial homeostasis. PMID:24349391

  7. 17β-Estradiol and Agonism of G-protein-Coupled Estrogen Receptor Enhance Hippocampal Memory via Different Cell-Signaling Mechanisms

    PubMed Central

    Kim, Jaekyoon; Szinte, Julia S.; Boulware, Marissa I.

    2016-01-01

    The ability of 17β-estradiol (E2) to enhance hippocampal object recognition and spatial memory depends on rapid activation of extracellular signal-regulated kinase (ERK) in the dorsal hippocampus (DH). Although this activation can be mediated by the intracellular estrogen receptors ERα and ERβ, little is known about the role that the membrane estrogen receptor GPER plays in regulating ERK or E2-mediated memory formation. In this study, post-training DH infusion of the GPER agonist G-1 enhanced object recognition and spatial memory in ovariectomized female mice, whereas the GPER antagonist G-15 impaired memory, suggesting that GPER activation, like E2, promotes hippocampal memory formation. However, unlike E2, G-1 did not increase ERK phosphorylation, but instead significantly increased phosphorylation of c-Jun N-terminal kinase (JNK) in the DH. Moreover, DH infusion of the JNK inhibitor SP600125 prevented G-1 from enhancing object recognition and spatial memory, but the ERK inhibitor U0126 did not. These data suggest that GPER enhances memory via different cell-signaling mechanisms than E2. This conclusion was supported by data showing that the ability of E2 to facilitate memory and activate ERK signaling was not blocked by G-15 or SP600125, which demonstrates that the memory-enhancing effects of E2 are not dependent on JNK or GPER activation in the DH. Together, these data indicate that GPER regulates memory independently from ERα and ERβ by activating JNK signaling, rather than ERK signaling. Thus, the findings suggest that GPER in the DH may not function as an estrogen receptor to regulate object recognition and spatial memory. SIGNIFICANCE STATEMENT Although 17β-estradiol has long been known to regulate memory function, the molecular mechanisms underlying estrogenic memory modulation remain largely unknown. Here, we examined whether the putative membrane estrogen receptor GPER acts like the classical estrogen receptors, ERα and ERβ, to facilitate

  8. Toluene exposure during brain growth spurt and adolescence produces differential effects on N-methyl-D-aspartate receptor-mediated currents in rat hippocampus.

    PubMed

    Chen, Hwei-Hsien; Lin, Yi-Ruu; Chan, Ming-Huan

    2011-09-10

    Toluene, an industrial organic solvent, is voluntarily inhaled as drug of abuse. Because inhibition of N-methyl-d-aspartate (NMDA) receptors is one of the possible mechanisms underlying developmental neurotoxicity of toluene, the purpose of the present study was to examine the effects of toluene exposure during two major neurodevelopmental stages, brain growth spurt and adolescence, on NMDA receptor-mediated current. Rats were administered with toluene (500 mg/kg, i.p.) or corn oil daily over postnatal days (PN) 4-9 (brain growth spurt) or PN 21-26 (early adolescence). Intracellular electrophysiological recordings employing in CA1 pyramidal neurons in the hippocampal slices were performed during PN 30-38. Toluene exposure during brain growth spurt enhanced NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) by electrical stimulation, but impaired the paired-pulse facilitation and NMDA response by exogenous application of NMDA. Toluene exposure during adolescence resulted in an increase in NMDA receptor-mediated EPSCs and a decrease in exogenous NMDA-induced currents, while lack of any effect on paired-pulse facilitation. These findings suggest that toluene exposure during brain growth spurt and adolescence might result in an increase in synaptic NMDA receptor responsiveness and a decrease in extrasynaptic NMDA receptor responsiveness, while only toluene exposure during brain growth spurt can produce presynaptic modulation in CA1 pyramidal neurons. The functional changes in NMDA receptor-mediated transmission underlying developmental toluene exposure may lead to the neurobehavioral disturbances.

  9. Receptor-mediated toxicity of pahutoxin, a marine trunkfish surfactant.

    PubMed

    Kalmanzon, Eliahu; Rahamim, Yocheved; Barenholz, Yechezkel; Carmeli, Shmuel; Zlotkin, Eliahu

    2003-07-01

    Pahutoxin (PHN, choline chloride ester of 3-acetoxypalmitic acid) is a natural fish-killing (ichthyotoxic) agent derived from the defensive secretions of trunkfish. In spite of its obvious structural resemblance to synthetic cationic long-chain quaternary ammonium detergents, we show that PHN's action does not rely on its surfactant properties and is in fact, receptor-mediated. The above conclusion is supported by the following data: 1. Ichthyotoxicity is not related to its detergency or surfactivity, as indicated by the fact that the lethal concentration is about 1.5 orders of magnitude below its critical micelle concentration value (69 microM) and its liposomal/seawater partition coefficient is low (62-85); 2. The trunkfish is tolerant to its own pahutoxin; 3. Ichthyotoxicity occurs only upon application to the surrounding water, suggesting the existence of externally located receptors; 4. The receptor hypothesis was supported by the aid of equilibrium saturation binding assays revealing the presence of specific binding sites to PHN on the fish gill membranes; 5. The PHN tolerant trunkfish was shown to be devoid of PHN-binding sites. Some chemo-ecological, and environmental implications are discussed.

  10. Glutamate receptor-mediated toxicity in optic nerve oligodendrocytes

    PubMed Central

    Matute, Carlos; Sánchez-Gómez, M. Victoria; Martínez-Millán, Luis; Miledi, Ricardo

    1997-01-01

    In cultured oligodendrocytes isolated from perinatal rat optic nerves, we have analyzed the expression of ionotropic glutamate receptor subunits as well as the effect of the activation of these receptors on oligodendrocyte viability. Reverse transcription–PCR, in combination with immunocytochemistry, demonstrated that most oligodendrocytes differentiated in vitro express the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR3 and GluR4 and the kainate receptor subunits GluR6, GluR7, KA1 and KA2. Acute and chronic exposure to kainate caused extensive oligodendrocyte death in culture. This effect was partially prevented by the AMPA receptor antagonist GYKI 52466 and was completely abolished by the non-N-methyl-d-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), suggesting that both AMPA and kainate receptors mediate the observed kainate toxicity. Furthermore, chronic application of kainate to optic nerves in vivo resulted in massive oligodendrocyte death which, as in vitro, could be prevented by coinfusion of the toxin with CNQX. These findings suggest that excessive activation of the ionotropic glutamate receptors expressed by oligodendrocytes may act as a negative regulator of the size of this cell population. PMID:9238063

  11. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications.

    PubMed

    D'Souza, Anisha A; Devarajan, Padma V

    2015-04-10

    Hepatocyte resident afflictions continue to affect the human population unabated. The asialoglycoprotein receptor (ASGPR) is primarily expressed on hepatocytes and minimally on extra-hepatic cells. This makes it specifically attractive for receptor-mediated drug delivery with minimum concerns of toxicity. ASGPR facilitates internalization by clathrin-mediated endocytosis and exhibits high affinity for carbohydrates specifically galactose, N-acetylgalactosamine and glucose. Isomeric forms of sugar, galactose density and branching, spatial geometry and galactose linkages are key factors influencing ligand-receptor binding. Popular ligands for ASGPR mediated targeting are carbohydrate polymers, arabinogalactan and pullulan. Other ligands include galactose-bearing glycoproteins, glycopeptides and galactose modified polymers and lipids. Drug-ligand conjugates provide a viable strategy; nevertheless ligand-anchored nanocarriers provide an attractive option for ASGPR targeted delivery and are widely explored. The present review details various ligands and nanocarriers exploited for ASGPR mediated delivery of drugs to hepatocytes. Nanocarrier properties affecting ASGPR mediated uptake are discussed at length. The review also highlights the clinical relevance of ASGPR mediated targeting and applications in diagnostics. ASGPR mediated hepatocyte targeting provides great promise for improved therapy of hepatic afflictions.

  12. Visualization of Receptor-mediated Endocytosis in Yeast

    PubMed Central

    Mulholland, Jon; Konopka, James; Singer-Kruger, Birgit; Zerial, Marino; Botstein, David

    1999-01-01

    We studied the ligand-induced endocytosis of the yeast α-factor receptor Ste2p by immuno-electron microscopy. We observed and quantitated time-dependent loss of Ste2p from the plasma membrane of cells exposed to α-factor. This ligand-induced internalization of Ste2p was blocked in the well-characterized endocytosis-deficient mutant sac6Δ. We provide evidence that implicates furrow-like invaginations of the plasma membrane as the site of receptor internalization. These invaginations are distinct from the finger-like plasma membrane invaginations within actin cortical patches. Consistent with this, we show that Ste2p is not located within the cortical actin patch before and during receptor-mediated endocytosis. In wild-type cells exposed to α-factor we also observed and quantitated a time-dependent accumulation of Ste2p in intracellular, membrane-bound compartments. These compartments have a characteristic electron density but variable shape and size and are often located adjacent to the vacuole. In immuno-electron microscopy experiments these compartments labeled with antibodies directed against the rab5 homologue Ypt51p (Vps21p), the resident vacuolar protease carboxypeptidase Y, and the vacuolar H+-ATPase Vph1p. Using a new double-labeling technique we have colocalized antibodies against Ste2p and carboxypeptidase Y to this compartment, thereby identifying these compartments as prevacuolar late endosomes. PMID:10069819

  13. Purine receptor mediated actin cytoskeleton remodeling of human fibroblasts

    PubMed Central

    Goldman, Nanna; Chandler-Militello, Devin; Langevin, Helene; Nedergaard, Maiken; Takano, Takahiro

    2013-01-01

    Earlier studies have shown that activation of adenosine A1 receptors on peripheral pain fibers contributes to acupuncture-induced suppression of painful input. In addition to adenosine, acupuncture triggers the release of other purines, including ATP and ADP that may bind to purine receptors on nearby fibroblasts. We here show that purine agonists trigger increase in cytosolic Ca 2+ signaling in a cultured human fibroblasts cell line. The profile of agonist-induced Ca2+ increases indicates that the cells express functional P2yR2 and P2yR4 receptors, as well as P2yR1 and P2xR7 receptors. Unexpectedly, purine-induced Ca2+ signaling was associated with a remodeling of the actin cytoskeleton. ATP induced a transient loss in F-actin stress fiber. The changes of actin cytoskeleton occurred slowly and peaked at 10 min after agonist exposure. Inhibition of ATP-induced increases in Ca2+ by cyclopiazonic acid blocked receptor-mediated cytoskeleton remodeling. The Ca2+ ionophore failed to induce cytoskeletal remodeling despite triggering robust increases in cytosolic Ca2+. These observations indicate that purine signaling induces transient changes in fibroblast cytoarchitecture that could be related to the beneficial effects of acupuncture. PMID:23462235

  14. SPHINGOSINE-1-PHOSPHATE RECEPTORS MEDIATE NEUROMODULATORY FUNCTIONS IN THE CNS

    PubMed Central

    Sim-Selley, Laura J.; Goforth, Paulette B.; Mba, Mba U.; Macdonald, Timothy L.; Lynch, Kevin R.; Milstien, Sheldon; Spiegel, Sarah; Satin, Leslie S.; Welch, Sandra P.; Selley, Dana E.

    2009-01-01

    Sphingosine-1-phosphate is a ubiquitous, lipophilic cellular mediator that acts in part by activation of G-protein-coupled receptors. Modulation of S1P signaling is an emerging pharmacotherapeutic target for immunomodulatory drugs. Although multiple S1P receptor types exist in the CNS, little is known about their function. Here we report that S1P stimulated G- protein activity in the CNS, and results from [35S]GTPγS autoradiography using the S1P1-selective agonist SEW2871 and the S1P1/3-selective antagonist VPC44116 show that in several regions a majority of this activity is mediated by S1P1 receptors. S1P receptor activation inhibited glutamatergic neurotransmission as determined by electrophysiological recordings in cortical neurons in vitro, and this effect was mimicked by SEW2871 and inhibited by VPC44116. Moreover, central administration of S1P produced in vivo effects resembling the actions of cannabinoids, including thermal antinociception, hypothermia, catalepsy and hypolocomotion, but these actions were independent of CB1 receptors. At least one of the central effects of S1P, thermal antinociception, is also at least partly S1P1 receptor mediated because it was produced by SEW2871 and attenuated by VPC44116. These results indicate that CNS S1P receptors are part of a physiologically relevant and widespread neuromodulatory system, and that the S1P1 receptor contributes to S1P-mediated antinociception. PMID:19493165

  15. Biologically bounded risk assessment for receptor-mediated nongenotoxic carcinogens.

    PubMed

    Gastel, J A; Sutter, T R

    1995-12-01

    We have developed a biologically bounded marginal effect model for use in risk assessment of human exposure to receptor-mediated nongenotoxic carcinogens. Schematically this model can be reduced to four components: CI, the absence of an observable biological response; CII, observable biochemical responses but no observable pathology; CIII, observable pathology; and CIV, both observable pathology and lethality. The inflection point in the marginal response curve between CI and CII is defined as the biologically evaluated scientifically tested no observable effect level (BESTNOEL). We demonstrate the utility of this approach by applying it to the well-studied nongenotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Using a well-developed mechanistic understanding of the initial interactions of TCDD with the cell, we justify the selection of the minimal effective dose for CYP1A1 mRNA induction as the BESTNOEL. With allowance for variation in human sensitivity to TCDD, the BESTNOEL is assigned a human liver tissue burden of approximately 0.25-25 ppt and an allowable daily intake level in the range of 15-1500 fg/kg/day. In the future, the BESTNOEL can help establish a lower boundary for acceptable extrapolation when using either statistical or biologically based attributable risk models.

  16. G-protein-coupled receptor 30-mediated antiapoptotic effect of estrogen on spinal motor neurons following injury and its underlying mechanisms.

    PubMed

    Chen, Jingyu; Hu, Rong; Ge, Hongfei; Duanmu, Wangsheng; Li, Yuhong; Xue, Xingseng; Hu, Shengli; Feng, Hua

    2015-08-01

    Spinal cord injury (SCI) may result in severe dysfunction of motor neurons. G-protein-coupled receptor 30 (GPR30) expression in the motor neurons of the ventral horn of the spinal cord mediates neuroprotection through estrogen signaling. The present study explored the antiapoptotic effect of estrogen, mediated by GPR30 following SCI, and the mechanisms underlying this effect. Spinal motor neurons from rats were cultured in vitro in order to establish cell models of oxygen and glucose deprivation (OGD). The effects of estrogen, the estrogen agonist, G1, and the estrogen inhibitor, G15, on motor neurons were observed using MTT assays. The effects of E2, G1 and G15 on spinal motor neuron apoptosis following OGD, were detected using flow cytometry. The role of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) inhibitor, LY294002, was also determined using flow cytometry. Rat SCI models were established. E2, G1 and E2+LY294002 were administered in vivo. Motor function was scored at 3, 7, 14, 21 and 28 d following injury, using Basso-Beattie-Bresnahan (BBB) standards. Cell activity in the estrogen and G1 groups was higher than that in the solvent group, whereas cell activity in the E2+G15 group was lower than that in the E2 group (P<0.05). Following OGD, the proportion of apoptotic cells significantly increased (P<0.05). The proportion in the estrogen group was significantly lower than that in the solvent group, whereas the proportion of apoptotic cells in the E2+G15 and E2+LY294002 groups was higher than that in the E2 group (P<0.05). Treatment with E2 and G1 led to upregulation of P-Akt expression in normal cells and post-OGD cells. The BBB scores of rats in the E2 and G1 groups were higher than those in the placebo group (P<0.05). The BBB scores of the E2+LY294002 group were lower than those of the E2 group (P<0.05). Estrogen thus appears to exert a protective effect on spinal motor neurons following OGD, via GPR30. The PI3K/Akt pathway may be one of those

  17. Cellular mechanisms involved in iso-osmotic high K+ solutions-induced contraction of the estrogen-primed rat myometrium.

    PubMed

    Trujillo, M M; Ausina, P; Savineau, J P; Marthan, R; Strippoli, G; Advenier, C; Pinto, F M; Candenas, M L

    2000-01-01

    The aim of the present study was to investigate the mechanisms involved in the contraction evoked by iso-osmotic high K+ solutions in the estrogen-primed rat uterus. In Ca2+-containing solution, iso-osmotic addition of KCl (30, 60 or 90 mM K+) induced a rapid, phasic contraction followed by a prolonged sustained plateau (tonic component) of smaller amplitude. The KCl (60 mM)-induced contraction was unaffected by tetrodotoxin (3 microM), omega-conotoxin MVIIC (1 microM), GF 109203X (1 microM) or calphostin C (3 microM) but was markedly reduced by tissue treatment with neomycin (1 mM), mepacrine (10 microM) or U-73122 (10 microM). Nifedipine (0.01-0.1 microM) was significantly more effective as an inhibitor of the tonic component than of the phasic component. After 60 min incubation in Ca2+-free solution containing 3 mM EGTA, iso-osmotic KCl did not cause any increase in tension but potentiated contractions evoked by oxytocin (1 microM), sodium orthovanadate (160 micrM) or okadaic acid (20 microM) in these experimental conditions. In freshly dispersed myometrial cells maintained in Ca2+-containing solution and loaded with indo 1, iso-osmotic KCl (60 mM) caused a biphasic increase in the intracellular Ca2+ concentration ([Ca2+]i). In cells superfused for 60 min in Ca2+-free solution containing EGTA (1 mM), KCl did not increase [Ca2+]i. In Ca2+-containing solution, KCl (60 mM) produced a 76.0 +/- 16.2% increase in total [3H]inositol phosphates above basal levels and increased the intracellular levels of free arachidonic acid. These results suggest that, in the estrogen-primed rat uterus, iso-osmotic high K+ solutions, in addition to their well known effect on Ca2+ influx, activate other cellular processes leading to an increase in the Ca2+ sensitivity of the contractile machinery by a mechanism independent of extracellular Ca2+.

  18. VASCULAR ACTIONS OF ESTROGENS: FUNCTIONAL IMPLICATIONS

    PubMed Central

    Miller, Virginia M.; Duckles, Sue P.

    2009-01-01

    The impact of estrogen exposure in preventing or treating cardiovascular disease is controversial. But it is clear that estrogen has important effects on vascular physiology and pathophysiology, with potential therapeutic implications. Therefore, it is the goal of this review to summarize, using an integrated approach, current knowledge of the vascular effects of estrogen, both in humans and in experimental animals. Aspects of estrogen synthesis and receptors, as well as general mechanisms of estrogenic action are reviewed with an emphasis on issues particularly relevant to the vascular system. Recent understanding of the impact of estrogen on mitochondrial function suggests that the longer lifespan of women compared to men may depend in part on the ability of estrogen to decrease production of reactive oxygen species in mitochondria. Mechanisms by which estrogen increases endothelial vasodilator function, promotes angiogenesis and modulates autonomic function are summarized. Key aspects of the relevant pathophysiology of inflammation, atherosclerosis, stroke, migraine and thrombosis are reviewed concerning current knowledge of estrogenic effects. A number of emerging concepts are addressed throughout. These include the importance of estrogenic formulation and route of administration and the impact of genetic polymorphisms, either in estrogen receptors or in enzymes responsible for estrogen metabolism, on responsiveness to hormone treatment. The importance of local metabolism of estrogenic precursors and the impact of timing for initiation of treatment and its duration are also considered. While consensus opinions are emphasized, controversial views are presented in order to stimulate future research. PMID:18579753

  19. Phenobarbital but Not Diazepam Reduces AMPA/kainate Receptor Mediated Currents and Exerts Opposite Actions on Initial Seizures in the Neonatal Rat Hippocampus

    PubMed Central

    Nardou, Romain; Yamamoto, Sumii; Bhar, Asma; Burnashev, Nail; Ben-Ari, Yehezkel; Khalilov, Ilgam

    2011-01-01

    Diazepam (DZP) and phenobarbital (PB) are extensively used as first and second line drugs to treat acute seizures in neonates and their actions are thought to be mediated by increasing the actions of GABAergic signals. Yet, their efficacy is variable with occasional failure or even aggravation of recurrent seizures questioning whether other mechanisms are not involved in their actions. We have now compared the effects of DZP and PB on ictal-like events (ILEs) in an in vitro model of mirror focus (MF). Using the three-compartment chamber with the two immature hippocampi and their commissural fibers placed in three different compartments, kainate was applied to one hippocampus and PB or DZP to the contralateral one, either after one ILE, or after many recurrent ILEs that produce an epileptogenic MF. We report that in contrast to PB, DZP aggravated propagating ILEs from the start, and did not prevent the formation of MF. PB reduced and DZP increased the network driven giant depolarizing potentials suggesting that PB may exert additional actions that are not mediated by GABA signaling. In keeping with this, PB but not DZP reduced field potentials recorded in the presence of GABA and NMDA receptor antagonists. These effects are mediated by a direct action on AMPA/kainate receptors since PB: (i) reduced AMPA/kainate receptor mediated currents induced by focal applications of glutamate; (ii) reduced the amplitude and the frequency of AMPA but not NMDA receptor mediated miniature excitatory postsynaptic currents (EPSCs); (iii) augmented the number of AMPA receptor mediated EPSCs failures evoked by minimal stimulation. These effects persisted in MF. Therefore, PB exerts its anticonvulsive actions partly by reducing AMPA/kainate receptors mediated EPSCs in addition to the pro-GABA effects. We suggest that PB may have advantage over DZP in the treatment of initial neonatal seizures since the additional reduction of glutamate receptors mediated signals may reduce the severity

  20. Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD)

    PubMed Central

    Okada, Yasunobu; Maeno, Emi; Shimizu, Takahiro; Dezaki, Katsuya; Wang, Jun; Morishima, Shigeru

    2001-01-01

    A fundamental property of animal cells is the ability to regulate their own cell volume. Even under hypotonic stress imposed by either decreased extracellular or increased intracellular osmolarity, the cells can re-adjust their volume after transient osmotic swelling by a mechanism known as regulatory volume decrease (RVD). In most cell types, RVD is accomplished mainly by KCl efflux induced by parallel activation of K+ and Cl− channels. We have studied the molecular mechanism of RVD in a human epithelial cell line (Intestine 407). Osmotic swelling results in a significant increase in the cytosolic Ca2+ concentration and thereby activates intermediate-conductance Ca2+-dependent K+ (IK) channels. Osmotic swelling also induces ATP release from the cells to the extracellular compartment. Released ATP stimulates purinergic ATP (P2Y2) receptors, thereby inducing phospholipase C-mediated Ca2+ mobilization. Thus, RVD is facilitated by stimulation of P2Y2 receptors due to augmentation of IK channels. In contrast, stimulation of another G protein-coupled Ca2+-sensing receptor (CaR) enhances the activity of volume-sensitive outwardly rectifying Cl− channels, thereby facilitating RVD. Therefore, it is possible that Ca2+ efflux stimulated by swelling-induced and P2Y2 receptor-mediated intracellular Ca2+ mobilization activates the CaR, thereby secondarily upregulating the volume-regulatory Cl− conductance. On the other hand, the initial process towards apoptotic cell death is coupled to normotonic cell shrinkage, called apoptotic volume decrease (AVD). Stimulation of death receptors, such as TNFα receptor and Fas, induces AVD and thereafter biochemical apoptotic events in human lymphoid (U937), human epithelial (HeLa), mouse neuroblastoma × rat glioma hybrid (NG108-15) and rat phaeochromocytoma (PC12) cells. In those cells exhibiting AVD, facilitation of RVD is always observed. Both AVD induction and RVD facilitation as well as succeeding apoptotic events can be

  1. Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD).

    PubMed

    Okada, Y; Maeno, E; Shimizu, T; Dezaki, K; Wang, J; Morishima, S

    2001-04-01

    A fundamental property of animal cells is the ability to regulate their own cell volume. Even under hypotonic stress imposed by either decreased extracellular or increased intracellular osmolarity, the cells can re-adjust their volume after transient osmotic swelling by a mechanism known as regulatory volume decrease (RVD). In most cell types, RVD is accomplished mainly by KCl efflux induced by parallel activation of K+ and Cl- channels. We have studied the molecular mechanism of RVD in a human epithelial cell line (Intestine 407). Osmotic swelling results in a significant increase in the cytosolic Ca2+ concentration and thereby activates intermediate-conductance Ca2+-dependent K+ (IK) channels. Osmotic swelling also induces ATP release from the cells to the extracellular compartment. Released ATP stimulates purinergic ATP (P2Y2) receptors, thereby inducing phospholipase C-mediated Ca2+ mobilization. Thus, RVD is facilitated by stimulation of P2Y2 receptors due to augmentation of IK channels. In contrast, stimulation of another G protein-coupled Ca2+-sensing receptor (CaR) enhances the activity of volume-sensitive outwardly rectifying Cl- channels, thereby facilitating RVD. Therefore, it is possible that Ca2+ efflux stimulated by swelling-induced and P2Y2 receptor-mediated intracellular Ca2+ mobilization activates the CaR, thereby secondarily upregulating the volume-regulatory Cl- conductance. On the other hand, the initial process towards apoptotic cell death is coupled to normotonic cell shrinkage, called apoptotic volume decrease (AVD). Stimulation of death receptors, such as TNF receptor and Fas, induces AVD and thereafter biochemical apoptotic events in human lymphoid (U937), human epithelial (HeLa), mouse neuroblastoma x rat glioma hybrid (NG108-15) and rat phaeochromocytoma (PC12) cells. In those cells exhibiting AVD, facilitation of RVD is always observed. Both AVD induction and RVD facilitation as well as succeeding apoptotic events can be abolished by

  2. QSAR classification of estrogen receptor binders and pre-screening of potential pleiotropic EDCs.

    PubMed

    Li, J; Gramatica, P

    2010-10-01

    Endocrine disrupting chemicals (EDCs) are suspected of posing serious threats to human and wildlife health through a variety of mechanisms, these being mainly receptor-mediated modes of action. It is reported that some EDCs exhibit dual activities as estrogen receptor (ER) and androgen receptor (AR) binders. Indeed, such compounds can affect the normal endocrine system through a dual complex mechanism, so steps should be taken not only to identify them a priori from their chemical structure, but also to prioritize them for experimental tests in order to reduce and even forbid their usage. To date, very few EDCs with dual activities have been identified. The present research uses QSARs, to investigate what, so far, is the largest and most heterogeneous ER binder data set (combined METI and EDKB databases). New predictive classification models were derived using different modelling methods and a consensus approach, and these were used to virtually screen a large AR binder data set after strict validation. As a result, 46 AR antagonists were predicted from their chemical structure to also have potential ER binding activities, i.e. pleiotropic EDCs. In addition, 48 not yet recognized ER binders were in silico identified, which increases the number of potential EDCs that are substances of very high concern (SVHC) in REACH. Thus, the proposed screening models, based only on structure information, have the main aim to prioritize experimental tests for the highlighted compounds with potential estrogenic activities and also to design safer alternatives.

  3. Bombesin receptor-mediated imaging and cytotoxicity: review and current status

    PubMed Central

    Sancho, Veronica; Di Florio, Alessia; Moody, Terry W.; Jensen, Robert T.

    2010-01-01

    The three mammalian bombesin (Bn) receptors (gastrin-releasing peptide [GRP] receptor, neuromedin B [NMB] receptor, BRS-3) are one of the classes of G protein-coupled receptors that are most frequently over-express/ectopically expressed by common, important malignancies. Because of the clinical success of somatostatin receptor-mediated imaging and cytotoxicity with neuroendocrine tumors, there is now increasing interest in pursuing a similar approach with Bn receptors. In the last few years then have been more than 200 studies in this area. In the present paper, the in vitro and in vivo results, as well as results of human studies from many of these studies are reviewed and the current state of Bn receptor-mediated imaging or cytotoxicity is discussed. Both Bn receptor-mediated imaging studies as well as Bn receptor-mediated tumoral cytotoxic studies using radioactive and non-radioactive Bn-based ligands are covered. PMID:21034419

  4. Histamine H3 receptor-mediated inhibition of noradrenaline release in the human brain.

    PubMed

    Schlicker, E; Werthwein, S; Zentner, J

    1999-01-01

    Stimulation-evoked 3H-noradrenaline release in human cerebrocortical slices was inhibited by histamine (in a manner sensitive to clobenpropit) and by imetit, suggesting H3 receptor-mediated inhibition of noradrenaline release in human brain.

  5. Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats.

    PubMed

    Starlard-Davenport, Athena; Tryndyak, Volodymyr P; James, Smitha R; Karpf, Adam R; Latendresse, John R; Beland, Frederick A; Pogribny, Igor P

    2010-03-01

    Breast cancer, the most common malignancy in women, emerges through a multistep process, encompassing the progressive sequential evolution of morphologically distinct stages from a normal cell to hyperplasia (with and without atypia), carcinoma in situ, invasive carcinoma and metastasis. The success of treatment of breast cancer could be greatly improved by the detection at early stages of cancer. In the present study, we investigated the underlying molecular mechanisms involved in breast carcinogenesis in Augustus and Copenhagen-Irish female rats, a cross between the ACI strains, induced by continuous exposure to 17beta-estradiol. The results of our study demonstrate that early stages of estrogen-induced breast carcinogenesis are characterized by altered global DNA methylation, aberrant expression of proteins responsible for the proper maintenance of DNA methylation pattern and epigenetic silencing of the critical Rassf1a (Ras-association domain family 1, isoform A) tumor suppressor gene. Interestingly, transcriptional repression of the Rassf1a gene in mammary glands during early stages of breast carcinogenesis was associated with an increase in trimethylation of histones H3 lysine 9 and H3 lysine 27 and de novo CpG island methylation and at the Rassf1a promoter and first exon. In conclusion, we demonstrate that epigenetic alterations precede formation of preneoplastic lesions indicating the significance of epigenetic events in induction of oncogenic pathways in early stages of carcinogenesis.

  6. Molecular Mechanism of Action of Genistein and Related Phytoestrogens in Estrogen Receptor Dependent & Independent Growth of Breast Cancer Cells.

    DTIC Science & Technology

    1998-07-01

    During the first year of my pre-doctoral fellowship, I have demonstrated that genistein binds weakly to the estrogen receptor (ER) with an IC50 value...of 900 nM (task 1). Quercetin and genistein also bind to the type-II estrogen binding sites, and exert cell growth inhibition in MCF-7 and MDA-MB-468...In addition, we show that cell growth inhibition by genistein and quercetin is associated with decreased polyamine levels. Our results provide

  7. Enzyme induction and histopathology elucidate aryl hydrocarbon receptor-mediated versus non-aryl hydrocarbon receptor-mediated effects of Aroclor 1268 in American mink (Neovison vison).

    PubMed

    Folland, William R; Newsted, John L; Fitzgerald, Scott D; Fuchsman, Phyllis C; Bradley, Patrick W; Kern, John; Kannan, Kurunthachalam; Zwiernik, Matthew J

    2016-03-01

    Polychlorinated biphenyl (PCB) concentrations reported in preferred prey and blubber of bottlenose dolphins from the Turtle-Brunswick River estuary (Georgia, USA) suggest the potential for adverse effects. However, PCBs in Turtle-Brunswick River estuary dolphins are primarily derived from Aroclor 1268, and predicting toxic effects of Aroclor 1268 is uncertain because of the mixture's unique composition and associated physiochemical characteristics. These differences suggest that toxicity benchmarks for other PCB mixtures may not be relevant to dolphins exposed to Aroclor 1268. American mink (Neovison vison) were used as a surrogate model for cetaceans to characterize mechanisms of action associated with Aroclor 1268 exposure. Mink share similarities in phylogeny and life history with cetaceans and are characteristically sensitive to PCBs, making them an attractive surrogate species for marine mammals in ecotoxicity studies. Adult female mink and a subsequent F1 generation were exposed to Aroclor 1268 through diet, and effects on enzyme induction, histopathology, thyroid hormone regulation, hematology, organ weights, and body condition index were compared to a negative control and a 3,3',4,4',5-pentachlorobiphenyl (PCB 126)-positive control. Aroclor 1268 dietary exposure concentrations ranged from 1.8 µg/g wet weight to 29 µg/g wet weight. Anemia, hypothyroidism, and hepatomegaly were observed in mink exposed to Aroclor 1268 beyond various dietary thresholds. Cytochrome P450 induction and squamous epithelial proliferation jaw lesions were low in Aroclor 1268 treatments relative to the positive control. Differences in enzyme induction and the development of squamous epithelial proliferation jaw lesions between Aroclor 1268 treatments and the positive control, coupled with effects observed in Aroclor 1268 treatments not observed in the positive control, indicate that mechanisms additional to the aryl hydrocarbon receptor-mediated pathway are associated with

  8. An NO Donor Approach to Neuroprotective and Procognitive Estrogen Therapy Overcomes Loss of NO Synthase Function and Potentially Thrombotic Risk

    PubMed Central

    VandeVrede, Lawren; Abdelhamid, Ramy; Qin, Zhihui; Choi, Jaewoo; Piyankarage, Sujeewa; Luo, Jia; Larson, John; Bennett, Brian M.; Thatcher, Gregory R. J.

    2013-01-01

    Selective estrogen receptor modulators (SERMs) are effective therapeutics that preserve favorable actions of estrogens on bone and act as antiestrogens in breast tissue, decreasing the risk of vertebral fractures and breast cancer, but their potential in neuroprotective and procognitive therapy is limited by: 1) an increased lifetime risk of thrombotic events; and 2) an attenuated response to estrogens with age, sometimes linked to endothelial nitric oxide synthase (eNOS) dysfunction. Herein, three 3rd generation SERMs with similar high affinity for estrogen receptors (ERα, ERβ) were studied: desmethylarzoxifene (DMA), FDMA, and a novel NO-donating SERM (NO-DMA). Neuroprotection was studied in primary rat neurons exposed to oxygen glucose deprivation; reversal of cholinergic cognitive deficit was studied in mice in a behavioral model of memory; long term potentiation (LTP), underlying cognition, was measured in hippocampal slices from older 3×Tg Alzheimer's transgenic mice; vasodilation was measured in rat aortic strips; and anticoagulant activity was compared. Pharmacologic blockade of GPR30 and NOS; denudation of endothelium; measurement of NO; and genetic knockout of eNOS were used to probe mechanism. Comparison of the three chemical probes indicates key roles for GPR30 and eNOS in mediating therapeutic activity. Procognitive, vasodilator and anticoagulant activities of DMA were found to be eNOS dependent, while neuroprotection and restoration of LTP were both shown to be dependent upon GPR30, a G-protein coupled receptor mediating estrogenic function. Finally, the observation that an NO-SERM shows enhanced vasodilation and anticoagulant activity, while retaining the positive attributes of SERMs even in the presence of NOS dysfunction, indicates a potential therapeutic approach without the increased risk of thrombotic events. PMID:23976955

  9. An NO donor approach to neuroprotective and procognitive estrogen therapy overcomes loss of NO synthase function and potentially thrombotic risk.

    PubMed

    VandeVrede, Lawren; Abdelhamid, Ramy; Qin, Zhihui; Choi, Jaewoo; Piyankarage, Sujeewa; Luo, Jia; Larson, John; Bennett, Brian M; Thatcher, Gregory R J

    2013-01-01

    Selective estrogen receptor modulators (SERMs) are effective therapeutics that preserve favorable actions of estrogens on bone and act as antiestrogens in breast tissue, decreasing the risk of vertebral fractures and breast cancer, but their potential in neuroprotective and procognitive therapy is limited by: 1) an increased lifetime risk of thrombotic events; and 2) an attenuated response to estrogens with age, sometimes linked to endothelial nitric oxide synthase (eNOS) dysfunction. Herein, three 3(rd) generation SERMs with similar high affinity for estrogen receptors (ERα, ERβ) were studied: desmethylarzoxifene (DMA), FDMA, and a novel NO-donating SERM (NO-DMA). Neuroprotection was studied in primary rat neurons exposed to oxygen glucose deprivation; reversal of cholinergic cognitive deficit was studied in mice in a behavioral model of memory; long term potentiation (LTP), underlying cognition, was measured in hippocampal slices from older 3×Tg Alzheimer's transgenic mice; vasodilation was measured in rat aortic strips; and anticoagulant activity was compared. Pharmacologic blockade of GPR30 and NOS; denudation of endothelium; measurement of NO; and genetic knockout of eNOS were used to probe mechanism. Comparison of the three chemical probes indicates key roles for GPR30 and eNOS in mediating therapeutic activity. Procognitive, vasodilator and anticoagulant activities of DMA were found to be eNOS dependent, while neuroprotection and restoration of LTP were both shown to be dependent upon GPR30, a G-protein coupled receptor mediating estrogenic function. Finally, the observation that an NO-SERM shows enhanced vasodilation and anticoagulant activity, while retaining the positive attributes of SERMs even in the presence of NOS dysfunction, indicates a potential therapeutic approach without the increased risk of thrombotic events.

  10. Enhancement of postsynaptic GABAA and extrasynaptic NMDA receptor-mediated responses in the barrel cortex of Mecp2-null mice.

    PubMed

    Lo, Fu-Sun; Blue, Mary E; Erzurumlu, Reha S

    2016-03-01

    Rett syndrome (RTT) is a neurodevelopmental disorder that results from mutations in the X-linked gene for methyl-CpG-binding protein 2 (MECP2). The underlying cellular mechanism for the sensory deficits in patients with RTT is largely unknown. This study used the Bird mouse model of RTT to investigate sensory thalamocortical synaptic transmission in the barrel cortex of Mecp2-null mice. Electrophysiological results showed an excitation/inhibition imbalance, biased toward inhibition, due to an increase in efficacy of postsynaptic GABAA receptors rather than alterations in inhibitory network and presynaptic release properties. Enhanced inhibition impaired the transmission of tonic sensory signals from the thalamus to the somatosensory cortex. Previous morphological studies showed an upregulation of NMDA receptors in the neocortex of both RTT patients and Mecp2-null mice at early ages [Blue ME, Naidu S, Johnston MV. Ann Neurol 45: 541-545, 1999; Blue ME, Kaufmann WE, Bressler J, Eyring C, O'Driscoll C, Naidu S, Johnston MV. Anat Rec (Hoboken) 294: 1624-1634, 2011]. Although AMPA and NMDA receptor-mediated excitatory synaptic transmission was not altered in the barrel cortex of Mecp2-null mice, extrasynaptic NMDA receptor-mediated responses increased markedly. These responses were blocked by memantine, suggesting that extrasynaptic NMDA receptors play an important role in the pathogenesis of RTT. The results suggest that enhancement of postsynaptic GABAA and extrasynaptic NMDA receptor-mediated responses may underlie impaired somatosensation and that pharmacological blockade of extrasynaptic NMDA receptors may have therapeutic value for RTT.

  11. Enhancement of postsynaptic GABAA and extrasynaptic NMDA receptor-mediated responses in the barrel cortex of Mecp2-null mice

    PubMed Central

    Lo, Fu-Sun; Blue, Mary E.

    2015-01-01

    Rett syndrome (RTT) is a neurodevelopmental disorder that results from mutations in the X-linked gene for methyl-CpG-binding protein 2 (MECP2). The underlying cellular mechanism for the sensory deficits in patients with RTT is largely unknown. This study used the Bird mouse model of RTT to investigate sensory thalamocortical synaptic transmission in the barrel cortex of Mecp2-null mice. Electrophysiological results showed an excitation/inhibition imbalance, biased toward inhibition, due to an increase in efficacy of postsynaptic GABAA receptors rather than alterations in inhibitory network and presynaptic release properties. Enhanced inhibition impaired the transmission of tonic sensory signals from the thalamus to the somatosensory cortex. Previous morphological studies showed an upregulation of NMDA receptors in the neocortex of both RTT patients and Mecp2-null mice at early ages [Blue ME, Naidu S, Johnston MV. Ann Neurol 45: 541–545, 1999; Blue ME, Kaufmann WE, Bressler J, Eyring C, O'Driscoll C, Naidu S, Johnston MV. Anat Rec (Hoboken) 294: 1624–1634, 2011]. Although AMPA and NMDA receptor-mediated excitatory synaptic transmission was not altered in the barrel cortex of Mecp2-null mice, extrasynaptic NMDA receptor-mediated responses increased markedly. These responses were blocked by memantine, suggesting that extrasynaptic NMDA receptors play an important role in the pathogenesis of RTT. The results suggest that enhancement of postsynaptic GABAA and extrasynaptic NMDA receptor-mediated responses may underlie impaired somatosensation and that pharmacological blockade of extrasynaptic NMDA receptors may have therapeutic value for RTT. PMID:26683074

  12. Activation of innate antiviral immune response via double-stranded RNA-dependent RLR receptor-mediated necroptosis

    PubMed Central

    Wang, Wei; Wang, Wei-Hua; Azadzoi, Kazem M.; Su, Ning; Dai, Peng; Sun, Jianbin; Wang, Qin; Liang, Ping; Zhang, Wentao; Lei, Xiaoying; Yan, Zhen; Yang, Jing-Hua

    2016-01-01

    Viruses induce double-stranded RNA (dsRNA) in the host cells. The mammalian system has developed dsRNA-dependent recognition receptors such as RLRs that recognize the long stretches of dsRNA as PAMPs to activate interferon-mediated antiviral pathways and apoptosis in severe infection. Here we report an efficient antiviral immune response through dsRNA-dependent RLR receptor-mediated necroptosis against infections from different classes of viruses. We demonstrated that virus-infected A549 cells were efficiently killed in the presence of a chimeric RLR receptor, dsCARE. It measurably suppressed the interferon antiviral pathway but promoted IL-1β production. Canonical cell death analysis by morphologic assessment, phosphatidylserine exposure, caspase cleavage and chemical inhibition excluded the involvement of apoptosis and consistently suggested RLR receptor-mediated necroptosis as the underlying mechanism of infected cell death. The necroptotic pathway was augmented by the formation of RIP1-RIP3 necrosome, recruitment of MLKL protein and the activation of cathepsin D. Contributing roles of RIP1 and RIP3 were confirmed by gene knockdown. Furthermore, the necroptosis inhibitor necrostatin-1 but not the pan-caspase inhibitor zVAD impeded dsCARE-dependent infected cell death. Our data provides compelling evidence that the chimeric RLR receptor shifts the common interferon antiviral responses of infected cells to necroptosis and leads to rapid death of the virus-infected cells. This mechanism could be targeted as an efficient antiviral strategy. PMID:26935990

  13. Receptor-Mediated Endocytosis of Two-Dimensional Nanomaterials Undergoes Flat Vesiculation and Occurs by Revolution and Self-Rotation.

    PubMed

    Mao, Jian; Chen, Pengyu; Liang, Junshi; Guo, Ruohai; Yan, Li-Tang

    2016-01-26

    Two-dimensional nanomaterials, such as graphene and transitional metal dichalcogenide nanosheets, are promising materials for the development of antimicrobial surfaces and the nanocarriers for intracellular therapy. Understanding cell interaction with these emerging materials is an urgently important issue to promoting their wide applications. Experimental studies suggest that two-dimensional nanomaterials enter cells mainly through receptor-mediated endocytosis. However, the detailed molecular mechanisms and kinetic pathways of such processes remain unknown. Here, we combine computer simulations and theoretical derivation of the energy within the system to show that the receptor-mediated transport of two-dimensional nanomaterials, such as graphene nanosheet across model lipid membrane, experiences a flat vesiculation event governed by the receptor density and membrane tension. The graphene nanosheet is found to undergo revolution relative to the membrane and, particularly, unique self-rotation around its normal during membrane wrapping. We derive explicit expressions for the formation of the flat vesiculation, which reveals that the flat vesiculation event can be fundamentally dominated by a dimensionless parameter and a defined relationship determined by complicated energy contributions. The mechanism offers an essential understanding on the cellular internalization and cytotoxicity of the emerging two-dimensional nanomaterials.

  14. Monoacylglycerol lipase promotes Fcγ receptor-mediated phagocytosis in microglia but does not regulate LPS-induced upregulation of inflammatory cytokines.

    PubMed

    Kouchi, Zen

    2015-08-21

    Monoacylglycerol lipase (MAGL) is important for neuroinflammation. However, the regulatory mechanisms underlying its expression and function remain unknown. Lipopolysaccharide (LPS) treatment post-translationally upregulated MAGL expression, whereas it downregulated MAGL transcription through a Stat6-mediated mechanism in microglia. Neither MAGL knockdown nor JZL-184, a selective MAGL inhibitor, suppressed LPS-induced upregulation of inflammatory cytokines in microglia. Moreover, exogenous expression of MAGL in BV-2 microglial cell line, which lacks endogenous MAGL, did not promote the induction of inflammatory cytokines by LPS treatment. Interestingly, MAGL knockdown reduced Fcγ receptor-mediated phagocytosis in primary microglia, and introduction of MAGL into the BV-2 cells increased Fcγ receptor-mediated phagocytosis. Collectively, these results suggest that MAGL regulates phagocytosis, but not LPS-mediated cytokine induction in microglia.

  15. A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat.

    PubMed Central

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1982-01-01

    Native human carcinoembryonic antigen is rapidly removed from the circulation by the rat liver Kupffer cell after intravenous injection. The molecule is subsequently transferred to the hepatocyte in an immunologically identifiable form. Carcinoembryonic antigen has a circulatory half-life of 3.7 (+/- 0.8) min, and cellular entry is by receptor-mediated endocytosis. Non-specific fluid pinocytosis and phagocytosis can be excluded as possible mechanisms by the kinetics of clearance and failure of colloidal carbon to inhibit uptake. Substances with known affinity for the hepatic receptors for mannose, N-acetylglucosamine, fucose and galactose all fail to inhibit carcinoembryonic antigen clearance. After two cycles of the Smith degradation, carcinoembryonic antigen is still able to inhibit clearance of the native molecule. Receptor specificity is apparently not dependent on those non-reducing terminal sugars of the native molecule. Performic acid-oxidized carcinoembryonic antigen also inhibits clearance of carcinoembryonic antigen in vivo. Receptor binding is not dependent on tertiary protein conformation. Non-specific cross-reacting antigen, a glycoprotein structurally similar to carcinoembryonic antigen, is cleared by the same mechanism. PMID:6896821

  16. A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat.

    PubMed

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1982-05-15

    Native human carcinoembryonic antigen is rapidly removed from the circulation by the rat liver Kupffer cell after intravenous injection. The molecule is subsequently transferred to the hepatocyte in an immunologically identifiable form. Carcinoembryonic antigen has a circulatory half-life of 3.7 (+/- 0.8) min, and cellular entry is by receptor-mediated endocytosis. Non-specific fluid pinocytosis and phagocytosis can be excluded as possible mechanisms by the kinetics of clearance and failure of colloidal carbon to inhibit uptake. Substances with known affinity for the hepatic receptors for mannose, N-acetylglucosamine, fucose and galactose all fail to inhibit carcinoembryonic antigen clearance. After two cycles of the Smith degradation, carcinoembryonic antigen is still able to inhibit clearance of the native molecule. Receptor specificity is apparently not dependent on those non-reducing terminal sugars of the native molecule. Performic acid-oxidized carcinoembryonic antigen also inhibits clearance of carcinoembryonic antigen in vivo. Receptor binding is not dependent on tertiary protein conformation. Non-specific cross-reacting antigen, a glycoprotein structurally similar to carcinoembryonic antigen, is cleared by the same mechanism.

  17. Non-genomic actions of estrogens and their interaction with genomic actions in the brain.

    PubMed

    Vasudevan, Nandini; Pfaff, Donald W

    2008-05-01

    Ligands for the nuclear receptor superfamily have at least two mechanisms of action: (a) classical transcriptional regulation of target genes (genomic mechanisms); and (b) non-genomic actions, which are initiated at the cell membrane, which could also impact transcription. Though transcriptional mechanisms are increasingly well understood, membrane-initiated actions of these ligands are incompletely understood. This has led to considerable debate over the physiological relevance of membrane-initiated actions of hormones versus genomic actions of hormones, with genomic actions predominating in the endocrine field. There is good evidence that the membrane-limited actions of hormones, particularly estrogens, involve the rapid activation of kinases and the release of calcium and that these are linked to physiologically relevant scenarios in the brain. We show evidence in this review, that membrane actions of estrogens, which activate these rapid signaling cascades, can also potentiate nuclear transcription in both the central nervous system and in non-neuronal cell lines. We present a theoretical scenario which can be used to understand this phenomenon. These signaling cascades may occur in parallel or in series but subsequently, converge at the modification of transcriptionally relevant molecules such as nuclear receptors and/or coactivators. In addition, other non-cognate hormones or neurotransmitters may also activate cascades to crosstalk with estrogen receptor-mediated transcription, though the relevance of this is less clear. The idea that coupling between membrane-initiated and genomic actions of hormones is a novel idea in neuroendocrinology and provides us with a unified view of hormone action in the central nervous system.

  18. The regulation of Hh/Gli1 signaling cascade involves Gsk3β- mediated mechanism in estrogen-derived endometrial hyperplasia.

    PubMed

    Kaushal, Jyoti Bala; Sankhwar, Pushplata; Kumari, Suparna; Popli, Pooja; Shukla, Vinay; Hussain, Mohd Kamil; Hajela, Kanchan; Dwivedi, Anila

    2017-07-26

    The present study was undertaken to explore the functional involvement of Hh signaling and its regulatory mechanism in endometrial hyperplasia. Differential expression of Hh signaling molecules i.e., Ihh, Shh, Gli1 or Gsk3β was observed in endometrial hyperplasial (EH) cells as compared to normal endometrial cells. Estradiol induced the expression of Hh signaling molecules and attenuated the expression of Gsk3β whereas anti-estrogen (K1) or progestin (MPA) suppressed these effects in EH cells. Cyclopamine treatment or Gli1 siRNA knockdown suppressed the growth of EH cells and reduced the expression of proliferative markers. Estradiol also induced the nuclear translocation of Gli1 which was suppressed by both MPA and K1 in EH cells. While exploring non-canonical mechanism, LY-294002 (Gsk3β activator) caused a decrease in Gli1 expression indicating the involvement of Gsk3β in Gli1 regulation. Further, Gsk3β silencing promoted the expression and nuclear translocation of Gli1 demonstrating that Gsk3β serves as a negative kinase regulator of Gli1 in EH cells. Similar attenuation of Hh signaling molecules was observed in rats with uterine hyperplasia undergoing anti-estrogen treatment. The study suggested that Hh/Gli1 cascade (canonical pathway) as well as Gsk3β-Gli1 crosstalk (non-canonical pathway) play crucial role in estrogen-dependent cell proliferation in endometrial hyperplasia.

  19. Estradiol Rapidly Attenuates ORL-1 Receptor-Mediated Inhibition of Proopiomelanocortin Neurons via Gq-Coupled, Membrane-Initiated Signaling.

    PubMed

    Conde, Kristie; Meza, Cecilia; Kelly, Martin J; Sinchak, Kevin; Wagner, Edward J

    2016-01-01

    Estradiol rapidly regulates the activity of arcuate nucleus (ARH) proopiomelanocortin (POMC) neurons that project to the medial preoptic nucleus (MPN) to regulate lordosis. Orphanin FQ/nociceptin (OFQ/N) acts via opioid receptor-like (ORL)-1 receptors to inhibit these POMC neurons. Therefore, we tested the hypothesis that estradiol excites POMC neurons by rapidly attenuating inhibitory ORL-1 signaling in these cells. Hypothalamic slices through the ARH were prepared from ovariectomized rats injected with Fluorogold into the MPN. Electrophysiological recordings were generated in ARH neurons held at or near -60 mV, and neuronal phenotype was determined post hoc by immunohistofluorescence. OFQ/N application induced robust outward currents and hyperpolarizations via G protein-gated, inwardly rectifying K+ (GIRK) channels that were attenuated by pretreatment with either 17-β estradiol (E2) or E2 conjugated to bovine serum albumin. This was blocked by the estrogen receptor (ER) antagonist ICI 182,780 and mimicked by the Gq-coupled membrane ER (Gq-mER) ligand STX and the ERα agonist PPT. Inhibiting phosphatidylinositol-3-kinase (PI3K) blocked the estrogenic attenuation of ORL-1/GIRK currents. Antagonizing either phospholipase C (PLC), protein kinase C (PKC), protein kinase A (PKA) or neuronal nitric oxide synthase (nNOS) also abrogated E2 inhibition of ORL-1/GIRK currents, whereas activation of PKC, PKA, protein kinase B (Akt) and nNOS substrate L-arginine all attenuated the OFQ/N response. This was observed in 92 MPN-projecting, POMC-positive ARH neurons. Thus, ORL-1 receptor-mediated inhibition of POMC neurons is rapidly and negatively modulated by E2, an effect which is stereoselective and membrane initiated via Gq-mER and ERα activation that signals through PLC, PKC, PKA, PI3K and nNOS.

  20. Estradiol rapidly attenuates ORL-1 receptor-mediated inhibition of proopiomelanocortin neurons via Gq-coupled, membrane-initiated signaling

    PubMed Central

    Conde, Kristie; Meza, Cecilia; Kelly, Martin J.; Sinchak, Kevin; Wagner, Edward J.

    2016-01-01

    Estradiol rapidly regulates the activity of arcuate nucleus (ARH) proopiomelanocortin (POMC) neurons that project to the medial preoptic nucleus (MPN) to regulate lordosis. Orphanin FQ/nociceptin (OFQ/N) acts via opioid receptor-like (ORL)-1 receptors to inhibit these POMC neurons. Therefore, we tested the hypothesis that estradiol excites POMC neurons by rapidly attenuating inhibitory ORL-1 signaling in these cells. Hypothalamic slices through the ARH were prepared from ovariectomized rats injected with Fluorogold into the MPN. Electrophysiologic recordings were generated in ARH neurons held at or near −60 mV, and neuronal phenotype was determined posthoc by immunohistofluorescence. OFQ/N application induced robust outward currents and hyperpolarizations via GIRK channels that were attenuated by pretreatment with either 17-β estradiol (E2) or E2 conjugated to bovine serum albumin. This was blocked by the estrogen receptor (ER) antagonist ICI 182,780, and mimicked by the Gq-coupled, membrane ER (Gq-mER) ligand STX and the ERα agonist PPT. Inhibiting phosphatidylinositol-3-kinase (PI3K) blocked the estrogenic attenuation of ORL-1/GIRK currents. Antagonizing either phospholipase C (PLC), protein kinase C (PKC), protein kinase A (PKA) or neuronal nitric oxide synthase (nNOS) also abrogated E2 inhibition of ORL-1/GIRK currents, whereas activation of PKC, PKA, protein kinase B (Akt) and nNOS substrate L-arginine all attenuated the OFQ/N response. This was observed in 92 MPN-projecting, POMC-positive ARH neurons. Thus, ORL-1 receptor-mediated inhibition of POMC neurons is rapidly and negatively modulated by E2, an effect which is stereoselective and membrane initiated via Gq-coupled mER and ERα activation that signals through PLC, PKC, PKA, PI3K and nNOS. PMID:26765570

  1. Nicotine impairs cyclooxygenase-2-dependent kinin-receptor-mediated murine airway relaxations

    SciTech Connect

    Xu, Yuan Cardell, Lars-Olaf

    2014-02-15

    Introduction: Cigarette smoke induces local inflammation and airway hyperreactivity. In asthmatics, it worsens the symptoms and increases the risk for exacerbation. The present study investigates the effects of nicotine on airway relaxations in isolated murine tracheal segments. Methods: Segments were cultured for 24 h in the presence of vehicle, nicotine (10 μM) and/or dexamethasone (1 μM). Airway relaxations were assessed in myographs after pre-contraction with carbachol (1 μM). Kinin receptors, cyclooxygenase (COX) and inflammatory mediator expressions were assessed by real-time PCR and confocal-microscopy-based immunohistochemistry. Results: The organ culture procedure markedly increased bradykinin- (selective B{sub 2} receptor agonist) and des-Arg{sup 9}-bradykinin- (selective B{sub 1} receptor agonist) induced relaxations, and slightly increased relaxation induced by isoprenaline, but not that induced by PGE{sub 2}. The kinin receptor mediated relaxations were epithelium-, COX-2- and EP2-receptor-dependent and accompanied by drastically enhanced mRNA levels of kinin receptors, as well as inflammatory mediators MCP-1 and iNOS. Increase in COX-2 and mPGES-1 was verified both at mRNA and protein levels. Nicotine selectively suppressed the organ-culture-enhanced relaxations induced by des-Arg{sup 9}-bradykinin and bradykinin, at the same time reducing mPGES-1 mRNA and protein expressions. α7-nicotinic acetylcholine receptor inhibitors α-bungarotoxin and MG624 both blocked the nicotine effects on kinin B{sub 2} receptors, but not those on B{sub 1}. Dexamethasone completely abolished kinin-induced relaxations. Conclusion: It is tempting to conclude that a local inflammatory process per se could have a bronchoprotective component by increasing COX-2 mediated airway relaxations and that nicotine could impede this safety mechanism. Dexamethasone further reduced airway inflammation together with relaxations. This might contribute to the steroid resistance seen in

  2. Histamine H4 receptor mediates eosinophil chemotaxis with cell shape change and adhesion molecule upregulation

    PubMed Central

    Ling, Ping; Ngo, Karen; Nguyen, Steven; Thurmond, Robin L; Edwards, James P; Karlsson, Lars; Fung-Leung, Wai-Ping

    2004-01-01

    During mast cell degranulation, histamine is released in large quantities. Human eosinophils were found to express histamine H4 but not H3 receptors. The possible effects of histamine on eosinophils and the receptor mediating these effects were investigated in our studies. Histamine (0.01–30 μM) induced a rapid and transient cell shape change in human eosinophils, but had no effects on neutrophils. The maximal shape change was at 0.3 μM histamine with EC50 at 19 nM. After 60 min incubation with 1 μM histamine, eosinophils were desensitized and were refractory to shape change response upon histamine restimulation. Histamine (0.01–1 μM) also enhanced the eosinophil shape change induced by other chemokines. Histamine-induced eosinophil shape change was mediated by the H4 receptor. This effect was completely inhibited by H4 receptor-specific antagonist JNJ 7777120 (IC50 0.3 μM) and H3/H4 receptor antagonist thioperamide (IC50 1.4 μM), but not by selective H1, H2 or H3 receptor antagonists. H4 receptor agonists imetit (EC50 25 nM) and clobenpropit (EC50 72 nM) could mimic histamine effect in inducing eosinophil shape change. Histamine (0.01–100 μM) induced upregulation of adhesion molecules CD11b/CD18 (Mac-1) and CD54 (ICAM-1) on eosinophils. This effect was mediated by the H4 receptor and could be blocked by H4 receptor antagonists JNJ 7777120 and thioperamide. Histamine (0.01–10 μM) induced eosinophil chemotaxis with an EC50 of 83 nM. This effect was mediated by the H4 receptor and could be blocked by H4 receptor antagonists JNJ 7777120 (IC50 86 nM) and thioperamide (IC50 519 nM). Histamine (0.5 μM) also enhanced the eosinophil shape change induced by other chemokines. In conclusion, we have demonstrated a new mechanism of eosinophil recruitment driven by mast cells via the release of histamine. Using specific histamine receptor ligands, we have provided a definitive proof that the H4 receptor mediates eosinophil chemotaxis, cell shape change and

  3. Loss of progesterone receptor-mediated actions induce preterm cellular and structural remodeling of the cervix and premature birth.

    PubMed

    Yellon, Steven M; Dobyns, Abigail E; Beck, Hailey L; Kurtzman, James T; Garfield, Robert E; Kirby, Michael A

    2013-01-01

    A decline in serum progesterone or antagonism of progesterone receptor function results in preterm labor and birth. Whether characteristics of premature remodeling of the cervix after antiprogestins or ovariectomy are similar to that at term was the focus of the present study. Groups of pregnant rats were treated with vehicle, a progesterone receptor antagonist (onapristone or mifepristone), or ovariectomized on day 17 postbreeding. As expected, controls given vehicle delivered at term while rats delivered preterm after progesterone receptor antagonist treatment or ovariectomy. Similar to the cervix before term, the preterm cervix of progesterone receptor antagonist-treated rats was characterized by reduced cell nuclei density, decreased collagen content and structure, as well as a greater presence of macrophages per unit area. Thus, loss of nuclear progesterone receptor-mediated actions promoted structural remodeling of the cervix, increased census of resident macrophages, and preterm birth much like that found in the cervix at term. In contrast to the progesterone receptor antagonist-induced advance in characteristics associated with remodeling, ovariectomy-induced loss of systemic progesterone did not affect hypertrophy, extracellular collagen, or macrophage numbers in the cervix. Thus, the structure and macrophage census in the cervix appear sufficient for premature ripening and birth to occur well before term. With progesterone receptors predominantly localized on cells other than macrophages, the findings suggest that interactions between cells may facilitate the loss of progesterone receptor-mediated actions as part of a final common mechanism that remodels the cervix in certain etiologies of preterm and with parturition at term.

  4. Kainate Receptors Mediate Signaling in Both Transient and Sustained OFF Bipolar Cell Pathways in Mouse Retina

    PubMed Central

    Looger, Loren L.; Tomita, Susumu

    2014-01-01

    A fundamental question in sensory neuroscience is how parallel processing is implemented at the level of molecular and circuit mechanisms. In the retina, it has been proposed that distinct OFF cone bipolar cell types generate fast/transient and slow/sustained pathways by the differential expression of AMPA- and kainate-type glutamate receptors, respectively. However, the functional significance of these receptors in the intact circuit during light stimulation remains unclear. Here, we measured glutamate release from mouse bipolar cells by two-photon imaging of a glutamate sensor (iGluSnFR) expressed on postsynaptic amacrine and ganglion cell dendrites. In both transient and sustained OFF layers, cone-driven glutamate release from bipolar cells was blocked by antagonists to kainate receptors but not AMPA receptors. Electrophysiological recordings from bipolar and ganglion cells confirmed the essential role of kainate receptors for signaling in both transient and sustained OFF pathways. Kainate receptors mediated responses to contrast modulation up to 20 Hz. Light-evoked responses in all mouse OFF bipolar pathways depend on kainate, not AMPA, receptors. PMID:24790183

  5. Muscarinic receptor-mediated inositol tetrakisphosphate response in bovine adrenal chromaffin cells

    SciTech Connect

    Sanborn, B.B.; Schneider, A.S. )

    1990-01-01

    Inositol trisphosphate (IP{sub 3}), a product of the phosphoinositide cycle, mobilizes intracellular Ca{sup 2+} in many cell types. New evidence suggests that inositol tetrakisphosphate (IP{sub 4}), an IP{sub 3} derivative, may act as another second messenger to further alter calcium homeostasis. However, the function and mechanism of action of IP{sub 4} are presently unresolved. We now report evidence of muscarinic receptor-mediated accumulation of IP{sub 4} in bovine adrenal chromaffin cells, a classic neurosecretory system in which calcium movements have been well studied. Muscarine stimulated an increase in ({sup 3}H)IP{sub 4} and ({sup 3}H)IP{sub 3} accumulation in chromaffin cells and this effect was completely blocked by atropine. ({sup 3}H)IP{sub 4} accumulation was detectable within 15 sec, increased to a maximum by 30 sec and thereafter declined. 2,3-diphosphoglycerate, an inhibitor of IP{sub 3} and IP{sub 4} hydrolysis, enhanced accumulation of these inositol polyphosphates. The results provide the first evidence of a rapid inositol tetrakisphosphate response in adrenal chromaffin cells, which should facilitate the future resolution of the relationship between IP{sub 4} and calcium homeostasis.

  6. Cloned M1 muscarinic receptors mediate both adenylate cyclase inhibition and phosphoinositide turnover.

    PubMed Central

    Stein, R; Pinkas-Kramarski, R; Sokolovsky, M

    1988-01-01

    The rat M1 muscarinic receptor gene was cloned and expressed in a rat cell line lacking endogenous muscarinic receptors. Assignment of the cloned receptors to the M1 class was pharmacologically confirmed by their high affinity for the M1-selective muscarinic antagonist pirenzepine and low affinity for the M2-selective antagonist AF-DX-116. Guanylyl imidodiphosphate [Gpp(NH)p] converted agonist binding sites on the receptor, from high-affinity to the low-affinity state, thus indicating that the cloned receptors couple to endogenous G-proteins. The cloned receptors mediated both adenylate cyclase inhibition and phosphoinositide hydrolysis, but by different mechanisms. Pertussis toxin blocked the inhibition of adenylate cyclase (indicating coupling of the receptor to inhibitory G-protein), but did not affect phosphoinositide turnover. Furthermore, the stimulation of phosphoinositide hydrolysis was less efficient than the inhibition of adenylate cyclase. These findings demonstrate that cloned M1 receptors are capable of mediating multiple responses in the cell by coupling to different effectors, possibly to different G-proteins. Images PMID:2846274

  7. Dual effects of anandamide on NMDA receptor-mediated responses and neurotransmission.

    PubMed

    Hampson, A J; Bornheim, L M; Scanziani, M; Yost, C S; Gray, A T; Hansen, B M; Leonoudakis, D J; Bickler, P E

    1998-02-01

    Anandamide is an endogenous ligand of cannabinoid receptors that induces pharmacological responses in animals similar to those of cannabinoids such as delta9-tetrahydrocannabinol (THC). Typical pharmacological effects of cannabinoids include disruption of pain, memory formation, and motor coordination, systems that all depend on NMDA receptor mediated neurotransmission. We investigated whether anandamide can influence NMDA receptor activity by examining NMDA-induced calcium flux (deltaCa2+NMDA) in rat brain slices. The presence of anandamide reduced deltaCa2+NMDA and the inhibition was disrupted by cannabinoid receptor antagonist, pertussis toxin treatment, and agatoxin (a calcium channel inhibitor). Whereas these treatments prevented anandamide inhibiting deltaCa2+NMDA, they also revealed another, underlying mechanism by which anandamide influences deltaCa2+NMDA. In the presence of cannabinoid receptor antagonist, anandamide potentiated deltaCa2+NMDA in cortical, cerebellar, and hippocampal slices. Anandamide (but not THC) also augmented NMDA-stimulated currents in Xenopus oocytes expressing cloned NMDA receptors, suggesting a capacity to directly modulate NMDA receptor activity. In a similar manner, anandamide enhanced neurotransmission across NMDA receptor-dependent synapses in hippocampus in a manner that was not mimicked by THC and was unaffected by cannabinoid receptor antagonist. These data demonstrate that anandamide can modulate NMDA receptor activity in addition to its role as a cannabinoid receptor ligand.

  8. Receptor-Mediated Entry of Pristine Octahedral DNA Nanocages in Mammalian Cells.

    PubMed

    Vindigni, Giulia; Raniolo, Sofia; Ottaviani, Alessio; Falconi, Mattia; Franch, Oskar; Knudsen, Birgitta R; Desideri, Alessandro; Biocca, Silvia

    2016-06-28

    DNA offers excellent programming properties for the generation of nanometer-scaled polyhedral structures with a broad variety of potential applications. Translation to biomedical applications requires improving stability in biological fluids, efficient and selective cell binding, and/or internalization of the assembled DNA nanostructures. Here, we report an investigation on the selective mechanism of cellular uptake of pristine DNA nanocages in cells expressing the receptor "oxidized low-density lipoprotein receptor-1" (LOX-1), a scavenger receptor associated with cardiovascular diseases and, more recently, identified as a tumor marker. For this purpose a truncated octahedral DNA nanocage functionalized with a single biotin molecule, which allows DNA cage detection through the biotin-streptavidin assays, was constructed. The results indicate that DNA nanocages are stable in biological fluids, including human serum, and are selectively bound and very efficiently internalized in vesicles only in LOX-1-expressing cells. The amount of internalized cages is 30 times higher in LOX-1-expressing cells than in normal fibroblasts, indicating that the receptor-mediated uptake of pristine DNA nanocages can be pursued for a selective cellular internalization. These results open the route for a therapeutic use of pristine DNA cages targeting LOX-1-overexpressing tumor cells.

  9. Receptor mediated endocytosis of vicilin in Callosobruchus maculatus (Coleoptera: Chrysomelidae) larval midgut epithelial cells.

    PubMed

    Kunz, Daniele; Oliveira, Gabriel B; Uchôa, Adriana F; Samuels, Richard I; Macedo, Maria Lígia R; Silva, Carlos P

    2017-08-01

    The transport of proteins across the intestinal epithelium of insects is still not well understood. There is evidence that vicilin, a major storage protein of cowpea seeds (Vigna unguiculata), is internalized in larvae of the seed-beetle Callosobruchus maculatus. It has been reported that this vicilin interacts with proteins present in the microvillar membranes of columnar cells along the digestive tract of the larvae. In the present work, we studied the cellular pathway involved in endocytosis of vicilin in larval C. maculatus by employing ex vivo experiments. In the ex vivo approach, we incubated FITC-labelled vicilin with isolated midgut wholemounts in the absence or in the presence of endocytosis inhibitors. The fate of labelled or non-labelled globulins was monitored by confocal microscopy and fluorescence measurement. Our results suggest that the internalization of vicilins is due to receptor-mediated endocytosis. Here we report the identity of a microvillar vicilin-binding protein that was purified using affinity chromatography on a vicilin-sepharose column. The putative vicilin receptor showed high homology to proteins with the CRAL-TRIO domain, specifically the Sec14 superfamily member α-tocopherol transfer protein. The precise mechanism involved in vicilin internalization was defined through the use of specific inhibitors of the endocytosis pathway. The inhibitors filipin III and nystatin significantly inhibited the endocytosis of vicilin, while chlorpromazine and phenylarsine oxide had a much lower effect on endocytosis, suggesting that the endocytic pathway is predominantly mediated by caveolin. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Lactate modulates the activity of primary cortical neurons through a receptor-mediated pathway.

    PubMed

    Bozzo, Luigi; Puyal, Julien; Chatton, Jean-Yves

    2013-01-01

    Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism.

  11. Lactate Modulates the Activity of Primary Cortical Neurons through a Receptor-Mediated Pathway

    PubMed Central

    Bozzo, Luigi; Puyal, Julien; Chatton, Jean-Yves

    2013-01-01

    Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism. PMID:23951229

  12. Estrogen Injection

    MedlinePlus

    ... carefully for side effects.tell your doctor what herbal products you are taking, especially St. John's wort.tell your doctor if you have or have ever had yellowing of the skin or eyes during pregnancy or during your treatment with an estrogen product, endometriosis (a condition in ...

  13. E2/Estrogen Receptor/Sjogren Syndrome-Associated Autoantigen Relieves Coactivator Activator-Induced G1/S Arrest To Promote Breast Tumorigenicity

    PubMed Central

    Kang, Yun Kyoung; Jung, Sung Yun; Qin, Jun; Li, Chao; Tsai, Sophia Y.; Tsai, Ming-Jer

    2014-01-01

    Coactivator activator (CoAA) is a dual-functional coregulator that regulates steroid receptor-mediated transcription and alternative splicing. Previously, we have shown that CoAA has tumor-suppressive potential in tumorigenic human kidney cells. Here, we uncover a molecular mechanism by which Sjogren syndrome-associated autoantigen (SSA), an estrogen receptor (ER) coactivator, induces MYC oncogene by removing repressive CoAA through E2-dependent degradation of CoAA and promotes G1/S transition of the cell cycle as well as anchorage-independent growth capability of breast cancer cells. We also show that E2 and ER enhance the E3 ligase activity of SSA to modulate CoAA through splicing isoform-selective ubiquitylation. We propose this as one potential molecular basis for the reduced tumor incidence in autoimmune disease patients and suggest SSA as a potential therapeutic target to treat breast cancer. PMID:24567374

  14. Self-Assembly into Nanoparticles Is Essential for Receptor Mediated Uptake of Therapeutic Antisense Oligonucleotides.

    PubMed

    Ezzat, Kariem; Aoki, Yoshitsugu; Koo, Taeyoung; McClorey, Graham; Benner, Leif; Coenen-Stass, Anna; O'Donovan, Liz; Lehto, Taavi; Garcia-Guerra, Antonio; Nordin, Joel; Saleh, Amer F; Behlke, Mark; Morris, John; Goyenvalle, Aurelie; Dugovic, Branislav; Leumann, Christian; Gordon, Siamon; Gait, Michael J; El-Andaloussi, Samir; Wood, Matthew J A

    2015-07-08

    Antisense oligonucleotides (ASOs) have the potential to revolutionize medicine due to their ability to manipulate gene function for therapeutic purposes. ASOs are chemically modified and/or incorporated within nanoparticles to enhance their stability and cellular uptake, however, a major challenge is the poor understanding of their uptake mechanisms, which would facilitate improved ASO designs with enhanced activity and reduced toxicity. Here, we study the uptake mechanism of three therapeutically relevant ASOs (peptide-conjugated phosphorodiamidate morpholino (PPMO), 2'Omethyl phosphorothioate (2'OMe), and phosphorothioated tricyclo DNA (tcDNA) that have been optimized to induce exon skipping in models of Duchenne muscular dystrophy (DMD). We show that PPMO and tcDNA have high propensity to spontaneously self-assemble into nanoparticles. PPMO forms micelles of defined size and their net charge (zeta potential) is dependent on the medium and concentration. In biomimetic conditions and at low concentrations, PPMO obtains net negative charge and its uptake is mediated by class A scavenger receptor subtypes (SCARAs) as shown by competitive inhibition and RNAi silencing experiments in vitro. In vivo, the activity of PPMO was significantly decreased in SCARA1 knockout mice compared to wild-type animals. Additionally, we show that SCARA1 is involved in the uptake of tcDNA and 2'OMe as shown by competitive inhibition and colocalization experiments. Surface plasmon resonance binding analysis to SCARA1 demonstrated that PPMO and tcDNA have higher binding profiles to the receptor compared to 2'OMe. These results demonstrate receptor-mediated uptake for a range of therapeutic ASO chemistries, a mechanism that is dependent on their self-assembly into nanoparticles.

  15. Fc receptor-mediated immune responses: new tools but increased complexity in HIV prevention.

    PubMed

    Vargas-Inchaustegui, Diego A; Robert-Guroff, Marjorie

    2013-07-01

    The modest success of the RV144 HIV vaccine trial in Thailand and the ensuing suggestion that a Fc-receptormediated antibody activity might have played a role in the protection observed have intensified investigations on Fcrelated immune responses. HIV neutralizing antibodies have been and continue to be the focal point of research into humoral immune protection. However, recent knowledge that their protective efficacy can be augmented by Fc-FcR interactions has increased the complexity of identifying immune correlates of protection. If anything, continued studies of both humoral and cellular immune mechanisms point to the lack of a single protective anti-HIV immune response. Here we focus on humoral immunity, analyzing the role played by Fc receptor-related responses and discussing how new knowledge of their interactions requires further investigation, but may also spur novel vaccination approaches. We initially address classical Fc-receptor mediated anti-viral mechanisms including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell mediated viral inhibition (ADCVI), and antibody-dependent cellular phagocytosis (ADCP), as well as the effector cells that mediate these functions. Next, we summarize key aspects of FcR-Fc interactions that are important for potential control of HIV/SIV such as FcR polymorphisms and post-transcriptional modifications. Finally we discuss less commonly studied non-mechanistic anti-HIV immune functions: antibody avidity and envelopespecific B cell memory. Overall, a spectrum of immune responses, reflecting the immune system's redundancy, will likely be needed to prevent HIV infection and/or disease progression. Aside from elicitation of critical immune mechanisms, a successful vaccine will need to induce mature B cell responses and long-lasting immune memory.

  16. Receptor-mediated endocytosis for drug delivery in African trypanosomes: fulfilling Paul Ehrlich's vision of chemotherapy.

    PubMed

    Alsford, Sam; Field, Mark C; Horn, David

    2013-05-01

    Bloodstream-form cells of Trypanosoma brucei exhibit massively increased endocytic activity relative to the insect midgut stage, enabling rapid recycling of variant surface glycoprotein and antibody clearance from the surface. In addition, recent advances have identified a role for receptor-mediated endocytosis in the uptake of the antitrypanosomal drug, suramin, via invariant surface glycoprotein 75, and in the uptake of trypanosome lytic factor 1 via haptoglobin-haemoglobin receptor. Here, we argue that receptor-mediated endocytosis represents both a validated drug target and a promising route for the delivery of novel therapeutics into trypanosomes.

  17. Menopause, estrogens and frailty.

    PubMed

    Nedergaard, Anders; Henriksen, Kim; Karsdal, Morten Asser; Christiansen, Claus

    2013-05-01

    The controversy surrounding the results from the Women's Health Initiative (WHI) trials published a decade ago caused a significant decline in the use of menopausal hormone replacement therapy. However, these results have been vehemently contested and several lines of evidence suggest that in perimenopausal and non-obese women, estrogen therapy may indeed be of benefit. There is ample proof that menopause causes a loss of musculoskeletal tissue mass and quality, thereby causing a loss of health and quality of life. There is also solid evidence that hormone replacement therapy in itself prevents most of these effects in connective tissue in itself. Besides the independent, direct effects on the musculoskeletal tissues, estrogen deficiency also reduces the ability to adequately respond and adapt to external mechanical and metabolic stressors, e.g. exercise, which are otherwise the main stimuli that should maintain musculoskeletal integrity and metabolic function. Thus, normophysiological estrogen levels appear to exert a permissive effect on musculoskeletal adaptations to loading, thereby likely improving the outcome of rehabilitation following critical illness, musculoskeletal trauma or orthopedic surgical therapy. These effects add to the evidence supporting the use of estrogen therapy, particularly accelerated gain of functional capacity and independence following musculoskeletal disuse.

  18. EP2 receptor mediates PGE2-induced cystogenesis of human renal epithelial cells.

    PubMed

    Elberg, Gerard; Elberg, Dorit; Lewis, Teresa V; Guruswamy, Suresh; Chen, Lijuan; Logan, Charlotte J; Chan, Michael D; Turman, Martin A

    2007-11-01

    Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by formation of cysts from tubular epithelial cells. Previous studies indicate that secretion of prostaglandin E2 (PGE2) into cyst fluid and production of cAMP underlie cyst expansion. However, the mechanism by which PGE2 directly stimulates cAMP formation and modulates cystogenesis is still unclear, because the particular E-prostanoid (EP) receptor mediating the PGE2 effect has not been characterized. Our goal is to define the PGE2 receptor subtype involved in ADPKD. We used a three-dimensional cell-culture system of human epithelial cells from normal and ADPKD kidneys in primary cultures to demonstrate that PGE2 induces cyst formation. Biochemical evidence gathered by using real-time RT-PCR mRNA analysis and immunodetection indicate the presence of EP2 receptor in cystic epithelial cells in ADPKD kidney. Pharmacological evidence obtained by using PGE2-selective analogs further demonstrates that EP2 mediates cAMP formation and cystogenesis. Functional evidence for a role of EP2 receptor in mediating cAMP signaling was also provided by inhibiting EP2 receptor expression with transfection of small interfering RNA in cystic epithelial cells. Our results indicate that PGE2 produced in cyst fluid binds to adjacent EP2 receptors located on the apical side of cysts and stimulates EP2 receptor expression. PGE2 binding to EP2 receptor leads to cAMP signaling and cystogenesis by a mechanism that involves protection of cystic epithelial cells from apoptosis. The role of EP2 receptor in mediating the PGE2 effect on stimulating cyst formation may have direct pharmacological implications for the treatment of polycystic kidney disease.

  19. Receptor-Mediated Enhancement of Beta Adrenergic Drug Activity by Ascorbate In Vitro and In Vivo

    PubMed Central

    Dillon, Patrick F.; Root-Bernstein, Robert; Robinson, N. Edward; Abraham, William M.; Berney, Catherine

    2010-01-01

    Rationale Previous in vitro research demonstrated that ascorbate enhances potency and duration of activity of agonists binding to alpha 1 adrenergic and histamine receptors. Objectives Extending this work to beta 2 adrenergic systems in vitro and in vivo. Methods Ultraviolet spectroscopy was used to study ascorbate binding to adrenergic receptor preparations and peptides. Force transduction studies on acetylcholine-contracted trachealis preparations from pigs and guinea pigs measured the effect of ascorbate on relaxation due to submaximal doses of beta adrenergic agonists. The effect of inhaled albuterol with and without ascorbate was tested on horses with heaves and sheep with carbachol-induced bronchoconstriction. Measurements Binding constants for ascorbate binding to beta adrenergic receptor were derived from concentration-dependent spectral shifts. Dose- dependence curves were obtained for the relaxation of pre-contracted trachealis preparations due to beta agonists in the presence and absence of varied ascorbate. Tachyphylaxis and fade were also measured. Dose response curves were determined for the effect of albuterol plus-and-minus ascorbate on airway resistance in horses and sheep. Main Results Ascorbate binds to the beta 2 adrenergic receptor at physiological concentrations. The receptor recycles dehydroascorbate. Physiological and supra-physiological concentrations of ascorbate enhance submaximal epinephrine and isoproterenol relaxation of trachealis, producing a 3–10-fold increase in sensitivity, preventing tachyphylaxis, and reversing fade. In vivo, ascorbate improves albuterol's effect on heaves and produces a 10-fold enhancement of albuterol activity in “asthmatic” sheep. Conclusions Ascorbate enhances beta-adrenergic activity via a novel receptor-mediated mechanism; increases potency and duration of beta adrenergic agonists effective in asthma and COPD; prevents tachyphylaxis; and reverses fade. These novel effects are probably caused by a

  20. Bradykinin B2-receptor-mediated modulation of membrane currents in guinea-pig cardiomyocytes

    PubMed Central

    Sakamoto, Naoya; Uemura, Hiroko; Hara, Yukio; Saito, Toshihiro; Masuda, Yoshiaki; Nakaya, Haruaki

    1998-01-01

    In order to define the electrophysiological mechanism(s) responsible for bradykinin (BK)-induced positive inotropic and chronotropic responses in isolated guinea-pig atria, effects of BK on the membrane currents were examined in isolated atrial cells using patch clamp techniques.BK (0.1–1000 nM) increased the L-type Ca2+ current (ICa), which was recorded from enzymatically-dissociated atrial myocytes by the nystatin-perforated patch method, in a concentration-dependent fashion, and the calculated EC50 value for increasing ICa was 5.2 nM. In conventional ruptured patch experiments, BK inhibited the muscarinic acetylcholine receptor-operated K+ current (IK.ACh) that was activated by the muscarinic agonist carbachol (1 μM) with an EC50 value of 0.57 nM. Both the increase in ICa and the decrease in IK.ACh were blocked by HOE140, a selective bradykinin B2 receptor antagonist.The BK-induced inhibition of IK.ACh was significantly attenuated by staurosporine and calphostin C, protein kinase C inhibitors. In addition, the IK.ACh inhibition by BK was also attenuated by the tyrosine kinase inhibitor genistein or tyrphostin but not by daidzein, an inactive analogue of genistein. However, neither protein kinase C inhibitor nor tyrosine kinase inhibitor affected the BK-induced increase in ICa.In the presence and absence of muscarinic stimulation, BK prolonged the action potential recorded from the atrial cells in the current clamp mode.We conclude that BK increases ICa and decreases IK.ACh in atrial cells, resulting in positive inotropic and chronotropic responses in atrial preparations. Protein kinase C activation, and possibly tyrosine kinase activation, may be involved in the B2-receptor-mediated IK.ACh inhibition. PMID:9786500

  1. Functional evaluation of the receptors mediating vasoconstriction of rat aorta by trace amines and amphetamines.

    PubMed

    Broadley, Kenneth J; Fehler, Martina; Ford, William R; Kidd, Emma J

    2013-09-05

    Trace amines including β-phenylethylamine (β-PEA) and amphetamines classically exert pharmacological actions via indirect sympathomimetic mechanisms. However, there is evidence for other mechanisms and this study explores the receptors mediating vasoconstriction in rat aorta. β-PEA, d-amphetamine, MDMA, cathinone and methylphenidate caused concentration-dependent contractions of rat isolated aortic rings which were unaffected by prazosin (1 μM), ICI-118,551 (1 μM), cocaine (10 μM) and pargyline (10 μM), to inhibit α1- and β2-adrenoceptors, neuronal transport and monoamine oxidase (MAO), respectively. Octopamine concentration-response curves, however, were shifted to the right. In the presence of the inhibitors, the rate of onset of octopamine contractions was slowed. Lineweaver-Burk analysis of the kinetics of the response generated different KM values for octopamine in the absence (2.35 × 10(-6)M) and presence (6.09 × 10(-5)M) of inhibitors, indicating mediation by different receptors. Tryptamine-induced vasoconstriction also resisted blockade by adrenergic inhibitors and the 5-HT1A, 1B, 1D and 5-HT2A receptor antagonists, methiothepin (50 nM) and ketanserin (30 nM), respectively. Trace amines and amphetamines therefore exert vasoconstriction independently of adrenoceptors, neuronal transport and 5-HT receptor activation. There was no evidence of tachyphylaxis or cross-tachyphylaxis of the vasoconstriction to these amines. Tyramine was a partial agonist and in its presence, β-PEA, d-amphetamine and octopamine were antagonised indicating that they all act through a common receptor for which tyramine serves as an antagonist. We conclude that the vasoconstriction is via TAAR-1, because of structural similarities between amines, ability to stimulate recombinant trace amine-associated receptor 1 (TAAR-1) and the presence of TAAR-1 in rat aorta. © 2013 Elsevier B.V. All rights reserved.

  2. Nandrolone and stanozolol induce Leydig cell tumor proliferation through an estrogen-dependent mechanism involving IGF-I system.

    PubMed

    Chimento, Adele; Sirianni, Rosa; Zolea, Fabiana; De Luca, Arianna; Lanzino, Marilena; Catalano, Stefania; Andò, Sebastiano; Pezzi, Vincenzo

    2012-05-01

    Several substances such as anabolic androgenic steroids (AAS), peptide hormones like insulin-like growth factor-I (IGF-I), aromatase inhibitors and estrogen antagonists are offered via the Internet, and are assumed without considering the potential deleterious effects that can be caused by their administration. In this study we aimed to determine if nandrolone and stanozolol, two commonly used AAS, could have an effect on Leydig cell tumor proliferation and if their effects could be potentiated by the concomitant use of IGF-I. Using a rat Leydig tumor cell line, R2C cells, as experimental model we found that nandrolone and stanozolol caused a dose-dependent induction of aromatase expression and estradiol (E2) production. When used in combination with IGF-I they were more effective than single molecules in inducing aromatase expression. AAS exhibited estrogenic activity and induced rapid estrogen receptor (ER)-dependent pathways involving IGF1R, AKT, and ERK1/2 phosphorylation. Inhibitors for these kinases decreased AAS-dependent aromatase expression. Up-regulated aromatase levels and related E2 production increased cell proliferation as a consequence of increased cyclin E expression. The observation that ER antagonist ICI182,780 was also able to significantly reduce ASS- and AAS + IGF-induced cell proliferation, confirmed a role for estrogens in AAS-dependent proliferative effects. Taken together these data clearly indicate that the use of high doses of AAS, as it occurs in doping practice, enhances Leydig cell proliferation, increasing the risk of tumor development. This risk is higher when AAS are used in association with IGF-I. To our knowledge this is the first report directly associating AAS and testicular cancer.

  3. Disruption of estrogen homeostasis as a mechanism for uterine toxicity in Wistar Han rats treated with tetrabromobisphenol A.

    PubMed

    Sanders, J Michael; Coulter, Sherry J; Knudsen, Gabriel A; Dunnick, June K; Kissling, Grace E; Birnbaum, Linda S

    2016-05-01

    Chronic oral treatment of tetrabromobisphenol A (TBBPA) to female Wistar Han rats resulted in increased incidence of cell proliferation at 250mg/kg and tumor formation in the uterus at higher doses. The present study was designed to test the hypothesis that disruption of estrogen homeostasis was a major mode-of-action for the observed effects. Biological changes were assessed in serum, liver, and the proximal (nearest the cervix) and distal (nearest the ovaries) sections of the uterine horn of Wistar Han rats 24h following administration of the last of five daily oral doses of 250mg/kg. Expression of genes associated with receptors, biosynthesis, and metabolism of estrogen was altered in the liver and uterus. TBBPA treatment also resulted in changes in expression of genes associated with cell division and growth. Changes were also observed in the concentration of thyroxine in serum and in expression of genes in the liver and uterus associated with thyroid hormone receptors. Differential expression of some genes was tissue-dependent or specific to tissue location in the uterus. The biological responses observed in the present study support the hypothesis that perturbation of estrogen homeostasis is a major mode-of-action for TBBPA-mediated cell proliferation and tumorigenesis previously observed in the uterus of TBBPA-treated Wistar Han rats.

  4. Differential Regulation of Progesterone Receptor-Mediated Transcription by CDK2 and DNA-PK.

    PubMed

    Treviño, Lindsey S; Bolt, Michael J; Grimm, Sandra L; Edwards, Dean P; Mancini, Michael A; Weigel, Nancy L

    2016-02-01

    Progesterone receptor (PR) function is altered by cell signaling, but the mechanisms of kinase-specific regulation are not well defined. To examine the role of cell signaling in the regulation of PR transcriptional activity, we have utilized a previously developed mammalian-based estrogen-response element promoter array cell model and automated cell imaging and analysis platform to visualize and quantify effects of specific kinases on different mechanistic steps of PR-mediated target gene activation. For these studies, we generated stable estrogen-response element array cell lines expressing inducible chimeric PR that contains a swap of the estrogen receptor-α DNA-binding domain for the DNA-binding domain of PR. We have focused on 2 kinases important for steroid receptor activity: cyclin-dependent kinase 2 and DNA-dependent protein kinase. Treatment with either a Cdk1/2 inhibitor (NU6102) or a DNA-dependent protein kinase inhibitor (NU7441) decreased hormone-mediated chromatin decondensation and transcriptional activity. Further, we observed a quantitative reduction in the hormone-mediated recruitment of select coregulator proteins with NU6102 that is not observed with NU7441. In parallel, we determined the effect of kinase inhibition on hormone-mediated induction of primary and mature transcripts of endogenous genes in T47D breast cancer cells. Treatment with NU6102 was much more effective than NU7441, in inhibiting induction of PR target genes that exhibit a rapid increase in primary transcript expression in response to hormone. Taken together, these results indicate that the 2 kinases regulate PR transcriptional activity by distinct mechanisms.

  5. Mechanical stimulation enhanced estrogen receptor expression and callus formation in diaphyseal long bone fracture healing in ovariectomy-induced osteoporotic rats.

    PubMed

    Chow, S K H; Leung, K S; Qin, J; Guo, A; Sun, M; Qin, L; Cheung, W H

    2016-10-01

    Estrogen receptor (ER) in ovariectomy-induced osteoporotic fracture was reported to exhibit delayed expression. Mechanical stimulation enhanced ER-α expression in osteoporotic fracture callus at the tissue level. ER was also found to be required for the effectiveness of vibrational mechanical stimulation treatment in osteoporotic fracture healing. Estrogen receptor(ER) is involved in mechanical signal transduction in bone metabolism. Its expression was reported to be delayed in osteoporotic fracture healing. The purpose of this study was to investigate the roles played by ER during osteoporotic fracture healing enhanced with mechanical stimulation. Ovariectomy-induced osteoporotic SD rats that received closed femoral fractures were divided into five groups, (i) SHAM, (ii) SHAM-VT, (iii) OVX, (iv) OVX-VT, and (v) OVX-VT-ICI, where VT stands for whole-body vibration treatment and ICI for ER antagonization by ICI 182,780. Callus formation and gene expression were assessed at 2, 4, and 8 weeks postfracture. In vitro osteoblastic differentiation, mineralization, and ER-α expression were assessed. The delayed ER expression was found to be enhanced by vibration treatment. Callus formation enhancement was shown by callus morphometry and micro-CT analysis. Enhancement effects by vibration were partially abolished when ER was modulated by ICI 182,780, in terms of callus formation capacity at 2-4 weeks and ER gene and protein expression at all time points. In vitro, ER expression in osteoblasts was not enhanced by VT treatment, but osteoblastic differentiation and mineralization were enhanced under estrogen-deprived condition. When osteoblastic cells were modulated by ICI 182,780, enhancement effects of VT were eliminated. Vibration was able to enhance ER expression in ovariectomy-induced osteoporotic fracture healing. ER was essential in mechanical signal transduction and enhancement in callus formation effects during osteoporotic fracture healing enhanced by vibration

  6. Distinct Effects of Estrogen on Mouse Maternal Behavior: The Contribution of Estrogen Synthesis in the Brain.

    PubMed

    Murakami, Gen

    2016-01-01

    Estrogen surge following progesterone withdrawal at parturition plays an important role in initiating maternal behavior in various rodent species. Systemic estrogen treatment shortens the latency to onset of maternal behavior in nulliparous female rats that have not experienced parturition. In contrast, nulliparous laboratory mice show rapid onset of maternal behavior without estrogen treatment, and the role of estrogen still remains unclear. Here the effect of systemic estrogen treatment (for 2 h, 1 day, 3 days, and 7 days) after progesterone withdrawal was examined on maternal behavior of C57BL/6 mice. This estrogen regimen led to different effects on nursing, pup retrieval, and nest building behaviors. Latency to nursing was shortened by estrogen treatment within 2 h. Moreover, pup retrieval and nest building were decreased. mRNA expression was also investigated for estrogen receptor α (ERα) and for genes involved in regulating maternal behavior, specifically, the oxytocin receptor (OTR) and vasopressin receptor in the medial amygdala (MeA) and medial preoptic area (MPOA). Estrogen treatment led to decreased ERα mRNA in both regions. Although OTR mRNA was increased in the MeA, OTR and vasopressin receptor mRNA were reduced in the MPOA, showing region-dependent transcription regulation. To determine the mechanisms for the actions of estrogen treatment, the contribution of estrogen synthesis in the brain was examined. Blockade of estrogen synthesis in the brain by systemic letrozole treatment in ovariectomized mice interfered with pup retrieval and nest building but not nursing behavior, indicating different contributions of estrogen synthesis to maternal behavior. Furthermore, letrozole treatment led to an increase in ERα mRNA in the MeA but not in the MPOA, suggesting that involvement of estrogen synthesis is brain region dependent. Altogether, these results suggest that region-dependent estrogen synthesis leads to differential transcriptional activation due

  7. Distinct Effects of Estrogen on Mouse Maternal Behavior: The Contribution of Estrogen Synthesis in the Brain

    PubMed Central

    Murakami, Gen

    2016-01-01

    Estrogen surge following progesterone withdrawal at parturition plays an important role in initiating maternal behavior in various rodent species. Systemic estrogen treatment shortens the latency to onset of maternal behavior in nulliparous female rats that have not experienced parturition. In contrast, nulliparous laboratory mice show rapid onset of maternal behavior without estrogen treatment, and the role of estrogen still remains unclear. Here the effect of systemic estrogen treatment (for 2 h, 1 day, 3 days, and 7 days) after progesterone withdrawal was examined on maternal behavior of C57BL/6 mice. This estrogen regimen led to different effects on nursing, pup retrieval, and nest building behaviors. Latency to nursing was shortened by estrogen treatment within 2 h. Moreover, pup retrieval and nest building were decreased. mRNA expression was also investigated for estrogen receptor α (ERα) and for genes involved in regulating maternal behavior, specifically, the oxytocin receptor (OTR) and vasopressin receptor in the medial amygdala (MeA) and medial preoptic area (MPOA). Estrogen treatment led to decreased ERα mRNA in both regions. Although OTR mRNA was increased in the MeA, OTR and vasopressin receptor mRNA were reduced in the MPOA, showing region-dependent transcription regulation. To determine the mechanisms for the actions of estrogen treatment, the contribution of estrogen synthesis in the brain was examined. Blockade of estrogen synthesis in the brain by systemic letrozole treatment in ovariectomized mice interfered with pup retrieval and nest building but not nursing behavior, indicating different contributions of estrogen synthesis to maternal behavior. Furthermore, letrozole treatment led to an increase in ERα mRNA in the MeA but not in the MPOA, suggesting that involvement of estrogen synthesis is brain region dependent. Altogether, these results suggest that region-dependent estrogen synthesis leads to differential transcriptional activation due

  8. PKCɛ mediates substance P inhibition of GABAA receptors-mediated current in rat dorsal root ganglion.

    PubMed

    Li, Li; Zhao, Lei; Wang, Yang; Ma, Ke-tao; Shi, Wen-yan; Wang, Ying-zi; Si, Jun-qiang

    2015-02-01

    The mechanism underlying the modulatory effect of substance P (SP) on GABA-activated response in rat dorsal root ganglion (DRG) neurons was investigated. In freshly dissociated rat DRG neurons, whole-cell patch-clamp technique was used to record GABA-activated current and sharp electrode intracellular recording technique was used to record GABA-induced membrane depolarization. Application of GABA (1-1000 μmol/L) induced an inward current in a concentration-dependent manner in 114 out of 127 DRG neurons (89.8 %) examined with whole-cell patch-clamp recordings. Bath application of GABA (1-1000 μmol/L) evoked a depolarizing response in 236 out of 257 (91.8%) DRG neurons examined with intracellular recordings. Application of SP (0.001-1 μmol/L) suppressed the GABA-activated inward current and membrane depolarization. The inhibitory effects were concentration-dependent and could be blocked by the selective neurokinin 1 (NK1) receptors antagonist spantide but not by L659187 and SR142801 (1 μmol/L, n=7), selective antagonists of NK2 and NK3. The inhibitory effect of SP was significantly reduced by the calcium chelator BAPTA-AM, phospholipase C (PLC) inhibitor U73122, and PKC inhibitor chelerythrine, respectively. The PKA inhibitor H-89 did not affect the SP effect. Remarkably, the inhibitory effect of SP on GABA-activated current was nearly completely removed by a selective PKCε inhibitor epilon-V1-2 but not by safingol and LY333531, selective inhibitors of PKCα and PKCβ. Our results suggest that NK1 receptor mediates SP-induced inhibition of GABA-activated current and membrane depolarization by activating intracellular PLC-Ca²⁺-PKCε cascade. SP might regulate the excitability of peripheral nociceptors through inhibition of the "pre-synaptic inhibition" evoked by GABA, which may explain its role in pain and neurogenic inflammation.

  9. Direct muscarinic and nicotinic receptor-mediated excitation of rat medial vestibular nucleus neurons in vitro

    NASA Technical Reports Server (NTRS)

    Phelan, K. D.; Gallagher, J. P.

    1992-01-01

    We have utilized intracellular recording techniques to investigate the cholinoceptivity of rat medial vestibular nucleus (MVN) neurons in a submerged brain slice preparation. Exogenous application of the mixed cholinergic agonists, acetylcholine (ACh) or carbachol (CCh), produced predominantly membrane depolarization, induction of action potential firing, and decreased input resistance. Application of the selective muscarinic receptor agonist muscarine (MUSC), or the selective nicotinic receptor agonists nicotine (NIC) or 1,1-dimethyl-4-phenylpiperazinium (DMPP) also produced membrane depolarizations. The MUSC-induced depolarization was accompanied by decreased conductance, while an increase in conductance appeared to underlie the NIC- and DMPP-induced depolarizations. The muscarinic and nicotinic receptor mediated depolarizations persisted in tetrodotoxin and/or low Ca2+/high Mg2+ containing media, suggesting direct postsynaptic receptor activation. The MUSC-induced depolarization could be reversibly blocked by the selective muscarinic-receptor antagonist, atropine, while the DMPP-induced depolarization could be reversibly suppressed by the selective ganglionic nicotinic-receptor antagonist, mecamylamine. Some neurons exhibited a transient membrane hyperpolarization during the depolarizing response to CCh or MUSC application. This transient inhibition could be reversibly blocked by the gamma-aminobutyric acid (GABA) antagonist, bicuculline, suggesting that the underlying hyperpolarization results indirectly from the endogenous release of GABA acting at GABA receptors. This study confirms the cholinoceptivity of MVN neurons and establishes that individual MVN cells possess muscarinic as well as nicotinic receptors. The data provide support for a prominent role of cholinergic mechanisms in the direct and indirect regulation of the excitability of MVN neurons.

  10. Direct muscarinic and nicotinic receptor-mediated excitation of rat medial vestibular nucleus neurons in vitro

    NASA Technical Reports Server (NTRS)

    Phelan, K. D.; Gallagher, J. P.

    1992-01-01

    We have utilized intracellular recording techniques to investigate the cholinoceptivity of rat medial vestibular nucleus (MVN) neurons in a submerged brain slice preparation. Exogenous application of the mixed cholinergic agonists, acetylcholine (ACh) or carbachol (CCh), produced predominantly membrane depolarization, induction of action potential firing, and decreased input resistance. Application of the selective muscarinic receptor agonist muscarine (MUSC), or the selective nicotinic receptor agonists nicotine (NIC) or 1,1-dimethyl-4-phenylpiperazinium (DMPP) also produced membrane depolarizations. The MUSC-induced depolarization was accompanied by decreased conductance, while an increase in conductance appeared to underlie the NIC- and DMPP-induced depolarizations. The muscarinic and nicotinic receptor mediated depolarizations persisted in tetrodotoxin and/or low Ca2+/high Mg2+ containing media, suggesting direct postsynaptic receptor activation. The MUSC-induced depolarization could be reversibly blocked by the selective muscarinic-receptor antagonist, atropine, while the DMPP-induced depolarization could be reversibly suppressed by the selective ganglionic nicotinic-receptor antagonist, mecamylamine. Some neurons exhibited a transient membrane hyperpolarization during the depolarizing response to CCh or MUSC application. This transient inhibition could be reversibly blocked by the gamma-aminobutyric acid (GABA) antagonist, bicuculline, suggesting that the underlying hyperpolarization results indirectly from the endogenous release of GABA acting at GABA receptors. This study confirms the cholinoceptivity of MVN neurons and establishes that individual MVN cells possess muscarinic as well as nicotinic receptors. The data provide support for a prominent role of cholinergic mechanisms in the direct and indirect regulation of the excitability of MVN neurons.

  11. N-Methyl-D-Aspartate Receptor-Mediated Axonal Injury in Adult Rat Corpus Callosum

    PubMed Central

    Zhang, Jingdong; Liu, Jianuo; Fox, Howard S.; Xiong, Huangui

    2013-01-01

    Damage to white matter such as corpus callosum (CC) is a pathological characteristic in many brain disorders. Glutamate (Glut) excitotoxicity through AMPA receptors on oligodendrocyte (OL) was previously considered as a mechanism for white matter damage. Recent studies have shown that N-methyl-D-aspartate receptors (NMDARs) are expressed on myelin sheath of neonatal rat OL processes and that activation of these receptors mediated demyelization. Whether NMDARs are expressed in the adult CC and are involved in excitotoxic axonal injury remains to be determined. In this study, we demonstrate the presence of NMDARs in the adult rat CC and their distributions in myelinated nerve fibers and OL somata by means of immunocytochemical staining and Western blot. Incubation of the CC slices with Glut or NMDA induced axonal injury as revealed by analyzing amplitude of CC fiber compound action potentials (CAPs) and input–output response. Both Glut and NMDA decreased the CAP amplitude and input–output responses, suggesting an involvement of NMDARs in Glut- and NMDA-induced axonal injury. The involvement of NMDAR in Glut-induced axonal injury was further assayed by detection of β-amyloid precursor protein (β-APP) in the CC axonal fibers. Treatment of the CC slices with Glut resulted in β-APP accumulation in the CC fibers as detected by Western blot, reflecting an impairment of axonal transport function. This injurious effect of Glut on CC axonal transport was significantly blocked by MK801. Taken together, these results show that NMDARs are expressed in the adult CC and are involved in excitotoxic activity in adult CC slices in vitro. PMID:23161705

  12. Differential estradiol and selective estrogen receptor modulator (SERM) regulation of Keratin 13 gene expression and its underlying mechanism in breast cancer cells.

    PubMed

    Sheng, Shubin; Barnett, Daniel H; Katzenellenbogen, Benita S

    2008-12-16

    Expression of the Keratin 13 (KRT13) gene, which encodes a cytoskeletal protein thought to play important roles in breast cancer growth and metastasis, is differentially regulated by estradiol (E2) and the selective estrogen receptor modulators (SERMs) tamoxifen and raloxifene. While stimulation of KRT13 by tamoxifen is robust and prolonged, stimulation by E2 is more transient and raloxifene has virtually no effect. To investigate the mechanistic basis for the differential ligand regulation of KRT13, we have defined the regulatory regions of KRT13, compared gene expression by E2 and SERMs, and explored the magnitudes and time courses of estrogen receptor (ER) and cofactor recruitment patterns on these regions. Using a ChIP scanning approach and reporter transactivation assays, we identified a 2.5 kb upstream ER-binding regulatory region for KRT13. Directed composite mutations in this region revealed that three estrogen response elements and three Sp1 sites were involved in its ligand-dependent regulation. Differential recruitment of ERalpha and cofactors to the KRT13 regulatory sites paralleled the different time course and magnitude of regulation by these ligands: there was almost no ERalpha or cofactor recruitment with raloxifene, whereas there was strong, prolonged ER recruitment and histone acetylation with tamoxifen, and an early and more transient recruitment with E2. Taken together, our results suggest that the different ligand regulations of KRT13 are due to ligand-differential recruitment of ER and coactivators, and they provide insight into the mechanisms responsible for the different agonistic activities and differential gene regulation by estradiol and the SERMs tamoxifen and raloxifene.

  13. Quercetin and epigallocatechin gallate inhibit glucose uptake and metabolism by breast cancer cells by an estrogen receptor-independent mechanism

    SciTech Connect

    Moreira, Liliana; Araújo, Isabel; Costa, Tito; Correia-Branco, Ana; Faria, Ana; Martel, Fátima; Keating, Elisa

    2013-07-15

    In this study we characterized {sup 3}H-2-deoxy-D-glucose ({sup 3}H -DG) uptake by the estrogen receptor (ER)-positive MCF7 and the ER-negative MDA-MB-231 human breast cancer cell lines and investigated the effect of quercetin (QUE) and epigallocatechin gallate (EGCG) upon {sup 3}H-DG uptake, glucose metabolism and cell viability and proliferation. In both MCF7 and MDA-MB-231 cells {sup 3}H-DG uptake was (a) time-dependent, (b) saturable with similar capacity (V{sub max}) and affinity (K{sub m}), (c) potently inhibited by cytochalasin B, an inhibitor of the facilitative glucose transporters (GLUT), (d) sodium-independent and (e) slightly insulin-stimulated. This suggests that {sup 3}H-DG uptake by both cell types is mediated by members of the GLUT family, including the insulin-responsive GLUT4 or GLUT12, while being independent of the sodium-dependent glucose transporter (SGLT1). QUE and EGCG markedly and concentration-dependently inhibited {sup 3}H-DG uptake by MCF7 and by MDA-MB-231 cells, and both compounds blocked lactate production by MCF7 cells. Additionally, a 4 h-treatment with QUE or EGCG decreased MCF7 cell viability and proliferation, an effect that was more potent when glucose was available in the extracellular medium. Our results implicate QUE and EGCG as metabolic antagonists in breast cancer cells, independently of estrogen signalling, and suggest that these flavonoids could serve as therapeutic agents/adjuvants even for ER-negative breast tumors. -- Highlights: • Glucose uptake by MCF7 and MDA-MB-231 cells is mainly mediated by GLUT1. • QUE and EGCG inhibit cellular glucose uptake thus abolishing the Warburg effect. • This process induces cytotoxicity and proliferation arrest in MCF7 cells. • The flavonoids’ effects are independent of estrogen receptor signalling.

  14. Serum estrogen receptor bioactivity and breast cancer risk among postmenopausal women

    PubMed Central

    Lim, Vanessa W; Li, Jun; Gong, Yinhan; Jin, Aizhen; Yuan, Jian-Min; Yong, Eu Leong; Koh, Woon-Puay

    2014-01-01

    The estrogen levels of Asian women are different from those of Western women, and this could affect estrogen receptor (ER) bioactivity and breast cancer risk. We conducted a case-control study of 169 postmenopausal breast cancer cases and 426 matched controls nested within a population-based prospective cohort, The Singapore Chinese Health Study, to evaluate serum levels of estrogens and their receptor (ERα and ERβ)-mediated estrogenic activities in relation to breast cancer risk. Breast cancer cases had higher levels of estrogens and estrogen receptor mediated bioactivities in baseline serum than controls. Compared to the lowest quartile, women in the highest quartile for estrone or ERα-mediated bioactivity had increased breast cancer risk. After additional adjustment for ERβ bioactivity, free E2 and estrone; serum ERα-mediated estrogenic activity remained associated with increased breast cancer risk. Compared to the lowest quartile, women in the highest quartile for ERα-mediated bioactivity had an odds ratio of 2.39 (95% confidence interval=1.17–4.88, p for trend=0.016). Conversely, the positive association between estrone and cancer risk became null after adjustment for ERα-mediated estrogenic activity, suggesting that the effect of estrone could be mediated through ERα. Identification of the factor(s) contributing to increased ERα-mediated estrogenic bioactivity in sera, and its role as a predictor for breast cancer risk needs to be validated in future studies. PMID:24322303

  15. Molecular mechanisms mediating the neuroprotective role of the selective estrogen receptor modulator, bazedoxifene, in acute ischemic stroke: A comparative study with 17β-estradiol.

    PubMed

    Jover-Mengual, Teresa; Castelló-Ruiz, María; Burguete, María C; Jorques, María; López-Morales, Mikahela A; Aliena-Valero, Alicia; Jurado-Rodríguez, Andrés; Pérez, Salvador; Centeno, José M; Miranda, Francisco J; Alborch, Enrique; Torregrosa, Germán; Salom, Juan B

    2017-07-01

    As the knowledge on the estrogenic system in the brain grows, the possibilities to modulate it in order to afford further neuroprotection in brain damaging disorders so do it. We have previously demonstrated the ability of the selective estrogen receptor modulator, bazedoxifene (BZA), to reduce experimental ischemic brain damage. The present study has been designed to gain insight into the molecular mechanisms involved in such a neuroprotective action by investigating: 1) stroke-induced apoptotic cell death; 2) expression of estrogen receptors (ER) ERα, ERβ and the G-protein coupled estrogen receptor (GPER); and 3) modulation of MAPK/ERK1/2 and PI3K/Akt signaling pathways. For comparison, a parallel study was done with 17β-estradiol (E2)-treated animals. Male Wistar rats subject to transient right middle cerebral artery occlusion (tMCAO, intraluminal thread technique, 60min), were distributed in vehicle-, BZA- (20.7±2.1ng/mL in plasma) and E2- (45.6±7.8pg/mL in plasma) treated groups. At 24h from the onset of tMCAO, RT-PCR, Western blot and histochemical analysis were performed on brain tissue samples. Ischemia-reperfusion per se increased apoptosis as assessed by both caspase-3 activity and TUNEL-positive cell counts, which were reversed by both BZA and E2. ERα and ERβ expression, but not that of GPER, was reduced by the ischemic insult. BZA and E2 had different effects: while BZA increased both ERα and ERβ expression, E2 increased ERα expression but did not change that of ERβ. Both MAPK/ERK1/2 and PI3K/Akt pathways were stimulated under ischemic conditions. While BZA strongly reduced the increased p-ERK1/2 levels, E2 did not. Neither BZA nor E2 modified ischemia-induced increase in p-Akt levels. These results show that modulation of ERα and ERβ expression, as well as of the ERK1/2 signaling pathway accounts, at least in part, for the inhibitory effect of BZA on the stroke-induced apoptotic cell death. This lends mechanistic support to the

  16. [Uterine estrogen sulfotransferase and estrogen sulfatase in embryo implantation].

    PubMed

    Loza Arredondo, M C; González Juarez, N A

    1994-11-01

    The relation conjugated/unconjugated estrogens associated with reproductive processes has brought about the interest to study the biological role and regulation of the estrogen sulfotransferase and estrogen sulfatase which participate in the formation and hydrolysis of estrogen 3-sulfates, respectively. In this paper, both activities were measured through the reciprocal conversion of 3H-estrone sulfate and 3H-unconjugated estrogen during in vitro incubation with implantation sites (SI) and non-implanted sites (SNI) from the rat uterus, during the process of embryo implantation. Contrasting enzyme activities were found in these tissues. While sulfotransferase activity was higher in SI than in SNI (0.205 vs 0.144 pmol of E1S formed/mg protein/h, the inverse was found for the sulfatase (1.470 vs 1.977 pmol of E1 formed/mg protein/h). These results indicate the presence of both enzymes in the rat uterus and suggest the existence of a mechanism in SI that locally regulate the concentration of free and sulfoconjugated estrogens in which these enzymes participate.

  17. Loss of Progesterone Receptor-Mediated Actions Induce Preterm Cellular and Structural Remodeling of the Cervix and Premature Birth

    PubMed Central

    Yellon, Steven M.; Dobyns, Abigail E.; Beck, Hailey L.; Kurtzman, James T.; Garfield, Robert E.; Kirby, Michael A.

    2013-01-01

    A decline in serum progesterone or antagonism of progesterone receptor function results in preterm labor and birth. Whether characteristics of premature remodeling of the cervix after antiprogestins or ovariectomy are similar to that at term was the focus of the present study. Groups of pregnant rats were treated with vehicle, a progesterone receptor antagonist (onapristone or mifepristone), or ovariectomized on day 17 postbreeding. As expected, controls given vehicle delivered at term while rats delivered preterm after progesterone receptor antagonist treatment or ovariectomy. Similar to the cervix before term, the preterm cervix of progesterone receptor antagonist-treated rats was characterized by reduced cell nuclei density, decreased collagen content and structure, as well as a greater presence of macrophages per unit area. Thus, loss of nuclear progesterone receptor-mediated actions promoted structural remodeling of the cervix, increased census of resident macrophages, and preterm birth much like that found in the cervix at term. In contrast to the progesterone receptor antagonist-induced advance in characteristics associated with remodeling, ovariectomy-induced loss of systemic progesterone did not affect hypertrophy, extracellular collagen, or macrophage numbers in the cervix. Thus, the structure and macrophage census in the cervix appear sufficient for premature ripening and birth to occur well before term. With progesterone receptors predominantly localized on cells other than macrophages, the findings suggest that interactions between cells may facilitate the loss of progesterone receptor-mediated actions as part of a final common mechanism that remodels the cervix in certain etiologies of preterm and with parturition at term. PMID:24339918

  18. Adenosine A2 receptor-mediated regulation of renal hemodynamics and glomerular filtration rate is abolished in diabetes.

    PubMed

    Persson, Patrik; Hansell, Peter; Palm, Fredrik

    2013-01-01

    Alterations in glomerular filtration rate (GFR) are one of the earliest indications of altered kidney function in diabetes. Adenosine regulates GFR through tubuloglomerular feedback mechanism acting on adenosine A1 receptor. In addition, adenosine can directly regulate vascular tone by acting on A1 and A2 receptors expressed in afferent and efferent arterioles. Opposite to A1 receptors, A2 receptors mediate vasorelaxation. This study investigates the involvement of adenosine A2 receptors in regulation of renal blood flow (RBF) and GFR in control and diabetic kidneys. GFR was measured by inulin clearance and RBF by a transonic flow probe placed around the renal artery. Measurements were performed in isoflurane-anesthetized normoglycemic and alloxan-diabetic C57BL/6 mice during baseline and after acute administration of 3,7-dimethyl-1-propargylxanthine (DMPX), a selective A2 receptor antagonist. GFR and RBF were lower in diabetic mice compared to control (258 ± 61 vs. 443 ± 33 μl min(-1) and 1,083 ± 51 vs. 1,405 ± 78 μl min(-1)). In control animals, DMPX decreased RBF by -6%, whereas GFR increased +44%. DMPX had no effects on GFR and RBF in diabetic mice. Sodium excretion increased in diabetic mice after A2 receptor blockade (+78%). In conclusion, adenosine acting on A2 receptors mediates an efferent arteriolar dilatation which reduces filtration fraction (FF) and maintains GFR within normal range in normoglycemic mice. However, this regulation is absent in diabetic mice, which may contribute to reduced oxygen availability in the diabetic kidney.

  19. The P2Y2 Nucleotide Receptor Mediates Tissue Factor Expression in Human Coronary Artery Endothelial Cells*

    PubMed Central

    Ding, Ling; Ma, Wanshu; Littmann, Timothy; Camp, Riley; Shen, Jianzhong

    2011-01-01

    The discovery of the role of P2Y12 receptor in platelet aggregation leads to a new anti-thrombotic drug Plavix; however, little is known about non-platelet P2Y receptors in thrombosis. This study tested the hypothesis that endothelial P2Y receptor(s) mediates up-regulation of tissue factor (TF), the initiator of coagulation cascade. Stimulation of human coronary artery endothelial cells (HCAEC) by UTP/ATP increased the mRNA level of TF but not of its counterpart-tissue factor pathway inhibitor, which was accompanied by up-regulation of TF protein and cell surface activity. RT-PCR revealed a selective expression of P2Y2 and P2Y11 receptors in HCAEC. Consistent with this, TF up-regulation was inhibited by suramin or by siRNA silencing of P2Y2 receptor, but not by NF-157, a P2Y11-selective antagonist, suggesting a role for the P2Y2 receptor. In addition, P2Y2 receptor activated ERK1/2, JNK, and p38 MAPK pathways without affecting the positive NF-κB and negative AKT regulatory pathways of TF expression. Furthermore, TF up-regulation was abolished or partially suppressed by inhibition of p38 or JNK but not ERK1/2. Interestingly, blockade of the PLC/Ca2+ pathway did not affect P2Y2 receptor activation of p38, JNK, and TF induction. However, blockade of Src kinase reduced phosphorylation of p38 but not JNK, eliminating TF induction. In contrast, inhibition of Rho kinase reduced phosphorylation of JNK but not p38, decreasing TF expression. These findings demonstrate that P2Y2 receptor mediates TF expression in HCAEC through new mechanisms involving Src/p38 and Rho/JNK pathways, possibly contributing to a pro-thrombotic status after vascular injury. PMID:21652710

  20. Chronic Neuropathic Pain in Mice Reduces μ-Opioid Receptor-Mediated G-protein Activity in the Thalamus

    PubMed Central

    Hoot, Michelle R.; Sim-Selley, Laura J.; Selley, Dana E.; Scoggins, Krista L.; Dewey, William L.

    2011-01-01

    Neuropathic pain is a debilitating condition that is often difficult to treat using conventional pharmacological interventions and the exact mechanisms involved in the establishment and maintenance of this type of chronic pain have yet to be fully elucidated. The present studies examined the effect of chronic nerve injury on μ-opioid receptors and receptor-mediated G-protein activity within the supraspinal brain regions involved in pain processing of mice. Chronic constriction injury (CCI) reduced paw withdrawal latency, which was maximal at 10 days post-injury. [d-Ala2,(N-Me)Phe4, Gly5-OH] enkephalin (DAMGO)-stimulated [35S]GTPγS binding was then conducted at this time point in membranes prepared from the rostral ACC (rACC), thalamus and periaqueductal grey (PAG) of CCI and sham-operated mice. Results showed reduced DAMGO-stimulated [35S]GTPγS binding in the thalamus and PAG of CCI mice, with no change in the rACC. In thalamus, this reduction was due to decreased maximal stimulation by DAMGO, with no difference in EC50 values. In PAG, however, DAMGO Emax values did not significantly differ between groups, possibly due to the small magnitude of the main effect. [3H]Naloxone binding in membranes of the thalamus showed no significant differences in Bmax values between CCI and sham-operated mice, indicating that the difference in G-protein activation did not result from differences in μ-opioid receptor levels. These results suggest that CCI induced a region-specific adaptation of μ-opioid receptor-mediated G-protein activity, with apparent desensitization of the μ-opioid receptor in the thalamus and PAG and could have implications for treatment of neuropathic pain. PMID:21762883

  1. Low responsiveness to agents evoking 5-HT2 receptor-mediated behaviors in Sardinian alcohol-preferring rats.

    PubMed

    Ciccocioppo, R; Panocka, I; Stefanini, E; Gessa, G L; Massi, M

    1995-05-01

    The selective NK3 tachykinin agonist senktide evokes in rodents 5-HT mediated behaviors, including 5-HT2 receptor-mediated wet dog shakes (WDS) and head shakes (HS). It was observed previously that genetically selected Sardinian alcohol-preferring (sP) rats show a small number of WDS and HS following intracerebroventricular (ICV) injection of senktide. The present study was aimed at confirming these observations and at providing information on the reasons accounting for the anomalous response of sP rats. Senktide (500-2000 ng/rat, ICV) produced a much lower number of WDS and HS in sP rats than in nonselected Wistar (nsW) rats. Both behaviors were suppressed by the 5-HT2 antagonist ritanserin (1 mg/kg, subcutaneously), confirming that 5-HT2 receptors mediate the response. HS induced by the ICV injection of 5-HT agonists endowed with marked activity at 5-HT2 receptors, such as quipazine (1500-6000 ng/rat) or DOI (500-3500 ng/rat), were much less pronounced in sP rats than in nsW rats. Moreover, WDS following peripheral injection of 5-hydroxytryptophan, 25-100 mg/kg, and carbidopa, 12.5 mg/kg, were less intense in sP and in ethanol-naive sP rats than in nsW and in Sardinian alcohol-nonpreferring rats. These findings suggest that sP rats have an inherent different regulation of central 5-HT2 mechanisms.

  2. Receptor-mediated Ca2+ and PKC signaling triggers the loss of cortical PKA compartmentalization through the redistribution of gravin.

    PubMed

    Schott, Micah B; Grove, Bryon

    2013-11-01

    A-Kinase Anchoring Proteins (AKAPs) direct the flow of cellular information by positioning multiprotein signaling complexes into proximity with effector proteins. However, certain AKAPs are not stationary but can undergo spatiotemporal redistribution in response to stimuli. Gravin, a 300kD AKAP that intersects with a diverse signaling array, is localized to the plasma membrane but has been shown to translocate to the cytosol following the elevation of intracellular calcium ([Ca(2+)]i). Despite the potential for gravin redistribution to impact multiple signaling pathways, the dynamics of this event remain poorly understood. In this study, quantitative microscopy of cells expressing gravin-EGFP revealed that Ca(2+) elevation caused the complete translocation of gravin from the cell cortex to the cytosol in as little as 60s of treatment with ionomycin or thapsigargin. In addition, receptor mediated signaling was also shown to cause gravin redistribution following ATP treatment, and this event required both [Ca(2+)]i elevation and PKC activation. To understand the mechanism for Ca(2+) mediated gravin dynamics, deletion of calmodulin-binding domains revealed that a fourth putative calmodulin binding domain called CB4 (a.a. 670-694) is critical for targeting gravin to the cell cortex despite its location downstream of gravin's membrane-targeting domains, which include an N-terminal myristoylation site and three polybasic domains. Finally, confocal microscopy of cells co-transfected with gravin-EYFP and PKA RII-ECFP revealed that gravin redistribution mediated by ionomycin, thapsigargin, and ATP each triggered the gravin-dependent loss of PKA localized at the cell cortex. Our results support the hypothesis that gravin redistribution regulates cross-talk between PKA-dependent signaling and receptor-mediated events involving Ca(2+) and PKC. © 2013.

  3. Insulin-Independent GABAA Receptor-Mediated Response in the Barrel Cortex of Mice with Impaired Met Activity

    PubMed Central

    Lo, Fu-Sun; Erzurumlu, Reha S.

    2016-01-01

    Autism spectrum disorder (ASD) is a neurodevelopmental disorder caused by genetic variants, susceptibility alleles, and environmental perturbations. The autism associated gene MET tyrosine kinase has been implicated in many behavioral domains and endophenotypes of autism, including abnormal neural signaling in human sensory cortex. We investigated somatosensory thalamocortical synaptic communication in mice deficient in Met activity in cortical excitatory neurons to gain insights into aberrant somatosensation characteristic of ASD. The ratio of excitation to inhibition is dramatically increased due to decreased postsynaptic GABAA receptor-mediated inhibition in the trigeminal thalamocortical pathway of mice lacking active Met in the cerebral cortex. Furthermore, in contrast to wild-type mice, insulin failed to increase GABAA receptor-mediated response in the barrel cortex of mice with compromised Met signaling. Thus, lacking insulin effects may be a risk factor in ASD pathogenesis. SIGNIFICANCE STATEMENT A proposed common cause of neurodevelopmental disorders is an imbalance in excitatory neural transmission, provided by the glutamatergic neurons, and the inhibitory signals from the GABAergic interneurons. Many genes associated with autism spectrum disorders impair synaptic transmission in the expected cell type. Previously, inactivation of the autism-associated Met tyrosine kinase receptor in GABAergic interneurons led to decreased inhibition. In thus report, decreased Met signaling in glutamatergic neurons had no effect on excitation, but decimated inhibition. Further experiments indicate that loss of Met activity downregulates GABAA receptors on glutamatergic neurons in an insulin independent manner. These data provide a new mechanism for the loss of inhibition and subsequent abnormal excitation/inhibition balance and potential molecular candidates for treatment or prevention. PMID:27030755

  4. H1 and H2 receptors mediate postexercise hyperemia in sedentary and endurance exercise-trained men and women.

    PubMed

    McCord, Jennifer L; Halliwill, John R

    2006-12-01

    In sedentary individuals, H(1) receptors mediate the early portion of postexercise skeletal muscle hyperemia, whereas H(2) receptors mediate the later portion. It is not known whether postexercise hyperemia also presents in endurance-trained individuals. We hypothesized that the postexercise skeletal muscle hyperemia would also exist in endurance-trained individuals and that combined blockade of H(1) and H(2) receptors would abolish the long-lasting postexercise hyperemia in trained and sedentary individuals. We studied 28 sedentary and endurance trained men and women before and through 90 min after a 60-min bout of cycling at 60% peak O(2) uptake on control and combined H(1)- and H(2)-receptor antagonist days (fexofenadine and ranitidine). We measured arterial pressure (brachial auscultation) and femoral blood flow (Doppler ultrasound). On the control day, femoral vascular conductance (calculated as flow/pressure) was elevated in all groups 60 min after exercise (sedentary men: Delta86 +/- 35%, trained men, Delta65 +/- 18%; sedentary women, Delta61 +/- 19%, trained women: Delta59 +/- 23%, where Delta is change; all P < 0.05 vs. preexercise). In contrast, on the histamine antagonist day, femoral vascular conductance was not elevated in any of the groups after exercise (sedentary men: Delta21 +/- 17%, trained men: Delta9 +/- 5%, sedentary women: Delta19 +/- 4%, trained women: Delta11 +/- 11%; all P > 0.16 vs. preexercise; all P < 0.05 vs. control day). These data suggest postexercise skeletal muscle hyperemia exists in endurance trained men and women. Furthermore, histaminergic mechanisms produce the long-lasting hyperemia in sedentary and endurance-trained individuals.

  5. Opioid receptor agonists may favorably affect bone mechanical properties in rats with estrogen deficiency-induced osteoporosis.

    PubMed

    Janas, Aleksandra; Folwarczna, Joanna

    2017-02-01

    The results of epidemiological, clinical, and in vivo and in vitro experimental studies on the effect of opioid analgesics on bone are inconsistent. The aim of the present study was to investigate the effect of morphine (an agonist of opioid receptors), buprenorphine (a partial μ opioid receptor agonist and κ opioid receptor antagonist), and naloxone (an antagonist of opioid receptors) on the skeletal system of female rats in vivo. The experiments were carried out on 3-month-old Wistar rats, divided into two groups: nonovariectomized (intact; NOVX) rats and ovariectomized (OVX) rats. The bilateral ovariectomy was performed 7 days before the start of drug administration. Morphine hydrochloride (20 mg/kg/day s.c.), buprenorphine (0.05 mg/kg/day s.c.), or naloxone hydrochloride dihydrate (2 mg/kg/day s.c.) were administered for 4 weeks to NOVX and OVX rats. In OVX rats, the use of morphine and buprenorphine counteracted the development of osteoporotic changes in the skeletal system induced by estrogen deficiency. Morphine and buprenorphine beneficially affected also the skeletal system of NOVX rats, but the effects were much weaker than those in OVX rats. Naloxone generally did not affect the rat skeletal system. The results confirmed the role of opioid receptors in the regulation of bone remodeling processes and demonstrated, in experimental conditions, that the use of opioid analgesics at moderate doses may exert beneficial effects on the skeletal system, especially in estrogen deficiency.

  6. Mechanism of estrogen biosynthesis. Stereochemistry of C-1 hydrogen elimination in the aromatization of 2 beta-hydroxy-19-oxoandrostenedione.

    PubMed

    Fishman, J; Raju, M S

    1981-05-10

    19-Hydroxy[1 alpha-3H]androstenedione was synthesized and its specific activity was accurately determined. Upon aromatization of the above material by placental microsomal aromatase preparation, a process involving 1 beta hydrogen elimination, only 7.4% of the isotope was lost establishing the alpha orientation of the 3H at C-1 in the substrate. The 19-hydroxy[1 alpha]3H]androstenedione was used as the starting material in the synthesis of 2 beta-hydroxy-19-oxo[1 alpha-3H]androstenedione which therefore had the same specific activity and isotope orientation as its precursor. The nonenzymatic collapse of 2 beta-hydroxy-19-oxo[1 alpha-3H]androstenedione in pH 7.1 buffer to estrone was associated with the elimination of only 2.6% of the isotope indicating that this process proceeds also with stereospecific 1 beta hydrogen elimination. The stereochemistry of hydrogen loss in the nonenzymatic aromatization of the 2 beta-hydroxy-19-oxo derivative is therefore beta and identical with that of estrogen biosynthesis. This provides further evidence in support of the hypothesis that the final enzymatic hydroxylation of the aromatization sequence takes place at position 2 beta of the androgen substrate and that its product, the 2 beta-hydroxy-19-aldehyde, is the proximate precursor of estrogen with the final conversion occurring nonenzymatically.

  7. Estrogen receptor-α is required for the osteogenic response to mechanical loading in a ligand-independent manner involving its activation function 1 but not 2

    PubMed Central

    Windahl, Sara H; Saxon, Leanne; Börjesson, Anna E; Lagerquist, Marie K; Frenkel, Baruch; Henning, Petra; Lerner, Ulf H; Galea, Gabriel L; Meakin, Lee B; Engdahl, Cecilia; Sjögren, Klara; Antal, Maria C; Krust, Andrée; Chambon, Pierre; Lanyon, Lance E; Price, Joanna S; Ohlsson, Claes

    2013-01-01

    Estrogen receptor-α (ERα) is crucial for the adaptive response of bone to loading but the role of endogenous estradiol (E2) for this response is unclear. To determine in vivo the ligand dependency and relative roles of different ERα domains for the osteogenic response to mechanical loading, gene-targeted mouse models with (1) a complete ERα inactivation (ERα−/−), (2) specific inactivation of activation function 1 (AF-1) in ERα (ERαAF-10), or (3) specific inactivation of ERαAF-2 (ERαAF-20) were subjected to axial loading of tibia, in the presence or absence (ovariectomy [ovx]) of endogenous E2. Loading increased the cortical bone area in the tibia mainly as a result of an increased periosteal bone formation rate (BFR) and this osteogenic response was similar in gonadal intact and ovx mice, demonstrating that E2 (ligand) is not required for this response. Female ERα−/− mice displayed a severely reduced osteogenic response to loading with changes in cortical area (−78% ± 15%, p < 0.01) and periosteal BFR (−81% ± 9%, p < 0.01) being significantly lower than in wild-type (WT) mice. ERαAF-10 mice also displayed a reduced response to mechanical loading compared with WT mice (cortical area −40% ± 11%, p < 0.05 and periosteal BFR −41% ± 8%, p < 0.01), whereas the periosteal osteogenic response to loading was unaffected in ERαAF-20 mice. Mechanical loading of transgenic estrogen response element (ERE)-luciferase reporter mice did not increase luciferase expression in cortical bone, suggesting that the loading response does not involve classical genomic ERE-mediated pathways. In conclusion, ERα is required for the osteogenic response to mechanical loading in a ligand-independent manner involving AF-1 but not AF-2. © 2013 American Society for Bone and Mineral Research PMID:22972752

  8. Bisphosphonates do not inhibit periosteal bone formation in estrogen deficient animals and allow enhanced bone modeling in response to mechanical loading.

    PubMed

    Feher, Anthony; Koivunemi, Andrew; Koivunemi, Mark; Fuchs, Robyn K; Burr, David B; Phipps, Roger J; Reinwald, Susan; Allen, Matthew R

    2010-01-01

    The suppressive effects of bisphosphonates (BPs) on bone remodeling are clear yet there is conflicting data concerning the effects of BPs on modeling (specifically formation modeling on the periosteal surface). The normal periosteal expansion that occurs during aging has significant benefits to maintaining/improving the bones' mechanical properties and thus it is important to understand whether BPs affect this bone surface. Therefore, the purpose of this study was to determine the effects of BPs on periosteal bone formation modeling induced by ovariectomy (OVX) and mechanical loading. Six-month-old Sprague-Dawley OVX rats (n=60; 12/group) were administered vehicle, risedronate, alendronate, or zoledronate at doses used clinically for treatment of post-menopausal osteoporosis. Three weeks after initiating BP treatment, all animals underwent in vivo ulnar loading of the right limb every other day for 1 week (3 total sessions). Periosteal surface mineral apposition rate, mineralizing surface, and bone formation rate were determined at the mid-diaphysis of both loaded (right) and non-loaded (left) ulnae. There was no significant effect of any of the BPs on periosteal bone formation parameters compared to VEH-treated animals in the non-loaded limb, suggesting that BP treatment does not compromise the normal periosteal expansion associated with estrogen loss. Mechanical loading significantly increased BFR in the loaded limb compared to the non-loaded limb in all BP-treated groups, with no difference in the magnitude of this effect among the various BPs. Collectively, these data show that BP treatment, at doses comparable to those used for treatment of post-menopausal osteoporosis, (1) does not alter the periosteal formation activity that occurs in the absence of estrogen and (2) allows normal stimulation of periosteal bone formation in response to the anabolic stimulation of mechanical loading.

  9. The endocrine disrupting alkylphenols and 4,4'-DDT interfere with estrogen conversion and clearance by mouse liver cytosol.

    PubMed

    El-Hefnawy, Talal; Hernandez, Claudia; Stabile, Laura P

    2017-09-01

    Endocrine disrupting chemicals (EDCs) are ubiquitous compounds known for negative impacts on reproductive functions and for increasing cancer risk. EDCs are believed to cause the harmful effects in part through their inappropriate low-affinity binding to steroid receptors and other possible non-receptor mediated paradigms, however there is a need to further elucidate other mechanisms involving the direct and indirect impact of EDCs on reproductive functions. We examined the metabolism of 17β-estradiol (E2) and estrone (E1) by cell-free hepatic cytosol in the presence of alkylphenols (nonylphenol/NP and 4-tert-octylphenol/tOP), Dichlorodiphenyltrichloroethane (4,4'-DDT) and other EDCs. Tandem liquid chromatography mass spectrometry was utilized to quantitatively assess the impact of each EDC on estrogen clearance, inter-conversions and downstream metabolism by mouse liver cytosol. The results revealed that NP and tOP (0.1-3μg/mL) significantly reduced the hepatic cytosol clearance and biotransformation of estrogens with inclination for accumulating E2, the stronger estrogen form, than E1. Alkylphenols also caused up to a 34-fold increase in the E2/E1 ratio possibly by suppressing the hepatic E2→E1 conversion by 17β-hydroxysteroid dehydrogenase (17βHSD) types 2, 4 while displaying a weaker inhibition of E1→E2 conversion by type 1, 17βHSD. On the other hand, the pesticide 4,4'-DDT was a weaker inhibitor of clearance of estrogens by the cytosol preparations when compared to alkylphenols, whereas chemicals such as phthalates and atrazine were ineffective. Our data suggest that exposure to NP, tOP and DDT can indirectly increase the estrogenic load by suppressing the hepatic clearance of estrogens and by elevating the E2/1 ratio and could therefore increase the risk of reproductive lesions. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier

  10. Estrogen receptor beta and truncated variants enhance the expression of transfected MMP-1 promoter constructs in response to specific mechanical loading

    PubMed Central

    2014-01-01

    Background Joint diseases such as osteoarthritis (OA) predominantly afflict post-menopausal women, suggesting a pertinent role for female hormones. Estrogen receptor beta (ER-β) has been detected in connective tissues of the knee joint suggesting that these tissues are responsive to the hormone estrogen. Matrix metalloproteinase-1 (MMP-1) activity contributes to cartilage degradation, a key factor leading to OA development in synovial joints. Two polymorphic forms of MMP-1 exist due to a deletion/insertion of the guanine residue in the promoter, and the 2G allelic variant of MMP-1 exhibits more activity than the 1G allele. Previous studies have demonstrated that the polymorphic forms of the human MMP-1 are influenced by the modulating effects of estrogen receptor isoforms. In addition to hormonal influences, physiological factors such as altered mechanical loading are also contributory features of OA. In the present study, the combined influence of biomechanical and hormonal variables on the activity of MMP-1 isoforms was evaluated. We hypothesized that the combined effects of ER-β and sheer stress will differentially activate the two allelic forms of MMP-1 in a hormone-independent manner. Methods HIG-82 synoviocytes were transiently transfected with 1G or 2G alleles (±) ER-β and subjected to either shear or equibiaxial stress. Next, 1G/2G promoter activity was measured to determine the combined influence of physiological stimuli. Truncated ER-β constructs were used to determine the importance of different domains of ER-β on 1G/2G activation. Results The 2G allele exhibited a constitutively higher activity than the 1G allele, which was further increased when the transfected cells were subject to shear stress, but not equibiaxial stress. Moreover, the combination of ER-β and shear stress further increased the activity levels of the 1G/2G allelic variants. Additionally, select AF-2 truncated ER-β variants led to increased activity levels for the 2G allele

  11. Disturbed estrogen and progesterone action in ovarian endometriosis.

    PubMed

    Smuc, Tina; Hevir, Neli; Ribic-Pucelj, Martina; Husen, Bettina; Thole, Hubert; Rizner, Tea Lanisnik

    2009-03-25

    Endometriosis is a very common disease in pre-menopausal women, where defective metabolism of steroid hormones plays an important role in its development and promotion. In the present study, we have examined the expression of 11 estrogen and progesterone metabolizing enzymes and their corresponding receptors in samples of ovarian endometriomas and control endometrium. Expression analysis revealed significant up-regulation of enzymes involved in estradiol formation (aromatase, sulfatase and all reductive 17beta-hydroxysteroid dehydrogenases) and in progesterone inactivation (AKR1C1 and AKR1C3). Among the estrogen and progesterone receptors, ERalpha was down-regulated, ERbeta was up-regulated, and there was no significant difference in expression of progesterone receptors A and B (PRAB). Our data indicate that several enzymes of estrogen and progesterone metabolism are aberrantly expressed in endometriosis, which can lead to increased local levels of mitogenic estradiol and decreased levels of protective progesterone. Changes in estrogen receptor expression suggest that estradiol may also act via non-estrogen receptor-mediated pathways, while expression of progesterone receptors still needs further investigation.

  12. Ryanodine receptor-mediated arrhythmias and sudden cardiac death

    PubMed Central

    Blayney, Lynda M.; Lai, F. Anthony

    2009-01-01

    The cardiac ryanodine receptor-Ca2+ release channel (RyR2) is an essential sarcoplasmic reticulum (SR) transmembrane protein that plays a central role in excitation–contraction coupling (ECC) in cardiomyocytes. Aberrant spontaneous, diastolic Ca2+ leak from the SR due to dysfunctional RyR2 contributes to the formation of delayed after-depolarisations, which are thought to underlie the fatal arrhythmia that occurs in both heart failure (HF) and in catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT is an inherited disorder associated with mutations in either the RyR2 or a SR luminal protein, calsequestrin. RyR2 shows normal function at rest in CPVT but the RyR2 dysfunction is unmasked by physical exercise or emotional stress, suggesting abnormal RyR2 activation as an underlying mechanism. Several potential mechanisms have been advanced to explain the dysfunctional RyR2 observed in HF and CPVT, including enhanced RyR2 phosphorylation status, altered RyR2 regulation at luminal/cytoplasmic sites and perturbed RyR2 intra/inter-molecular interactions. This review considers RyR2 dysfunction in the context of the structural and functional modulation of the channel, and potential therapeutic strategies to stabilise RyR2 function in cardiac pathology. PMID:19345240

  13. Ionotropic Chemosensory Receptors Mediate the Taste and Smell of Polyamines.

    PubMed

    Hussain, Ashiq; Zhang, Mo; Üçpunar, Habibe K; Svensson, Thomas; Quillery, Elsa; Gompel, Nicolas; Ignell, Rickard; Grunwald Kadow, Ilona C

    2016-05-01

    The ability to find and consume nutrient-rich diets for successful reproduction and survival is fundamental to animal life. Among the nutrients important for all animals are polyamines, a class of pungent smelling compounds required in numerous cellular and organismic processes. Polyamine deficiency or excess has detrimental effects on health, cognitive function, reproduction, and lifespan. Here, we show that a diet high in polyamine is beneficial and increases reproductive success of flies, and we unravel the sensory mechanisms that attract Drosophila to polyamine-rich food and egg-laying substrates. Using a combination of behavioral genetics and in vivo calcium imaging, we demonstrate that Drosophila uses multisensory detection to find and evaluate polyamines present in overripe and fermenting fruit, their favored feeding and egg-laying substrate. In the olfactory system, two coexpressed ionotropic receptors (IRs), IR76b and IR41a, mediate the long-range attraction to the odor. In the gustatory system, multimodal taste sensation by IR76b receptor and GR66a bitter receptor neurons is used to evaluate quality and valence of the polyamine providing a mechanism for the fly's high attraction to polyamine-rich and sweet decaying fruit. Given their universal and highly conserved biological roles, we propose that the ability to evaluate food for polyamine content may impact health and reproductive success also of other animals including humans.

  14. Ionotropic Chemosensory Receptors Mediate the Taste and Smell of Polyamines

    PubMed Central

    Üçpunar, Habibe K.; Svensson, Thomas; Quillery, Elsa; Gompel, Nicolas; Ignell, Rickard; Grunwald Kadow, Ilona C.

    2016-01-01

    The ability to find and consume nutrient-rich diets for successful reproduction and survival is fundamental to animal life. Among the nutrients important for all animals are polyamines, a class of pungent smelling compounds required in numerous cellular and organismic processes. Polyamine deficiency or excess has detrimental effects on health, cognitive function, reproduction, and lifespan. Here, we show that a diet high in polyamine is beneficial and increases reproductive success of flies, and we unravel the sensory mechanisms that attract Drosophila to polyamine-rich food and egg-laying substrates. Using a combination of behavioral genetics and in vivo calcium imaging, we demonstrate that Drosophila uses multisensory detection to find and evaluate polyamines present in overripe and fermenting fruit, their favored feeding and egg-laying substrate. In the olfactory system, two coexpressed ionotropic receptors (IRs), IR76b and IR41a, mediate the long-range attraction to the odor. In the gustatory system, multimodal taste sensation by IR76b receptor and GR66a bitter receptor neurons is used to evaluate quality and valence of the polyamine providing a mechanism for the fly’s high attraction to polyamine-rich and sweet decaying fruit. Given their universal and highly conserved biological roles, we propose that the ability to evaluate food for polyamine content may impact health and reproductive success also of other animals including humans. PMID:27145030

  15. Dynamics of Receptor-Mediated Nanoparticle Internalization into Endothelial Cells

    PubMed Central

    Gonzalez-Rodriguez, David; Barakat, Abdul I.

    2015-01-01

    Nanoparticles offer a promising medical tool for targeted drug delivery, for example to treat inflamed endothelial cells during the development of atherosclerosis. To inform the design of such therapeutic strategies, we develop a computational model of nanoparticle internalization into endothelial cells, where internalization is driven by receptor-ligand binding and limited by the deformation of the cell membrane and cytoplasm. We specifically consider the case of nanoparticles targeted against ICAM-1 receptors, of relevance for treating atherosclerosis. The model computes the kinetics of the internalization process, the dynamics of binding, and the distribution of stresses exerted between the nanoparticle and the cell membrane. The model predicts the existence of an optimal nanoparticle size for fastest internalization, consistent with experimental observations, as well as the role of bond characteristics, local cell mechanical properties, and external forces in the nanoparticle internalization process. PMID:25901833

  16. Legume LysM receptors mediate symbiotic and pathogenic signalling.

    PubMed

    Kelly, Simon; Radutoiu, Simona; Stougaard, Jens

    2017-10-01

    Legume-rhizobia symbiosis is coordinated through the production and perception of signal molecules by both partners with legume LysM receptor kinases performing a central role in this process. Receptor complex formation and signalling outputs derived from these are regulated through ligand binding and further modulated by a diverse variety of interactors. The challenge now is to understand the molecular mechanisms of these reported interactors. Recently attributed roles of LysM receptors in the perception of rhizobial exopolysaccharide, distinguishing between pathogens and symbionts, and assembly of root and rhizosphere communities expand on the importance of these receptors. These studies also highlight challenges, such as identification of cognate ligands, formation of responsive receptor complexes and separation of downstream signal transduction pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Effect of estrogen on iron metabolism in mammals.

    PubMed

    Yang, Xiao; Xu, Man-Man; Wang, Jun; Xie, Jun-Xia

    2016-10-25

    Estrogen is a steroid hormone produced mainly by the ovaries. It combines with the nuclear receptors to exert the biological effects influencing the metabolism of body. Elevated levels of estrogen are often associated with altered iron levels in mammals. Furthermore, the findings of estrogen response element (ERE) have demonstrated that estrogen affects iron metabolism directly in peripheral tissues. In this review, we will briefly summarize the effect of estrogen on iron metabolism in mammals, and discuss recent progress in the mechanisms of estrogen on some iron related proteins in order to provide guidance for clinical use of estrogen. Estrogen and iron metabolism are closely related, but the exact regulatory mechanisms still need further exploration.

  18. NFAT regulates calcium-sensing receptor-mediated TNF production

    SciTech Connect

    abdullah, huda ismail; Pedraza, Paulina L.; Hao, Shoujin; Rodland, Karin D.; McGiff, John C.; Ferreri, Nicholas R.

    2006-05-01

    Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca2+ (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca2+ were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.

  19. NFAT regulates calcium-sensing receptor-mediated TNF production.

    PubMed

    Abdullah, Huda Ismail; Pedraza, Paulina L; Hao, Shoujin; Rodland, Karin D; McGiff, John C; Ferreri, Nicholas R

    2006-05-01

    Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca(2+) (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca(2+) were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.

  20. Ethanol inhibits epileptiform activity and NMDA receptor-mediated synaptic transmission in rat amygdaloid slices

    SciTech Connect

    Gean, P.W. )

    1992-02-26

    The effect of ethanol on the epileptiform activity induced by Mg{sup ++}-free solution was studied in rat amygdalar slices using intracellular recording techniques. The spontaneous and evoked epileptiform discharges consisting of an initial burst followed by afterdischarges were observed 20-30 min after switching to Mg{sup ++}-free medium. Superfusion with ethanol reversibly reduced the duration of spontaneous and evoked bursting discharges in a concentration-dependent manner. Synaptic response mediated by N-methyl-D-aspartate (NMDA) receptor activation was isolated by application of a solution containing the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and either in Mg{sup ++}-free solution or in the presence of 50 {mu}M bicuculline. Application of ethanol reversibly suppressed the duration of NMDA receptor-mediated synaptic response. These results suggest that intoxicating concentrations of ethanol possess anticonvulsant activity through blocking the NMDA receptor-mediated synaptic excitation.

  1. Receptor-mediated signalling in plants: molecular patterns and programmes

    PubMed Central

    Tör, Mahmut; Lotze, Michael T.; Holton, Nicholas

    2009-01-01

    A highly evolved surveillance system in plants is able to detect a broad range of signals originating from pathogens, damaged tissues, or altered developmental processes, initiating sophisticated molecular mechanisms that result in defence, wound healing, and development. Microbe-associated molecular pattern molecules (MAMPs), damage-associated molecular pattern molecules (DAMPs), virulence factors, secreted proteins, and processed peptides can be recognized directly or indirectly by this surveillance system. Nucleotide binding-leucine rich repeat proteins (NB-LRR) are intracellular receptors and have been targeted by breeders for decades to elicit resistance to crop pathogens in the field. Receptor-like kinases (RLKs) or receptor like proteins (RLPs) are membrane bound signalling molecules with an extracellular receptor domain. They provide an early warning system for the presence of potential pathogens and activate protective immune signalling in plants. In addition, they act as a signal amplifier in the case of tissue damage, establishing symbiotic relationships and effecting developmental processes. The identification of several important ligands for the RLK-type receptors provided an opportunity to understand how plants differentiate, how they distinguish beneficial and detrimental stimuli, and how they co-ordinate the role of various types of receptors under varying environmental conditions. The diverse roles of extra-and intracellular plant receptors are examined here and the recent findings on how they promote defence and development is reviewed. PMID:19628572

  2. Metabotropic glutamate receptor-mediated signaling in neuroglia

    PubMed Central

    Loane, David J.; Stoica, Bogdan A.; Faden, Alan I.

    2011-01-01

    Metabotropic glutamate (mGlu) receptors are G-protein-coupled receptors, which include eight subtypes that have been classified into three groups (I–III) based upon sequence homology, signal transduction mechanism and pharmacological profile. Although most studied with regard to neuronal function and modulation, mGlu receptors are also expressed by neuroglia-including astrocytes, microglia and oligodendrocytes. Activation of mGlu receptors on neuroglia under both physiologic and pathophysiologic conditions mediates numerous actions that are essential for intrinsic glial cell function, as well as for glial–neuronal interactions. Astrocyte mGlu receptors play important physiological roles in regulating neurotransmission and maintaining neuronal homeostasis. However, mGlu receptors on astrocytes and microglia also serve to modulate cell death and neurological function in a variety of pathophysiological conditions such as acute and chronic neurodegenerative disorders. The latter effects are complex and bi-directional, depending on which mGlu receptor sub-types are activated. PMID:22662309

  3. Menthol enhances phasic and tonic GABAA receptor-mediated currents in midbrain periaqueductal grey neurons

    PubMed Central

    Lau, Benjamin K; Karim, Shafinaz; Goodchild, Ann K; Vaughan, Christopher W; Drew, Geoffrey M

    2014-01-01

    Background and Purpose Menthol, a naturally occurring compound in the essential oil of mint leaves, is used for its medicinal, sensory and fragrant properties. Menthol acts via transient receptor potential (TRPM8 and TRPA1) channels and as a positive allosteric modulator of recombinant GABAA receptors. Here, we examined the actions of menthol on GABAA receptor-mediated currents in intact midbrain slices. Experimental Approach Whole-cell voltage-clamp recordings were made from periaqueductal grey (PAG) neurons in midbrain slices from rats to determine the effects of menthol on GABAA receptor-mediated phasic IPSCs and tonic currents. Key Results Menthol (150–750 μM) produced a concentration-dependent prolongation of spontaneous GABAA receptor-mediated IPSCs, but not non-NMDA receptor-mediated EPSCs throughout the PAG. Menthol actions were unaffected by TRPM8 and TRPA1 antagonists, tetrodotoxin and the benzodiazepine antagonist, flumazenil. Menthol also enhanced a tonic current, which was sensitive to the GABAA receptor antagonists, picrotoxin (100 μM), bicuculline (30 μM) and Zn2+ (100 μM), but unaffected by gabazine (10 μM) and a GABAC receptor antagonist, 1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid hydrate (TPMPA; 50 μM). In addition, menthol potentiated currents induced by the extrasynaptic GABAA receptor agonist THIP/gaboxadol (10 μM). Conclusions and Implications These results suggest that menthol positively modulates both synaptic and extrasynaptic populations of GABAA receptors in native PAG neurons. The development of agents that potentiate GABAA-mediated tonic currents and phasic IPSCs in a manner similar to menthol could provide a basis for novel GABAA-related pharmacotherapies. PMID:24460753

  4. Effects of the phytoestrogen, genistein, and protein tyrosine kinase inhibitor-dependent mechanisms on steroidogenesis and estrogen receptor expression in porcine granulosa cells of medium follicles.

    PubMed

    Nynca, A; Nynca, J; Wąsowska, B; Kolesarova, A; Kołomycka, A; Ciereszko, R E

    2013-01-01

    The use of soy-based products in pig diets had raised concerns regarding the reproductive toxicity of genistein, the predominant isoflavone in soybeans. Genistein was reported to exhibit weak estrogenic activity but its mechanism of action is not fully recognized. The aim of the study was to examine the in vitro effects of genistein on (1) progesterone (P(4)) and estradiol (E(2)) secretion by porcine granulosa cells harvested from medium follicles, (2) the viability of cultured granulosa cells, and (3) the mRNA and protein expression of estrogen receptors α and β (ERα and ERβ) in these cells. In addition, to verify the role of protein tyrosine kinase (PTK)-dependent mechanisms possibly involved in genistein biological action, we tested the effects of lavendustin C, the nonsteroidal PTK inhibitor, on granulosa cell steroidogenesis. We found that genistein inhibited (P < 0.05) basal P(4) secretion by granulosa cells harvested from medium follicles of pigs. In contrast, lavendustin C did not affect basal P(4) secretion by the cells. Moreover, genistein increased (P < 0.05) basal granulosal secretion of E(2). In contrast, lavendustin C did not alter basal E(2) secretion by porcine granulosa cells. In addition, we demonstrated that genistein increased mRNA and protein expression of ERβ (P < 0.05) in the examined cells. The expression of ERα mRNA was not affected by genistein and ERα protein was not detected in the cultured granulosa cells of pigs. In summary, the genistein action on follicular steroidogenesis in pigs involved changes in the granulosal expression of ERβ. However, the genistein action on P(4) and E(2) production by granulosa cells harvested from medium follicles did not seem to be associated with PTK. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Muscarinic receptor-mediated excitation of rat intracardiac ganglion neurons.

    PubMed

    Hirayama, Michiko; Ogata, Masanori; Kawamata, Tomoyuki; Ishibashi, Hitoshi

    2015-08-01

    Modulation of the membrane excitability of rat parasympathetic intracardiac ganglion neurons by muscarinic receptors was studied using an amphotericin B-perforated patch-clamp recording configuration. Activation of muscarinic receptors by oxotremorine-M (OxoM) depolarized the membrane, accompanied by repetitive action potentials. OxoM evoked inward currents under voltage-clamp conditions at a holding potential of -60 mV. Removal of extracellular Ca(2+) markedly increased the OxoM-induced current (IOxoM). The inward IOxoM in the absence of extracellular Ca(2+) was fully inhibited by removal of extracellular Na(+), indicating the involvement of non-selective cation channels. The IOxoM was inhibited by organic cation channel antagonists including SKF-96365 and ML-204. The IOxoM was antagonized by muscarinic receptor antagonists with the following potency: 4-DAMP > pirenzepine = darifenacin > methoctramine. Muscarinic toxin 7 (MT-7), a highly selective inhibitor for M1 receptor, produced partial inhibition of the IOxoM. In the presence of MT-7, concentration-inhibition curve of the M3-preferring antagonist darifenacin was shifted to the left. These results suggest the contribution of M1 and M3 receptors to the OxoM response. The IOxoM was inhibited by U-73122, a phospholipase C inhibitor. The membrane-permeable IP3 receptor blocker xestospongin C also inhibited the IOxoM. Furthermore, pretreatment with thapsigargin and BAPTA-AM inhibited the IOxoM, while KN-62, a blocker of Ca(2+)/calmodulin-dependent protein kinase II, had no effect. These results suggest that the activation mechanism involves a PLC pathway, release of Ca(2+) from intracellular Ca(2+) stores and calmodulin. The cation channels activated by muscarinic receptors may play an important role in neuronal membrane depolarization in rat intracardiac ganglion neurons.

  6. Cannabinoid CB1 receptors mediate the effects of dipyrone.

    PubMed

    Crunfli, Fernanda; Vilela, Fabiana C; Giusti-Paiva, Alexandre

    2015-03-01

    Dipyrone is a non-steroidal anti-inflammatory drug used primarily as an analgesic and antipyretic. Some hypothesize that dipyrone activity can modulate other pathways, including endocannabinoid signalling. Thus, the aim of the present study was to evaluate the possible role of endocannabinoids in mediating dipyrone activity. This study is based on the tetrad effects of cannabinoids, namely an antinociceptive and cataleptic state, hypolocomotion and hypothermia. Dipyrone (500 mg/kg, i.p.) treatment decreased locomotor activity, increased the latency to a thermal analgesic response and induced a cataleptic and hypothermic state. These reactions are similar to the tetrad effects caused by the cannabinoid agonist WIN 55,212-2 (3 mg/kg, i.p.). The cannabinoid CB1 receptor antagonist AM251 (10 mg/kg, i.p.) reversed the effects of dipyrone on locomotor activity, the cataleptic response and thermal analgesia. Both AM251 (10 mg/kg, i.p.) and the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine (10 mg/kg, i.p.) accentuated the reduction in body temperature caused by dipyrone. However, the CB2 receptor antagonist AM630 did not alter the hypothermic response to dipyrone. These results indicate involvement of the endocannabinoid system, especially CB1 receptors, in the analgesic and cataleptic effects of dipyrone, as well as hypolocomotion. However, cannabinoid receptors and TRPV1 were not involved in the hypothermic effects of dipyrone. We hypothesize that the mechanism of action of dipyrone may involve inhibition of cyclo-oxygenase and fatty acid amide hydrolase, which together provide additional arachidonic acid as substrate for endocannabinoid synthesis or other related molecules. This increase in endocannabinoid availability enhances CB1 receptor stimulation, contributing to the observed effects. © 2014 Wiley Publishing Asia Pty Ltd.

  7. In vitro estrogenicity of ambient particulate matter: contribution of hydroxylated polycyclic aromatic hydrocarbons.

    PubMed

    Wenger, Daniela; Gerecke, Andreas C; Heeb, Norbert V; Schmid, Peter; Hueglin, Christoph; Naegeli, Hanspeter; Zenobi, Renato

    2009-04-01

    Atmospheric particulate matter (PM1) was collected at an urban and a rural site in Switzerland during a hibernal high air pollution episode and was investigated for estrogenicity using an estrogen-sensitive reporter gene assay (ER-CALUX). All samples that were tested induced estrogen receptor-mediated gene expression in T47D human breast adenocarcinoma cells. Observed estrogenic activities corresponded to 17beta-estradiol (E2) CALUX equivalent concentrations ranging from 2 to 23 ng E2-CEQ per gram of PM1 (particulate matter of < or = 1 microm aerodynamic diameter) and from 0.07 to 1.25 pg E2-CEQ per m(3) of sampled air. There was a strong correlation between the PM1 estrogenicity of the urban and rural sites (r = 0.92). Five hydroxylated polycyclic aromatic hydrocarbons (hydroxy-PAHs), which show structural similarities to E2, were assessed for their estrogenic activity. The following order of estrogenic potency was found: 2-hydroxychrysene > 2-hydroxyphenanthrene > 1-hydroxypyrene > 2-hydroxynaphthalene > 1-hydroxynaphthalene. Three of these hydroxy-PAHs, namely 2-hydroxyphenanthrene, 2-hydroxynaphthalene and 1-hydroxynaphthalene, were detected in all PM1 extracts. However, they contributed only 0.01-0.2% to the overall estrogenic activity. Hence, mainly other estrogenic compounds not yet identified by chemical analysis must be responsible for the observed activity. The temporal trend of PM1 estrogenicity at the urban and rural site, respectively, was compared with the time course of several air pollutants (NO2, NO, SO2, O3, CO) and meteorological parameters (temperature, humidity, air pressure, solar irradiation, wind velocity). However, specific emission sources and formation processes of atmospheric xenoestrogens could not be elucidated. This study showed that ambient particulate matter contains compounds that are able to interact with estrogen receptors in vitro and potentially also interfere with estrogen-regulated pathways in vivo.

  8. Mannose receptor-mediated gene delivery into antigen presenting dendritic cells.

    PubMed

    Diebold, Sandra S; Plank, Christian; Cotten, Matt; Wagner, Ernst; Zenke, Martin

    2002-11-01

    Dendritic cells are professional antigen presenting cells and are unique in their ability to prime naïve T cells. Gene modification of dendritic cells is of particular interest for immunotherapy of diseases where the immune system has failed or is aberrantly regulated, such as in cancer or autoimmune disease, respectively. Dendritic cells abundantly express mannose receptor and mannose receptor-related receptors, and receptor-mediated gene transfer via mannose receptor offers a versatile tool for targeted gene delivery into these cells. Accordingly, mannose polyethylenimine DNA transfer complexes were generated and used for gene delivery into dendritic cells. Mannose receptor belongs to the group of scavenger receptors that allow dendritic cells to take up pathogenic material, which is directed for degradation and MHC class II presentation. Therefore, a limiting step of transgene expression by mannose receptor-mediated gene delivery is endosomal degradation of DNA. Several strategies have been explored to overcome this limitation including the addition of endosomolytic components to DNA transfer complexes like adenovirus particles and influenza peptides. Here, we review the current understanding of mannose receptor-mediated gene delivery into dendritic cells and discuss strategies to identify appropriate endosomolytic agents to improve DNA transfer efficacy.

  9. Size of single-wall carbon nanotube affects the folate receptor-mediated cancer cell targeting.

    PubMed

    Charbgoo, Fahimeh; Nikkhah, Maryam; Behmanesh, Mehrdad

    2017-08-30

    Advances in nanobiotechnology and targeting strategy could improve the delivery of therapeutic molecules into cancer cells, leading to improved treatment efficiency with minimal side effects on normal cells. To design an efficient nanocarrier, consideration of parameters that facilitate direct drug delivery into the target cells is important. We studied the effect of single-wall carbon nanotubes (SWNTs) size on their cell internalization level via the folate receptor-mediated pathway through folic acid targeting. Folate-SWNTs were covalently synthesized and characterized. Folate-SWNTs ≤ 450 nm had lower cell internalization level than folate-SWNTs >450 nm with a P value of ≤0.01. This indicated that using folate-SWNT with an average length of ≤450 nm was not suitable for receptor-mediated cancer cell targeting. Receptor-mediated uptake of folate-SWNTs is dependent on the nanoparticle length. However, sub-450 nm SWNTs could serve as a vehicle to transfer nucleic acids into the cells due to direct cell penetrance based on their needle-like structure. We find that SWNTs larger than 450 nm were suitable to target the cells through receptors. These results might provide a promising approach for designing more effective targeted delivery systems based on SWNTs. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  10. Nucleus accumbens μ-opioid receptors mediate social reward.

    PubMed

    Trezza, Viviana; Damsteegt, Ruth; Achterberg, E J Marijke; Vanderschuren, Louk J M J

    2011-04-27

    Positive social interactions are essential for emotional well-being and proper behavioral development of young individuals. Here, we studied the neural underpinnings of social reward by investigating the involvement of opioid neurotransmission in the nucleus accumbens (NAc) in social play behavior, a highly rewarding social interaction in adolescent rats. Intra-NAc infusion of morphine (0.05-0.1 μg) increased pinning and pouncing, characteristic elements of social play behavior in rats, and blockade of NAc opioid receptors with naloxone (0.5 μg) prevented the play-enhancing effects of systemic morphine (1 mg/kg, s.c.) administration. Thus, stimulation of opioid receptors in the NAc was necessary and sufficient for morphine to increase social play. Intra-NAc treatment with the selective μ-opioid receptor agonist [D-Ala(2),N-MePhe(4),Gly(5)-ol]enkephalin (DAMGO) (0.1-10 ng) and the μ-opioid receptor antagonist Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP) (0.3-3 μg) increased and decreased social play, respectively. The δ-opioid receptor agonist DPDPE ([D-Pen(2),D-Pen(5)]-enkephalin) (0.3-3 μg) had no effects, whereas the κ-opioid receptor agonist U69593 (N-methyl-2-phenyl-N-[(5R,7S,8S)-7-(pyrrolidin-1-yl)-1-oxaspiro[4.5]dec-8-yl]acetamide) (0.01-1 μg) decreased social play. Intra-NAc treatment with β-endorphin (0.01-1 μg) increased social play, but met-enkephalin (0.1-5 μg) and the enkephalinase inhibitor thiorphan (0.1-1 μg) were ineffective. DAMGO (0.1-10 ng) increased social play after infusion into both the shell and core subregions of the NAc. Last, intra-NAc infusion of CTAP (3 μg) prevented the development of social play-induced conditioned place preference. These findings identify NAc μ-opioid receptor stimulation as an important neural mechanism for the attribution of positive value to social interactions in adolescent rats. Altered NAc μ-opioid receptor function may underlie social impairments in psychiatric disorders such as autism

  11. Ligand Receptor-Mediated Regulation of Growth in Plants.

    PubMed

    Haruta, Miyoshi; Sussman, Michael R

    2017-01-01

    Growth and development of multicellular organisms are coordinately regulated by various signaling pathways involving the communication of inter- and intracellular components. To form the appropriate body patterns, cellular growth and development are modulated by either stimulating or inhibiting these pathways. Hormones and second messengers help to mediate the initiation and/or interaction of the various signaling pathways in all complex multicellular eukaryotes. In plants, hormones include small organic molecules, as well as larger peptides and small proteins, which, as in animals, act as ligands and interact with receptor proteins to trigger rapid biochemical changes and induce the intracellular transcriptional and long-term physiological responses. During the past two decades, the availability of genetic and genomic resources in the model plant species, Arabidopsis thaliana, has greatly helped in the discovery of plant hormone receptors and the components of signal transduction pathways and mechanisms used by these immobile but highly complex organisms. Recently, it has been shown that two of the most important plant hormones, auxin and abscisic acid (ABA), act through signaling pathways that have not yet been recognized in animals. For example, auxins stimulate cell elongation by bringing negatively acting transcriptional repressor proteins to the proteasome to be degraded, thus unleashing the gene expression program required for increasing cell size. The "dormancy" inducing hormone, ABA, binds to soluble receptor proteins and inhibits a specific class of protein phosphatases (PP2C), which activates phosphorylation signaling leading to transcriptional changes needed for the desiccation of the seeds prior to entering dormancy. While these two hormone receptors have no known animal counterparts, there are also many similarities between animal and plant signaling pathways. For example, in plants, the largest single gene family in the genome is the protein kinase

  12. Brain prostanoid TP receptor-mediated adrenal noradrenaline secretion and EP3 receptor-mediated sympathetic noradrenaline release in rats.

    PubMed

    Yokotani, Keiko; Okada, Shoshiro; Nakamura, Kumiko; Yamaguchi-Shima, Naoko; Shimizu, Takahiro; Arai, Junichi; Wakiguchi, Hiroshi; Yokotani, Kunihiko

    2005-04-04

    Sympathetic nerves release noradrenaline, whereas adrenal medullary chromaffin cells secrete noradrenaline and adrenaline. Therefore, plasma noradrenaline reflects the secretion from adrenal medulla in addition to the release from sympathetic nerves, however the exact mechanisms of adrenal noradrenaline secretion remain to be elucidated. The present study was designated to characterize the source of plasma noradrenaline induced by intracerebroventricularly (i.c.v.) administered bombesin and prostaglandin E2 in urethane-anesthetized rats. Bombesin (1.0 nmol/animal, i.c.v.) elevated plasma noradrenaline and adrenaline, while prostaglandin E2 (0.3 nmol/animal, i.c.v.) elevated only plasma noradrenaline. The bombesin-induced elevations of both catecholamines were attenuated by pretreatments with furegrelate (an inhibitor of thromboxane A2 synthase) [250 and 500 microg (0.9 and 1.8 micromol)/animal, i.c.v.)] and [(+)-S-145] [(+)-(1R,2R,3S,4S)-(5Z)-7-(3-[4-3H]-phenylsulphonyl-aminobicyclo[2.2.1]hept-2-yl)hept-5-enoic acid sodium salt] (an antagonist of prostanoid TP receptors) [100 and 250 microg (250 and 625 nmol)/animal)], and abolished by acute bilateral adrenalectomy. On the other hand, the prostaglandin E2-induced elevation of plasma noradrenaline was not influenced by acute bilateral adrenalectomy. These results suggest that adrenal noradrenaline secretion and sympathetic noradrenaline release are mediated by differential central mechanisms; brain prostanoid TP receptors activated by bombesin are involved in the adrenal noradrenaline secretion, while brain prostanoid EP (probably EP3) receptors activated by prostaglandin E2 are involved in the sympathetic noradrenaline release in rats. Brain prostanoid TP receptors activated by bombesin are also involved in the adrenal adrenaline secretion.

  13. Characterization of prejunctional 5-HT receptors mediating inhibition of sympathetic vasopressor responses in the pithed rat.

    PubMed Central

    Villalón, C. M.; Contreras, J.; Ramírez-San Juan, E.; Castillo, C.; Perusquía, M.; Terrón, J. A.

    1995-01-01

    1. It has recently been shown that continuous infusions of 5-hydroxytryptamine (5-HT) are able to inhibit, in a dose-dependent manner, the pressor responses induced by preganglionic (T7-T9) sympathetic stimulation in pithed rats pretreated with desipramine (50 micrograms kg-1, i.v.). This inhibitory effect, besides being significantly more pronounced at lower frequencies of stimulation (0.03-I Hz) and devoid of tachyphylaxis, is reversible after interrupting the infusions of 5-HT (up to 5.6 micrograms kg-1 min-1). In the present study we have characterized the pharmacological profile of the receptors mediating the above inhibitory effect of 5-HT. 2. The inhibition induced by 5.6 micrograms kg-1 min-1 of 5-HT on sympathetically-induced pressor responses was not blocked after i.v. treatment with physiological saline (1 ml kg-1), ritanserin (0.1 mg kg-1), MDL 72222 (0.15 mg kg-1) or tropisetron (3 mg kg-1), which did not modify the sympathetically-induced pressor responses per se, but was significantly antagonized by the 5-HT1-like and 5-HT2 receptor antagonist, methysergide (0.3 mg kg-1), which also produced a slight attenuation of the pressor responses to 0.03 and 0.1 Hz per se. 3. Unexpectedly and contrasting with methysergide, the 5-HT1-like and 5-HT2 receptor antagonists, methiothepin (0.01, 0.03 and 0.1 mg kg-1) and metergoline (1 and 3 mg kg-1), apparently failed to block the above 5-HT-induced inhibition. Nevertheless, it is noteworthy that these antagonists also blocked the electrically-induced pressor responses per se, presumably by blockade of vascular alpha 1-adrenoceptors and, indeed, this property might have masked their potential antagonism at the inhibitory 5-HT1-like receptors. 4. Consistent with the above findings, 5-carboxamidotryptamine (5-CT, a potent 5-HT1-like receptor agonist), metergoline and methysergide mimicked the inhibitory action of 5-HT with the following rank order of agonist potency: 5CT > > 5-HT > metergoline > or = methysergide. 5

  14. The Impact of Hyperthermia on Receptor-Mediated Interleukin-6 Regulation in Mouse Skeletal Muscle

    PubMed Central

    Welc, Steven S.; Morse, Deborah A.; Mattingly, Alex J.; Laitano, Orlando; King, Michelle A.; Clanton, Thomas L.

    2016-01-01

    In inflammatory cells, hyperthermia inhibits lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) gene expression and protein secretion. Since hyperthermia alone stimulates IL-6 in skeletal muscle, we hypothesized that it would amplify responses to other receptor-mediated stimuli. IL-6 regulation was tested in C2C12 myotubes and in soleus during treatment with epinephrine (EPI) or LPS. In EPI-treated myotubes (100 ng/ml), 1 h exposure at 40.5°C-42°C transiently increased IL-6 mRNA compared to EPI treatment alone at 37°C. In LPS-treated myotubes (1 μg/ml), exposure to 41°C-42°C also increased IL-6 mRNA. In isolated mouse soleus, similar amplifications of IL-6 gene expression were observed in 41°C, during both low (1 ng/ml) and high dose (100 ng/ml) EPI, but only in high dose LPS (1 μg/ml). In myotubes, heat increased IL-6 secretion during EPI exposure but had no effect or inhibited secretion with LPS. In soleus there were no effects of heat on IL-6 secretion during either EPI or LPS treatment. Mechanisms for the effects of heat on IL-6 mRNA were explored using a luciferase-reporter in C2C12 myotubes. Overexpression of heat shock factor-1 (HSF-1) had no impact on IL-6 promoter activity during EPI stimulation, but elevated IL-6 promoter activity during LPS stimulation. In contrast, when the activator protein-1 (AP-1) element was mutated, responses to both LPS and EPI were suppressed in heat. Using siRNA against activating transcription factor-3 (ATF-3), a heat-stress-induced inhibitor of IL-6, no ATF-3-dependent effects were observed. The results demonstrate that, unlike inflammatory cells, hyperthermia in muscle fibers amplifies IL-6 gene expression to EPI and LPS. The effect appears to reflect differential engagement of HSF-1 and AP-1 sensitive elements on the IL-6 gene, with no evidence for involvement of ATF-3. The functional significance of increased IL-6 mRNA expression during heat may serve to overcome the well-known suppression of protein synthetic

  15. Effect of taurine deficiency on adenosine receptor-mediated relaxation of the rat aorta.

    PubMed

    Abebe, Worku; Mozaffari, Mahmood S

    2003-11-01

    not altered by taurine deficiency. The results indicate that endogenous taurine deficiency causes differential inhibitory effects on adenosine receptor-mediated vasorelaxation, depending upon the agonists used. Given the recognized role of adenosine in the vasculature, these alterations suggest taurine-mediated modulation of blood flow regulation.

  16. Estrogen increases Nrf2 activity through activation of the PI3K pathway in MCF-7 breast cancer cells

    SciTech Connect

    Wu, Juanjuan; Williams, Devin; Walter, Grant A.; Thompson, Winston E.; Sidell, Neil

    2014-11-01

    The actions of the transcription factor Nuclear factor erythroid 2-related factor (Nrf2) in breast cancer have been shown to include both pro-oncogenic and anti-oncogenic activities which is influenced, at least in part, by the hormonal environment. However, direct regulation of Nrf2 by steroid hormones (estrogen and progesterone) has received only scant attention. Nrf2 is known to be regulated by its cytosolic binding protein, Kelch-like ECH-associated protein 1 (Keap1), and by a Keap1-independent mechanism involving a series of phosphorylation steps mediated by phosphatidylinositol 3-kinase (PI3K) and glycogen synthase kinase 3 beta (GSK3β). Here, we report that estrogen (E2) increases Nrf2 activity in MCF7 breast cancer cells through activation of the PI3K/GSK3β pathway. Utilizing antioxidant response element (ARE)-containing luciferase reporter constructs as read-outs for Nrf2 activity, our data indicated that E2 increased ARE activity >14-fold and enhanced the action of the Nrf2 activators, tertiary butylhydroquinone (tBHQ) and sulforaphane (Sul) 4 to 9 fold compared with cells treated with tBHQ or Sul as single agents. This activity was shown to be an estrogen receptor-mediated phenomenon and was antagonized by progesterone. In addition to its action on the reporter constructs, mRNA and protein levels of heme oxygenase 1, an endogenous target gene of Nrf2, was markedly upregulated by E2 both alone and in combination with tBHQ. Importantly, E2-induced Nrf2 activation was completely suppressed by the PI3K inhibitors LY294002 and Wortmannin while the GSK3β inhibitor CT99021 upregulated Nrf2 activity. Confirmation that E2 was, at least partly, acting through the PI3K/GSK3β pathway was indicated by our finding that E2 increased the phosphorylation status of both GSK3β and Akt, a well-characterized downstream target of PI3K. Together, these results demonstrate a novel mechanism by which E2 can regulate Nrf2 activity in estrogen receptor-positive breast cancer

  17. Slowly developing depression of N-methyl-D-aspartate receptor mediated responses in young rat hippocampi

    PubMed Central

    Dozmorov, Mikhail; Li, Rui; Xu, Hui-Ping; Jilderos, Barbro; Wigström, Holger

    2004-01-01

    Background Activation of N-methyl-D-aspartate (NMDA) type glutamate receptors is essential in triggering various forms of synaptic plasticity. A critical issue is to what extent such plasticity involves persistent changes of glutamate receptor subtypes and many prior studies have suggested a main role for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors in mediating the effect. Our previous work in hippocampal slices revealed that, under pharmacological unblocking of NMDA receptors, both AMPA and NMDA receptor mediated responses undergo a slowly developing depression. In the present study we have further adressed this phenomenon, focusing on the contribution via NMDA receptors. Pharmacologically isolated NMDA receptor mediated excitatory postsynaptic potentials (EPSPs) were recorded for two independent synaptic pathways in CA1 area using perfusion with low Mg2+ (0.1 mM) to unblock NMDA receptors. Results Following unblocking of NMDA receptors, there was a gradual decline of NMDA receptor mediated EPSPs for 2–3 hours towards a stable level of ca. 60–70 % of the maximal size. If such an experimental session was repeated twice in the same pathway with a period of NMDA receptor blockade in between, the depression attained in the first session was still evident in the second one and no further decay occurred. The persistency of the depression was also validated by comparison between pathways. It was found that the responses of a control pathway, unstimulated in the first session of receptor unblocking, behaved as novel responses when tested in association with the depressed pathway under the second session. In similar experiments, but with AP5 present during the first session, there was no subsequent difference between NMDA EPSPs. Conclusions Our findings show that merely evoking NMDA receptor mediated responses results in a depression which is input specific, induced via NMDA receptor activation, and is maintained for several hours through

  18. Site of action of the general anesthetic propofol in muscarinic M1 receptor-mediated signal transduction.

    PubMed

    Murasaki, Osamu; Kaibara, Muneshige; Nagase, Yoshihisa; Mitarai, Sayaka; Doi, Yoshiyuki; Sumikawa, Koji; Taniyama, Kohtaro

    2003-12-01

    Although a potential target site of general anesthetics is primarily the GABA A receptor, a chloride ion channel, a previous study suggested that the intravenous general anesthetic propofol attenuates the M1 muscarinic acetylcholine receptor (M1 receptor)-mediated signal transduction. In the present study, we examined the target site of propofol in M1 receptor-mediated signal transduction. Two-electrode voltage-clamp method was used in Xenopus oocytes expressing both M1 receptors and associated G protein alpha subunits (Gqalpha). Propofol inhibited M1 receptor-mediated signal transduction in a dose-dependent manner (IC50 = 50 nM). Injection of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) into oocytes overexpressing Gqalpha was used to investigate direct effects of propofol on G protein coupled with the M1 receptor. Propofol did not affect activation of Gqalpha-mediated signal transduction with the intracellular injection of GTPgammaS. We also studied effects of propofol on l-[N-methyl-3H]scopolamine methyl chloride ([3H]NMS) binding and M1 receptor-mediated signal transduction in mammalian cells expressing M1 receptor. Propofol inhibited the M1 receptor-mediated signal transduction but did not inhibit binding of [3H]NMS. Effects of propofol on Gs- and Gi/o-coupled signal transduction were investigated, using oocytes expressing the beta2 adrenoceptor (beta2 receptor)/cystic fibrosis transmembrane conductance regulator or oocytes expressing the M2 muscarinic acetylcholine receptor (M2 receptor)/Kir3.1 (a member of G protein-gated inwardly rectifying K(+) channels). Neither beta2 receptor-mediated nor M2 receptor-mediated signal transduction was inhibited by a relatively high concentration of propofol (50 microM). These results indicate that propofol inhibits M1 receptor-mediated signal transduction by selectively disrupting interaction between the receptor and associated G protein.

  19. Cryptococcus neoformans Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

    PubMed Central

    Guerra, Caroline Rezende; Seabra, Sergio Henrique; de Souza, Wanderley; Rozental, Sonia

    2014-01-01

    Cryptococcosis by the encapsulated yeast Cryptococcus neoformans affects mostly immunocompromised individuals and is a frequent neurological complication in AIDS patients. Recent studies support the idea that intracellular survival of Cryptococcus yeast cells is important for the pathogenesis of cryptococcosis. However, the initial steps of Cryptococcus internalization by host cells remain poorly understood. Here, we investigate the mechanism of Cryptococcus neoformans phagocytosis by peritoneal macrophages using confocal and electron microscopy techniques, as well as flow cytometry quantification, evaluating the importance of fungal capsule production and of host cell cytoskeletal elements for fungal phagocytosis. Electron microscopy analyses revealed that capsular and acapsular strains of C. neoformans are internalized by macrophages via both ‘zipper’ (receptor-mediated) and ‘trigger’ (membrane ruffle-dependent) phagocytosis mechanisms. Actin filaments surrounded phagosomes of capsular and acapsular yeasts, and the actin depolymerizing drugs cytochalasin D and latrunculin B inhibited yeast internalization and actin recruitment to the phagosome area. In contrast, nocodazole and paclitaxel, inhibitors of microtubule dynamics decreased internalization but did not prevent actin recruitment to the site of phagocytosis. Our results show that different uptake mechanisms, dependent on both actin and tubulin dynamics occur during yeast internalization by macrophages, and that capsule production does not affect the mode of Cryptococcus uptake by host cells. PMID:24586631

  20. Mechanisms of estradiol-induced insulin secretion by the G protein-coupled estrogen receptor GPR30/GPER in pancreatic beta-cells.

    PubMed

    Sharma, Geetanjali; Prossnitz, Eric R

    2011-08-01

    Sexual dimorphism and supplementation studies suggest an important role for estrogens in the amelioration of glucose intolerance and diabetes. Because little is known regarding the signaling mechanisms involved in estradiol-mediated insulin secretion, we investigated the role of the G protein-coupled receptor 30, now designated G protein-coupled estrogen receptor (GPER), in activating signal transduction cascades in β-cells, leading to secretion of insulin. GPER function in estradiol-induced signaling in the pancreatic β-cell line MIN6 was assessed using small interfering RNA and GPER-selective ligands (G-1 and G15) and in islets isolated from wild-type and GPER knockout mice. GPER is expressed in MIN6 cells, where estradiol and the GPER-selective agonist G-1 mediate calcium mobilization and activation of ERK and phosphatidylinositol 3-kinase. Both estradiol and G-1 induced insulin secretion under low- and high-glucose conditions, which was inhibited by pretreatment with GPER antagonist G15 as well as depletion of GPER by small interfering RNA. Insulin secretion in response to estradiol and G-1 was dependent on epidermal growth factor receptor and ERK activation and further modulated by phosphatidylinositol 3-kinase activity. In islets isolated from wild-type mice, the GPER antagonist G15 inhibited insulin secretion induced by estradiol and G-1, both of which failed to induce insulin secretion in islets obtained from GPER knockout mice. Our results indicate that GPER activation of the epidermal growth factor receptor and ERK in response to estradiol treatment plays a critical role in the secretion of insulin from β-cells. The results of this study suggest that the activation of downstream signaling pathways by the GPER-selective ligand G-1 could represent a novel therapeutic strategy in the treatment of diabetes.

  1. Presynaptic BK type Ca(2+)-activated K(+) channels are involved in prostanoid TP receptor-mediated inhibition of noradrenaline release from the rat gastric sympathetic nerves.

    PubMed

    Nakamura, Kumiko; Yokotani, Kunihiko

    2010-03-10

    Previously, we reported that prostanoid TP receptor mediates the inhibition of electrically evoked noradrenaline release from gastric sympathetic nerves in rats. Prostanoid TP receptor has been shown to activate phospholipase C (PLC), which catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-triphosphate (IP(3)) and diacylglycerol; IP(3) triggers the release of Ca(2+) from intracellular stores and diacylglycerol activates protein kinase C. In the present study, therefore, we examined whether these PLC-mediated mechanisms are involved in the TP receptor-mediated inhibition of gastric noradrenaline release using an isolated, vascularly perfused rat stomach. U-46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy PGF(2alpha)) (a prostanoid TP receptor agonist)-induced inhibition of noradrenaline release from the stomach was reduced by U-73122 [1-[6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]-amino]hexyl]-1H-pyrrole-2,5-dine] (a PLC inhibitor) and ET-18-OCH(3) (1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine) (a phosphatidylinositol-specific PLC inhibitor), respectively. 2-APB (2-aminoethyldiphenyl borate) (a putative IP(3) receptor antagonist) also abolished the U-46619-induced inhibition of noradrenaline release, but Ro 31-8220 [2-{1-[3-(amidinothio)propyl]-1H-indol-3-yl}-3-(1-methylindol-3-yl)-maleimide] (a protein kinase C inhibitor) had no effect. Furthermore, a small dose of tetraethylammonium and charybdotoxin [blockers of BK type Ca(2+)-activated K(+) channel] abolished the U-46619-induced inhibition, but apamin (a blocker of SK-type Ca(2+)-activated K(+) channel) had no effect. These results suggest that BK type Ca(2+)-activated K(+) channels are involved in prostanoid TP receptor-mediated inhibition of electrically evoked noradrenaline release from the gastric sympathetic nerve terminals in rats.

  2. Mechanisms for the activity of heterocyclic cyclohexanone curcumin derivatives in estrogen receptor negative human breast cancer cell lines.

    PubMed

    Somers-Edgar, Tiffany J; Taurin, Sebastien; Larsen, Lesley; Chandramouli, Anupama; Nelson, Mark A; Rosengren, Rhonda J

    2011-02-01

    Estrogen receptor (ER)-negative breast cancer is an aggressive form that currently requires more drug treatment options. Thus, we have further modified cyclohexanone derivatives of curcumin and examined them for cytotoxicity towards ER-negative human breast cancer cells. Two of the analogs screened elicited increased cytotoxic potency compared to curcumin and other previously studied derivatives. Specifically, 2,6-bis(pyridin-3-ylmethylene)-cyclohexanone (RL90) and 2,6-bis(pyridin-4-ylmethylene)-cyclohexanone (RL91) elicited EC(50) values of 1.54 and 1.10 µM, respectively, in MDA-MB-231 cells and EC(50) values of 0.51 and 0.23 in SKBr3 cells. All other new compounds examined were less potent than curcumin, which elicited EC(50) values of 7.6 and 2.4 µM in MDA-MB-231 and SKBr3 cells, respectively. Mechanistic analyses demonstrated that RL90 and RL91 significantly induced G(2)/M-phase cell cycle arrest and apoptosis. RL90 and RL91 also modulated the expression of key cell signaling proteins, specifically, in SKBr3 cells, protein levels of Her-2, Akt, and NFκB were decreased in a time-dependent manner, while activity of stress kinases JNK1/2 and P38 MAPK were increased. Signaling events in MDA-MB-231 cells were differently implicated, as EGFR protein levels were decreased and activity of GSK-3β transiently decreased, while β-catenin protein level and activity of P38 MAPK, Akt, and JNK1/2 were transiently increased. In conclusion replacement of the phenyl group of cyclohexanone derived curcumin derivatives with heterocyclic rings forms a class of second-generation analogs that are more potent than both curcumin and other derivatives. These new derivatives provide a platform for the further development of drugs for the treatment of ER-negative breast cancer.

  3. [Exon 5 alternative splicing of the cytochrome P450 aromatase could be a regulatory mechanism for estrogen production in humans].

    PubMed

    Pepe, Carolina M; Saraco, Nora I; Baquedano, María Sonia; Guercio, Gabriela; Vaiani, Elisa; Berensztein, Esperanza; Rivarola, Marco A; Belgorosky, Alicia

    2007-01-01

    P450 aromatase (P450Aro), involved in androgen to estrogen conversion, is encoded by the CYP19 gene. P450Aro c655G>A mutation described in heterozygous form in a girl and in homozygous form in an adult male with P450Aro deficiency results in an aberrant splicing due to disruption of a donor splice site. A truncated inactive protein would be expected if intron5 is retained. Surprisingly, the girl described with this mutation showed spontaneous breast development and pubertal estradiol (E2) levels suggesting residual P450Aro activity (AA). Formerly, we postulate the in frame E5 skipping as a consequence of this mutation generating a protein with some degree of activity. When P450Aro mRNA expression was analysed from patient's lymphocytes, an aberrant spliced mRNA lacking E5 (-E5mRNA) was detected, suggesting an association between E5 skipping and the presence of the mutation. Splicing assays in Y1 cells confirmed this association. -Ex5 cDNA expression in Y1 cells resulted in an inactive protein that could not explain patient's phenotype. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to splicing mutations and physiological alternative splicing (AS) events. Therefore, -Ex5mRNA was assessed as a natural occurring alternative transcript in normal human steroidogenic tissues. As P450Aro -E5mRNA expression was detected in human term placenta, prepubertal testis and prepubertal adrenal, we might speculate that AS of P450Aro coding region would occur in humans and would be involved in the complex AA regulation. Furthermore, tissue specific regulation of AS might suggest low expression of +E5mRNA from the c655G>A allele explaining residual AA evidenced in the affected girl.

  4. Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays.

    PubMed

    Legler, Juliette; Dennekamp, Martine; Vethaak, A Dick; Brouwer, Abraham; Koeman, Jan H; van der Burg, Bart; Murk, Albertinka J

    2002-07-03

    Sediments may be the ultimate sink for persistent (xeno-)estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-CALUX assay was more sensitive to 17beta-estradiol (E2) than the recombinant yeast screen, with an EC50 of 6 pM E2 compared to 100 pM in the yeast screen. Yeast cells were unable to distinguish the anti-estrogens ICI 182,780 and (4-hydroxy)tamoxifen, which were agonistic in the yeast. Acetone-soluble fractions of hexane/acetone extracts of sediments showed higher estrogenic potency than hexane-soluble extracts in the ER-CALUX assay. Sediments obtained from industrialized areas such as the Port of Rotterdam showed the highest estrogenic potency of the 12 marine sediments tested (up to 40 pmol estradiol equivalents per gram sediment). The estrogenic activity of individual chemicals that can be found in sediments including: alkylphenol ethoxylates and carboxylates; phthalates; and pesticides, was tested. Increasing sidechain length of various nonylphenol ethoxylates resulted in decreased estrogenic activity. Of the phthalates tested, butylbenzylphthalate was the most estrogenic, though with a potency approximately 100,000 times less than E2. The organochlorine herbicides atrazine and simazine failed to induce reporter gene activity. As metabolic activation may be required to induce estrogenic activity, a metabolic transformation step was added to the ER-CALUX assay using incubation of compounds with liver microsomes obtained from PCB-treated rats. Results indicate that metabolites of E2, NP and bisphenol A were less active than the parent compounds, while metabolites of methoxychlor were more estrogenic following microsomal incubations.

  5. Acamprosate enhances N-methyl-D-apartate receptor-mediated neurotransmission but inhibits presynaptic GABA(B) receptors in nucleus accumbens neurons.

    PubMed

    Berton, F; Francesconi, W G; Madamba, S G; Zieglgänsberger, W; Siggins, G R

    1998-02-01

    Acamprosate (calcium acetylhomotaurine) is used therapeutically in Europe to reduce relapse in weaned alcoholics. However, the mechanisms of acamprosate action in the central nervous system are still obscure, although early studies suggested an action on GABA receptors. The nucleus accumbens (NAcc) is a brain region thought to underlie ethanol reinforcement. Recent studies from our laboratory have demonstrated that ethanol inhibits both N-methyl-D-aspartate (NMDA) and non-NMDA types of glutamatergic synaptic transmission in the NAcc. In the present study, we used voltage- and current-clamp intracellular recording of NAcc core neurons in a slice preparation to examine acamprosate actions on resting membrane properties and pharmacologically isolated synaptic responses. We isolated NMDA and non-NMDA receptor-mediated excitatory postsynaptic potentials or currents (EPSP/Cs) with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and DL-2-amino-5-phosphonovalerate (d-APV), respectively. Bicuculline was also included to block GABA(A) receptors. Superfusion of acamprosate (5, 50, and 300 microM) did not alter the resting membrane properties of NAcc neurons. However, 300 microM acamprosate significantly increased the NMDA receptor-mediated components of EPSP/Cs (NMDA-EPSP/Cs) with recovery on washout. In contrast, 300 microM acamprosate had no significant effect on the non-NMDA receptor component of the EPSP/Cs (non-NMDA-EPSP/Cs). To test acamprosate actions on the GABA system, we superfused 60 microM d-APV and 20 microM CNQX to block glutamatergic transmission and evoked monosynaptic GABA(A) receptor-mediated synaptic responses within the NAcc. Acamprosate (300 microM) did not change these monosynaptic GABA(A)-IPSCs. We also used a paired-pulse paradigm to test whether acamprosate could act on presynaptic GABA(B) autoreceptors, in the presence of d-APV and CNQX to block glutamatergic transmission. Like 0.5 microM CGP 34358 (a GABA[B] receptor blocker), acamprosate significantly

  6. Functional interaction of heat shock protein 90 and Beclin 1 modulates Toll-like receptor-mediated autophagy

    PubMed Central

    Xu, Congfeng; Liu, Jin; Hsu, Li-Chung; Luo, Yunping; Xiang, Rong; Chuang, Tsung-Hsien

    2011-01-01

    Autophagy is one of the downstream effector mechanisms for elimination of intracellular microbes following activation of the Toll-like receptors (TLRs). Although the detailed molecular mechanism for this cellular process is still unclear, Beclin 1, a key molecule for autophagy, has been suggested to play a role. Heat shock protein 90 (Hsp90) is a molecular chaperone that regulates the stability of signaling proteins. Herein, we show that Hsp90 forms a complex with Beclin 1 through an evolutionarily conserved domain to maintain the stability of Beclin 1. In monocytic cells, geldanamycin (GA), an Hsp90 inhibitor, effectively promoted proteasomal degradation of Beclin 1 in a concentration-dependent (EC50 100 nM) and time-dependent (t50 2 h) manner. In contrast, KNK437/Hsp inhibitor I had no effect. Hsp90 specifically interacted with Beclin 1 but not with other adapter proteins in the TLR signalsome. Treatment of cells with GA inhibited TLR3- and TLR4-mediated autophagy. In addition, S. typhimurium infection-induced autophagy was blocked by GA treatment. This further suggested a role of the Hsp90/Beclin 1 in controlling autophagy in response to microbial infections. Taken together, our data revealed that by maintaining the homeostasis of Beclin 1, Hsp90 plays a novel role in TLR-mediated autophagy.—Xu, C., Liu, J., Hsu, L. -C., Luo, Y., Xiang, R., Chuang, T. -H. Functional interaction of Hsp90 and Beclin 1 modulates Toll-like receptor-mediated autophagy. PMID:21543763

  7. Estrogens and Stem Cells in Thyroid Cancer

    PubMed Central

    Zane, Mariangela; Catalano, Veronica; Scavo, Emanuela; Bonanno, Marco; Pelizzo, Maria Rosa; Todaro, Matilde; Stassi, Giorgio

    2014-01-01

    Recent discoveries highlight the emerging role of estrogens in the initiation and progression of different malignancies through their interaction with stem cell (SC) compartment. Estrogens play a relevant role especially for those tumors bearing a gender disparity in incidence and aggressiveness, as occurs for most thyroid diseases. Although several experimental lines suggest that estrogens promote thyroid cell proliferation and invasion, their precise contribution in SC compartment still remains unclear. This review underlines the interplay between hormones and thyroid function, which could help to complete the puzzle of gender discrepancy in thyroid malignancies. Defining the association between estrogen receptors’ status and signaling pathways by which estrogens exert their effects on thyroid cells is a potential tool that provides important insights in pathogenetic mechanisms of thyroid tumors. PMID:25120531

  8. [Estrogen receptors and the mammary gland].

    PubMed

    Barrón, A; Bermejo, L; Castro, I

    1997-01-01

    For several decades it has been known that steroid hormones, estrogen and progesterone, regulate some genes involved in the growth, proliferation and differentiation of the mammary-gland in animals and humans. In the last years, the presence or absence of the nuclear estrogen receptor has been used by clinicians as a marker for tumor malignancy, as a prognostic index or as an important parameter for hormonal therapy with anti-estrogenic compounds of some hormone-dependent breast cancers. This review shows some advances in the knowledge of the structure, function, molecular mechanisms of estrogenic activity, and interaction with proteins like protooncogenes and growth factors. Also, we refer to the role of the estrogen receptor in the physiophatology of breast cancer.

  9. Fenretinide prevents obesity in aged female mice in association with increased retinoid and estrogen signaling.

    PubMed

    Shearer, Kirsty D; Morrice, Nicola; Henderson, Claire; Reekie, Jenny; Mcilroy, George D; McCaffery, Peter J; Delibegovic, Mirela; Mody, Nimesh

    2015-08-01

    The synthetic retinoid fenretinide (FEN) inhibits adiposity in male mice fed a high-fat diet (HFD) in association with alterations in retinoic acid (RA) signaling. Young female mice are protected from obesity via estrogen signaling. We, therefore, investigated whether FEN also influences adiposity in aged female mice differing in parity and whether such effects are mediated by retinoid and estrogen signaling. Aged nulliparous and parous female mice were maintained on HFD ± FEN, and adiposity was assessed. Quantitative polymerase chain reaction was performed on white adipose tissue (WAT), liver, and 3T3-L1 adipocytes treated with RA or FEN ± estrogen. Parous females were more obese than nulliparous mice independent of age. FEN-HFD prevented the HFD-induced increase in adiposity and leptin levels independently of parity. FEN-HFD induced retinoid-responsive genes in WAT and liver. Parous females had reduced expression of hepatic estrogen-responsive genes, but FEN-HFD up-regulated WAT Cyp19a1 and Esr2 in parous mice. Estrogen and RA acted synergistically to increase RA receptor-mediated gene expression in 3T3-L1 adipocytes. FEN increased Cyp19a1 and Esr2, similar to our findings in vivo. The prevention of adiposity by FEN in response to HFD in female mice seems to involve increased retinoid signaling in association with induction of local estrogen production and estrogen signaling in WAT. © 2015 The Obesity Society.

  10. Estrogens and postcoital contraception.

    PubMed

    Notelovitz, M

    1981-07-01

    The contraceptive effect of large doses of estrogens administered postcoitally is not fully understood, although numerous reports have described the use of a 4 to 6 day course of high dose oral diethylstilbestrol (DES), ethinyl estradiol, conjugated estrogens, and combinations of estrogen and progestogen. Because estrogens are effective postovulatory rather than postcoital contraceptives, it is necessary to know the exact time of unprotected intercouse in relation to a woman's menstrual cycle. Depending on the frequency and timing of intercouse, a 5-day course of postcoital estrogen, introduced within 72 hours, yields a pregancy rate of .03-.3%. Failures are usually due to inadequate doses of estrogen, errors in timing, or multiple exposures. A lowering of basal body temperature after postovulatory administration of high doses of estrogen indicates successful intervention. Existence of various conditions such as hypertension and migraine contraindicate the use of postcoital estrogens. DES and possibly other estrogens are associated with teratogenic and potentially carcinogenic effects. 70 to 80% of women taking postcoital estrogens report side effects such as nausea, weight gain and headache. No randomized studies have compared the efficacy, side effects, or safety of the available estrogens. The use of informed coinsent procedures is advised because of the potency of high dose estrogens.

  11. Chloride transporters and receptor-mediated endocytosis in the renal proximal tubule

    PubMed Central

    Devuyst, Olivier; Luciani, Alessandro

    2015-01-01

    Abstract The epithelial cells lining the proximal tubules of the kidney reabsorb a large amount of filtered ions and solutes owing to receptor-mediated endocytosis and polarized transport systems that reflect final cell differentiation. Dedifferentiation of proximal tubule cells and dysfunction of receptor-mediated endocytosis characterize Dent’s disease, a rare disorder caused by inactivating mutations in the CLCN5 gene that encodes the endosomal chloride–proton exchanger, ClC-5. The disease is characterized by a massive urinary loss of solutes (renal Fanconi syndrome), with severe metabolic complications and progressive renal failure. Investigations of mutations affecting the gating of ClC-5 revealed that the proximal tubule dysfunction may occur despite normal endosomal acidification. In addition to defective endocytosis, proximal tubule cells lacking ClC-5 show a trafficking defect in apical receptors and transporters, as well as lysosomal dysfunction and typical features of dedifferentiation, proliferation and oxidative stress. A similar but milder defect is observed in mouse models with defective CFTR, a chloride channel that is also expressed in the endosomes of proximal tubule cells. These data suggest a major role for endosomal chloride transport in the maintenance of epithelial differentiation and reabsorption capacity of the renal proximal tubule. Key points The reabsorptive activity of renal proximal tubule cells is mediated by receptor-mediated endocytosis and polarized transport systems that reflect final cell differentiation. Loss-of-function mutations of the endosomal chloride–proton exchanger ClC-5 (Dent’s disease) cause a major trafficking defect in proximal tubule cells, associated with lysosomal dysfunction, oxidative stress and dedifferentiation/proliferation. A similar but milder defect is associated with mutations in CFTR (cystic fibrosis transmembrane conductance regulator). Vesicular chloride transport appears to be important for

  12. Combined estrogenic and anti-estrogenic properties of estetrol on breast cancer may provide a safe therapeutic window for the treatment of menopausal symptoms

    PubMed Central

    Gérard, Céline; Mestdagt, Mélanie; Tskitishvili, Ekaterine; Communal, Laudine; Gompel, Anne; Silva, Elisabete; Arnal, Jean-François; Lenfant, Françoise; Noel, Agnès; Foidart, Jean-Michel; Péqueux, Christel

    2015-01-01

    Increased risk of breast cancer is a critical side effect associated with the use of a menopausal hormone therapy (MHT). Estetrol (E4) is a natural estrogen produced by the human fetal liver and is a promising compound for clinical use in MHT. However, its impact on breast cancer is controversial and poorly defined. In this preclinical study, we show that E4 acts as a weak estrogen by stimulating the growth of hormone-dependent breast cancer only at concentrations exceeding menopausal therapeutic needs. E4 presents also an antitumor activity by decreasing the strong proliferative effect of estradiol (E2). While estrogen receptor alpha (ERα) is the predominant receptor mediating its effects, the dual weak-estrogenic/anti-estrogenic feature of E4 results from differential signaling pathways activation. Both nuclear and rapid extra-nuclear signaling pathway are necessary for a complete estrogenic effect of E4. However, the antitumor action of E4 is not due to a capacity to antagonize E2-induced nuclear activity. Altogether, our results highlight that E4 has a limited impact on breast cancer and may offer a safe therapeutic window for the treatment of menopausal symptoms. PMID:26056044

  13. Combined estrogenic and anti-estrogenic properties of estetrol on breast cancer may provide a safe therapeutic window for the treatment of menopausal symptoms.

    PubMed

    Gérard, Céline; Mestdagt, Mélanie; Tskitishvili, Ekaterine; Communal, Laudine; Gompel, Anne; Silva, Elisabete; Arnal, Jean-François; Lenfant, Françoise; Noel, Agnès; Foidart, Jean-Michel; Péqueux, Christel

    2015-07-10

    Increased risk of breast cancer is a critical side effect associated with the use of a menopausal hormone therapy (MHT). Estetrol (E4) is a natural estrogen produced by the human fetal liver and is a promising compound for clinical use in MHT. However, its impact on breast cancer is controversial and poorly defined. In this preclinical study, we show that E4 acts as a weak estrogen by stimulating the growth of hormone-dependent breast cancer only at concentrations exceeding menopausal therapeutic needs. E4 presents also an antitumor activity by decreasing the strong proliferative effect of estradiol (E2). While estrogen receptor alpha (ERα) is the predominant receptor mediating its effects, the dual weak-estrogenic/anti-estrogenic feature of E4 results from differential signaling pathways activation. Both nuclear and rapid extra-nuclear signaling pathway are necessary for a complete estrogenic effect of E4. However, the antitumor action of E4 is not due to a capacity to antagonize E2-induced nuclear activity. Altogether, our results highlight that E4 has a limited impact on breast cancer and may offer a safe therapeutic window for the treatment of menopausal symptoms.

  14. Receptor-Mediated Drug Delivery to Macrophages in Chemotherapy of Leishmaniasis

    NASA Astrophysics Data System (ADS)

    Mukhopadhyay, Amitabha; Chaudhuri, Gautam; Arora, Sunil K.; Sehgal, Shobha; Basu, Sandip K.

    1989-05-01

    Methotrexate coupled to maleylated bovine serum albumin was taken up efficiently through the ``scavenger'' receptors present on macrophages and led to selective killing of intracellular Leishmania mexicana amazonensis amastigotes in cultured hamster peritoneal macrophages. The drug conjugate was nearly 100 times as effective as free methotrexate in eliminating the intracellular parasites. Furthermore, in a model of experimental cutaneous leishmaniasis in hamsters, the drug conjugate brought about more than 90% reduction in the size of footpad lesions within 11 days. In contrast, the free drug at a similar concentration did not significantly affect lesion size. These studies demonstrate the potential of receptor-mediated drug delivery in the therapy of macrophage-associated diseases.

  15. Investigations of receptor-mediated phagocytosis by hormone-induced (imprinted) Tetrahymena pyriformis.

    PubMed

    Kovács, P; Sundermann, C A; Csaba, G

    1996-08-15

    Receptor-mediated endocytosis by Tetrahvmena pyriformis was studied using tetramethylrhodamine isothiocyanate-labeled concanavalin A (TRITC-Con A) with fluorescence and confocal microscopy. In the presence of insulin, or 24 h after insulin pretreatment (hormonal imprinting), the binding and uptake of TRITC-Con A increased when compared to controls, owing to the binding of TRITC-Con A to sugar oligomers of insulin receptors. Mannose inhibited the binding of Con A, thus demonstrating the specificity of binding. Histamine, a phagocytosis-promoting factor in mammals and Tetrahymena, and galactose, did not influence the uptake of TRITC-Con A.

  16. Paroxysmal beta-adrenergic receptor-mediated alterations in ventricular repolarization at rapid heart rates during inhibition of delayed rectifier currents.

    PubMed

    Overholser, Brian R; Zheng, Xiaomei; Tisdale, James E

    2009-09-01

    The contribution of the slow component of the delayed rectifier current (IKs) to ventricular repolarization is increased during rapid heart rates and prolonged repolarization. The objective was to characterize physiologically relevant paroxysmal beta-adrenergic receptor-mediated alterations on ventricular repolarization under these conditions. Paced guinea pig hearts were perfused with (1) control, (2) sparfloxacin (IKr inhibitor), or (3) sparfloxacin and HMR 1556 (IKs inhibitor). The mean +/- standard error of the mean epicardial action potential duration at 90% repolarization (APD90) increased from baseline with IKr inhibition (12.9% +/- 4.7%) and dual IKr/IKs inhibition (25.1% +/- 5.3). Paroxysmal isoproterenol (0.01 and 1.0 nM) significantly decreased APD90 in the presence of IKr inhibition but was attenuated with the addition of IKs inhibition. Spontaneous episodes of polymorphic ventricular tachycardia were observed with isoproterenol during dual IKr and IKs inhibition. The endocardial expression of KCNQ1 increased greater than 2-fold after exposure to IKr and dual IKr/IKs inhibition relative to control but was not altered in epicardial tissue. The beta-adrenergic receptor-mediated decrease in APD90 during IKr inhibition is reversed in the presence of IKs inhibition at rapid heart rates. IKs may serve as an important compensatory mechanism to protect against adrenergically induced arrhythmias when the repolarization reserve is depleted.

  17. Additive estrogenic effects of mixtures of frequently used UV filters on pS2-gene transcription in MCF-7 cells.

    PubMed

    Heneweer, Marjoke; Muusse, Martine; van den Berg, Martin; Sanderson, J Thomas

    2005-10-15

    In order to protect consumers from ultraviolet (UV) radiation and enhance light stability of the product, three to eight UV filters are usually added to consumer sunscreen products. High lipophilicity of the UV filters has been shown to cause bioaccumulation in fish and humans, leading to environmental levels of UV filters that are similar to those of PCBs and DDT. In this paper, estrogen-regulated pS2 gene transcription in the human mammary tumor cell line MCF-7 was used as a measure of estrogenicity of four individual UV filters. Since humans are exposed to more than one UV filter at a time, an equipotent binary mixture of 2-hydroxy-4-methoxy-benzophenone (BP-3) and its metabolite 2,4-dihydroxy benzophenone (BP-1), as well as an equipotent multi-component mixture of BP-1, BP-3, octyl methoxy cinnamate (OMC) and 3-(4-methylbenzylidene) camphor (4-MBC), were also evaluated for their ability to induce pS2 gene transcription in order to examine additivity. An estrogen receptor-mediated mechanism of action was expected for all UV filters. Therefore, our null-hypothesis was that combined estrogenic responses, measured as increased pS2 gene transcription in MCF-7 cells after exposure to mixtures of UV filters, are additive, according to a concentration-addition model. Not all UV filters produced a full concentration-response curve within the concentration range tested (100 nM-1 microM). Therefore, instead of using EC50 values for comparison, the concentration at which each compound caused a 50% increase of basal pS2 gene transcription was defined as the C50 value for that compound and used to calculate relative potencies. For comparison, the EC50 value of a compound is the concentration at which the compound elicits an effect that is 50% of its maximal effect. Individual UV filters increased pS2 gene transcription concentration-dependently with C50 values of 0.12 microM, 0.5 microM, 1.9 microM, and 1.0 microM for BP-1, BP-3, 4-MBC and OMC, respectively. Estradiol (E2

  18. Additive estrogenic effects of mixtures of frequently used UV filters on pS2-gene transcription in MCF-7 cells

    SciTech Connect

    Heneweer, Marjoke . E-mail: M.Heneweer@iras.uu.nl; Muusse, Martine; Berg, Martin van den; Sanderson, J. Thomas

    2005-10-15

    In order to protect consumers from ultraviolet (UV) radiation and enhance light stability of the product, three to eight UV filters are usually added to consumer sunscreen products. High lipophilicity of the UV filters has been shown to cause bioaccumulation in fish and humans, leading to environmental levels of UV filters that are similar to those of PCBs and DDT. In this paper, estrogen-regulated pS2 gene transcription in the human mammary tumor cell line MCF-7 was used as a measure of estrogenicity of four individual UV filters. Since humans are exposed to more than one UV filter at a time, an equipotent binary mixture of 2-hydroxy-4-methoxy-benzophenone (BP-3) and its metabolite 2,4-dihydroxy benzophenone (BP-1), as well as an equipotent multi-component mixture of BP-1, BP-3, octyl methoxy cinnamate (OMC) and 3-(4-methylbenzylidene) camphor (4-MBC), were also evaluated for their ability to induce pS2 gene transcription in order to examine additivity. An estrogen receptor-mediated mechanism of action was expected for all UV filters. Therefore, our null-hypothesis was that combined estrogenic responses, measured as increased pS2 gene transcription in MCF-7 cells after exposure to mixtures of UV filters, are additive, according to a concentration-addition model. Not all UV filters produced a full concentration-response curve within the concentration range tested (100 nM-1 {mu}M). Therefore, instead of using EC{sub 50} values for comparison, the concentration at which each compound caused a 50% increase of basal pS2 gene transcription was defined as the C50 value for that compound and used to calculate relative potencies. For comparison, the EC{sub 50} value of a compound is the concentration at which the compound elicits an effect that is 50% of its maximal effect. Individual UV filters increased pS2 gene transcription concentration-dependently with C50 values of 0.12 {mu}M, 0.5 {mu}M, 1.9 {mu}M, and 1.0 {mu}M for BP-1, BP-3, 4-MBC and OMC, respectively. Estradiol

  19. Resveratrol attenuates acute kidney injury by inhibiting death receptor-mediated apoptotic pathways in a cisplatin-induced rat model

    PubMed Central

    Hao, Qiufa; Xiao, Xiaoyan; Zhen, Junhui; Feng, Jinbo; Song, Chun; Jiang, Bei; Hu, Zhao

    2016-01-01

    Acute kidney injury is a clinical syndrome characterized by a loss of renal function and acute tubular necrosis. Resveratrol exerts a wide range of pharmacological effects based on its anti-inflammatory, antioxidant and cytoprotective properties. The present study aimed to evaluate whether resveratrol attenuates acute kidney injury in a cisplatin-induced rat model and to investigate the potential mechanisms involved. Rats were randomly divided into four treatment groups: Control, cisplatin, resveratrol, and cisplatin plus resveratrol. Rats exposed to cisplatin displayed acute kidney injury, identified by analysis of renal function and histopathological observation. Resveratrol significantly ameliorated the increased serum creatinine, blood urea nitrogen, renal index and histopathological damage induced by cisplatin. Furthermore, compared with untreated control animals, cisplatin lead to significantly increased expression of Fas ligand, tumor necrosis factor-α (TNF-α), caspase-8 and Bcl-2 associated protein X apoptosis regulator (Bax), and decreased expression of anti-apoptosis regulators, BH3 interacting domain death agonist (BID) and B cell lymphoma 2 apoptosis regulator (Bcl-2). Administration of resveratrol significantly reversed the cisplatin-induced alteration in these apoptosis-associated proteins. In conclusion, these findings suggest that resveratrol attenuates cisplatin-induced acute kidney injury through inactivation of the death receptor-mediated apoptotic pathway, and may provide a new therapeutic strategy to ameliorate the process of acute kidney injury. PMID:27600998

  20. A whole-genome RNAi screen identifies an 8q22 gene cluster that inhibits death receptor-mediated apoptosis.

    PubMed

    Dompe, Nicholas; Rivers, Celina Sanchez; Li, Li; Cordes, Shaun; Schwickart, Martin; Punnoose, Elizabeth A; Amler, Lukas; Seshagiri, Somasekar; Tang, Jerry; Modrusan, Zora; Davis, David P

    2011-10-25

    Deregulation of apoptosis is a common occurrence in cancer, for which emerging oncology therapeutic agents designed to engage this pathway are undergoing clinical trials. With the aim of uncovering strategies to activate apoptosis in cancer cells, we used a pooled shRNA screen to interrogate death receptor signaling. This screening approach identified 16 genes that modulate the sensitivity to ligand induced apoptosis, with several genes exhibiting frequent overexpression and/or copy number gain in cancer. Interestingly, two of the top hits, EDD1 and GRHL2, are found 50 kb apart on chromosome 8q22, a region that is frequently amplified in many cancers. By using a series of silencing and overexpression studies, we show that EDD1 and GRHL2 suppress death-receptor expression, and that EDD1 expression is elevated in breast, pancreas, and lung cancer cell lines resistant to death receptor-mediated apoptosis. Supporting the relevance of EDD1 and GRHL2 as therapeutic candidates to engage apoptosis in cancer cells, silencing the expression of either gene sensitizes 8q22-amplified breast cancer cell lines to death receptor induced apoptosis. Our findings highlight a mechanism by which cancer cells may evade apoptosis, and therefore provide insight in the search for new targets and functional biomarkers for this pathway.

  1. Paired-pulse depression of the N-methyl-D-aspartate receptor-mediated synaptic potentials in the amygdala.

    PubMed Central

    Huang, C C; Gean, P W

    1994-01-01

    1. An in vitro slice preparation of rat amygdala was used to study the paired-pulse depression of the N-methyl-D-aspartate (NMDA) receptor-mediated synaptic potential e.p.s.p.NMDA. 2. The e.p.s.p.NMDA was isolated pharmacologically by applying a solution containing the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the gamma-aminobutyric acidA (GABAA) blocker picrotoxin and increasing the stimulus intensity. 3. When two stimuli of identical strength were applied in close succession, the second e.p.s.p.NMDA was depressed. This paired-pulse depression was seen with interstimulus intervals of between 100 ms and 2000 ms; the maximal depression was observed at interval of 200 ms. 4. Superfusion of phaclofen or 2-hydroxy-saclofen inhibited the paired-pulse depression indicating the involvement of GABAB receptors. 5. Bath applications of Ba2+ or intracellular injection of Cs+ to block post- but not presynaptic GABAB receptors failed to inhibit the paired-pulse depression (PPD). 6. Incubation of slices with pertussis toxin prevented the postsynaptic hyperpolarization induced by baclofen. The PPD of e.p.s.p.NMDA, however, was not affected by pertussis toxin treatment. 7. These results suggest that GABA released by the first stimulus acts on GABAB receptors to suppress the second e.p.s.p.NMDA via mechanisms other than activation of a postsynaptic GABAB receptor-coupled K+ conductance. PMID:7858845

  2. Homocysteine-NMDA receptor mediated activation of extracellular-signal regulated kinase leads to neuronal cell death

    PubMed Central

    Poddar, Ranjana; Paul, Surojit

    2009-01-01

    Hyper-homocysteinemia is an independent risk factor for stroke and neurological abnormalities. However the underlying cellular mechanisms by which elevated homocysteine can promote neuronal death is not clear. In the present study we have examined the role of NMDA receptor mediated activation of the extracellular-signal regulated mitogen activated protein (ERK MAP) kinase pathway in homocysteine-dependent neurotoxicity. The study demonstrates that in neurons L-homocysteine-induced cell death is mediated through activation of NMDA receptors. The study also shows that homocysteine-dependent NMDA receptor stimulation and resultant Ca2+ influx leads to rapid and sustained phosphorylation of ERK MAP kinase. Inhibition of ERK phosphorylation attenuates homocysteine mediated neuronal cell death thereby demonstrating that activation of ERK MAP kinase signaling pathway is an intermediate step that couples homocysteine mediated NMDA receptor stimulation to neuronal death. The findings also show that cAMP response-element binding protein (CREB), a pro-survival transcription factor and a downstream target of ERK, is only transiently activated following homocysteine exposure. The sustained activation of ERK but a transient activation of CREB together suggest that exposure to homocysteine initiates a feedback loop that shuts off CREB signaling without affecting ERK phosphorylation and thereby facilitates homocysteine mediated neurotoxicity. PMID:19508427

  3. Postmenopausal skin and estrogen.

    PubMed

    Archer, David F

    2012-10-01

    The aging global population continues to drive increasing demand for cosmaceuticals and cosmetic surgery among older men and women. Since the discovery in the 1990s that estrogen receptors are present in skin cells and decline in number from the onset of menopause in women, researchers have explored a number of ways in which estrogen can improve skin condition. Skin is estrogen responsive, and several studies now exist to support the antiaging properties of estrogen replacement therapies in postmenopausal women. Both systemic and topical estrogens appear to have positive effects on hormonal aging, increasing skin collagen content, thickness, elasticity and hydration. Estrogen therapies may also improve wound healing and reduce the incidence of wound complications. This review explores the potential for targeted estrogen replacement as a therapeutic option for long-term skin management in postmenopausal women.

  4. The Molecular, Cellular and Clinical Consequences of Targeting the Estrogen Receptor Following Estrogen Deprivation Therapy

    PubMed Central

    Fan, Ping; Maximov, Philipp Y.; Curpan, Ramona F.; Abderrahman, Balkees; Jordan, V. Craig

    2015-01-01

    During the past twenty years our understanding of the control of breast tumor development, growth and survival has changed dramatically. The once long forgotten application of high dose synthetic estrogen therapy as the first chemical therapy to treat any cancer has been resurrected, refined and reinvented as the new biology of estrogen-induced apoptosis. High dose estrogen therapy was cast aside once tamoxifen, from its origins as a failed “morning after pill”, was reinvented as the first targeted therapy to treat any cancer. The current understanding of the mechanism of estrogen-induced apoptosis is described as a consequence of acquired resistance to long term antihormone therapy in estrogen receptor (ER) positive breast cancer. The ER signal transduction pathway remains a target for therapy in breast cancer despite “antiestrogen” resistance, but becomes a regulator of resistance. Multiple mechanisms of resistance come into play: Selective ER Modulator (SERM) stimulated growth, growth factor/ER crosstalk, estrogen-induced apoptosis and mutations of ER. But it is with the science of estrogen-induced apoptosis that the next innovation in women’s health will be developed. Recent evidence suggests that the glucocorticoid properties of medroxyprogesterone acetate blunt estrogen-induced apoptosis in estrogen deprived breast cancer cell populations. As a result breast cancer develops during long-term Hormone Replacement Therapy (HRT). A new synthetic progestin with estrogen-like properties, such as the 19 nortestosterone derivatives used in oral contraceptives, will continue to protect the uterus from unopposed estrogen stimulation but at the same time, reinforce apoptosis in vulnerable populations of nascent breast cancer cells. PMID:26052034

  5. 5-HT1-like receptor-mediated contraction in the human internal mammary artery.

    PubMed

    Yildiz, O; Ciçek, S; Ay, I; Tatar, H; Tuncer, M

    1996-07-01

    We wished to characterize the 5-hydroxytryptamine (5-HT) receptors mediating vasoconstriction in the human internal mammary artery (IMA). Segments of the IMA obtained from patients undergoing coronary by-pass surgery were suspended in an organ bath and exposed to 5-HT and sumatriptan (SUM), a 5-HT1-like receptor agonist, in the presence and absence of potassium chloride (KCl) and angiotensin II. 5-HT induced concentration-dependent contractions in all quiescent and pre-contracted preparations. SUM induced small contractions in 70% of quiescent IMA rings, whereas it elicited marked and concentration-dependent contractions in all of the preparations given a moderate tone by a threshold concentration of KCl and angiotensin II. The efficacy of SUM was higher in precontracted arteries. Concentration-effect curves (CEC) of 5-HT and SUM were not affected by the 5-HT3-receptor antagonist tropisetron (1 microM). The nonselective antagonist, methiothepin (30 nM), shifted the CEC of SUM to the right. 5-HT2A-receptor antagonist, ketanserin (1 microM) inhibited responses to 5-HT, whereas it affected only the responses to the smaller concentrations of SUM. When methiothepin (30 nM) was applied in the presence of ketanserin (1 microM), a further inhibition in the responses to 5-HT was observed. These results suggest that 5-HT1-like receptors mediate the contractile action of SUM and contribute to that of 5-HT in IMA.

  6. Receptor-mediated binding and uptake of GnRH agonist and antagonist by pituitary cells

    SciTech Connect

    Jennes, L.; Stumpf, W.E.; Conn, P.M.

    1984-01-01

    The intracellular pathway of an enzyme resistant GnRH agonist (D- Lys6 -GnRH) conjugated to ferritin or to colloidal gold was followed in cultured pituitary cells. After an initial uniform distribution over the cell surface of gonadotropes, the electrondense marker was internalized, either individually or in small groups. After longer incubation times, the marker appeared in the lysosomal compartment and the Golgi apparatus, where it could be found in the vesicular as well as cisternal portion. In addition, the receptor-mediated endocytosis of the GnRH antagonist D-p-Glu1-D-Phe2-D-Trp3-D- Lys6 -GnRH was studied by light and electron microscopic autoradiography after 30 and 60 min of incubation to ensure uptake. At both time points, in in vitro as well as in vivo studies, silver grains were localized over cytoplasmic organelles of castration cells, including dilated endoplasmic reticulum, lysosomes, and clear vesicles. No consistent association with cell nuclei, mitochondria, or secretory vesicles could be observed. The results suggest that both agonist and antagonist are binding selectively to the plasma membrane of gonadotropes and subsequently are taken up via receptor-mediated endocytosis for degradation or possible action on synthetic processes.

  7. Folate receptor mediated intracellular protein delivery using PLL-PEG-FOL conjugate.

    PubMed

    Hwa Kim, Sun; Hoon Jeong, Ji; Joe, Cheol O; Gwan Park, Tae

    2005-04-18

    To develop a receptor-mediated intracellular delivery system that can transport therapeutic proteins or other bioactive macromolecules into a specific cell, a di-block copolymer conjugate, poly(L-lysine)-poly(ethylene glycol)-folate (PLL-PEG-FOL), was synthesized. The PLL-PEG-FOL conjugate was physically complexed with fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) in an aqueous phase by ionic interactions. Cellular uptake of PLL-PEG-FOL/FITC-BSA complexes was greatly enhanced against a folate receptor over-expressing cell line (KB cells) compared to a folate receptor deficient cell line (A549 cells). The presence of an excess amount of free folate (1 mM) in the medium inhibited the intracellular delivery of PLL-PEG-FOL/FITC-BSA complexes. This suggests that the enhanced cellular uptake of FITC-BSA by KB cells in a specific manner was attributed to folate receptor-mediated endocytosis of the complexes having folate moieties on the surface. The PLL-PEG-FOL di-block copolymer could be potentially applied for intracellular delivery of a wide range of other biological active agents that have negative charges on the surface.

  8. Understanding magnetic nanoparticle osteoblast receptor-mediated endocytosis using experiments and modeling

    NASA Astrophysics Data System (ADS)

    Tran, Nhiem; Webster, Thomas J.

    2013-05-01

    Iron oxide nanoparticles are promising candidates for controlling drug delivery through an external magnetic force to treat a wide range of diseases, including osteoporosis. Previous studies have demonstrated that in the presence of hydroxyapatite coated magnetite (Fe3O4) nanoparticles, osteoblast (or bone forming cell) proliferation and long-term functions (such as calcium deposition) were significantly enhanced. Hydroxyapatite is the major inorganic component of bone. As a further attempt to understand why, in the current study, the uptake of such nanoparticles into osteoblasts was experimentally investigated and mathematically modeled. Magnetite nanoparticles were synthesized using a co-precipitation method and were coated with hydroxyapatite. A cellular uptake experiment at low temperatures indicated that receptor-mediated endocytosis contributed to the internalization of the magnetic nanoparticles into osteoblasts. A model was further developed to explain the uptake of magnetic nanoparticles into osteoblasts using receptor-mediated endocytosis. This model may explain the internalization of hydroxyapatite into osteoblasts to elevate intracellular calcium levels necessary to promote osteoblast functions to treat a wide range of orthopedic problems, including osteoporosis.

  9. Regulation and ontogeny of subtypes of muscarinic receptors and muscarinic receptor-mediated

    SciTech Connect

    Lee, W.

    1989-01-01

    The densities of total and M1 muscarinic receptors were measured using the muscarinic receptor antagonists {sup 3}H-quinuclidinyl benzilate and {sup 3}H-pirenzepine, respectively. Thus, the difference between the density of {sup 3}H-quinuclidinyl benzilate and {sup 3}H-pirenzepine binding sites represents the density of M2 sites. In addition, there is no observable change in either acetylcholine-stimulated phosphoinositide breakdown (suggested to be an M1 receptor-mediated response) or in carbachol-mediated inhibition of cyclic AMP accumulation (suggested to be an M2 receptor-mediated response) in slices of cortex+dorsal hippocampus following chronic atropine administration. In other experiments, it has been shown that the M1 and M2 receptors in rat cortex have different ontogenetic profiles. The M2 receptor is present at adult levels at birth, while the M1 receptor develops slowly from low levels at postnatal week 1 to adult levels at postnatal week 3. The expression of acetylcholine-stimulated phosphoinositide breakdown parallels the development of M1 receptors, while the development of carbachol-mediated inhibition of cyclic AMP accumulation occurs abruptly between weeks 2 and 3 postnatally.

  10. G protein-coupled estrogen receptor 1 (GPER1)/GPR30 increases ERK1/2 activity through PDZ motif-dependent and -independent mechanisms.

    PubMed

    Gonzalez de Valdivia, Ernesto; Broselid, Stefan; Kahn, Robin; Olde, Björn; Leeb-Lundberg, L M Fredrik

    2017-06-16

    G protein-coupled receptor 30 (GPR30), also called G protein-coupled estrogen receptor 1 (GPER1), is thought to play important roles in breast cancer and cardiometabolic regulation, but many questions remain about ligand activation, effector coupling, and subcellular localization. We showed recently that GPR30 interacts through the C-terminal type I PDZ motif with SAP97 and protein kinase A (PKA)-anchoring protein (AKAP) 5, which anchor the receptor in the plasma membrane and mediate an apparently constitutive decrease in cAMP production independently of Gi/o Here, we show that GPR30 also constitutively increases ERK1/2 activity. Removing the receptor PDZ motif or knocking down specifically AKAP5 inhibited the increase, showing that this increase also requires the PDZ interaction. However, the increase was inhibited by pertussis toxin as well as by wortmannin but not by AG1478, indicating that Gi/o and phosphoinositide 3-kinase (PI3K) mediate the increase independently of epidermal growth factor receptor transactivation. FK506 and okadaic acid also inhibited the increase, implying that a protein phosphatase is involved. The proposed GPR30 agonist G-1 also increased ERK1/2 activity, but this increase was only observed at a level of receptor expression below that required for the constitutive increase. Furthermore, deleting the PDZ motif did not inhibit the G-1-stimulated increase. Based on these results, we propose that GPR30 increases ERK1/2 activity via two Gi/o-mediated mechanisms, a PDZ-dependent, apparently constitutive mechanism and a PDZ-independent G-1-stimulated mechanism. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Validation and application of a rapid in vitro assay for assessing the estrogenic potency of halogenated phenolic chemicals.

    PubMed

    Körner, W; Hanf, V; Schuller, W; Bartsch, H; Zwirner, M; Hagenmaier, H

    1998-01-01

    The E-Screen assay serves as an in vitro tool for the detection of estrogenic activity of chemicals and extracts of environmental samples. Based on the induction of proliferation in human estrogen receptor-positive MCF-7 breast cancer cells we could substantially simplify the assay. As one important step of validation we applied the modified assay for testing nine known xenoestrogens. We could confirm the results of other groups assuring the reproducibility of the E-Screen assay. The results provide evidence that the E-Screen assay is suitable for determination of estradiol equivalency factors (EEFs) for environmental estrogens to rank their estrogenic potency relative to the natural estrogen 17 beta-estradiol. Further, we used the optimized proliferation test to screen nine halogenated phenolic compounds for their possible estrogenic potency. Three widely applied chemicals expressed a weak receptor-mediated estrogenic activity: the flame retardant Tetrabromo-Bisphenol-A, the disinfectant 4-chloro-3-methylphenol, and the herbicide educt 4-chloro-2-methylphenol. Their estrogenic potencies were five to six orders of magnitude lower than that of 17 beta-estradiol.

  12. Biological measurement of estrogenic activity in urine and bile conjugates with the in vitro ER-CALUX reporter gene assay.

    PubMed

    Legler, Juliette; Jonas, Arjen; Lahr, Joost; Vethaak, A Dick; Brouwer, Abraham; Murk, Albertinka J

    2002-03-01

    Although estrogens are excreted as biologically inactive conjugates, they can be reconverted to an active form, possibly by bacteria. A simple method was developed to deconjugate estrogen metabolites present in human urine and fish bile back to active estrogens by enzymatic hydrolysis with beta-glucuronidase or live Escherichia coli cells. Deconjugated extracts were tested for estrogenic activity in the in vitro stable estrogen receptor-mediated chemical-activated luciferase gene expression (ER-CALUX) assay. Estrogen glucuronides in urine obtained from human males and females were effectively converted to active forms after incubation with beta-glucuronidase or E. coli. The highest estrogenic activity was found in deconjugated metabolites from urine of a pregnant woman, in which levels up to 3,000 nmol estradiol equivalents per liter of urine were found after overnight incubation of urine with E. coli. Bile sampled from male bream and flounder from various freshwater and marine locations was also deconjugated and a good correlation was found between high biliary estrogenic activity and elevated levels of xenoestrogenic activity in surface water as well as in plasma vitellogenin. Therefore, the measurement of deconjugated bile could form a useful (indirect) biomarker for internal dose of xenoestrogens in male fish.

  13. Interactions of dietary estrogens with human estrogen receptors and the effect on estrogen receptor-estrogen response element complex formation.

    PubMed Central

    Nikov, G N; Hopkins, N E; Boue, S; Alworth, W L

    2000-01-01

    Epidemiologic and experimental studies support the hypothesis that dietary estrogens from plant sources (phytoestrogens) may play a role in the prevention of breast and prostate cancer. The molecular mechanisms for such chemopreventive effect are still unclear. We investigated the possibility that phytoestrogens may bind differentially to estrogen receptor proteins (ER[alpha] and ERss) and affect the interactions of the ligand-ER complexes with different estrogen response element (ERE) sequences. We used fluorescence polarization to measure the binding affinities of genistein, coumestrol, daidzein, glyceollin, and zearalenone for human ER[alpha] and ERss. Competition binding experiments revealed higher affinity of the phytoestrogens for ERss than for ER[alpha]. Genistein [median inhibitory concentration 12nM] is the most potent and has the same relative binding affinity for ERss as 17ss-estradiol. We also studied the effect of these phytoestrogens on the ability of ER[alpha] and ERss to associate with specific DNA sequences (EREs). The direct binding of human recombinant estrogen receptors to fluorescein-labeled EREs indicates that phytoestrogens can cause conformational changes in both human ERs, which results in altered affinities of the complexes for the ERE from the Xenopus vitellogenin A2 gene and an ERE from the human pS2 gene. PMID:11017892

  14. Low Concentrations of o,p’-DDT Inhibit Gene Expression and Prostaglandin Synthesis by Estrogen Receptor-Independent Mechanism in Rat Ovarian Cells

    PubMed Central

    Liu, Jing; Zhao, Meirong; Zhuang, Shulin; Yang, Yan; Yang, Ye; Liu, Weiping

    2012-01-01

    o,p’-DDT is an infamous xenoestrogen as well as a ubiquitous and persistent pollutant. Biomonitoring studies show that women have been internally exposed to o,p’-DDT at range of 0.3–500 ng/g (8.46×10−10 M−1.41×10−6 M) in blood and other tissues. However, very limited studies have investigated the biological effects and mechanism(s) of o,p’-DDT at levels equal to or lower than current exposure levels in human. In this study, using primary cultures of rat ovarian granulosa cells, we determined that very low doses of o,p’-DDT (10−12−10−8 M) suppressed the expression of ovarian genes and production of prostaglandin E2 (PGE2). In vivo experiments consistently demonstrated that o,p’-DDT at 0.5–1 mg/kg inhibited the gene expression and PGE2 levels in rat ovary. The surprising results from the receptor inhibitors studies showed that these inhibitory effects were exerted independently of either classical estrogen receptors (ERs) or G protein-coupled receptor 30 (GPR30). Instead, o,p’-DDT altered gene expression or hormone action via inhibiting the activation of protein kinase A (PKA), rather than protein kinase C (PKC). We further revealed that o,p’-DDT directly interfered with the PKA catalytic subunit. Our novel findings support the hypothesis that exposure to low concentrations of o,p’-DDT alters gene expression and hormone synthesis through signaling mediators beyond receptor binding, and imply that the current exposure levels of o,p’-DDT observed in the population likely poses a health risk to female reproduction. PMID:23209616

  15. Low concentrations of o,p'-DDT inhibit gene expression and prostaglandin synthesis by estrogen receptor-independent mechanism in rat ovarian cells.

    PubMed

    Liu, Jing; Zhao, Meirong; Zhuang, Shulin; Yang, Yan; Yang, Ye; Liu, Weiping

    2012-01-01

    o,p'-DDT is an infamous xenoestrogen as well as a ubiquitous and persistent pollutant. Biomonitoring studies show that women have been internally exposed to o,p'-DDT at range of 0.3-500 ng/g (8.46×10(-10) M-1.41×10(-6) M) in blood and other tissues. However, very limited studies have investigated the biological effects and mechanism(s) of o,p'-DDT at levels equal to or lower than current exposure levels in human. In this study, using primary cultures of rat ovarian granulosa cells, we determined that very low doses of o,p'-DDT (10(-12)-10(-8) M) suppressed the expression of ovarian genes and production of prostaglandin E2 (PGE2). In vivo experiments consistently demonstrated that o,p'-DDT at 0.5-1 mg/kg inhibited the gene expression and PGE2 levels in rat ovary. The surprising results from the receptor inhibitors studies showed that these inhibitory effects were exerted independently of either classical estrogen receptors (ERs) or G protein-coupled receptor 30 (GPR30). Instead, o,p'-DDT altered gene expression or hormone action via inhibiting the activation of protein kinase A (PKA), rather than protein kinase C (PKC). We further revealed that o,p'-DDT directly interfered with the PKA catalytic subunit. Our novel findings support the hypothesis that exposure to low concentrations of o,p'-DDT alters gene expression and hormone synthesis through signaling mediators beyond receptor binding, and imply that the current exposure levels of o,p'-DDT observed in the population likely poses a health risk to female reproduction.

  16. Vascular Effects of Estrogenic Menopausal Hormone Therapy

    PubMed Central

    Reslan, Ossama M.; Khalil, Raouf A.

    2011-01-01

    Cardiovascular disease (CVD) is more common in men and postmenopausal women (Post-MW) than premenopausal women (Pre-MW). Despite recent advances in preventive measures, the incidence of CVD in women has shown a rise that matched the increase in the Post-MW population. The increased incidence of CVD in Post-MW has been related to the decline in estrogen levels, and hence suggested vascular benefits of endogenous estrogen. Experimental studies have identified estrogen receptor ERα, ERβ and a novel estrogen binding membrane protein GPR30 (GPER) in blood vessels of humans and experimental animals. The interaction of estrogen with vascular ERs mediates both genomic and non-genomic effects. Estrogen promotes endothelium-dependent relaxation by increasing nitric oxide, prostacyclin, and hyperpolarizing factor. Estrogen also inhibits the mechanisms of vascular smooth muscle (VSM) contraction including [Ca2+]i, protein kinase C and Rho-kinase. Additional effects of estrogen on the vascular cytoskeleton, extracellular matrix, lipid profile and the vascular inflammatory response have been reported. In addition to the experimental evidence in animal models and vascular cells, initial observational studies in women using menopausal hormonal therapy (MHT) have suggested that estrogen may protect against CVD. However, randomized clinical trials (RCTs) such as the Heart and Estrogen/progestin Replacement Study (HERS) and the Women’s Health Initiative (WHI), which examined the effects of conjugated equine estrogens (CEE) in older women with established CVD (HERS) or without overt CVD (WHI), failed to demonstrate protective vascular effects of estrogen treatment. Despite the initial set-back from the results of MHT RCTs, growing evidence now supports the ‘timing hypothesis’, which suggests that MHT could increase the risk of CVD if started late after menopause, but may produce beneficial cardiovascular effects in younger women during the perimenopausal period. The choice of

  17. Inhibition of carbonic anhydrase augments GABAA receptor-mediated analgesia via a spinal mechanism of action

    PubMed Central

    Asiedu, Marina N.; Mejia, Galo L.; Hübner, Christian A.; Kaila, Kai; Price, Theodore J.

    2014-01-01

    Peripheral nerve injury negatively influences spinal GABAergic networks via a reduction in the neuron-specific K+-Cl- cotransporter KCC2. This process has been linked to the emergence of neuropathic allodynia. In vivo pharmacological and modeling studies show that a loss of KCC2 function results in a decrease in the efficacy of GABAA -mediated spinal inhibition. One potential strategy to mitigate this effect entails inhibition of carbonic anhydrase activity to reduce HCO3- -dependent depolarization via GABAA receptors when KCC2 function is compromised. We have tested this hypothesis here. Our results show that, similarly to when KCC2 is pharmacologically blocked, peripheral nerve injury causes a loss of analgesic effect for neurosteroid GABAA allosteric modulators at maximally effective doses in naïve mice in the tail flick test. Remarkably, inhibition of carbonic anhydrase activity with intrathecal acetazolamide rapidly restores an analgesic effect for these compounds suggesting an important role of carbonic anhydrase activity in regulating GABAA -mediated analgesia after peripheral nerve injury. Moreover, spinal acetazolamide administration leads to a profound reduction in the mouse formalin pain test indicating that spinal carbonic anhydrase inhibition produces analgesia when primary afferent activity is driven by chemical mediators. Finally, we demonstrate that systemic administration of acetazolamide to rats with peripheral nerve injury produces an anti-allodynia effect by itself and an enhancement of the peak analgesic effect with a change in the shape of the dose response curve of the α1-sparing benzodiazepine L-838,417. Thus, carbonic anhydrase inhibition mitigates the negative effects of loss of KCC2 function after nerve injury in multiple species and through multiple administration routes resulting in an enhancement of analgesic effects for several GABAA allosteric modulators. We suggest that carbonic anhydrase inhibitors, many of which are clinically available, might be advantageously employed for the treatment of pathological pain states. PMID:24412803

  18. Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock

    PubMed Central

    Van Amersfoort, Edwin S.; Van Berkel, Theo J. C.; Kuiper, Johan

    2003-01-01

    Bacterial sepsis and septic shock result from the overproduction of inflammatory mediators as a consequence of the interaction of the immune system with bacteria and bacterial wall constituents in the body. Bacterial cell wall constituents such as lipopolysaccharide, peptidoglycans, and lipoteichoic acid are particularly responsible for the deleterious effects of bacteria. These constituents interact in the body with a large number of proteins and receptors, and this interaction determines the eventual inflammatory effect of the compounds. Within the circulation bacterial constituents interact with proteins such as plasma lipoproteins and lipopolysaccharide binding protein. The interaction of the bacterial constituents with receptors on the surface of mononuclear cells is mainly responsible for the induction of proinflammatory mediators by the bacterial constituents. The role of individual receptors such as the toll-like receptors and CD14 in the induction of proinflammatory cytokines and adhesion molecules is discussed in detail. In addition, the roles of a number of other receptors that bind bacterial compounds such as scavenger receptors and their modulating role in inflammation are described. Finally, the therapies for the treatment of bacterial sepsis and septic shock are discussed in relation to the action of the aforementioned receptors and proteins. PMID:12857774

  19. Potential insight for drug discovery from high fidelity receptor-mediated transduction mechanisms in insects

    PubMed Central

    Raffa, Robert B.; Raffa, Kenneth F.

    2011-01-01

    Introduction There is a pervasive and growing concern about the small number of new pharmaceutical agents. There are many proposed explanations for this trend that do not involve the drug-discovery process per se, but the discovery process itself has also come under scrutiny. If the current paradigms are indeed not working, where are novel ideas to come from? Perhaps it is time to look to novel sources. Areas covered The receptor-signaling and 2nd-messenger transduction processes present in insects are quite similar to those in mammals (involving G proteins, ion channels, etc.). However, a review of these systems reveals an unprecedented degree of high potency and receptor selectivity to an extent greater than that modeled in most current drug-discovery approaches. Expert opinion A better understanding of insect receptor pharmacology could stimulate novel theoretical and practical ideas in mammalian pharmacology (drug discovery) and, conversely, the application of pharmacology and medicinal chemistry principles could stimulate novel advances in entomology (safer and more targeted control of pest species). PMID:21984882

  20. Cannabinoid-induced stimulation of motor activity in planaria through an opioid receptor-mediated mechanism.

    PubMed

    Buttarelli, Francesca R; Pontieri, Francesco E; Margotta, Vito; Palladini, Guido

    2002-01-01

    Planaria, the most primitive example of centralization and cephalization of the nervous system along phylogeny, shows specific stereotyped behavioral patterns following exposure to drugs acting on neural transmission. In this study, the authors investigated the effects of exposure to the synthetic cannabinoid receptor agonist WTN55212.2 on motor activity in planaria. WTN55212.2 produced dose-dependent stimulation of motor behavior. High doses of the drug caused stereotyped activities identical to those seen previously with opioid agonists. These effects were antagonized by coexposure to cannabinoid or opioid receptor antagonists. The results indicate that functional interactions between cannabinoid and opioid systems are highly conserved along phylogeny, at least at the behavioral level.

  1. Norgestrel and gestodene stimulate breast cancer cell growth through an oestrogen receptor mediated mechanism.

    PubMed Central

    Catherino, W. H.; Jeng, M. H.; Jordan, V. C.

    1993-01-01

    There is great concern over the long-term influence of oral contraceptives on the development of breast cancer in women. Oestrogens are known to stimulate the growth of human breast cancer cells, and this laboratory has previously reported (Jeng & Jordan, 1991) that the 19-norprogestin norethindrone could stimulate the proliferation of MCF-7 human breast cancer cells. We studied the influence of the 19-norprogestins norgestrel and gestodene compared to a 'non' 19-norprogestin medroxyprogesterone acetate (MPA) on MCF-7 cell proliferation. The 19-norprogestins stimulated proliferation at a concentration of 10(-8) M, while MPA could not stimulate proliferation at concentrations as great as 3 x 10(-6) M. The stimulatory activity of the 19-norprogestins could be blocked by the antioestrogen ICI 164,384, but not by the antiprogestin RU486. Transfection studies with the reporter plasmids containing an oestrogen response element or progesterone response element (vitERE-CAT, pS2ERE-CAT, and PRE15-CAT) were performed to determine the intracellular action of norgestrel and gestodene. The 19-norprogestins stimulated the vitERE-CAT activity maximally at 10(-6) M, and this stimulation was inhibited by the addition of ICI 164,384. MPA did not stimulate vitERE-CAT activity. A single base pair alteration in the palindromic sequence of vitERE (resulting in the pS2ERE) led to a dramatic decrease in CAT expression by the 19-norprogestins, suggesting that the progestin activity required specific response element base sequencing. PRE15-CAT activity was stimulated by norgestrel, gestodene and MPA at concentrations well below growth stimulatory activity. This stimulation could be blocked by RU486. These studies suggest that the 19-norprogestins norgestrel and gestodene stimulate MCF-7 breast cancer cell growth by activating the oestrogen receptor. PMID:8494728

  2. Estrogenic effects of natural and synthetic compounds including tibolone assessed in Saccharomyces cerevisiae expressing the human estrogen alpha and beta receptors.

    PubMed

    Hasenbrink, Guido; Sievernich, André; Wildt, Ludwig; Ludwig, Jost; Lichtenberg-Fraté, Hella

    2006-07-01

    The human estrogen receptors (hER)alpha and hERbeta, differentially expressed and localized in various tissues and cell types, mediate transcriptional activation of target genes. These encode a variety of physiological reproductive and nonreproductive functions involved in energy metabolism, salt balance, immune system, development, and differentiation. As a step toward developing a screening assay for the use in applications where significant numbers of compounds or complex matrices need to be tested for (anti) estrogenic bioactivity, hERalpha and hERbeta were expressed in a genetically modified Saccharomyces cerevisiae strain, devoid of three endogenous xenobiotic transporters (PDR5, SNQ2, and YOR1). By using receptor-mediated transcriptional activation of the green fluorescent protein optimized for expression in yeast (yEGFP) as reporter 17 natural, comprising estrogens and phytoestrogens or synthetic compounds among which tibolone with its metabolites, gestagens, and antiestrogens were investigated. The reporter assay deployed a simple and robust protocol for the rapid detection of estrogenic effects within a 96-well microplate format. Results were expressed as effective concentrations (EC50) and correlated to other yeast based and cell line assays. Tibolone and its metabolites exerted clear estrogenic effects, though considerably less potent than all other natural and synthetic compounds. For the blood serum of two volunteers, considerable higher total estrogenic bioactivity than single estradiol concentrations as determined by immunoassay was found. Visualization of a hERalpha/GFP fusion protein in yeast revealed a sub cellular cytosolic localization. This study demonstrates the versatility of (anti) estrogenic bioactivity determination using sensitized S. cerevisiae cells to assess estrogenic exposure and effects.

  3. Effects of Estrogens and Estrogenic Disrupting Compounds on Fish Mineralized Tissues

    PubMed Central

    Pinto, Patricia I. S.; Estêvão, Maria D.; Power, Deborah M.

    2014-01-01

    Estrogens play well-recognized roles in reproduction across vertebrates, but also intervene in a wide range of other physiological processes, including mineral homeostasis. Classical actions are triggered when estrogens bind and activate intracellular estrogen receptors (ERs), regulating the transcription of responsive genes, but rapid non-genomic actions initiated by binding to plasma membrane receptors were recently described. A wide range of structurally diverse compounds from natural and anthropogenic sources have been shown to interact with and disrupt the normal functions of the estrogen system, and fish are particularly vulnerable to endocrine disruption, as these compounds are frequently discharged or run-off into waterways. The effect of estrogen disruptors in fish has mainly been assessed in relation to reproductive endpoints, and relatively little attention has been given to other disruptive actions. This review will overview the actions of estrogens in fish, including ER isoforms, their expression, structure and mechanisms of action. The estrogen functions will be considered in relation to mineral homeostasis and actions on mineralized tissues. The impact of estrogenic endocrine disrupting compounds on fish mineralized tissues will be reviewed, and the potential adverse outcomes of exposure to such compounds will be discussed. Current lacunae in knowledge are highlighted along with future research priorities. PMID:25196834

  4. Effects of estrogens and estrogenic disrupting compounds on fish mineralized tissues.

    PubMed

    Pinto, Patricia I S; Estêvão, Maria D; Power, Deborah M

    2014-08-15

    Estrogens play well-recognized roles in reproduction across vertebrates, but also intervene in a wide range of other physiological processes, including mineral homeostasis. Classical actions are triggered when estrogens bind and activate intracellular estrogen receptors (ERs), regulating the transcription of responsive genes, but rapid non-genomic actions initiated by binding to plasma membrane receptors were recently described. A wide range of structurally diverse compounds from natural and anthropogenic sources have been shown to interact with and disrupt the normal functions of the estrogen system, and fish are particularly vulnerable to endocrine disruption, as these compounds are frequently discharged or run-off into waterways. The effect of estrogen disruptors in fish has mainly been assessed in relation to reproductive endpoints, and relatively little attention has been given to other disruptive actions. This review will overview the actions of estrogens in fish, including ER isoforms, their expression, structure and mechanisms of action. The estrogen functions will be considered in relation to mineral homeostasis and actions on mineralized tissues. The impact of estrogenic endocrine disrupting compounds on fish mineralized tissues will be reviewed, and the potential adverse outcomes of exposure to such compounds will be discussed. Current lacunae in knowledge are highlighted along with future research priorities.

  5. Asialoglycoprotein receptor mediates the toxic effects of an asialofetuin-diphtheria toxin fragment A conjugate on cultured rat hepatocytes.

    PubMed Central

    Cawley, D B; Simpson, D L; Herschman, H R

    1981-01-01

    We have constructed a toxic hybrid protein that is recognized by asialoglycoprotein (ASGP) receptors of cultured rat hepatocytes. The conjugate consists of fragment A of diphtheria toxin (DTA) linked by a disulfide bond to asialofetuin (ASF). This conjugate is highly toxic, inhibiting protein synthesis in primary rat hepatocytes at concentrations as low as 10 pM. The ASF-DTA conjugate was 600 and 1800 times as toxic as diphtheria toxin and DTA, respectively, on primary rat hepatocytes. The ASGP receptor recognizes galactose-terminated proteins. We tested a series of glycoproteins for their ability to block the action of the ASF-DTA conjugate. Fetuin and orosomucoid, two glycoproteins with terminal sialic acid on their oligosaccharide chains, did not block the action of the conjugate. Their galactose-terminated asialo derivatives, ASF and asialoorosomucoid, as expected, did block the action of the conjugate. The N-acetylglucosaminyl-terminated derivative (asialogalactoorsomucoid) had no appreciable effect on the activity of the conjugate. We tested the ASF-DTA conjugate on six cell types; except for primary rat hepatocytes, none of them were affected by a high concentration (10 nM) of ASF-DTA conjugate. A fetuin-DTA conjugate was less toxic by a factor of 300 than the ASF-DTA conjugate and exerted its effects primarily through non-receptor-mediated mechanisms. The highly toxic ASF-DTA conjugate is cell-type specific, and its action is mediated by a well-characterized receptor, whose mechanism of receptor-ligand internalization has been extensively investigated. Images PMID:6167984

  6. Asialoglycoprotein receptor mediates the toxic effects of an asialofetuin-diphtheria toxin fragment A conjugate on cultured rat hepatocytes

    SciTech Connect

    Cawley, D.B.; Simpson, D.L.; Herschman, H.R.

    1981-06-01

    We have constructed a toxic hybrid protein that is recognized by asialoglycoprotein (ASGP) receptors of cultured rat hepatocytes. The conjugate consists of fragment A of diphtheria toxin (DTA) linked by a disulfide bond to asialofetuin (ASF). This conjugate is highly toxic, inhibiting protein synthesis in primary rat hepatocytes at concentrations as low as 10 pM. The ASF-DTA conjugate was 600 and 1800 times as toxic as diphtheria toxin and DTA, respectively, on primary rat hepatocytes. The ASGP receptor recognizes galactose-terminated proteins. We tested a series of glycoproteins for their ability to block the action of the ASF-DTA conjugate. Fetuin and orosomucoid, two glycoproteins with terminal sialic acid on their oligosaccharide chains, did not block the action of the conjugate. Their galactose-terminated asialo derivatives, ASF and asialoorosomucoid, as expected, did block the action of the conjugate. The N-acetylglucosaminyl-terminated derivative (asialoagalactoorosomucoid) had no appreciable effect on the activity of the conjugate. We tested the ASF-DTA conjugate on six cell types; except for primary rat hepatocytes, none of them were affected by a high concentration (10 nM) of ASF-DTA conjugate. A fetuin-DTA conjugate was less toxic by a factor of 300 than the ASF-DTA conjugate and exerted its effects primarily through non-receptor-mediated mechanisms. The highly toxic ASF-DTA conjugate is cell-type specific, and its action is mediated by a well-characterized receptor, whose mechanism of receptor-ligand internalization has been extensively investigated.

  7. Role of estrogen in diastolic dysfunction.

    PubMed

    Zhao, Zhuo; Wang, Hao; Jessup, Jewell A; Lindsey, Sarah H; Chappell, Mark C; Groban, Leanne

    2014-03-01

    The prevalence of left ventricular diastolic dysfunction (LVDD) sharply increases in women after menopause and may lead to heart failure. While evidence suggests that estrogens protect the premenopausal heart from hypertension and ventricular remodeling, the specific mechanisms involved remain elusive. Moreover, whether there is a protective role of estrogens against cardiovascular disease, and specifically LVDD, continues to be controversial. Clinical and basic science have implicated activation of the renin-angiotensin-aldosterone system (RAAS), linked to the loss of ovarian estrogens, in the pathogenesis of postmenopausal diastolic dysfunction. As a consequence of increased tissue ANG II and low estrogen, a maladaptive nitric oxide synthase (NOS) system produces ROS that contribute to female sex-specific hypertensive heart disease. Recent insights from rodent models that mimic the cardiac phenotype of an estrogen-insufficient or -deficient woman (e.g., premature ovarian failure or postmenopausal), including the ovariectomized congenic mRen2.Lewis female rat, provide evidence showing that estrogen modulates the tissue RAAS and NOS system and related intracellular signaling pathways, in part via the membrane G protein-coupled receptor 30 (GPR30; also called G protein-coupled estrogen receptor 1). Complementing the cardiovascular research in this field, the echocardiographic correlates of LVDD as well as inherent limitations to its use in preclinical rodent studies will be briefly presented. Understanding the roles of estrogen and GPR30, their interactions with the local RAAS and NOS system, and the relationship of each of these to LVDD is necessary to identify new therapeutic targets and alternative treatments for diastolic heart failure that achieve the cardiovascular benefits of estrogen replacement without its side effects and contraindications.

  8. Role of estrogen in diastolic dysfunction

    PubMed Central

    Zhao, Zhuo; Wang, Hao; Jessup, Jewell A.; Lindsey, Sarah H.; Chappell, Mark C.

    2014-01-01

    The prevalence of left ventricular diastolic dysfunction (LVDD) sharply increases in women after menopause and may lead to heart failure. While evidence suggests that estrogens protect the premenopausal heart from hypertension and ventricular remodeling, the specific mechanisms involved remain elusive. Moreover, whether there is a protective role of estrogens against cardiovascular disease, and specifically LVDD, continues to be controversial. Clinical and basic science have implicated activation of the renin-angiotensin-aldosterone system (RAAS), linked to the loss of ovarian estrogens, in the pathogenesis of postmenopausal diastolic dysfunction. As a consequence of increased tissue ANG II and low estrogen, a maladaptive nitric oxide synthase (NOS) system produces ROS that contribute to female sex-specific hypertensive heart disease. Recent insights from rodent models that mimic the cardiac phenotype of an estrogen-insufficient or -deficient woman (e.g., premature ovarian failure or postmenopausal), including the ovariectomized congenic mRen2.Lewis female rat, provide evidence showing that estrogen modulates the tissue RAAS and NOS system and related intracellular signaling pathways, in part via the membrane G protein-coupled receptor 30 (GPR30; also called G protein-coupled estrogen receptor 1). Complementing the cardiovascular research in this field, the echocardiographic correlates of LVDD as well as inherent limitations to its use in preclinical rodent studies will be briefly presented. Understanding the roles of estrogen and GPR30, their interactions with the local RAAS and NOS system, and the relationship of each of these to LVDD is necessary to identify new therapeutic targets and alternative treatments for diastolic heart failure that achieve the cardiovascular benefits of estrogen replacement without its side effects and contraindications. PMID:24414072

  9. The CB₁ receptor-mediated endocannabinoid signaling and NGF: the novel targets of curcumin.

    PubMed

    Hassanzadeh, Parichehr; Hassanzadeh, Anna

    2012-05-01

    Increasing interest has recently been attracted towards the identification of natural compounds including those with antidepressant properties. Curcumin has shown promising antidepressant effect, however, its molecular target(s) have not been well defined. Based on the interaction between the neurotrophins and endocannabinoid system as well as their contribution to the emotional reactivity and antidepressant action, here we show that 4-week treatment with curcumin, similar to the classical antidepressant amitriptyline, results in the sustained elevation of brain nerve growth factor (NGF) and endocannabinoids in dose-dependent and brain region-specific fashion. Pretreatment with cannabinoid CB(1) receptor neutral antagonist AM4113, but not the CB(2) antagonist SR144528, prevents the enhancement of brain NGF contents. AM4113 exerts no effect by itself. Our findings by presenting the CB(1) receptor-mediated endocannabinoid signaling and NGF as novel targets for curcumin, suggest that more attention should be focused on the therapeutic potential of herbal medicines including curcumin.

  10. Using GFP--ligand fusions to measure receptor-mediated endocytosis in living cells.

    PubMed

    Medina-Kauwe, Lali K; Chen, Xinhua

    2002-01-01

    Recombinant DNA technology has enabled the production of many types of chimeric proteins containing heterologous functional domains that have served a variety of useful capacities for cell biology research. Among proteins gaining wide use as a fusion partner is Aequorea victoria green fluorescent protein (GFP). GFP has been employed by numerous groups as a reporter gene for cell transfection and as an autofluorescent tag by recombinant fusion to foreign sequences. Here we describe the use of GFP as a tag for ligands, and provide examples of how purified recombinant GFP-ligand fusion proteins may be used to detect ligand-receptor interactions, including receptor-mediated endocytosis. Both its utility and limitations are discussed.

  11. The C-kit receptor-mediated signal transduction and tumor-related diseases.

    PubMed

    Liang, Jing; Wu, Yan-Ling; Chen, Bing-Jia; Zhang, Wen; Tanaka, Yoshimasa; Sugiyama, Hiroshi

    2013-01-01

    As an important member of tyrosine kinase family, c-kit receptor causes specific expression of certain genes, regulates cell differentiation and proliferation, resists cell apoptosis, and plays a key role in tumor occurrence, development, migration and recurrence through activating the downstream signaling molecules following interaction with stem cell factor (SCF). The abnormality of SCF/c-kit signaling pathway is closely related to some certain tumors. The discovery of c-kit receptor-targeted drugs has promoted clinical-related cancer's diagnosis and treatment. In this paper, we review recent research progress on c-kit receptor-mediated signal transduction and its potential therapeutic application as a target in tumor-related diseases.

  12. Bicarbonate contributes to GABAA receptor-mediated neuronal excitation in surgically resected human hypothalamic hamartomas.

    PubMed

    Kim, Do-Young; Fenoglio, Kristina A; Kerrigan, John F; Rho, Jong M

    2009-01-01

    The role of bicarbonate (HCO(3)(-)) in GABA(A) receptor-mediated depolarization of human hypothalamic hamartoma (HH) neurons was investigated using cellular electrophysiological and calcium imaging techniques. Activation of GABA(A) receptors with muscimol (30 microM) provoked neuronal excitation in over 70% of large (18-22 microM) HH neurons in HCO(3)(-) buffer. Subsequent perfusion of HCO(3)(-)-free HEPES buffer produced partial suppression of muscimol-induced excitation. Additionally, 53% of large HH neurons under HCO(3)(-)-free conditions exhibited reduced intracellular calcium accumulation by muscimol. These results suggest that HCO(3)(-) efflux through GABA(A) receptors on a subpopulation of large HH neurons may contribute to membrane depolarization and subsequent activation of L-type calcium channels.

  13. The C-Kit Receptor-Mediated Signal Transduction and Tumor-Related Diseases

    PubMed Central

    Liang, Jing; Wu, Yan-Ling; Chen, Bing-Jia; Zhang, Wen; Tanaka, Yoshimasa; Sugiyama, Hiroshi

    2013-01-01

    As an important member of tyrosine kinase family, c-kit receptor causes specific expression of certain genes, regulates cell differentiation and proliferation, resists cell apoptosis, and plays a key role in tumor occurrence, development, migration and recurrence through activating the downstream signaling molecules following interaction with stem cell factor (SCF). The abnormality of SCF/c-kit signaling pathway is closely related to some certain tumors. The discovery of c-kit receptor-targeted drugs has promoted clinical-related cancer's diagnosis and treatment. In this paper, we review recent research progress on c-kit receptor-mediated signal transduction and its potential therapeutic application as a target in tumor-related diseases. PMID:23678293

  14. Steroid induction of therapy-resistant cytokeratin-5-positive cells in estrogen receptor-positive breast cancer through a BCL6-dependent mechanism

    PubMed Central

    Goodman, C R; Sato, T; Peck, A R; Girondo, M A; Yang, N; Liu, C; Yanac, A F; Kovatich, A J; Hooke, J A; Shriver, C D; Mitchell, E P; Hyslop, T; Rui, H

    2016-01-01

    Therapy resistance remains a major problem in estrogen receptor-α (ERα)-positive breast cancer. A subgroup of ERα-positive breast cancer is characterized by mosaic presence of a minor population of ERα-negative cancer cells expressing the basal cytokeratin-5 (CK5). These CK5-positive cells are therapy resistant and have increased tumor-initiating potential. Although a series of reports document induction of the CK5-positive cells by progestins, it is unknown if other 3-ketosteroids share this ability. We now report that glucocorticoids and mineralocorticoids effectively expand the CK5-positive cell population. CK5-positive cells induced by 3-ketosteroids lacked ERα and progesterone receptors, expressed stem cell marker, CD44, and displayed increased clonogenicity in soft agar and broad drug-resistance in vitro and in vivo. Upregulation of CK5-positive cells by 3-ketosteroids required induction of the transcriptional repressor BCL6 based on suppression of BCL6 by two independent BCL6 small hairpin RNAs or by prolactin. Prolactin also suppressed 3-ketosteroid induction of CK5+ cells in T47D xenografts in vivo. Survival analysis with recursive partitioning in node-negative ERα-positive breast cancer using quantitative CK5 and BCL6 mRNA or protein expression data identified patients at high or low risk for tumor recurrence in two independent patient cohorts. The data provide a mechanism by which common pathophysiological or pharmacologic elevations in glucocorticoids or other 3-ketosteroids may adversely affect patients with mixed ERα+/CK5+ breast cancer. The observations further suggest a cooperative diagnostic utility of CK5 and BCL6 expression levels and justify exploring efficacy of inhibitors of BCL6 and 3-ketosteroid receptors for a subset of ERα-positive breast cancers. PMID:26096934

  15. Bisphenol A depresses monosynaptic and polysynaptic reflexes in neonatal rat spinal cord in vitro involving estrogen receptor-dependent NO-mediated mechanisms.

    PubMed

    Pandey, A K; Deshpande, S B

    2015-03-19

    Bisphenol A (BPA), a toxic chemical from plastics, is known to produce locomotor abnormalities which may imply the alteration in synaptic activity at Ia-α motoneuron synapse also. However the effect of BPA on this synapse is not known. Therefore, this study was undertaken to examine the effect of BPA on reflexes originating at Ia-α motoneuron synapse in the spinal cord. The experiments were performed on isolated hemisected spinal cords from 4 to 6d rats. Stimulation of a dorsal root evoked segmental monosynaptic (MSR) and polysynaptic (PSR) reflex potentials in the corresponding ventral root. Nitrite content (indicator of NO activity) of cords was estimated in the presence of BPA with/without antagonists. Superfusion of BPA (3-100μM) depressed the reflexes in a concentration- and time-dependent manner. The depression was ∼20, ∼50 and ∼70% at 10, 30 and 100μM of BPA, respectively. The 50% depression occurred around 15min at 30μM of BPA. Pretreatment with estrogen receptor (ERα) antagonist, tamoxifen, blocked the BPA-induced depression of reflexes, whereas, 17β-estradiol, ER agonist, did not depress the reflexes even up to 10μM. Further, pretreatment with Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME) or hemoglobin (Hb) blocked the BPA-induced depression of spinal reflexes. Nitric oxide (NO) donor sodium-nitroprusside depressed the MSR and PSR in a concentration-dependent manner. The nitrite concentration of the cords exposed to BPA was 733μM/gm of tissue (three times the saline group). Pretreatment with tamoxifen/l-NAME/Hb blocked the BPA-induced increase of nitrite levels. The present observations indicate that BPA depressed spinal synaptic transmission through ERα-dependent NO-mediated mechanisms. The altered synaptic activity may implicate for neurobehavioral locomotor abnormalities after exposure to BPA.

  16. Steroid induction of therapy-resistant cytokeratin-5-positive cells in estrogen receptor-positive breast cancer through a BCL6-dependent mechanism.

    PubMed

    Goodman, C R; Sato, T; Peck, A R; Girondo, M A; Yang, N; Liu, C; Yanac, A F; Kovatich, A J; Hooke, J A; Shriver, C D; Mitchell, E P; Hyslop, T; Rui, H

    2016-03-17

    Therapy resistance remains a major problem in estrogen receptor-α (ERα)-positive breast cancer. A subgroup of ERα-positive breast cancer is characterized by mosaic presence of a minor population of ERα-negative cancer cells expressing the basal cytokeratin-5 (CK5). These CK5-positive cells are therapy resistant and have increased tumor-initiating potential. Although a series of reports document induction of the CK5-positive cells by progestins, it is unknown if other 3-ketosteroids share this ability. We now report that glucocorticoids and mineralocorticoids effectively expand the CK5-positive cell population. CK5-positive cells induced by 3-ketosteroids lacked ERα and progesterone receptors, expressed stem cell marker, CD44, and displayed increased clonogenicity in soft agar and broad drug-resistance in vitro and in vivo. Upregulation of CK5-positive cells by 3-ketosteroids required induction of the transcriptional repressor BCL6 based on suppression of BCL6 by two independent BCL6 small hairpin RNAs or by prolactin. Prolactin also suppressed 3-ketosteroid induction of CK5+ cells in T47D xenografts in vivo. Survival analysis with recursive partitioning in node-negative ERα-positive breast cancer using quantitative CK5 and BCL6 mRNA or protein expression data identified patients at high or low risk for tumor recurrence in two independent patient cohorts. The data provide a mechanism by which common pathophysiological or pharmacologic elevations in glucocorticoids or other 3-ketosteroids may adversely affect patients with mixed ERα+/CK5+ breast cancer. The observations further suggest a cooperative diagnostic utility of CK5 and BCL6 expression levels and justify exploring efficacy of inhibitors of BCL6 and 3-ketosteroid receptors for a subset of ERα-positive breast cancers.

  17. Chronic psychoemotional stress impairs cannabinoid-receptor-mediated control of GABA transmission in the striatum.

    PubMed

    Rossi, Silvia; De Chiara, Valentina; Musella, Alessandra; Kusayanagi, Hajime; Mataluni, Giorgia; Bernardi, Giorgio; Usiello, Alessandro; Centonze, Diego

    2008-07-16

    Exposure to stressful events has a myriad of consequences in animals and in humans, and triggers synaptic adaptations in many brain areas. Stress might also alter cannabinoid-receptor-mediated transmission in the brain, but no physiological study has addressed this issue so far. In the present study, we found that social defeat stress, induced in mice by exposure to aggression, altered cannabinoid CB(1)-receptor-mediated control of synaptic transmission in the striatum. In fact, the presynaptic inhibition of GABAergic IPSCs induced by the cannabinoid CB(1) receptor agonist HU210 [(6aR)-trans-3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol] was reduced after a single stressful episode and fully abolished after 3 and 7 d of stress exposure. Repeated psychoemotional stress also impaired the sensitivity of GABA synapses to endocannabinoids mobilized by group I metabotropic glutamate receptor stimulation, whereas the cannabinoid CB(1)-mediated control of glutamate transmission was unaffected by repeated exposure to an aggressor. Corticosteroids released in response to the activation of the hypothalamic-pituitary-adrenal axis played a major role in the synaptic defects observed in stressed animals, because these alterations were fully prevented by pharmacological blockade of glucocorticoid receptors and were mimicked by corticosterone injections. The recovery of stress-induced synaptic defects was favored when stressed mice were given access to a running wheel or to sucrose consumption, which function as potent natural rewards. A similar rescuing effect was obtained by a single injection of cocaine, a psychostimulant with strong rewarding properties. Targeting cannabinoid CB(1) receptors or endocannabinoid metabolism might be a valuable option to treat stress-associated neuropsychiatric conditions.

  18. Extracellular acidosis impairs P2Y receptor-mediated Ca(2+) signalling and migration of microglia.

    PubMed

    Langfelder, Antonia; Okonji, Emeka; Deca, Diana; Wei, Wei-Chun; Glitsch, Maike D

    2015-04-01

    Microglia are the resident macrophage and immune cell of the brain and are critically involved in combating disease and assaults on the brain. Virtually all brain pathologies are accompanied by acidosis of the interstitial fluid, meaning that microglia are exposed to an acidic environment. However, little is known about how extracellular acidosis impacts on microglial function. The activity of microglia is tightly controlled by 'on' and 'off' signals, the presence or absence of which results in generation of distinct phenotypes in microglia. Activation of G protein coupled purinergic (P2Y) receptors triggers a number of distinct behaviours in microglia, including activation, migration, and phagocytosis. Using pharmacological tools and fluorescence imaging of the murine cerebellar microglia cell line C8B4, we show that extracellular acidosis interferes with P2Y receptor-mediated Ca(2+) signalling in these cells. Distinct P2Y receptors give rise to signature intracellular Ca(2+) signals, and Ca(2+) release from stores and Ca(2+) influx are differentially affected by acidotic conditions: Ca(2+) release is virtually unaffected, whereas Ca(2+) influx, mediated at least in part by store-operated Ca(2+) channels, is profoundly inhibited. Furthermore, P2Y1 and P2Y6-mediated stimulation of migration is inhibited under conditions of extracellular acidosis, whereas basal migration independent of P2Y receptor activation is not. Taken together, our results demonstrate that an acidic microenvironment impacts on P2Y receptor-mediated Ca(2+) signalling, thereby influencing microglial responses and responsiveness to extracellular signals. This may result in altered behaviour of microglia under pathological conditions compared with microglial responses in healthy tissue. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Voltage-dependent inhibition of recombinant NMDA receptor-mediated currents by 5-hydroxytryptamine

    PubMed Central

    Kloda, Anna; Adams, David J

    2005-01-01

    The effect of 5-HT and related indolealkylamines on heteromeric recombinant NMDA receptors expressed in Xenopus oocytes was investigated using the two-electrode voltage-clamp recording technique. In the absence of external Mg2+ ions, 5-HT inhibited NMDA receptor-mediated currents in a concentration-dependent manner. The inhibitory effect of 5-HT was independent of the NR1a and NR2 subunit combination. The inhibition of glutamate-evoked currents by 5-HT was use- and voltage-dependent. The voltage sensitivity of inhibition for NR1a+NR2 subunit combinations by 5-HT was similar, exhibiting an e-fold change per ∼20 mV, indicating that 5-HT binds to a site deep within the membrane electric field. The inhibition of the open NMDA receptor by external Mg2+ and 5-HT was not additive, suggesting competition between Mg2+ and 5-HT for a binding site in the NMDA receptor channel. The concentration-dependence curves for 5-HT and 5-methoxytryptamine (5-MeOT) inhibition of NMDA receptor-mediated currents are shifted to the right in the presence of external Mg2+. The related indolealkylamines inhibited glutamate-evoked currents with the following order of inhibitory potency: 5-MeOT=5-methyltryptamine>tryptamine>7-methyltryptamine>5-HT≫tryptophan=melatonin. Taken together, these data suggest that 5-HT and related compounds can attenuate glutamate-mediated excitatory synaptic responses and may provide a basis for drug treatment of excitoxic neurodegeneration. PMID:15655527

  20. Current injection and receptor-mediated excitation produce similar maximal firing rates in hypoglossal motoneurons.

    PubMed

    Wakefield, Hilary E; Fregosi, Ralph F; Fuglevand, Andrew J

    2016-03-01

    The maximum firing rates of motoneurons (MNs), activated in response to synaptic drive, appear to be much lower than that elicited by current injection. It could be that the decrease in input resistance associated with increased synaptic activity (but not current injection) might blunt overall changes in membrane depolarization and thereby limit spike-frequency output. To test this idea, we recorded, in the same cells, maximal firing responses to current injection and to synaptic activation. We prepared 300 μm medullary slices in neonatal rats that contained hypoglossal MNs and used whole-cell patch-clamp electrophysiology to record their maximum firing rates in response to triangular-ramp current injections and to glutamate receptor-mediated excitation. Brief pressure pulses of high-concentration glutamate led to significant depolarization, high firing rates, and temporary cessation of spiking due to spike inactivation. In the same cells, we applied current clamp protocols that approximated the time course of membrane potential change associated with glutamate application and with peak current levels large enough to cause spike inactivation. Means (SD) of maximum firing rates obtained in response to glutamate application were nearly identical to those obtained in response to ramp current injection [glutamate 47.1 ± 12.0 impulses (imp)/s, current injection 47.5 ± 11.2 imp/s], even though input resistance was 40% less during glutamate application compared with current injection. Therefore, these data suggest that the reduction in input resistance associated with receptor-mediated excitation does not, by itself, limit the maximal firing rate responses in MNs. Copyright © 2016 the American Physiological Society.

  1. Estrogen modulates cognitive and cholinergic processes in surgically menopausal monkeys.

    PubMed

    Tinkler, Gregory Paul; Voytko, Mary Lou

    2005-03-01

    Estrogen deficiency in postmenopausal women is associated with changes in physiological processes. The extent to which estrogen loss is associated with cognitive changes noted by postmenopausal women has been more difficult to determine for a variety of reasons. Primate models of menopause are now being used to determine the effects of estrogen loss and replacement on cognitive abilities and to investigate the neural mechanisms by which estrogen may influence cognitive function. The present report presents data from cognitive and neurobiological studies in surgically menopausal monkeys that have examined how estrogen loss and replacement may be affecting cognitive abilities and the cholinergic system; a neural system that is known to influence memory and attention function. These studies are indicating that visuospatial attention function is especially sensitive to estrogen states in young monkeys, but that multiple cognitive domains are sensitive to estrogen states in middle-aged monkeys. In addition, anatomical and functional imaging studies indicate that the primate cholinergic system is modulated by estrogen, and pharmacological studies demonstrate that estrogen uses cholinergic muscarinic receptors to influence visuospatial attention. These studies demonstrate that estrogen influences cognitive abilities in monkey models of menopause and the cholinergic system may be one of the mechanisms by which estrogen modulates cognitive function. Given the current unknowns and concerns regarding the use of hormone replacement therapy in postmenopausal women, continued studies in monkey models of menopause are especially needed to further elucidate the effects of estrogen on cognitive and neurobiological processes, with particular emphasis on studies in middle-aged monkeys, determining the optimal aspects of ERT regimens, and identifying the relationships between estrogen effects on cognitive and neurobiological function.

  2. Estrogen and estrogen receptors in cardiovascular oxidative stress.

    PubMed

    Arias-Loza, Paula-Anahi; Muehlfelder, Melanie; Pelzer, Theo

    2013-05-01

    The cardiovascular system of a premenopausal woman is prepared to adapt to the challenges of increased cardiac output and work load that accompany pregnancy. Thus, it is tempting to speculate whether enhanced adaptability of the female cardiovascular system might be advantageous under conditions that promote cardiovascular disease. In support of this concept, 17β-estradiol as the major female sex hormone has been shown to confer protective cardiovascular effects in experimental studies. Mechanistically, these have been partially linked to the prevention and protection against oxidative stress. Current evidence indicates that estrogens attenuate oxidative stress at two levels: first, by preventing generation of reactive oxygen species (ROS) and, second, by scavenging ROS in the myocardium and in the vasculature. The purpose of this review is to give an overview on current concepts on conditions and mechanisms by which estrogens protect the cardiovascular system against ROS-mediated cellular injury.

  3. 2-Methoxyestradiol, an Endogenous Estrogen Metabolite, Sensitizes Radioresistant MCF-7/FIR Breast Cancer Cells Through Multiple Mechanisms

    SciTech Connect

    Salama, Salama; Diaz-Arrastia, Concepcion; Patel, Deepa; Botting, Shaleen; Hatch, Sandra

    2011-05-01

    Purpose: The requirement for a well-tolerated and highly effective radiosensitizer that preferentially sensitizes tumor cells at multiple levels of radioresistance remains largely unmet. 2-Methoxyestradiol (2ME) has polypharmacological profiles that target multiple signaling pathways involved in the development of radioresistance. In the current study, we investigated the radiosensitizing effect of 2ME on the radioresistant breast cancer MCF-7/FIR cell line and explored the underlying mechanisms. Methods and Materials: The radiosensitizing effect of 2ME was evaluated on the basis of cell death and clonogenic survival. Formation of reactive oxygen species (ROS), apoptosis, and cell cycle progression were assessed by flow cytometry. Radiation-induced DNA damage was evaluated on the basis of histone {gamma}-H2AX phosphorylation and foci formation. Immunoblotting was used to assess the effects of {gamma} radiation and/or 2ME on radioresistance pathways. Results: Our data demonstrate that MCF-7/FIR cells expressed higher levels of Bcl-2 and HIF-1{alpha} and displayed a lower ROS phenotype than the parental MCF-7 cells. Treatment of parental MCF-7 cells with 2ME (0.5 {mu}M) had minimal effect on {gamma} radiation-induced cell proliferation and surviving fractions. On the contrary, in MCF-7/FIR cells, treatment with 2ME significantly enhanced {gamma} radiation-induced reduction in cell proliferation and surviving fraction. This combination was effective in activating apoptosis, arresting the cell cycle at the G{sub 2}/M phase, and increasing the level of {gamma} radiation-induced ROS and the number of {gamma}-H2AX foci. In addition, 2ME significantly ameliorated {gamma} radiation-induced expression of the HIF-1{alpha} transcription factor and its downstream targets AKT/mTOR. Conclusion: 2ME preferentially sensitizes radioresistant MCF-7/FIR cells to {gamma} radiation by targeting multiple signaling pathways involved in the development of radioresistance. This

  4. Synthesis and mannose receptor-mediated uptake of clustered glycomimetics by human dendritic cells: effect of charge.

    PubMed

    Angyalosi, Gerhild; Grandjean, Cyrille; Lamirand, Mélanie; Auriault, Claude; Gras-Masse, Hélène; Melnyk, Oleg

    2002-10-07

    Effect of charge and shape of multivalent lysine-based cluster glycomimetics on their mannose receptor-mediated uptake by human dendritic cells has been evaluated: The capture is strongly affected by the shape of the ligands. The effect of charge is less pronounced although positive charges on the ligands seem to favor non-specific endocytosis capture.

  5. Human decay-accelerating factor and CEACAM receptor-mediated internalization and intracellular lifestyle of Afa/Dr diffusely adhering Escherichia coli in epithelial cells.

    PubMed

    Guignot, Julie; Hudault, Sylvie; Kansau, Imad; Chau, Ingrid; Servin, Alain L

    2009-01-01

    We used transfected epithelial CHO-B2 cells as a model to identify the mechanism mediating internalization of Afa/Dr diffusely adhering Escherichia coli. We provide evidence that neither the alpha5 or beta1 integrin subunits nor alpha5beta1 integrin functioned as a receptor mediating the adhesion and/or internalization of Dr or Afa-III fimbria-positive bacteria. We also demonstrated that (i) whether or not the AfaD or DraD invasin subunits were present, there was no difference in the cell association and entry of bacteria and that (ii) DraE or AfaE-III adhesin subunits are necessary and sufficient to promote the receptor-mediated bacterial internalization into epithelial cells expressing human decay-accelerating factor (DAF), CEACAM1, CEA, or CEACAM6. Internalization of Dr fimbria-positive E. coli within CHO-DAF, CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells occurs through a microfilament-independent, microtubule-dependent, and lipid raft-dependent mechanism. Wild-type Dr fimbria-positive bacteria survived better within cells expressing DAF than bacteria internalized within CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells. In DAF-positive cells, internalized Dr fimbria-positive bacteria were located in vacuoles that contained more than one bacterium, displaying some of the features of late endosomes, including the presence of Lamp-1 and Lamp-2, and some of the features of CD63 proteins, but not of cathepsin D, and were acidic. No interaction between Dr fimbria-positive-bacterium-containing vacuoles and the autophagic pathway was observed.

  6. AT1 receptor-mediated accumulation of extracellular angiotensin II in proximal tubule cells: role of cytoskeleton microtubules and tyrosine phosphatases

    PubMed Central

    Li, Xiao C.; Carretero, Oscar A.; Navar, L. Gabriel; Zhuo, Jia L.

    2008-01-01

    Long-term angiotensin II (ANG II) administration is associated with increased ANG II accumulation in the kidney, but intrarenal compartment(s) involved in this response remains to be determined. We tested the hypothesis that 1) extracellular ANG II is taken up by proximal tubule cells (PTCs) through AT1 receptor-mediated endocytosis, 2) this process is regulated by cytoskeleton microtubule- and tyrosine phosphatase-dependent mechanisms, and 3) AT1 receptor-mediated endocytosis of ANG II has a functional relevance by modulating intracellular cAMP signaling. In cultured PTCs, [125I]Tyr-labeled ANG II and fluorescein labeled-ANG II were internalized in a time-dependent manner and colocalized with the endosome marker Alexa Fluor 594-transferrin. Endocytosis of extracellular ANG II was inhibited by the AT1 receptor blocker losartan (16.5 ± 4.6%, P < 0.01 vs. ANG II, 78.3 ± 6.2%) and by the tyrosine phosphatase inhibitor phenylarsine oxide (PAO; 30.0 ± 3.5%, P < 0.05 vs. ANG II). Intracellular ANG II levels were increased by ~58% (basal, 229.8 ± 11.4 vs. ANG II, 361.3 ± 11.8 pg ANG II/mg protein, P < 0.01), and the responses were blocked by losartan (P < 0.01), the cytoskeleton microtubule inhibitor colchicine (P < 0.05), and PAO (P < 0.01), whereas depletion of clathrin-coated pits with hyperosmotic sucrose had no effect (356.1 ± 25.5 pg ANG II/mg protein, not significant). ANG II accumulation was associated with significant inhibition of both basal (control, 15.5 ± 2.8 vs. ANG II, 9.1 ± 2.4 pmol/mg protein, P < 0.05) and forskolin-stimulated cAMP signaling (forskolin, 68.7 ± 8.6 vs. forskolin + ANG II, 42.8 ± 13.8 pmol/mg protein, P < 0.01). These effects were blocked by losartan and PAO. We conclude that extracellular ANG II is internalized in PTCs through AT1 receptor-mediated endocytosis and that internalized ANG II may play a functional role in proximal tubule cells by inhibiting intracellular cAMP signaling. PMID:16478976

  7. Activation of a Gq-coupled membrane estrogen receptor rapidly attenuates α2-adrenoceptor-induced antinociception via an ERK I/II-dependent, non-genomic mechanism in the female rat

    PubMed Central

    Nag, Subodh; Mokha, Sukhbir S.

    2014-01-01

    Though sex differences in pain and analgesia are known, underlying mechanisms remain elusive. This study addresses the selective contribution of membrane estrogen receptors (mER) and mER-initiated non-genomic signaling mechanisms in our previously reported estrogen-induced attenuation of α2- adrenoceptor-mediated antinociception. By selectively targeting spinal mERs in ovariectomized female rats using E2BSA (membrane impermeant estradiol analogue), and ERα selective agonist PPT, ERβ selective agonist DPN, GPR 30 agonist G1 and Gq-coupled mER (Gq-mER) agonist STX, we provide strong evidence that Gq-mER activation may solely contribute to suppressing clonidine (an α2- adrenoceptor agonist)-induced antinociception, using the nociceptive tail flick test. Increased tail flick latencies (TFL) by intrathecal (i.t.) clonidine were not significantly altered by i.t. PPT, DPN, or G1. In contrast, E2BSA or STX rapidly and dose-dependently attenuated clonidine-induced increase in TFL. ICI 182,780, the ER antagonist, blocked this effect. Consistent with findings with the lack of effect of ERα and ERβ agonists that modulate receptor-regulated transcription, inhibition of de novo protein synthesis using anisomycin also failed to alter the effect of E2BSA or STX, arguing against a contribution of genomic mechanisms. Immunoblotting of spinal tissue revealed that mER activation increased levels of phosphorylated extracellular signal regulated kinase (ERK) but not of protein kinase A (PKA) or C (PKC). In vivo inhibition of ERK with U0126 blocked the effect of STX and restored clonidine antinociception. Although estrogen-induced delayed genomic mechanisms may still exist, data presented here indicate that Gq-mER may solely mediate estradiol-induced attenuation of clonidine antinociception via a rapid, reversible, and ERK-dependent, non-genomic mechanism, suggesting that Gq-mER blockade might provide improved analgesia in females. PMID:24613724

  8. Melatonin receptor-mediated protection against myocardial ischaemia/reperfusion injury: role of its anti-adrenergic actions.

    PubMed

    Genade, Sonia; Genis, Amanda; Ytrehus, Kirsti; Huisamen, Barbara; Lochner, Amanda

    2008-11-01

    Melatonin has potent cardioprotective properties. These actions have been attributed to its free radical scavenging and anti-oxidant actions, but may also be receptor mediated. Melatonin also exerts powerful anti-adrenergic actions based on its effects on contractility of isolated papillary muscles. The aims of this study were to determine whether melatonin also has anti-adrenergic effects on the isolated perfused rat heart, to determine the mechanism thereof and to establish whether these actions contribute to protection of the heart during ischaemia/reperfusion. The results showed that melatonin (50 microM) caused a significant reduction in both isoproterenol (10(-7) M) and forskolin (10(-6) M) induced cAMP production and that both these responses were melatonin receptor dependent, since the blocker, luzindole (5 x 10(-6) M) abolished this effect. Nitric oxide (NO), as well as guanylyl cyclase are involved, as L-NAME (50 microM), an NO synthase inhibitor and ODQ (20 microM), a guanylyl cyclase inhibitor, significantly counteracted the effects of melatonin. Protein kinase C (PKC), as indicated by the use of the inhibitor bisindolylmaleimide (50 microM), also play a role in melatonin's anti-adrenergic actions. These actions of melatonin are involved in its cardioprotection: simultaneous administration of L-NAME or ODQ with melatonin, before and after 35 min regional ischaemia, completely abolished its cardioprotection. PKC, on the other hand, had no effect on the melatonin-induced reduction in infarct size. Cardioprotection by melatonin was associated with a significant activation of PKB/Akt and attenuated activation of the pro-apoptotic kinase, p38MAPK during early reperfusion. In summary, the results show that melatonin-induced cardioprotection may be receptor dependent, and that its anti-adrenergic actions, mediated by NOS and guanylyl cyclase activation, are important contributors.

  9. Rab11-family interacting protein 2 and myosin Vb are required for CXCR2 recycling and receptor-mediated chemotaxis.

    PubMed

    Fan, Guo-Huang; Lapierre, Lynne A; Goldenring, James R; Sai, Jiqing; Richmond, Ann

    2004-05-01

    Agonist-stimulated internalization followed by recycling to the cell membrane play an important role in fine-tuning the activity of chemokine receptors. Because the recycling of chemokine receptors is critical for the reestablishment of the cellular responsiveness to ligand, it is crucial to understand the mechanisms underlying the receptor recycling and resensitization. In the present study, we have demonstrated that the chemokine receptor CXCR2 associated with myosin Vb and Rab11-family interacting protein 2 (FIP2) in a ligand-dependent manner. Truncation of the C-terminal domain of the receptor did not affect the association, suggesting that the interactions occur upstream of the C terminus of CXCR2. After ligand stimulation, the internalized CXCR2 colocalized with myosin Vb and Rab11-FIP2 in Rab11a-positive vesicles. The colocalization lasted for approximately 2 h, and little colocalization was observed after 4 h of ligand stimulation. CXCR2 also colocalized with myosin Vb tail or Rab11-FIP2 (129-512), the N-terminal-truncated mutants of myosin Vb and Rab11-FIP2, respectively, but in a highly condensed manner. Expression of the enhanced green fluorescent protein-tagged myosin Vb tail significantly retarded the recycling and resensitization of CXCR2. CXCR2 recycling was also reduced by the expression Rab11-FIP2 (129-512). Moreover, expression of the myosin Vb tail reduced CXCR2- and CXCR4-mediated chemotaxis. These data indicate that Rab11-FIP2 and myosin Vb regulate CXCR2 recycling and receptor-mediated chemotaxis and that passage of internalized CXCR2 through Rab11a-positive recycling system is critical for physiological response to a chemokine.

  10. Estrogenic Compounds, Estrogen Receptors and Vascular Cell Signaling in the Aging Blood Vessels

    PubMed Central

    Smiley, Dia A.; Khalil, Raouf A.

    2010-01-01

    The cardiovascular benefits of menopausal hormone therapy (MHT) remain controversial. The earlier clinical observations that cardiovascular disease (CVD) was less common in MHT users compared to non-users suggested cardiovascular benefits of MHT. Also, experimental studies have identified estrogen receptors ERα, ERβ and GPR30, which mediate genomic or non-genomic effects in vascular endothelium, smooth muscle, and extracellular matrix (ECM). However, data from randomized clinical trials (RCTs), most notably the Women's Health Initiative (WHI) study, have challenged the cardiovascular benefits and highlighted adverse cardiovascular events with MHT. The discrepancies have been attributed to the design of RCTs, the subjects' advanced age and preexisting CVD, and the form of estrogen used. The discrepancies may also stem from age-related changes in vascular ER amount, distribution, integrity, and post-receptor signaling pathways as well as structural changes in the vasculature. Age-related changes in other sex hormones such as testosterone may also alter the hormonal environment and influence the cardiovascular effects of estrogen. Investigating the chemical properties, structure-activity relationship and pharmacology of natural and synthetic estrogens should improve the effectiveness of conventional MHT. Further characterization of phytoestrogens, selective estrogen-receptor modulators (SERMs), and specific ER agonists may provide substitutes to conventional MHT. Conditions with excess or low estrogen levels such as polycystic ovary syndrome (PCOS) and Turner syndrome may provide insight into the development and regulation of ER and the mechanisms of aberrant estrogen-ER interactions. The lessons learned from previous RCTs have led to more directed studies such as the Kronos Early Estrogen Prevention Study (KEEPS). Careful design of experimental models and RCTs, coupled with the development of specific ER modulators, hold the promise of improving the actions of

  11. Estrogenic compounds, estrogen receptors and vascular cell signaling in the aging blood vessels.

    PubMed

    Smiley, Dia A; Khalil, Raouf A

    2009-01-01

    The cardiovascular benefits of menopausal hormone therapy (MHT) remain controversial. The earlier clinical observations that cardiovascular disease (CVD) was less common in MHT users compared to non-users suggested cardiovascular benefits of MHT. Also, experimental studies have identified estrogen receptors ERalpha, ERbeta and GPR30, which mediate genomic or non-genomic effects in vascular endothelium, smooth muscle, and extracellular matrix (ECM). However, data from randomized clinical trials (RCTs), most notably the Women's Health Initiative (WHI) study, have challenged the cardiovascular benefits and highlighted adverse cardiovascular events with MHT. The discrepancies have been attributed to the design of RCTs, the subjects' advanced age and preexisting CVD, and the form of estrogen used. The discrepancies may also stem from age-related changes in vascular ER amount, distribution, integrity, and post-receptor signaling pathways as well as structural changes in the vasculature. Age-related changes in other sex hormones such as testosterone may also alter the hormonal environment and influence the cardiovascular effects of estrogen. Investigating the chemical properties, structure-activity relationship and pharmacology of natural and synthetic estrogens should improve the effectiveness of conventional MHT. Further characterization of phytoestrogens, selective estrogen-receptor modulators (SERMs), and specific ER agonists may provide substitutes to conventional MHT. Conditions with excess or low estrogen levels such as polycystic ovary syndrome (PCOS) and Turner syndrome may provide insight into the development and regulation of ER and the mechanisms of aberrant estrogen-ER interactions. The lessons learned from previous RCTs have led to more directed studies such as the Kronos Early Estrogen Prevention Study (KEEPS). Careful design of experimental models and RCTs, coupled with the development of specific ER modulators, hold the promise of improving the actions of

  12. In vitro biomonitoring in polar extracts of solid phase matrices reveals the presence of unknown compounds with estrogenic activity.

    PubMed

    Legler, J; Leonards, P; Spenkelink, A; Murk, A J

    2003-01-01

    Determination of estrogenic activity has so far mainly concentrated on the assessment of compounds in surface water and effluent. This study is one of the first to biomonitor (xeno-)estrogens in sediment, suspended particulate matter and aquatic organisms. The relatively polar acetone extracts from these solid phase matrices do not contain the well-known estrogenic compounds such as hormones, alkylphenols and phthalates. An in vitro 'estrogen receptor-mediated chemical activated luciferase gene expression' (ER-CALUX) assay was applied to samples from various locations in the Netherlands. Estrogenic activity measured in polar fractions of particulate matter and sediment extracts ranged from below detection limit to up to 4.5 pmol estradiol equivalents (EEQ)/g dry weight. Estrogenic activity in freshwater river sediments was up to five times higher compared to sediments from large lakes and coastal locations. Tissue extracts EEQs were determined in bream (Abramis brama), flounder (Platichthysflesus), freshwater mussels (Dreissena polymorpha) and marine mussels (Mytilus edulis). The highest biota EEQ levels were found in the freshwater zebra mussel (30 pmol EEQ/g lipid). One sample site showed greatly elevated EEQs in sediment and biota, which correlated with effects found in the wild populations of bream. The EEQ activity of the unknown compounds in the polar fraction mostly was much higher than the calculated EEQ levels based on known estrogens in the non-polar fraction (previously published data).

  13. Extra-gonadal sites of estrogen biosynthesis and function

    PubMed Central

    Barakat, Radwa; Oakley, Oliver; Kim, Heehyen; Jin, Jooyoung; Ko, CheMyong Jay

    2016-01-01

    Estrogens are the key hormones regulating the development and function of reproductive organs in all vertebrates. Recent evidence indicates that estrogens play important roles in the immune system, cancer development, and other critical biological processes related to human well-being. Obviously, the gonads (ovary and testis) are the primary sites of estrogen synthesis, but estrogens synthesized in extra- gonadal sites play an equally important role in controlling biological activities. Understanding non-gonadal sites of estrogen synthesis and function is crucial and will lead to therapeutic interventions targeting estrogen signaling in disease prevention and treatment. Developing a rationale targeting strategy remains challenging because knowledge of extra-gonadal biosynthesis of estrogens, and the mechanism by which estrogen activity is exerted, is very limited. In this review, we will summarize recent discoveries of extra-gonadal sites of estrogen biosynthesis and their local functions and discuss the significance of the most recent novel discovery of intestinal estrogen biosynthesis. [BMB Reports 2016; 49(9): 488-496] PMID:27530684

  14. Circulating microparticles and endogenous estrogen in newly menopausal women.

    PubMed

    Jayachandran, M; Litwiller, R D; Owen, W G; Miller, V M

    2009-04-01

    Estrogen modulates antithrombotic characteristics of the vascular endothelium and the interaction of blood elements with the vascular surface. A marker of these modulatory activities is formation of cell-specific microparticles. This study examined the relationship between blood-borne microparticles and endogenous estrogen at menopause. Platelet activation and plasma microparticles were characterized from women being screened (n = 146) for the Kronos Early Estrogen Prevention Study. Women were grouped according to serum estrogen (< 20 pg/ml; low estrogen, n = 21 or > 40 pg/ml; high estrogen, n = 11). Age, body mass index, blood pressure and blood chemistries were the same in both groups. No woman was hypertensive, diabetic or a current smoker. Platelet counts, basal and activated expression of P-selectin on platelet membranes were the same, but activated expression of glycoprotein IIb/IIIa was greater in the high-estrogen group. Numbers of endothelium-, platelet-, monocyte- and granulocyte-derived microparticles were greater in the low-estrogen group. Of the total numbers of microparticles, those positive for phosphatidylserine and tissue factor were also greater in the low-estrogen group. These results suggest that, with declines in endogenous estrogen at menopause, numbers of procoagulant microparticles increase and thus may provide a means to explore mechanisms for cardiovascular risk development in newly menopausal women.

  15. Signaling by estrogens and tamoxifen in the human endometrium.

    PubMed

    Gielen, Susanne C J P; Santegoets, Lindy A M; Hanifi-Moghaddam, Payman; Burger, Curt W; Blok, Leen J

    2008-04-01

    Tamoxifen is used as adjuvant treatment for postmenopausal breast cancer patients. The mechanism of action of tamoxifen in breast cancer patients is that tamoxifen inhibits growth of cancer cells by competitive antagonism for estrogens at the estrogen receptor (ER). In the endometrium, tamoxifen has an effect that varies with the ambient concentration of estrogen: in premenopausal women (high estrogen levels), tamoxifen displays an estrogen-antagonistic effect, while in postmenopausal women (low estrogen levels), tamoxifen displays an estrogen-agonistic mode of action. Here, using microarray technology we have compared estrogen signaling with tamoxifen signaling in the human endometrium. It was observed that on the one hand tamoxifen-treatment results in modulation of expression of specific genes (370 genes) and on the other hand tamoxifen-treatment results in modulation of a set of genes which are also regulated by estrogen treatment (142 genes). Upon focusing on regulation of proliferation, we found that tamoxifen-induced endometrial proliferation is largely accomplished by using the same set of genes as are regulated by estradiol. So, as far as regulation of proliferation goes, tamoxifen seems to act as estrogen agonist. Furthermore, tamoxifen-specific gene regulation may explain why tamoxifen-induced endometrial tumors behave more aggressively than sporadic endometrial tumors.

  16. The Estrogen Hypothesis of Obesity

    PubMed Central

    Grantham, James P.; Henneberg, Maciej

    2014-01-01

    The explanation of obesity as a simple result of positive energy balance fails to account for the scope of variable responses to diets and lifestyles. It is postulated that individual physiological and anatomical variation may be responsible for developing obesity. Girls in poor families develop greater adiposity than their male siblings, a trend not present in richer environments. This indicates strong influence of estrogen on fat accumulation irrespective of poor socioeconomic conditions. Obesity rates in males and females of developed nations are similar, while in poorer nations obesity is much more prevalent in females. Female to male ratio of obesity correlates inversely with gross domestic product. Therefore, the parity of male and female obesity in developed countries may result from male exposure to environmental estrogen-like substances associated with affluence. These hormonally driven mechanisms may be equally active within both sexes in more developed areas, thereby increasing overall obesity. PMID:24915457

  17. Decreases in mitochondrial reactive oxygen species initiate GABAA receptor-mediated electrical suppression in anoxia-tolerant turtle neurons

    PubMed Central

    Hogg, David W; Pamenter, Matthew E; Dukoff, David J; Buck, Leslie T

    2015-01-01

    Key points Anoxia induces hyper-excitability and cell death in mammalian brain but in the western painted turtle (Chrysemys picta bellii) enhanced GABA transmission prevents injury. The mechanism responsible for increased GABA transmission is unknown; however, reactive oxygen species (ROS) generated by mitochondria may play a role because this is an oxygen-sensitive process. In this study, we show that inhibition of mitochondrial ROS production is sufficient to initiate a redox-sensitive GABA signalling cascade that suppresses pyramidal neuron action potential frequency. These results further our understanding of the turtle's unique strategy for reducing ATP consumption during anoxia and highlights a natural mechanism in which to explore therapies to protect mammalian brain from low-oxygen insults (e.g. cerebral stroke). Abstract Anoxia induces hyper-excitability and cell death in mammalian brain but in the anoxia-tolerant western painted turtle (Chrysemys picta bellii) neuronal electrical activity is suppressed (i.e. spike arrest), adenosine triphosphate (ATP) consumption is reduced, and cell death does not occur. Electrical suppression is primarily the result of enhanced γ-aminobutyric acid (GABA) transmission; however, the underlying mechanism responsible for initiating oxygen-sensitive GABAergic spike arrest is unknown. In turtle cortical pyramidal neurons there are three types of GABAA receptor-mediated currents: spontaneous inhibitory postsynaptic currents (IPSCs), giant IPSCs and tonic currents. The aim of this study was to assess the effects of reactive oxygen species (ROS) scavenging on these three currents since ROS levels naturally decrease with anoxia and may serve as a redox signal to initiate spike arrest. We found that anoxia, pharmacological ROS scavenging, or inhibition of mitochondrial ROS generation enhanced all three types of GABA currents, with tonic currents comprising ∼50% of the total current. Application of hydrogen peroxide inhibited

  18. [Effects of estrogen on epidermis growth of mice and proliferation of human epidermal cell line HaCaT and its mechanism].

    PubMed

    Zhou, Tao; Chen, Jing; Huang, Zongwei; Fang, Li; Chen, Yu; Chen, Yajie; Peng, Yizhi

    2016-05-01

    To observe the effects of estrogen on epidermis growth of mice and proliferation of keratinocytes (human epidermal cell line HaCaT), and to explore its mechanism. (1) Five adult C57BL/6 mice in estrus cycle were identified by vaginal exfoliative cytology diagnosis and set as estrus group, while another 5 adult C57BL/6 mice with ovary resected before sexual development were set as ovariectomized group. The full-thickness skin from the tail root of mice in two groups were collected. The thickness of epidermis was observed and measured after HE staining. The distribution of proliferating cell nuclear antigen (PCNA)-positive cells in epidermis was observed by immunohistochemical staining, the number of which was counted. (2) HaCaT cells in logarithmic growth phase were cultured with RPMI 1640 nutrient solution containing 10% fetal bovine serum, and they were divided into negative control group (NC), pure estradiol group (PE), protein kinase B (Akt) inhibitor group (AI), and extracellular signal-regulated kinase (ERK) inhibitor group (EI) according to the random number table, with 20 wells in each group. To nutrient solution of each group, 1 μL dimethyl sulfoxide, 1 μL 17β-estradiol (100 nmol/L), 1 μL LY294002 (10 μmol/L), and 1 μL PD98059 (30 μmol/L) were added in group NC, group PE, group AI, and group EI respectively, and the last two groups were added with 1 μL 17β-estradiol (100 nmol/L) in addition. At post culture hour (PCH) 0 (immediately after culture), 24, 48, 72, 5 wells of cells from each group were collected to detect the proliferation activity of cells by cell counting kit 8 and microplate reader. (3) HaCaT cells in logarithmic growth phase were collected, grouped, and treated with the above-mentioned methods, with 3 wells in each group. At PCH 72, cell cycle distribution was detected by flow cytometer to calculate proliferation index (PI) of cells. (4) HaCaT cells in logarithmic growth phase were collected, grouped, and treated with the above

  19. Kinetics of low-density lipoprotein receptor activity in Hep-G2 cells: derivation and validation of a Briggs-Haldane-based kinetic model for evaluating receptor-mediated endocytotic processes in which receptors recycle.

    PubMed Central

    Harwood, H J; Pellarin, L D

    1997-01-01

    The process of receptor-mediated endocytosis for receptors that recycle to the cell surface in an active form can be considered as being kinetically analogous to that of a uni-substrate, uni-product enzyme-catalysed reaction. In this study we have derived steady-state initial-velocity rate equations for this process, based on classical Briggs-Haldane and King-Altman kinetic approaches to multi-step reactions, and have evaluated this kinetic paradigm, using as a model system the low-density lipoprotein (LDL)-receptor-mediated endocytosis of the trapped label [14C]sucrose-LDL in uninduced, steady-state Hep-G2 cells. Using the derived rate equations, together with experimentally determined values for Bmax (123 fmol/mg of cell protein), Kd (14.3 nM), the endocytotic rate constant ke (analogous to kcat; 0.163 min-1), Km (80 nM) and maximal internalization velocity (26.4 fmol/min per mg), we have calculated the ratio ke/Km (0.00204 nM-1.min-1), the bimolecular rate constant for LDL and LDL-receptor association (0. 00248 nM-1.min-1), the first-order rate constant for LDL-LDL-receptor complex dissociation (0.0354 min-1), the total cellular content of LDL receptors (154 fmol/mg of cell protein), the intracellular LDL receptor concentration (30.7 fmol/mg of cell protein) and the pseudo-first-order rate constant for LDL receptor recycling (0.0653 min-1). Based on this mathematical model, the kinetic mechanism for the receptor-mediated endocytosis of [14C]sucrose-LDL by steady-state Hep-G2 cells is one of constitutive endocytosis via independent internalization sites that follows steady-state Briggs-Haldane kinetics, such that LDL-LDL-receptor interactions are characterized by a rapid-high-affinity ligand-receptor association, followed by ligand-receptor complex internalization that is rapid relative to complex dissociation, and by receptor recycling that is more rapid than complex internalization and that serves to maintain 80% of cellular LDL receptors on the cell surface in

  20. γ-Aminobutyric Acid B Receptor Mediated Inhibition of Gonadotropin-Releasing Hormone Neurons Is Suppressed by Kisspeptin-G Protein-Coupled Receptor 54 Signaling

    PubMed Central

    Zhang, Chunguang; Bosch, Martha A.; Rønnekleiv, Oline K.; Kelly, Martin J.

    2009-01-01

    γ-Aminobutyric acid (GABA) is one of the most important neurotransmitters that regulate the excitability of GnRH neurons. Numerous studies have shown that GABA activates Cl− currents in GnRH neurons, and these effects are antagonized by GABAA receptor antagonists. The GABAB receptor is a heterodimer composed of GABAB R1 and R2, and although both subunits have been localized in GnRH neurons, nothing is known about the cellular signaling of this Gαi,o-coupled receptor in GnRH neurons. Using whole-cell recordings from mouse enhanced green fluorescent protein-GnRH neurons, we found that the GABAB receptor agonist baclofen hyperpolarized GnRH neurons through activation of an inwardly rectifying K+ current in a concentration-dependent manner. The effects of baclofen were antagonized by the selective GABAB receptor antagonist CGP 52432 with a Ki (inhibitory constant) of 85 nm. Furthermore, in the presence of the GABAA receptor antagonist picrotoxin, GABA hyperpolarized GnRH neurons in a similar manner. Treatment with 17β-estradiol as compared with oil vehicle did not significantly alter either the EC50 for the baclofen-induced response (0.8 ± 0.1 vs. 1.0 ± 0.1 μm, respectively) or the maximal outward current (10.8 ± 1.7 pA vs. 11.4 ± 0.6 pA, respectively) in GnRH neurons. However, the outward current (and membrane hyperpolarization) was abrogated by submaximal concentrations of the G protein-coupled receptor 54 (GPR54) agonist kisspeptin-10 in both groups, indicating that Gαq-coupled (GPR54) can desensitize the GABAB receptor-mediated response. Therefore, the activation of GABAB receptors in GnRH neurons may provide increased inhibitory tone during estrogen-negative feedback states that is attenuated by kisspeptin during positive feedback. PMID:19164470

  1. Estrogen signaling and unfolded protein response in breast cancer.

    PubMed

    Rajapaksa, Gayani; Thomas, Christoforos; Gustafsson, Jan-Åke

    2016-10-01

    Activation of the unfolded protein response (UPR) confers resistance to anti-estrogens and chemotherapeutics in estrogen receptor α (ERα)-positive and triple-negative breast cancers. Among the regulators of the UPR in breast cancer is estrogen signaling. Estrogen regulates major components of the UPR and ER expression is associated with the sensitivity of tumor cells to UPR-regulated apoptosis. Recent studies have confirmed the crosstalk between the ERs and UPR and suggest novel therapeutic strategies that combine targeting of both signaling pathways. These remedies may be more effective in repressing oncogenic adaptive mechanisms and benefit patients with resistant disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Assessment of estrogen receptor--histone interactions.

    PubMed Central

    Kallos, J; Fasy, T M; Hollander, V P

    1981-01-01

    Several different in vitro binding assays show that the estrogen receptor from rabbit uterus interacts selectively with purified histones from calf thymus. The estrogen receptor binds strongly to histones H2B and H2A, moderately to histones H3 and H4, and poorly to histone H1. In the presence of histones H2B or H2A, the position at which the estrogen receptor focuses in an isoelectric gradient is shifted to a more basic zone. Kinetic experiments show that, if histone H2B is bound to a DNA, the estrogen receptor dissociates more slowly from that DNA. The portion of the estrogen receptor molecule required for binding to histone H2B is relatively stable to tryptic digestion; in contrast, the portion of the receptor molecule responsible for DNA binding is promptly lost during limited tryptic digestion. These in vitro findings suggest that the mechanism by which the estrogen receptor selectively alters gene expression may involve specific contacts with histone molecules. PMID:6942408

  3. Phosphatidylethanolamine-binding protein is not involved in μ-opioid receptor-mediated regulation of extracellular signal-regulated kinase

    PubMed Central

    BIAN, JIA-MING; WU, NING; SU, RUI-BIN; LI, JIN

    2015-01-01

    Stimulation of the μ-opioid receptor activates extracellular signal-regulated kinase (ERK), however, the mechanism by which this occurs remains to be elucidated. Phosphatidylethanolamine-binding protein (PEBP) has been reported to act as a negative regulator of the ERK cascade (Raf-MEK-ERK) by binding to Raf-1 kinase. In the present study, the role of PEBP in μ-opioid receptor-mediated ERK activation was investigated in Chinese hamster ovary/μ cells and SH-SY5Y cells, as well as in human embryonic kidney 293 cells expressing other types of G protein-coupled receptors. The acute activation of μ-opioid receptors by morphine or (D-Ala2, MePhe4, Gly5-ol) enkephalin induced a rapid activation of ERK. Prolonged morphine treatment did not affect the phosphorylation level of ERK compared with control cells, but the phosphorylation level of ERK decreased markedly when cells were precipitated with naloxone following chronic morphine treatment. For the phosphorylation of PEBP, no change was identified under the designated drug treatment and exposure duration. A total of two other types of G protein-coupled receptors, including Gs-coupled dopamine D1 receptors and Gq-coupled adrenergic α1A receptors were also investigated and only the activation of adrenergic α1A receptors induced an upregulated phosphorylation of PEBP, which was protein kinase C activity dependent. Thus, PEBP did not have a significant role in μ-opioid receptor-mediated regulation of ERK. PMID:25573435

  4. GABAA receptor-mediated feedforward and feedback inhibition differentially modulate the gain and the neural code transformation in hippocampal CA1 pyramidal cells.

    PubMed

    Jang, Hyun Jae; Park, Kyerl; Lee, Jaedong; Kim, Hyuncheol; Han, Kyu Hun; Kwag, Jeehyun

    2015-12-01

    Diverse variety of hippocampal interneurons exists in the CA1 area, which provides either feedforward (FF) or feedback (FB) inhibition to CA1 pyramidal cell (PC). However, how the two different inhibitory network architectures modulate the computational mode of CA1 PC is unknown. By investigating the CA3 PC rate-driven input-output function of CA1 PC using in vitro electrophysiology, in vitro-simulation of inhibitory network, and in silico computational modeling, we demonstrated for the first time that GABAA receptor-mediated FF and FB inhibition differentially modulate the gain, the spike precision, the neural code transformation and the information capacity of CA1 PC. Recruitment of FF inhibition buffered the CA1 PC spikes to theta-frequency regardless of the input frequency, abolishing the gain and making CA1 PC insensitive to its inputs. Instead, temporal variability of the CA1 PC spikes was increased, promoting the rate-to-temporal code transformation to enhance the information capacity of CA1 PC. In contrast, the recruitment of FB inhibition sub-linearly transformed the input rate to spike output rate with high gain and low spike temporal variability, promoting the rate-to-rate code transformation. These results suggest that GABAA receptor-mediated FF and FB inhibitory circuits could serve as network mechanisms for differentially modulating the gain of CA1 PC, allowing CA1 PC to switch between different computational modes using rate and temporal codes ad hoc. Such switch will allow CA1 PC to efficiently respond to spatio-temporally dynamic inputs and expand its computational capacity during different behavioral and neuromodulatory states in vivo.

  5. Different mechanisms for resistance to trastuzumab versus lapatinib in HER2- positive breast cancers -- role of estrogen receptor and HER2 reactivation

    PubMed Central

    2011-01-01

    Introduction The human epidermal growth factor receptor 2 (HER2)-targeted therapies trastuzumab (T) and lapatinib (L) show high efficacy in patients with HER2-positive breast cancer, but resistance is prevalent. Here we investigate resistance mechanisms to each drug alone, or to their combination using a large panel of HER2-positive cell lines made resistant to these drugs. Methods Response to L + T treatment was characterized in a panel of 13 HER2-positive cell lines to identify lines that were de novo resistant. Acquired resistant lines were then established by long-term exposure to increasing drug concentrations. Levels and activity of HER2 and estrogen receptor (ER) pathways were determined by qRT-PCR, immunohistochemistry, and immunoblotting assays. Cell growth, proliferation, and apoptosis in parental cells and resistant derivatives were assessed in response to inhibition of HER or ER pathways, either pharmacologically (L, T, L + T, or fulvestrant) or by using siRNAs. Efficacy of combined endocrine and anti-HER2 therapies was studied in vivo using UACC-812 xenografts. Results ER or its downstream products increased in four out of the five ER+/HER2+ lines, and was evident in one of the two intrinsically resistant lines. In UACC-812 and BT474 parental and resistant derivatives, HER2 inhibition by T reactivated HER network activity to promote resistance. T-resistant lines remained sensitive to HER2 inhibition by either L or HER2 siRNA. With more complete HER2 blockade, resistance to L-containing regimens required the activation of a redundant survival pathway, ER, which was up-regulated and promoted survival via various Bcl2 family members. These L- and L + T-resistant lines were responsive to fulvestrant and to ER siRNA. However, after prolonged treatment with L, but not L + T, BT474 cells switched from depending on ER as a survival pathway, to relying again on the HER network (increased HER2, HER3, and receptor ligands) to overcome L's effects. The combination

  6. 2-Methoxyestradiol, an endogenous 17β-estradiol metabolite, inhibits microglial proliferation and activation via an estrogen receptor-independent mechanism.

    PubMed

    Schaufelberger, Sara A; Rosselli, Marinella; Barchiesi, Federica; Gillespie, Delbert G; Jackson, Edwin K; Dubey, Raghvendra K

    2016-03-01

    17β-Estradiol (estradiol) inhibits microglia proliferation. 2-Methoxyestradiol (2-ME) is an endogenous metabolite of estradiol with little affinity for estrogen receptors (ERs). We hypothesize that 2-ME inhibits microglial proliferation and activation and contributes to estradiol's inhibitory effects on microglia. We compared the effects of estradiol, 2-hydroxyestradiol [2-OE; estradiol metabolite produced by cytochrome P450 (CYP450)], and 2-ME [formed by catechol-O-methyltransferase (COMT) acting upon 2-OE] on microglial (BV2 cells) DNA synthesis, cell proliferation, activation, and phagocytosis. 2-ME and 2-OE were approximately three- and 10-fold, respectively, more potent than estradiol in inhibiting microglia DNA synthesis. The antimitogenic effects of estradiol were reduced by pharmacological inhibitors of CYP450 and COMT. Inhibition of COMT blocked the conversion of 2-OE to 2-ME and the antimitogenic effects of 2-OE but not 2-ME. Microglia expressed ERβ and GPR30 but not ERα. 2,3-Bis(4-hydroxyphenyl)-propionitrile (ERβ agonist), but not 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (ERα agonist) or G1 (GPR30 agonist), inhibited microglial proliferation. The antiproliferative effects of estradiol, but not 2-OE or 2-ME, were partially reversed by ICI-182,780 (ERα/β antagonist) but not by 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole (ERα antagonist) or G15 (GPR30 antagonist). Lipopolysaccharide increased microglia iNOS and COX-2 expression and phagocytosing activity of microglia; these effects were inhibited by 2-ME. We conclude that in microglia, 2-ME inhibits proliferation, proinflammatory responses, and phagocytosis. 2-ME partially mediates the effects of estradiol via ER-independent mechanisms involving sequential metabolism of estradiol to 2-OE and 2-ME. 2-ME could be of potential therapeutic use in postischemic stroke injuries. Interindividual differences in estradiol metabolism might affect the

  7. Sample preparation method for the ER-CALUX bioassay screening of (xeno-)estrogenic activity in sediment extracts.

    PubMed

    Houtman, Corine J; Leonards, Pim E G; Kapiteijn, Wendy; Bakker, Joop F; Brouwer, Abraham; Lamoree, Marja H; Legler, Juliette; Klamer, Hans J C

    2007-11-01

    The application of bioassays to assess the occurrence of estrogenic compounds in the environment is increasing in both a scientific and statutory context. The availability of appropriate validated methods for sample pre-treatment and analysis is crucial for the successful implementation of bioassays. Here, we present a sample preparation method for the bioassay screening of estrogenic activity in sediment with the in vitro Estrogen Receptor mediated Chemical Activated LUciferase gene eXpression (ER-CALUX) assay. The method makes use of an Accelerated Solvent (ASE) or Soxhlet extraction with a mixture of dichloromethane and acetone (3:1, v/v), followed by clean up of the extract by Gel Permeation Chromatography (GPC). Recoveries of a panel of 17 pollutants differing largely in physical-chemical properties from spiked sediment were determined and appeared to be on average about 86%. Furthermore, the estrogenic potencies of all test compounds were individually assessed by determination of concentration-response relationships in the ER-CALUX assay. Concentration dependent estrogenic potency was found for 14 of the 17 compounds, with potencies of about 10(5) to 10(7) fold lower than the natural estrogenic hormone 17beta-estradiol. Anti-estrogenic potency was assessed by testing combinations of estradiol and individual test compounds, but was found for none of the compounds. The low estrogenic activity of the test compounds in the spiking mixture was well recovered during GPC treatment of the pure mixture, but did not contribute significantly to the background estrogenic activity present in the spiked sediment. Application of the method to field samples showed that estrogenic activity can be found at different types of locations, and demonstrated that levels between locations may vary considerably over relatively short distances.

  8. Estrogen, schizophrenia and neurodevelopment.

    PubMed

    Seeman, Mary V

    2006-07-01

    Women are relatively protected against schizophrenia. The illness has a similar rate in women and men, but it starts later in women and is less severe. It is tempting to attribute this to the neuroprotective effect of estrogen, but the story is not straightforward and contains many unknowns. Women begin their schizophrenia trajectory later in development compared with men and this probably accounts for their relatively superior prognosis. Estrogen agonists are potential therapeutic agents but need to be proven safe, and the timing of administration may be crucial. This article examines what is known about estrogen and the development of schizophrenia.

  9. Estrogen Induces c-myc Gene Expression via an Upstream Enhancer Activated by the Estrogen Receptor and the AP-1 Transcription Factor

    PubMed Central

    Wang, Chunyu; Mayer, Julie Ann; Mazumdar, Abhijit; Fertuck, Kirsten; Kim, Heetae; Brown, Myles

    2011-01-01

    c-myc oncogene is implicated in tumorigenesis of many cancers, including breast cancer. Although c-myc is a well-known estrogen-induced gene, its promoter has no estrogen-response element, and the underlying mechanism by which estrogen induces its expression remains obscure. Recent genome-wide studies by us and others suggested that distant elements may mediate estrogen induction of gene expression. In this study, we investigated the molecular mechanism by which estrogen induces c-myc expression with a focus on these distal elements. Estrogen rapidly induced c-myc expression in estrogen receptor (ER)-positive breast cancer cells. Although estrogen had little effect on c-myc proximal promoter activity, it did stimulate the activity of a luciferase reporter containing a distal 67-kb enhancer. Estrogen induction of this luciferase reporter was dependent upon both a half-estrogen response element and an activator protein 1 (AP-1) site within this enhancer, which are conserved across 11 different mammalian species. Small interfering RNA experiments and chromatin immunoprecipitation assays demonstrated the necessity of ER and AP-1 cross talk for estrogen to induce c-myc expression. TAM67, the AP-1 dominant negative, partially inhibited estrogen induction of c-myc expression and suppressed estrogen-induced cell cycle progression. Together, these results demonstrate a novel pathway of estrogen regulation of gene expression by cooperation between ER and AP-1 at the distal enhancer element and that AP-1 is involved in estrogen induction of the c-myc oncogene. These results solve the long-standing question in the field of endocrinology of how estrogen induces c-myc expression. PMID:21835891

  10. H2 receptor-mediated facilitation and H3 receptor-mediated inhibition of noradrenaline release in the guinea-pig brain.

    PubMed

    Timm, J; Marr, I; Werthwein, S; Elz, S; Schunack, W; Schlicker, E

    1998-03-01

    , hippocampal or hypothalamic slices were used instead of cortical slices. The Ca2+-induced tritium overflow in guinea-pig cortex slices was inhibited by histamine (in the presence of ranitidine); this effect was abolished by clobenpropit. In slices superfused in the presence of clobenpropit, impromidine failed to facilitate the Ca2+-evoked tritium overflow. The electrically evoked tritium overflow in mouse brain cortex slices was inhibited by histamine by about 60% (both in the absence or presence of ranitidine). The inhibitory effect of histamine was abolished (but not reversed) by clobenpropit. In conclusion, noradrenaline release in the guinea-pig brain cortex is inhibited via presynaptic H3 receptors and facilitated via H2 receptors not located presynaptically. In the mouse brain cortex, only inhibitory H3 receptors occur. The extent of the H3 receptor-mediated effect is more marked in the mouse than in the guinea-pig brain cortex.

  11. Bone marrow oxytocin mediates the anabolic action of estrogen on the skeleton

    USDA-ARS?s Scientific Manuscript database

    Estrogen withdrawal in women due to natural or artificial menopause is followed by rapid bone loss, osteoporosis, and a high fracture risk. Replacement with estrogen prevents this bone loss and reduces the risk of fracture. Estrogen uses two mechanisms to exert this effect: it inhibits bone resorpti...

  12. Estrogenic and anti-estrogenic activity of 23 commercial textile dyes.

    PubMed

    Bazin, Ingrid; Ibn Hadj Hassine, Aziza; Haj Hamouda, Yosra; Mnif, Wissem; Bartegi, Ahgleb; Lopez-Ferber, Miguel; De Waard, Michel; Gonzalez, Catherine

    2012-11-01

    The presence of dyes in wastewater effluent of textile industry is well documented. In contrast, the endocrine disrupting effects of these dyes and wastewater effluent have been poorly investigated. Herein, we studied twenty-three commercial dyes, usually used in the textile industry, and extracts of blue jean textile wastewater samples were evaluated for their agonistic and antagonistic estrogen activity. Total estrogenic and anti-estrogenic activities were measured using the Yeast Estrogen Screen bioassay (YES) that evaluates estrogen receptor binding-dependent transcriptional and translational activities. The estrogenic potencies of the dyes and wastewater samples were evaluated by dose-response curves and compared to the dose-response curve of 17β-estradiol (E2), the reference compound. The dose-dependent anti-estrogenic activities of the dyes and wastewater samples were normalized to the known antagonistic effect of 4-hydroxytamoxifen (4-OHT) on the induction of the lac Z reporter gene by E2. About half azo textile dyes have anti-estrogenic activity with the most active being Blue HFRL. Most azo dyes however have no or weak estrogenic activity. E2/dye or E2/waste water ER competitive binding assays show activity of Blue HFRL, benzopurpurine 4B, Everzol Navy Blue FBN, direct red 89 BNL 200% and waste water samples indicating a mechanism of action common to E2. Our results indicate that several textile dyes are potential endocrine disrupting agents. The presence of some of these dyes in textile industry wastewater may thus impact the aquatic ecosystem. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Receptor-mediated uptake and transport of macromolecules in the human placenta.

    PubMed

    Schneider, Henning; Miller, Richard K

    2010-01-01

    The human placenta is required to be the anchor, the conduit and the controller during pregnancy. The survival of the baby and its associated placenta is dependent upon the placenta shielding the embryo/fetus from harm, e.g., autoimmune disease - thrombophilia, antiphospholipid syndrome or infections, while simultaneously providing for the passage of critical nutrients (e.g., amino acids, vitamins) and beneficial immunoglobulins. In a number of instances, the movements of macromolecules into and through the placenta can result in the passage of the intact molecules into the fetal circulation or in the case of proteins - catabolism to amino acids which are utilized by the placenta and the fetus for continued growth and development. The transfer of two such macromolecules, immunoglobulin G (IgG) and vitamin B12 (cyanocobalamin or B12), are examined as to the unique receptor-mediated transfer capability of the human placenta, its transfer specificity as related to specific receptors and the role of endogeneous placental proteins (trancobalamins) in facilitating the recognition and transport of specifically B12. Brief comparisons will be made to other animal species and the differences in specific organ transfer capabilities.

  14. Multivalent ligand-receptor-mediated interaction of small filled vesicles with a cellular membrane

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2017-07-01

    The ligand-receptor-mediated contacts of small sub-100-nm-sized lipid vesicles (or nanoparticles) with the cellular membrane are of interest in the contexts of cell-to-cell communication, endocytosis of membrane-coated virions, and drug (RNA) delivery. In all these cases, the interior of vesicles is filled by biologically relevant content. Despite the diversity of such systems, the corresponding ligand-receptor interaction possesses universal features. One of them is that the vesicle-membrane contacts can be accompanied by the redistribution of ligands and receptors between the contact and contact-free regions. In particular, the concentrations of ligands and receptors may become appreciably higher in the contact regions and their composition may there be different compared to that in the suspended state in the solution. A statistical model presented herein describes the corresponding distribution of various ligands and receptors and allows one to calculate the related change of the free energy with variation of the vesicle-engulfment extent. The results obtained are used to clarify the necessary conditions for the vesicle-assisted pathway of drug delivery.

  15. Beta receptor-mediated modulation of the late positive potential in humans.

    PubMed

    de Rover, Mischa; Brown, Stephen B R E; Boot, Nathalie; Hajcak, Greg; van Noorden, Martijn S; van der Wee, Nic J A; Nieuwenhuis, Sander

    2012-02-01

    Electrophysiological studies have identified a scalp potential, the late positive potential (LPP), which is modulated by the emotional intensity of observed stimuli. Previous work has shown that the LPP reflects the modulation of activity in extrastriate visual cortical structures, but little is known about the source of that modulation. The present study investigated whether beta-adrenergic receptors are involved in the generation of the LPP. We used a genetic individual differences approach (experiment 1) and a pharmacological manipulation (experiment 2) to test the hypothesis that the LPP is modulated by the activation of β-adrenergic receptors. In experiment 1, we found that LPP amplitude depends on allelic variation in the β1-receptor gene polymorphism. In experiment 2, we found that LPP amplitude was modulated by the β-blocker propranolol in a direction dependent on subjects' level of trait anxiety: In participants with lower trait anxiety, propranolol led to a (nonsignificant) decrease in the LPP modulation; in participants with higher trait anxiety, propranolol increased the emotion-related LPP modulation. These results provide initial support for the hypothesis that the LPP reflects the downstream effects, in visual cortical areas, of β-receptor-mediated activation of the amygdala.

  16. Selective antagonism of AMPA receptors unmasks kainate receptor-mediated responses in hippocampal neurons.

    PubMed

    Paternain, A V; Morales, M; Lerma, J

    1995-01-01

    Although both protein and mRNAs for kainate receptor subunits are abundant in several brain regions, the responsiveness of AMPA receptors to kainate has made it difficult to demonstrate the presence of functional kainate-type receptors in native cells. Recently, however, we have shown that many hippocampal neurons in culture express glutamate receptors of the kainate type. The large nondesensitizing response that kainate induces at AMPA receptors precludes detection and analysis of smaller, rapidly desensitizing currents induced by kainate at kainate receptors. Consequently, the functional significance of these strongly desensitizing glutamate receptors remains enigmatic. We report here that the family of new noncompetitive antagonists of AMPA receptors (GYKI 52466 and 53655) minimally affects kainate-induced responses at kainate receptors while completely blocking AMPA receptor-mediated currents, making it possible to separate the responses mediated by each receptor. These compounds will allow determination of the role played by kainate receptors in synaptic transmission and plasticity in the mammalian brain, as well as evaluation of their involvement in neurotoxicity.

  17. Substance P selectively modulates GABA(A) receptor-mediated synaptic transmission in striatal cholinergic interneurons.

    PubMed

    Govindaiah, G; Wang, Yanyan; Cox, Charles L

    2010-02-01

    Substance P (SP) is co-localized and co-released with gamma-amino butyric acid (GABA) from approximately 50% of GABAergic medium spiny neurons (MSNs) in the striatum. MSNs innervate several cellular targets including neighboring MSNs and cholinergic interneurons via collaterals. However, the functional role of SP release onto striatal interneurons is unknown. Here we examined SP-mediated actions on inhibitory synaptic transmission in cholinergic interneurons using whole-cell recordings in mouse corticostriatal slices. We found that SP selectively suppressed GABA(A) receptor-mediated inhibitory post-synaptic currents (IPSCs), but not excitatory post-synaptic currents (EPSCs) in cholinergic interneurons. In contrast, SP did not alter IPSCs in fast-spiking interneurons and MSNs. SP suppressed IPSC amplitude in a concentration-dependent and reversible manner, and the NK1 receptor antagonist RP67580 attenuated the SP-mediated suppression. In addition, RP67580 alone enhanced the evoked IPSC amplitude in cholinergic interneurons, suggesting an endogenous action of SP on regulation of inhibitory synaptic transmission. SP did not alter the paired-pulse ratio, but reduced the amplitudes of GABA(A) agonist muscimol-induced outward currents and miniature IPSCs in cholinergic interneurons, suggesting SP exerts its effects primarily at the post-synaptic site. Our results indicate that the physiological effects of SP are to enhance the activity of striatal cholinergic interneurons and provide a rationale for designing potential new antiparkinsonian agents.

  18. S-nitrosylated SHP-2 contributes to NMDA receptor-mediated excitotoxicity in acute ischemic stroke

    PubMed Central

    Shi, Zhong-Qing; Sunico, Carmen R.; McKercher, Scott R.; Cui, Jiankun; Feng, Gen-Sheng; Nakamura, Tomohiro; Lipton, Stuart A.

    2013-01-01

    Overproduction of nitric oxide (NO) can cause neuronal damage, contributing to the pathogenesis of several neurodegenerative diseases and stroke (i.e., focal cerebral ischemia). NO can mediate neurotoxic effects at least in part via protein S-nitrosylation, a reaction that covalently attaches NO to a cysteine thiol (or thiolate anion) to form an S-nitrosothiol. Recently, the tyrosine phosphatase Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) and its downstream pathways have emerged as important mediators of cell survival. Here we report that in neurons and brain tissue NO can S-nitrosylate SHP-2 at its active site cysteine, forming S-nitrosylated SHP-2 (SNO–SHP-2). We found that NMDA exposure in vitro and transient focal cerebral ischemia in vivo resulted in increased levels of SNO–SHP-2. S-Nitrosylation of SHP-2 inhibited its phosphatase activity, blocking downstream activation of the neuroprotective physiological ERK1/2 pathway, thus increasing susceptibility to NMDA receptor-mediated excitotoxicity. These findings suggest that formation of SNO–SHP-2 represents a key chemical reaction contributing to excitotoxic damage in stroke and potentially other neurological disorders. PMID:23382182

  19. Fluid shear stress sensitizes cancer cells to receptor-mediated apoptosis via trimeric death receptors

    NASA Astrophysics Data System (ADS)

    Mitchell, Michael J.; King, Michael R.

    2013-01-01

    Cancer metastasis, the process of cancer cell migration from a primary to distal location, typically leads to a poor patient prognosis. Hematogenous metastasis is initiated by intravasation of circulating tumor cells (CTCs) into the bloodstream, which are then believed to adhere to the luminal surface of the endothelium and extravasate into distal locations. Apoptotic agents such as tumor necrosis factor apoptosis-inducing ligand (TRAIL), whether in soluble ligand form or expressed on the surface of natural killer cells, have shown promise in treating CTCs to reduce the probability of metastasis. The role of hemodynamic shear forces in altering the cancer cell response to apoptotic agents has not been previously investigated. Here, we report that human colon cancer COLO 205 and prostate cancer PC-3 cells exposed to a uniform fluid shear stress in a cone-and-plate viscometer become sensitized to TRAIL-induced apoptosis. Shear-induced sensitization directly correlates with the application of fluid shear stress, and TRAIL-induced apoptosis increases in a fluid shear stress force- and time-dependent manner. In contrast, TRAIL-induced necrosis is not affected by the application fluid shear stress. Interestingly, fluid shear stress does not sensitize cancer cells to apoptosis when treated with doxorubicin, which also induces apoptosis in cancer cells. Caspase inhibition experiments reveal that shear stress-induced sensitization to TRAIL occurs via caspase-dependent apoptosis. These results suggest that physiological fluid shear forces can modulate receptor-mediated apoptosis of cancer cells in the presence of apoptotic agents.

  20. Different receptors mediate the electrophysiological and growth cone responses of an identified neuron to applied dopamine.

    PubMed

    Dobson, K S; Dmetrichuk, J M; Spencer, G E

    2006-09-15

    Neurotransmitters are among the many cues that may guide developing axons toward appropriate targets in the developing nervous system. We have previously shown in the mollusk Lymnaea stagnalis that dopamine, released from an identified pre-synaptic cell, differentially affects growth cone behavior of its target and non-target cells in vitro. Here, we describe a group of non-target cells that also produce an inhibitory electrophysiological response to applied dopamine. We first determined, using pharmacological blockers, which receptors mediate this physiological response. We demonstrated that the dopaminergic electrophysiological responses of non-target cells were sensitive to a D2 receptor antagonist, as are known target cell responses. However, the non-target cell receptors were linked to different G-proteins and intracellular signaling pathways than the target cell receptors. Despite the presence of a D2-like receptor at the soma, the growth cone collapse of these non-target cells was mediated by D1-like receptors. This study shows that different dopamine receptor sub-types mediated the inhibitory physiological and growth cone responses of an identified cell type. We therefore not only provide further evidence that D2- and D1-like receptors can be present on the same neuron in invertebrates, but also show that these receptors are likely involved in very different cellular functions.

  1. Optimization of stress response through the nuclear receptor-mediated cortisol signalling network

    PubMed Central

    Kolodkin, Alexey; Sahin, Nilgun; Phillips, Anna; Hood, Steve R.; Bruggeman, Frank J.; Westerhoff, Hans V.; Plant, Nick

    2013-01-01

    It is an accepted paradigm that extended stress predisposes an individual to pathophysiology. However, the biological adaptations to minimize this risk are poorly understood. Using a computational model based upon realistic kinetic parameters we are able to reproduce the interaction of the stress hormone cortisol with its two nuclear receptors, the high-affinity glucocorticoid receptor and the low-affinity pregnane X-receptor. We demonstrate that regulatory signals between these two nuclear receptors are necessary to optimize the body’s response to stress episodes, attenuating both the magnitude and duration of the biological response. In addition, we predict that the activation of pregnane X-receptor by multiple, low-affinity endobiotic ligands is necessary for the significant pregnane X-receptor-mediated transcriptional response observed following stress episodes. This integration allows responses mediated through both the high and low-affinity nuclear receptors, which we predict is an important strategy to minimize the risk of disease from chronic stress. PMID:23653204

  2. M2-like macrophages are responsible for collagen degradation through a mannose receptor-mediated pathway.

    PubMed

    Madsen, Daniel H; Leonard, Daniel; Masedunskas, Andrius; Moyer, Amanda; Jürgensen, Henrik Jessen; Peters, Diane E; Amornphimoltham, Panomwat; Selvaraj, Arul; Yamada, Susan S; Brenner, David A; Burgdorf, Sven; Engelholm, Lars H; Behrendt, Niels; Holmbeck, Kenn; Weigert, Roberto; Bugge, Thomas H

    2013-09-16

    Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase-dependent manner and was subsequently routed to lysosomes for complete degradation. Collagen uptake was predominantly executed by a quantitatively minor population of M2-like macrophages, whereas more abundant Col1a1-expressing fibroblasts and Cx3cr1-expressing macrophages internalized collagen at lower levels. Genetic ablation of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor-associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies a key role of M2-like macrophages in this process.

  3. Endothelial adenosine A2a receptor-mediated glycolysis is essential for pathological retinal angiogenesis.

    PubMed

    Liu, Zhiping; Yan, Siyuan; Wang, Jiaojiao; Xu, Yiming; Wang, Yong; Zhang, Shuya; Xu, Xizhen; Yang, Qiuhua; Zeng, Xianqiu; Zhou, Yaqi; Gu, Xuejiao; Lu, Sarah; Fu, Zhongjie; Fulton, David J; Weintraub, Neal L; Caldwell, Ruth B; Zhang, Wenbo; Wu, Chaodong; Liu, Xiao-Ling; Chen, Jiang-Fan; Ahmad, Aftab; Kaddour-Djebbar, Ismail; Al-Shabrawey, Mohamed; Li, Qinkai; Jiang, Xuejun; Sun, Ye; Sodhi, Akrit; Smith, Lois; Hong, Mei; Huo, Yuqing

    2017-09-19

    Adenosine/adenosine receptor-mediated signaling has been implicated in the development of various ischemic diseases, including ischemic retinopathies. Here, we show that the adenosine A2a receptor (ADORA2A) promotes hypoxia-inducible transcription factor-1 (HIF-1)-dependent endothelial cell glycolysis, which is crucial for pathological angiogenesis in proliferative retinopathies. Adora2a expression is markedly increased in the retina of mice with oxygen-induced retinopathy (OIR). Endothelial cell-specific, but not macrophage-specific Adora2a deletion decreases key glycolytic enzymes and reduces pathological neovascularization in the OIR mice. In human primary retinal microvascular endothelial cells, hypoxia induces the expression of ADORA2A by activating HIF-2α. ADORA2A knockdown decreases hypoxia-induced glycolytic enzyme expression, glycolytic flux, and endothelial cell proliferation, sprouting and tubule formation. Mechanistically, ADORA2A activation promotes the transcriptional induction of glycolytic enzymes via ERK- and Akt-dependent translational activation of HIF-1α protein. Taken together, these findings advance translation of ADORA2A as a therapeutic target in the treatment of proliferative retinopathies and other diseases dependent on pathological angiogenesis.Pathological angiogenesis in the retina is a major cause of blindness. Here the authors show that adenosine receptor A2A drives pathological angiogenesis in the oxygen-induced retinopathy mouse model by promoting glycolysis in endothelial cells via the ERK/Akt/HIF-1α pathway, thereby suggesting new therapeutic targets for disease treatment.

  4. MAGI-1 acts as a scaffolding molecule for NGF receptor-mediated signaling pathway.

    PubMed

    Ito, Hidenori; Morishita, Rika; Iwamoto, Ikuko; Mizuno, Makoto; Nagata, Koh-ichi

    2013-10-01

    We have recently found that the membrane-associated guanylate kinase with inverted organization-1 (MAGI-1) was enriched in rat nervous tissues such as the glomeruli in olfactory bulb of adult rats and dorsal root entry zone in spinal cord of embryonic rats. In addition, we revealed the localization of MAGI-1 in the growth cone of the primary cultured rat dorsal root ganglion cells. These results point out the possibility that MAGI-1 is involved in the regulation of neurite extension or guidance. In this study, we attempted to reveal the physiological role(s) of MAGI-1 in neurite extension. We found that RNA interference (RNAi)-mediated knockdown of MAGI-1 caused inhibition of nerve growth factor (NGF)-induced neurite outgrowth in PC12 rat pheochromocytoma cells. To clarify the involvement of MAGI-1 in NGF-mediated signal pathway, we tried to identify binding partners for MAGI-1 and identified p75 neurotrophin receptor (p75NTR), a low affinity NGF receptor, and Shc, a phosphotyrosine-binding adaptor. These three proteins formed an immunocomplex in PC12 cells. Knockdown as well as overexpression of MAGI-1 caused suppression of NGF-stimulated activation of the Shc-ERK pathway, which is supposed to play important roles in neurite outgrowth of PC12 cells. These results indicate that MAGI-1 may act as a scaffolding molecule for NGF receptor-mediated signaling pathway.

  5. The effect of vanadate on receptor-mediated endocytosis of asialoorosomucoid in rat liver parenchymal cells

    SciTech Connect

    Kindberg, G.M.; Gudmundsen, O.; Berg, T. )

    1990-06-05

    Vanadate is a phosphate analogue that inhibits enzymes involved in phosphate release and transfer reactions. Since such reactions may play important roles in endocytosis, we studied the effects of vanadate on various steps in receptor-mediated endocytosis of asialoorosomucoid labeled with 125I-tyramine-cellobiose (125I-TC-AOM). The labeled degradation products formed from 125I-TC-AOM are trapped in the lysosomes and may therefore serve as lysosomal markers in subcellular fractionation studies. Vanadate reduced the amount of active surface asialoglycoprotein receptors approximately 70%, but had no effect on the rate of internalization and retroendocytosis of ligand. The amount of surface asialoglycoprotein receptors can be reduced by lowering the incubation temperature gradually from 37 to 15 degrees C; vanadate affected only the temperature--sensitive receptors. Vanadate inhibited degradation of 125I-TC-AOM 70-80%. Degradation was much more sensitive to vanadate than binding; half-maximal effects were seen at approximately 1 mM vanadate for binding and approximately 0.1 mM vanadate for degradation. By subcellular fractionation in sucrose and Nycodenz gradients, it was shown that vanadate completely prevented the transfer of 125I-TC-AOM from endosomes to lysosomes. Therefore, the inhibition of degradation by vanadate was indirect; in the presence of vanadate, ligand did not gain access to the lysosomes. The limited degradation in the presence of vanadate took place in a prelysosomal compartment. Vanadate did not affect cell viability and ATP content.

  6. FLIP ing the coin? Death receptor-mediated signals during skin tumorigenesis.

    PubMed

    Leverkus, Martin; Diessenbacher, Philip; Geserick, Peter

    2008-07-01

    Keratinocyte skin cancer is a multi-step process, during which a number of obstacles have to be overcome by the tumor cell to allow the development of a manifest tumor. Beside proliferation and immortality, apoptosis resistance is one additional and critical step during skin carcinogenesis. Over the past two decades, much has been learned about the prototypical membrane-bound inducers of apoptosis, namely the death receptors and their ligands, and the apoptosis signalling pathways activated by death receptors have been elucidated in great detail. In contrast, much less is known about the tissue-specific role of the death receptor/ligands systems during the development of skin cancer. Here, we summarize and discuss the role of this intriguing receptor family and the potential mechanistical impact of the intracellular caspase-8 inhibitor cFLIP for keratinocyte skin cancer. Given more recent data about cFLIP and its isoforms, a more complex regulatory role of cFLIP can be suspected. Indeed, cFLIP may not solely interfere with death receptor-mediated apoptosis signalling pathways, but may positively or negatively influence other, potential harmful signalling pathways such as the production of inflammatory cytokines, tumor cell migration or the activation of transcription factors such as NF-kappaB, considered crucial during skin tumorigenesis. In this respect, cFLIP may act to 'FLIP the coin' during the development of keratinocyte skin cancer.

  7. Hormone stimulation of androgen receptor mediates dynamic changes in DNA methylation patterns at regulatory elements

    PubMed Central

    Dhiman, Vineet K.; Attwood, Kristopher; Campbell, Moray J.; Smiraglia, Dominic J.

    2015-01-01

    DNA methylation is an epigenetic modification that contributes to stable gene silencing by interfering with the ability of transcriptional regulators to bind to DNA. Recent findings have revealed that hormone stimulation of certain nuclear receptors induces rapid, dynamic changes in DNA methylation patterns alongside transcriptional responses at a subset of target loci, over time. However, the ability of androgen receptor (AR) to dynamically regulate gene transcription is relatively under-studied and its role in the regulation of DNA methylation patterns remains to be elucidated. Here we demonstrate in normal prostate cells that hormone stimulated AR activity results in dynamic changes in the transcription rate and DNA methylation patterns at the AR target genes, TIPARP and SGK1. Time-resolved chromatin immunoprecipitation experiments on the SGK1 locus reveals dynamic recruitment of AR and RNA Polymerase II, as well as the recruitment of proteins involved in the DNA demethylation process, TET1 and TDG. Furthermore, the presence of DNA methylation at dynamic regions inhibits protein binding and transcriptional activity of SGK1. These findings establish AR activity as a contributing factor to the dynamic regulation of DNA methylation patterns at target genes in prostate biology and infer further complexity involved in nuclear receptor mediation of transcriptional regulation. PMID:26646795

  8. Spinal GABA receptors mediate the suppressive effect of electroacupuncture on cold allodynia in rats.

    PubMed

    Park, Jung-Hyun; Han, Jae-Bok; Kim, Sun-Kwang; Park, Jung-Hyuk; Go, Dong-Hyun; Sun, Boram; Min, Byung-Il

    2010-03-31

    This study was performed to determine whether spinal GABAergic systems mediate the relieving effects of low frequency electroacupuncture (EA) on cold allodynia in a rat tail model of neuropathic pain. For neuropathic surgery, the right superior caudal trunk was resected at the level between the S1 and S2 spinal nerves innervating the tail. Two weeks after the nerve injury, the intrathecal catheter was implanted. Five days after the catheterization, rats were intrathecally injected with gabazine (GABA(A) receptor antagonist, 0.0003, 0.001 or 0.003mug), or saclofen (GABA(B) receptor antagonist, 3, 10 or 30mug). Ten minutes after the injection, EA (2Hz) was applied to the ST36 acupoint for 30min. The cold allodynia was assessed by the tail immersion test (i.e. immersing the tail in cold (4 degrees C) water and measuring the latency of an abrupt tail movement) before and after the EA treatment. EA stimulation at ST36 significantly inhibited the cold allodynia sign, whereas EA at non-acupoint and plain acupuncture at ST36 (without electrical stimulation) did not show antiallodynic effects. Intrathecal administration of gabazine or saclofen blocked the relieving effects of ST36 EA stimulation on cold allodynia. These results suggest that spinal GABA(A) and GABA(B) receptors mediate the suppressive effect of low frequency EA on cold allodynia in the tail neuropathic rats.

  9. Accumbens shell AMPA receptors mediate expression of extinguished reward seeking through interactions with basolateral amygdala.

    PubMed

    Millan, E Zayra; McNally, Gavan P

    2011-07-01

    Extinction is the reduction in drug seeking when the contingency between drug seeking behavior and the delivery of drug reward is broken. Here, we investigated a role for the nucleus accumbens shell (AcbSh). Rats were trained to respond for 4% (v/v) alcoholic beer in one context (Context A) followed by extinction in a second context (Context B). Rats were subsequently tested in the training context, A (ABA), or the extinction context, B (ABB). Pre-test injections of the glutamate AMPA receptor antagonist, NBQX (1 µg) into AcbSh had no effect on renewal of alcoholic beer seeking when rats were returned to the training context (ABA). However, NBQX increased responding when rats were tested in the extinction context (ABB). In a second experiment, rats received training, extinction, and test in the same context. Pre-test injections of NBQX (0, 0.3, and 1 µg) into the AcbSh dose-dependently attenuated expression of extinction. We also found that NBQX in the AcbSh had no effect on initial acquisition of extinction or the motivation to respond for reward as measured by break point on a progressive ratio schedule. Finally, we show that pharmacological disconnection of a basolateral amygdala (BLA) → AcbSh pathway via NBQX in AcbSh combined with reversible inactivation